The Pineal Gland and Its Endocrine Role (PDFDrive)

NATO Advanced Science Institutes Series
J. Axelrod · F. Fraschini
G.P. Velo Editors
Pineal Gland and

Its Endocrine Role
The Pineal Gland and
Its Endocrine Role
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The Pineal Gland and
its Endocrine Role
Edited by
J. Axelrod
National Institute of Mental Health
Bethesda, Maryland
F. Fraschini
University of Milan
Milan, Italy
and
G. P. Vela
University of Verona
Verona, Italy
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Proceedings of a NATO Advanced Study Institute on
the Pineal Gland and lts Endocrine Role,
held June 21-July 2, 1982,
at the Ettore Majorana Center,
in Erice, Sicily, ltaly
Library of Congress Cataloging in Publication Data

NATO Advanced Study Institute on the Pineal Gland and lts Endocrine Role (1982: Ettore
Majorana International Centre for Scientific Culture)
The pineal gland and its endocrine role.
(NATO advanced science institutes role. Series A, Life science; v. 65)
"Published in cooperation with NATO Scientific Affairs Division."
"Proceedings of a NATO Advanced Study Institute on the Pineal Gland and lts Endocrine
Role, held June 21-July 2, 1982, at the Ettore Majorana Center, in Erice, Sicily, ltaly"
-T.p. verso.
lncludes bibliographical references and index.
1. Pineal body-Congresses. 2. Melatonin-Physiologi cal effect-Congresses. I. Ax-
elrod, Julius, 1912- II. Fraschini, F. 111. Velo, G. P. IV. North Atlantic Treaty
Organization. Scientific Affairs Division. V. Title. VI. Series. [DNLM: 1. Pineal body-Con-
gresses. 2. Melatonin-Congresse s. WK 350 N279p 1982)
[QP188.P55N37 1982) 599'01'42 83-8134
ISBN 978-1-4757-1453-1 ISBN 978-1-4757-1451-7 (eBook)
DOI 10.1007/978-1-4757- 1451-7
© 1983 Springer Science+Business Media New York

Originally published by Plenum Press, New York in 1983
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PREFACE
The pineal gland has been a subject of interest and speculation

for more than 2000 years. Greek anatomists were impressed by the ob-
servation that the pineal gland is an unpaired structure and they
believed that it regulated the flow of thoughts. The philosopher
Descartes proposed an important role for this organ in brain function.
At the beginning of the 20th century experiments by several investi-
gators indicated that the pineal influenced sexual function and skin
pigmentation and was also responsive to light signals. With the iso-
lation of melatonin from bovine pineal glands by Lerner and cowork-
ers in 1958 the modern era of pineal research was initiated.
Within a few years the pathway for the biosynthesis of melatonin

in the pineal was elucidated. Soon thereafter it was shown that the
formation of melatonin was influenced by environmental lighting. Ana-
tomists found that the pineal was innervated by sympathetic nerves
and that the gland had photoreceptor elements. It was also shown that
the gonads were influenced by light via the pineal gland. Research on
the pineal gland became of increasing interest to anatomists, bioche-
mists, pharmacologists and endocrinologists. With the expanding know-
ledge concerning the function of the pineal gland contributed by the
wide variety of disciplines, it was thought that a study workshop
would be timely.
For this study group, a limited number of investigators in anato-

my, biochemistry, pharmacology, endocrinology and clinical medicine
who were working on the pineal were asked to come together to tell
about their investigations, exchange ideas and to suggest new direc-
tions in research. Also attending the meeting were graduate students
and postdoctoral fellows. The Study Institute on "The Pineal and its
Endocrine Role" was held at Erice, Sicily at the "Centro Ettore Maj-
rana" and sponsored by NATO. The meeting fully satisfied our expecta-
tions. During 9 days of meeting, the pineal was discussed from many
v
vi PREFACE
points of view. There was an informative and stimulating exchange

by leading experts as well as junior investigators and graduate
students. The lecture and discussion provided provocative hypotheses
and suggestions for new research directions. Especially interesting
was the potential role of the pineal in clinical medicine. The pre-
sent volume includes the proceedings of this meeting as well as re-
views on anatomy, biochemistry, physiology and endocrinology of the
pineal gland and the most recent data concerning this gland.
The organizers and participants are grateful to NATO for their

support. We also thank the "Centro Ettore Majorana" for their gra-
cious hospitality. The lovely setting of Erice provided a peaceful
place for a fruitful scientific meeting.
J. Axelrod, F. Fraschini and G.P. Velo

CONTENTS
INTRODUCTORY LECTURE
Regulation of Circadian Rhythms of Indoleamines in

Pineal Gland. .. . . . . . . . . . . 1
Julius Axelrod
ANATOMY
Aspects of Evolution of the Pineal Organ. 15

Andreas Oksche
Comparative Gross and Fine Morphology of the Mammalian

Pineal Gland. . . . . . . . . . . . . . . . . . . 37
J. Ariens Kappers
Perspectives of Comparative Anatomy of the Mammalian

Pineal Gland. . . . . . . . . . . . . . . . . . 61
Lutz Vollrath
The Use of Electron Microscopy and Stereology in the

Study of the Mammalian Pineal Gland . . . . . . 71
Lutz Vollrath
Innervation of the Vertebrate Pineal Organ. . . . . . . . . . 87

J. Ariens Kappers
The Different Classes of Proteic and Peptidic Substances

Present in the Pineal Gland . . . . . . . . . . . . 113
Paul Pevet
BIOCHEMISTRY
Pteridines in the Pineal and Effects of These Substances

on the Indole Metabolism of This Organ. 151
I. Ebels, M.G.M. Balemans, J. van Benthem, H.P.J.M.
Noteborn, and A. de Moree
vii
viii CONTENTS
Peptidic and Proteic Substances Isolated from Pineals

and Their Relation to the Hypophysial-Hypothalamic-
Gonadal Axis . . . . . . . 179
I. Ebels
Molecular Aspects of Neuroendocrine Integrative Processes

in the Pineal Gland . . . . . . . . . . . . . . . . 199
D.P. Cardinali, M.N. Ritta, M.I. Vacas, P.R. Lowenstein,
P.V. Gejman, C. Gonzales Solveyra, and E. Pereyra
The Responses of Melatonin Rhythms to Environmental

Lighting. . . . . . . . . . . . . . 221
R.J. Wurtman, M.H. Deng, and P. Ronsheim
The Role of Light and Age in Determining Melatonin

Production in the Pineal Gland. . . . . . . . 227
Russel J. Reiter
Differential Regulation of the 24 Hour Pattern of Serum

Melatonin and N-Acetylserotonin . . . . . . . . 243
G.M. Brown, L.J. Grota, L. Harvey, H.W. Tsui, and
S.F. Pang
PHYSIOLOGY
Differential Localization of Melatonin and N-Acetylserotonin

in Brain. . . . . . . . . . . . . . . . . . . 257
G.M. Brown, O. Pulido, L.P. Niles, S. Psarakis,
A. Porietis, G.A. Bubenik, and L.J. Grota
Melatonin Action: Sites and Possible Mechanisms in Brain. 277

D.P. Cardinali, M.I. Vacas, M.I. Keller Sarmiento, and
E. Morguenstern
Seasonal Reproductive Events Related to the Pineal Gland . . , 303

Russel J. Reiter
Melatonin as the Hormone Which Mediates the Effects of the

Pineal Gland on Neuroendocrine-Reproductive Axis of
the Syrian Hamster. • . . . . . . . . . . . . . . . . 317
Russel J. Reiter
The 5-Methoxyindoles Different from Melatonin: Their

Effects on the Sexual Axis . . . . . . . . . . . 331
Paul Pevet
Actions of the Pineal Gland and Melatonin on the

Secretion of Cerebrospinal Fluid. .
W.B. Quay
CONTENTS ix
Pineal-Hypothalamic Interactions: Possible Role of the

Monoaminergic Neuron System. . . . . . . . . . . 361
B. Mess, G.P. Trentini, C. Ruzsas, and C.F. De
Gaetani
Humoral Interrelations of the Pineal Gland With Lateral

Eyes and Orbital Glands. . . . . . . . . . . . . 395
W.B. Quay
Modification of Hypothalamic Electrical Activity by

Pineal Indoles . . . . . . . . . . . . . . . . . . . 417
C. Demaine
Neurobiological Investigations of the Pineal Gland

and Its Hormone Melatonin. . . . . . . . ..... 437
P. Semm
Influence of the Pineal Gland on Tumor Growth in Mammalians:

a Reappraisal from A Biochemical Point of View . . . . . 467
M.E. Ferioli, G. Scalabrino, and F. Fraschini
Interactions Between Pineal and Non-reproductive Endocrine

Glands . . . . . . . . . . . . . . . . . . . . . . . . . 477
B. Mess
CLINICAL ASPECTS
The Pineal Gland and Sexual Function in Man. . . . . . . . . . 509

M. Poth, S. Higa, and S. Markey
Melatonin Secretion -- A Biological Marker for Human

Pineal Adrenergic Function . . . . . . . . . . 521
A.J. Lewy
Human Melatonin Secretion, Its Endogenous Circadian Pace-

maker and the Effects of Light . . . . . . . . . . . . . 535
A.J. Lewy
The Secretion and Effects of Melatonin in Humans . . . . . . . 551

R.J. Wurtman, F. Waldhauser, and H.R. Lieberman
Melatonin as a Chronobiological Marker in Health and

Disease. . . . . . . . . . . . . . . . " . . . . . 575
L. Wetterberg
Index. . '. . . . . . . . . . . . . . . . . . . . . . . . . . . 589

REGULATION OF CIRCADIAN RHYTHMS OF INDOLEAMINES IN THE PINEAL GLAND
Julius Axelrod
Section on Pharmacology, Laboratory of Clinical Science
National Institute of Mental Health, Bethesda, Maryland
20205
Although the pineal gland has been recognized for many centuries
it was the discovery of melatonin that opened the modern era of re-
search in this organ. McCord and Allen had observed in 1927 that an
extract of the bovine pineal can blanch the skin of tadpoles. This
prompted Lerner, a dermatologist and biochemist interested in pigmen-
ation, to isolate the active blanching factor of the pineal (Lerner
et al., 1978). The active blanching principle of the pineal organ
was isolated and identified as 5-methoxY-N-acetyltryamine (which
was named melatonin). Because of my interest in both indoles and
transmethylation reaction, I initiated studies on the biosynthesis
and regulation of melatonin metabolism in the pineal. Together with
Wurtman we found that the pineal gland can act as a neuroendocrine
transducer converting neuronal signals, which are controlled by
environmental lighting, into endocrine messages. This led to the
formul ati on of the "mel atonin hypothesi sOl in which we proposed that
the pineal gland is influenced by light-dark cycles to regulate the
synthesis of melatonin, a compound that acts at distant target
organs (Wurtman and Axelrod, 1965). Further work in our laboratory
led to the exploration of this gland to study circadian rhythms and
the beta-adrenergic receptors.
THE BIOSYNTHESIS AND METABOLISM OF MELATONIN IN THE PINEAL GLAND
Soon after the discovery of melatonin, Weissbach and I began
studies on the biosynthesis of this indole. We soon isolated and
purified an enzyme from bovine pineal which O-methylated several
indoles (Axelrod and Weissbach, 1961) (Table 1). Although many
hydroxy indoles could be O-methylated, N-acetylserotonin was by far
2 J. AXELROD
Table 1. Substrate Specificity of Hydroxyindole-O-Methyltransferase
Substrate Relative Activity

N-acetylserotonin 100
Serotonin 7
5-Hydroxydimethyltryptamine 14
5-Hydroxyindole acetic acid 12
4-Hydroxy-N-acetyltryptamine o
6-Hydroxydimethyltryptamine o
Purified enzyme obtained from bovine pineal was incubated with

[14C]methyl-S-adenosylmethionine and substrate at pH 7.9. After one
hour incubation the reaction mixture was examined for [14C]-labelled
methyl derivative after extraction into organic solvent (from Axelrod
and Weissbach, 1961).
the best substrate for this enzyme which we named hydroxyindole-

O-methyltransferase (HIOMT). The isolation of HIOMT was in great
part due to the synthesis of the radioactive methyl donor [14C]_
methyl-S-adenosyl-L-methionine. This made it possible to label
and isolate the transmethylated product [14C]-methoxymelatonin
from a bovine pineal gland preparation. HIOMT was found to be
highly local ized in the pineal gland of mammal s, birds and -amphib-
ians. This observation convinced me that this organ is biologi-
cally active and worthy of further studies. We also found an
enzyme in the pineal that converted serotonin to N-acetylserotonin
(Weissbach et al., 1961). In view of these findings it was propos-
ed that melatonin is synthesized almost exclusively in the pineal
gland as foll ows: tryptophan _ 5-hydroxytryptophan ..
serotonin .. N-acetylserotonin _ melatonin. To
study the fate of melatonin in the body Kopin and I synthesized
radioactive melatonin which was labelled on both the indole ring
and acetyl group (Kopin et al, 1961). After the injection,
melatonin was almost completely metabolized in the body. The
major route of melatonin was via hydroxylation to form 6-hydroxy-
melatonin. This transformation was carried out by an enzyme in
the liver microsomes that required NADPH and oxygen.
EFFECTS OF LIGHT ON INDOLEAMINE METABOLISM IN THE PINEAL
It has been long known that environmental lighting has an
effect on the pineal. Holmgren (1918) had found that the amphibian
pineal has photoreceptors and later it was reported that these re-
ceptors responded to light. Fiske et al. (1960) observed that
continuous light reduced the weight of the rat pineal and increased
CIRCADIAN RHYTHMS OF INDOLEAMINES IN PINEAL GLAND 3
that of the ovaries. Pineal extracts were then found to decrease

light induced hypertrophy of the ovaries and persistent estrus in
the rat (Wurtman et a1., 1961). These findings indicated a rela-
tionship between light, the pineal and ovaries. When Wurtman
joined my laboratory we found that injecting melatonin into rats
had the same effect on the rat gonads as bovine pineal extracts.
These results suggested that melatonin could be a putative gonadal
hormone secreted by the pineal and that light influences its forma-
tion. Since we had just described the route of biosynthesis of
melatonin in the pineal, the effect of light on the intermediate
metabolism of this indole was studied.
We first examined the effect of continuous light and darkness
on the melatonin forming enzyme, HIOMT (Wurtman et a1., 1963). Rats
kept in constant light showed a decrease in enzyme activity as
compared to those in darkness. This indicated that constant light
suppressed the activity of HIOMT and this would reduce the produc-
tion of the melatonin, a gonad inhibiting compound. How did environ-
mental lighting affect the enzymes in the pineal gland which lies
deep between two cerebral hemispheres? Kappers (1960) had, a
few years previously, found that the mammalian pineal gland is
innervated by sympathetic neurons arising in the superior cervical
ganglia. This provided an opportunity to trace that pathway by
which light signals relay their messages to the pineal gland
(Wurtman et a1., 1964). Blinded rats prevented the reduction of
pineal HIOMT in rats kept in constant light. This indicated that
the retina is necessary for environmental light signals to reach
the pineal gland. The sympathetic nerve terminals innervating the
pineal gland were then destroyed by the bilateral removal of the
superior cervical ganglia. Upon denervation there was no longer a
difference in HIOMT in rats kept in light or darkness. In another
experiment the pregang1 ion.ic nerves between the brain and the
superior cervical ganglia were cut. This also abolished the effect
of light and darkness on the melatonin forming enzyme. All these
experiments indicated that information about environmental lighting
that affects HIOMT reaches the brain via the retina, brain, superior
cervical glang1ia and sympathetic noradrenergic nerves. The above
observations led to the proposal that the pineal gland is a neuro-
endocrine transducer transforming neuronal signals concerning en-
vironmental lighting from noradrenergic sympathetic nerves to
endocrine signals (melatonin) acting on distant organs (gonads)
(Wurtman and Axelrod, 1965).
CIRCADIAN RHYTHMS OF INDOLEAMINES IN THE PINEAL GLAND
In 1963 Quay reported that the levels of serotonin in the
pineal were high during the daytime and low during the night. Since
acety1ated serotonin is the precursor of melatonin, this result
was consistent with our findings that the melatonin-forming
4 J. AXELROD
enzyme was higher at night than during the daytime. Together with
Snyder, a postdoctoral fellow in my laboratory, a extremely sensi-
tive assay for serotonin was developed (Snyder et a1., 1965) which
enabled us to explore the controlling mechanisms for the day-night
changes in pineal serotonin. We observed that if rats were held
in continuous darkness the diurnal rhythm in pineal serotonin
persisted (Snyder et a1., 1965) indicating that it was truly cir-
cadian driven by an internal clock. In rats kept in continuous
light the day-night differences in serotonin was suppressed.
Prompted by our previous findings that light and sympathetic nerves
affect HIOMT, the role of the retina and the sympathetic nervous
system on the circadian rhythm of pineal serotonin was examined.
In blinded rats the circadian rhythm in pineal serotonin persisted,
again supporting its circadian nature. The indo1eamine rhythm was
abolished when the superior cervical ganglia was removed. Depleting
the brain and sympathetic nerves of noradrenaline by the administra-
tion of reserpine suppressed the serotonin rhythm. Cutting the
nerves pregang1ionica11y and interrupting nerve connections from
the brain to the superior cervical ganglia also abolished the
pineal serotonin rhythm. These findings indicated that the circad-
ian rhythm of serotonin in the pineal is regulated by sympathetic
nerves innervating pineal cells presumably by the changes in the
release of noradrenaline. Later we showed that there were day-night
differences in the turnover and presumably the release of' noradren-
aline in the pineal (Brownstein and Axelrod, 1974). The rhythm in
pineal noradrenaline turnover persisted in blinded rats and was
suppressed by continuous light. These results suggested that more
of the neurotransmitter was released during the nighttime. Decen-
tralization experiments indicated that the circadian rhythm of
serotonin was generated by a clock in the brain (Snyder et a1.,
1965). Later, work by Moore (1974) showed that the site of
this clock in the brain is the suprachiasmatic nucleus.
In 1970, Klein and Weller found a circadian rhythm in N-
acety1transferase, in the rat pineal, which was 180 0 out of phase
with the serotonin rhythm (Fig. 1). Soon after the onset of dark-
ness there is a 30 to 50-fold increase in enzyme activity. Day-
night rhythms in the pineal melatonin was found which like N-acetyl-
transferase was highest during the night and lowest during the day
(Wurtman and Moskowitz, 1977). As was reported with serotonin,
pineal N-acety1serotonin rhythm was suppressed by removal of the
superior cervical ganglia or by decentralization. Bilateral lesions
in the suprachiasmatic nucleus also abolished the circadian rhythm
of N-acety1transferase.
SYNTHESIS OF MELATONIN IN PINEAL ORGAN CULTURE
In collaboration with the late Harvey Shein and Wurtman
(Axelrod et a1., 1968), it was observed that the rat pineal gland
in organ culture can synthesize [14C]melatonin from [14C]tryptophan.

The pathway of biosynthesis of melatonin in pineal organ culture
was similar to that observed in the intact rat pineal. This prep-
aration provided an opportunity to examine the role of the sympa-
thetic nerve neurotransmitter noradrenaline at a cellular level.
The addition of noradrenaline to the rat pineal gland in organ
culture markedly stimulated the formation of melatonin from tryp-
tophan. The addition of serotonin had little effect. To examine
whether noradrenaline stimulates melatonin synthesis via adrenergic
receptors, the effect of alpha and beta-adrenergic blocking agents
were examined. The beta-adrenergic blocking agent propranolol
prevented the stimulation of melatonin synthesis in organ culture
by noradrenaline while the alpha-blocking compound phenoxybenzamine
had no such effect (Wurtman et al., 1971). Dibutyryl cyclic AMP
also stimulated the synthesis of melatonin indicating that the
beta-adrenergic receptor involved in melatonin synthesis is coupled
to adenyl ate cyclase.
- N-Acetyl transferase
--- mekltonm
-_.- serotonin
....._-_._ ... _- .........

/ .•./' / ....._-_.... _-_.-
'--.. !
0000 0600 t200 1800 2400 0600 1200 1800 2400 0600 1200
CLOCK HOURS
dark light dark light
Fig. 1. Circadian rhythms of serotonin, N-acetyltransferase and

melatonin in the rat pineal gland (from Axelrod, 1974).
At about the same time Klein and Berg (1970) demonstrated a

marked stimulation of N-acetylserotonin transferase (NAT) by nor-
adrenaline in organ cultures. They also found an elevation of
cyclic AMP in rat pineal organ culture after the addition of nor-
6 J. AXELROD
ad rena 1i ne. Because of the '1 arge ampl itude of N':acety1 serotoni n
transferase rhythm in vivo and its capacity to be stimulated by a
beta-adrenergic receptor, work on the pineal in our laboratory and
others were directed mainly towards this enzyme. The development
of a simple assay for measuring N-acety1transferase (Deguchi and
Axelrod, 1972a) made it possible for ra~id advances in uncovering
the cellular and subcellular mechanisms driving the rhythms of
melatonin and other indole amines in the pineal gland.
ADRENERGIC REGULATION OF THE PINEAL CIRCADIAN RHYTHM
As mentioned above pineal N-acetyltransferase showed a marked
circadian rhythm in the rat. To determine the role of the adrenergic
system in regulating this rhythm a number of pharmacological manipu-
lations were tried (Deguchi and Axelrod, 1972b) (Fig. 2). The
administration of the beta-adrenergic blocking agent propranolol
blocked the nighttime rise in N-acety1transferase. Depletion of
noradrenaline from nerves by reserpine also prevented the marked
elevation of N-acetyltransferase at night. When cycloheximide, a
protein synthesis inhibitor, was injected before the onset of dark-
ness the circadian rhythm of N-aceyt1transferase was abolished.
Denervation of the superior cervical ganglia, decentralization or
lesions in the suprachiasmatic nucleus also abolished the nighttime
'i 400
]
..
'"
(I)
0:
'""-z
..
(I)
I"
200
...J
>-
I-
'"'1
z
10
1500 1800 2100 2400
isoproterenol CLOCK HOURS
light dark••••
Fig. 2. Effect of adrenergic drugs and other manipulations on

the circadian rhythms of N-acety1transferase in rat pineal.
elevation N-acetyltransferase. Thus indicating that the pacemaker

for circadian rhythm arises in the suprachiasmatic nucleus. The
circadian messages are then transmitted to preganglic fibers of
the superior cervical ganglia and on to the beta-adrenergic recep-
ors on the pineal cell surface. These experiments and others
indicated that with the onset of darkness there is an increased
release of noradrenaline onto the beta-adrenergic receptor of the
the pineal gland. This then initiates a series of events leading
to the synthesis of N-acetyltransferase intracellularly (Fig. 3).
Denervation of the superior ganglia, decentralization or lesions
in the suprachiasmatic nucleus also blocked the nighttime elevation
of N-acety1transferase.
When rats are exposed to a brief period of light during the
night when the pineal N-acety1transferase levels are highest there
is a rapid fall of this enzyme to the low daytime levels (Deguchi
and Axelrod, 1972c) (Fig. 2). Injection of isoproterenol blocked
the light induced fall of the enzyme. The administration of a
beta-blocking drug at night caused a precipitous fall in the
DNA
RNA
cAMP
(PKI ~ (Serotonin
Y
H NAT",.
ATP
N-Acetyl-
NATinact. serotonin
/HIOMT
Melatonin
/l-Adrenergic
Receptor
NERVE ENDING PINEAL CELL
Fig. 3. Intracellular events in the stimulation of melatonin

synthesis by the beta-adrenergic receptor. NAT act is
active form N~acetyltransferase, NATinact is inactive
form. HIOMT is hydroxyindole-O-methyltransferase •
• is noradrenaline. PK is protein kinase.
8 J. AXELROD
high levels of N-acetyltransferase. Thus the maintenance of the

elevated levels of N-acetyltransferase at night requires the contin-
uous stimulation of the pineal beta-adrenergic receptor.
SUPER- AND SUBSENSITIVITY OF THE PINEAL BETA-ADRENERGIC RECEPTOR AND

THE AMPLIFICATION OF CIRCADIAN RHYTHMS - -
Increased responsiveness (supersensitivity) after beta-
adrenergic stimulation by sympathetic nerve denervation ~ well-
known phenomena in many biological systems. The rat pineal gland
was found to be a useful model to study changes in responsiveness
in the beta-adrenergic receptor. Depriving the pineal gland of
beta-adrenergic stimulation by surgical denervation, chemical
sympathectomy with 6-hydroxydopamine or exposure of rats for long
periods of light caused a marked increase of N-acetyltransferase
synthesis, adenylate cyclase, accumulation of cyclic AMP after
the administration of the beta-adrenergic agonist isoproterenol
{Deguchi and Axelrod, 1973~These biochemical changes in beta-
adrenergic activity occurred rapidly and appeared to be a function
of the daily light-dark cycle. Pineal gland from rats housed
under diurnal lighting conditions are supersensitive to beta-
adrenergic stimulation as measured by induction of N-acetyltrans-
ferase by beta-adrenergic agonists at the end of the 12 hour light
period and subsensitive at the end of the dark period. These
rapid changes of sensitivity of the beta-adrenergic receptor are
presumably due to the decreased release of noradrenaline from
nerves innervating the pineal gland during the day and increased
release at night. Subsensitivity can occur very rapidly, a single
injection of isoproterenol causing a markedly decrease response in
the pineal to a challenging dose of the drug given five hours
1ater.
The role of catecholamines in producing super- and subsensi-
tivity was examined in pineal organ culture (Axelrod, 1974) (Table 2).
When the pineal gland was deprived of its neurotransmitter by
denervation it was five times more responsive to the induction of
N-acetyltransferase by a beta-adrenergic agonist in organ culture
than the intact gland. W~pineal glands of rats were exposed to
excessive amounts of catecholamines by repeated injections of
isoproterenol they were much less responsive to N-acetyltransferase
induction as the gland of the untreated rats. These experiments
indicated the activity of the post-synaptic beta-adrenergic receptor
and the consequent formation of the pineal ho~one depend on the
previous exposure of the receptor to the neurotransmitter.
The beta-adrenergic receptor can be measured by its ability
to specificaTTy bind [3H]dihydroalprenolol. a potent beta-adrenergic
antagonist (Zatz et al., 1976). Binding of [3H]dihydroalprenolol
Table 2. Supersensitivity and Subsensitivity in Rat Pineals.
N-Acetyltransferase (Units)
l-Isoproterenol
(M) l-Isoproterenol-
Intact Denervated treated
1 X 10- 9 13 330
5 X 10- 9 330 1330 26
2 X 10- 9 680 2190 70
1 X 10- 7 940 1180 320
1 X 10- 6 1720 1490 1380
Rats were denervated by bilateral removal of the superior

cervical ganglia 7 days before they were killed. Isoproterenol-
treated rats received the drug (2.0 mg/kg) 8, 16, and 24 hours
before they were killed. Pineals were cultured for 10 hours with
indicated concentrations of isoproterenol, and, N-acetyltransfera'se
activity was measured (from Deguchi and Axelrod, 1973).
to pineal membrane was found. This binding was found to be rapid,

reversable, saturable and stereospecific. A marked variation
in the number of available specific binding sites of [3H]dihydro.
alprenolol in the rat pineal was observed (Romero et al., 1975)
(Fig. 4). During the daytime with the decrease of sympathetic
nerve activity the number of receptor sites, as measured by binding
of the radioactive ligand, increased. During the night the number
of receptors fell and reached a minimum at the end of the dark
period.
The beta-adrenergic receptor is coupled to adenyl ate cyclase
(Fig. 3). Diurnal changes in the activity of the adenyl ate cyclase
as measured by the accumulation of cyclic AMP in the pineal was
found (Axelrod and Zatz, 1977). Pineals taken from light exposed
rats showed a greater accumulation of cyclic AMP in response to
isoproterenol than did glands taken from dark exposed animals.
Cholera toxin which bypasses the beta-adrenergic receptor and acts
directly on the adenyl ate cyclase produced a greater accumulation
of cyclic AMP in supersensitive glands. These differences in
cyclic AMP accumulation were reflected in the induction of N-
acetyl transferase activity.
Cyclic AMP acts to relay its biologic message (formation of
melatonin) via a protein kinase (Zatz and AxelrOd, 1978) (Fig. 3).
As in the case of the beta-adrenergic receptor and adenyl ate cyclase
the activity of pineal protein kinase varied with the activity of
10 J. AXELROD
DIURNAL VARIATION IN AVAILABILITY OF SPECIFIC
,.B-ADRENERGIC BINDING SITES IN RAT PINEAL
12
co
z
a
z 10
iii
..J
o~
~ : 8
z a.
'"a:: .,.e
~~ 6
<t 0
:i: -;
,., e
~ ~ 4
"- ~
u
'"
:J; 2 LIGHT f®OOWOARKi@i!ii!i!iij
0L,------.-----.-----~----~
0600 1200 1800 2400 0600
CLOCK HOURS
Fig. 4. Circadian rhythm in beta-adrenergic binding sites in

rat pineal (from Romero et al., 1975).
light-dependent sympathetic nerve activity. There was a greater

activity of protein kinase during the daytime than during the night.
Further experiments showed that cyclic AMP acts at three sites in
the biosynthesis of the protein, N-acetyltransferase; at the level
of transcription via messenger RNA at the level of translation of
the RNA message and at the level of degradation of NAT.
All these results indicate that there is a complex cascade of
cellular reactions that amplifies and dampens the neurotransmitter
signal to affect the activity of N-acetyltransferase, the enzyme that
generates the pineal hormone melatonin. During the daytime there
is a reduced release of noradrenaline but the amount of neurotrans-
mitter release is not sufficient to trigger the synthesis of N-
acetyl transferase. Because of the lower level of noradrenaline in-
teracting with the pineal cell surface there is a gradual increase
in the number of beta-adrenergic receptors, adenyl ate cyclase and
cyclic AMP dependent protein kinase. As night falls there is an
increased discharge of noradrenaline onto a supersensitive receptor

and because there is also an increased amount of adenyl ate cyclase,
cyclic AMP and protein kinase the neurotransmitter signal is ampli-
fied about 30- to 50-fold to stimulation in the synthesis of N-
acetyl transferase. As night progresses the beta-adrenergic
receptor number decreases and so does the cyclic AMP cascade and
the pineal becomes gradually desensitized and this is reflected in
the decreasing synthesis of N-acetyltransferase.
In the past few years there has been rapid advances in the
ability to directly measure melatonin in body fluids and tissues.
This makes it now possible to examine the role of pineal melatonin
in normal body functions and in disease in man. Recent evidence
indicates that the melatonin hypothesis that Wurtman and I proposed
in 1965 in animal studies also applies to man. Several investiga-
tors have found that there is a circadian rhythm in melatonin
synthesis controlled by sympathetic nerves and regulated by light.
The secreted pineal hormone melatonin then acts at distant organs
probably the gonads.
REFERENCES
Axelrod, J., 1974, The pineal gland: A neurochemical transducer.

Science, 184:1341.
Axelrod, J., Shein, H.M. and Wurtman, R.J., 1969, Stimulation of C14_
tryptophan by noradrenaline in rat pineal in organ culture.
Proc. Nat1. Acad. Sci. USA, 62:544, 1969.
Axelrod, J. and Weissbach, H, 1961, Purification and properties of
hydroxyindole-O-methyl transferase. J. Bio1. Chern., 236:
211.
Axelrod, J. and Zatz, M., 1977, The beta-adrenergic receptor and the
regulation of circadian rhythms-Tn the pineal gland. in:
"Biochemical Actions of Hormones," Vol. IV, Chapter 5,
G. Litwack, ed., Academic Press, New York.
Brownstein, M.J. and Axelrod, J., 1974, Pineal gland: 24-Hour rhythm
in norepinephrine turnover. Science, 184:163.
Deguchi, T. and Axelrod, J., 1972a, Sensitive assay for serotonin N-
acetyltransferase activity in rat pineal. Anal. Biochem. , 50:
174.
Deguchi, T. and Axelrod, J.• , 1972b, Induction and super-induction of
serotonin N-acetyltransferase by adrenergic drugs and dener-
vation in the rat pineal. Proc. Nat1. Acad. Sci. USA, 69::2208.
Deguchi, T. and Axelrod, J., 1972c, Control of circadian change of
serotonin N-acetyltransferase activity in the pineal organ by
the beta-adrenergic receptor. Proc. Nat1. Acad. Sci. USA,
69: 254"7:"
12 J. AXELROD
Deguchi, T. and Axelrod, J., 1973, Supersensitivity and subsensitiv-

ity of the beta-adrenergic receptor in pineal gland regulated
by catecholamine transmitter. Proc. Natl. Acad. Sci. USA,
70:2411.
Fiske, V.M., Bryant, K., Putnam, J., 1960, Effect of light on the
weight of the pineal in the rat. Endocrinology, 66:489.
Holmgren, N., 1918, Zur Kenntnis der Parietalogran von Rana temporari.
Ark. Zool., 11:1.
Kappers, J.A., 1960, The development, topographical relations and
innervation of the epiphysis cerebri in the albino rat.
Z. Zellforsch. Mikrosk. Anat. 5:163.
Klein, D.C. and Berg, G.R., 1970, Pineal gland: Stimulation of
melatonin production by norepinephrine involves cyclic AMP
mediated stimulation of N-acetyltransferase. Adv. Biochem.
Psychopharmacol., 3:241.
Klein, D.C. and Weller, J.L., 1970, Indole metabolism in the pineal
gland: A circadian rhythm in N-acety1transferase. Science,
169: 1093.
Kopin, I.J., Pare, C.M.B., Axelrod, J. and Weissbach, H., 1961,
The fate of melatonin in animals. J. Biol. Chern., 236:3072.
Lerner, A.B., Case, J.D., Takahashi, Y., Lee, T.H. and Mori, W.,
1958, Isolation of melatonin, the pineal gland factor that
lightens melanocytes. J. Amer. Chern. Soc., 80:2587.
McCord, C.P. and Allen, F.P., 1917, Evidences associating pineal
gland function with alterations in pigmentation. J. Exp.
Zool., 23:207.
Moore,~, 1979, Visual pathways and the central neural control of
diurnal rhythms. in: "The Neurosciences, Third Study Program,"
F.O. Schmitt, F.G. Worden, eds, MIT Press, Cambridge, Mass.
Quay, W.B., 1963, Circadian rhythm in rat pineal serotonin and its
modifications by estrous cycle and photoperiod. Gen. Compo
Endocrinol., 3:473.
Romero, J.A., Zatz, M., Kebabian, J.W and Axelrod, J., 1975,
Circadian cycles in binding of 3H-alprenolol to beta-adrenergic
receptor cites in rat pineal. Nature, 258:435.
Snyder, S.H., Axelrod, J. and Zweig, M., 1965, A sensitive and
specific fluorescence assay for tissue serotonin. Biochem.
Pharmacol., 14:831.
Snyder, S.H., Zweig, M., Axelrod, J. and Fischer, J.E., 1965, Control
of the circadian rhythm in serotonin content of the rat pineal
gland. Proc. Natl. Acad. Sci. USA, 53:301.
Weissbach, H., Redfield, B.G. and Axelrod, J., 1961, Enzymic acetyl-
ation of serotonin and other naturally occurring amines.
Biochim. Biophys. Acta, 54:190.
Wurtman, R.J. and Axelrod, J., 1965, The pineal gland. Sci. Amer.
213: 50.
Wurtman, R.J. Axelrod, J. and Fischer, E., 1964, Melatonin synthesis

in the pineal gland: Effect on light mediated by the sympathetic
nervous system. Science 143:1328.
Wurtman, R.J., Axelrod, J. and Phillips, l.S., 1963, Melatonin
synthesis in the pineal gland: Control by light. Science, 142:
1071.
Wurtman, R.J. and Moskowitz, M.A., 1977, The pineal organ. New Eng.
~ Med., 296:1329 and 1383.
Wurtman, R.J., Roth, W., Altschule, M.D. and Wurtman, J.J., 1961,
Interactions of the pineal and exposure to continuous light on
the organ weights of female rats. Acta Endocrino1. 36:617.
Wurtman, R.J., Shein, H.M. and Larin, F., 1971, Mediation by beta-
adrenergic receptors on the effect of norepinephrine in pineal
synthesis of [14C]serotonin and [14C]me1atonin. J.
Neurochem. 18: 1683. -
Zatz, M. and Axelrod, J., 1978, Regulation of sensitivity to
beta-adrenergic stimulation in the rat pineal. in: "Neuronal
Information Transfer," A. Karlin, M. Tennyson and H.J. Vogel,
eds., Academic Press, Inc., New York.
Zatz, M., Kebabian, J.W., Romero, J.A., lefkowitz, R.J. and Axelrod,
J, 1976, Pineal beta-adrenergic receptor: Correlation of
binding of 3H-1-alprenolol with stimulation of adenylate
cyclase. J. Pharmacol. Exp. Ther. 196:714.
ASPECTS OF EVOLUTION OF THE PINEAL ORGAN
Andreas Oksche
Department of Anatomy and Cytobiology

University of Giessen, 6300 Giessen
Federal Republic of Germany
INTRODUCTION
The pineal organ is an ancient derivative and integral

component of the brain. (Recent comprehensive reviews have
been published by Kappers and Pevet, 1979; Leonhardt, 1980;
Vollrath, 1981; Oksche and Pevet, 1981.) Phylogenetically
it has changed from a I third eye I endowed with photoreceptor
cells to an endocrine gland influenced by visual stimuli
from the retina (Fig. 1). The pineal organ has the capacity
to translate photic (and apparently also other sensory and
neural) information into a neuroendocrine response (cf. Wurtman
et al., 1968. In a subsequent step this neuroendocrine message
acts on various target organs. The unusual anatomical and
physiological characteristics of the pineal organ make it
an interesting subject of analysis both in brain research
and endocrinology. In evolutionary terms the pineal organ
displays features of a photoreceptor, biological clock, and
endocrine gland (cf. Oksche and Pevet, 1981). It is a member
of the group of circumventricular organs (Vigh and Vigh-Teichmann,
1981) and a component of the photoneuroendocrine systems
(Oksche and Hartwig, 1979). The pinealocyte can be considered
as a paraneuron (Ueck and Wake, 1979) originating from a
special type of cerebrospinal fluid (CSF)-contacting neuron
(Vigh and Vigh-Teichmann, 1981; see for references) displaying
both sensory and secretory properties (i.e. a sensoneuroendocrine
cell) .
The di versi ty of structure and function of the vertebrate

pineal complex reflects a surprisingly high degree of
evolutionary and adaptive capacity (cf. Ralph, 1970; Quay,
15
16 A.OKSCHE
'""'iil''''',,,,''''',,''II"
eCYCLOSTOMATA. PETROMYZONTIDAE
8ANURA Ci) LACERTILIA
o MAMMALIA
Fig. 1. Comparative representation of pineal complexes. Digram-
matic midsagittal views {after Studnicka, 1905}
in relation to the respective features of the pineal-
ocytes of the receptor line (modified after Oksche,
1980). Single star, pineal organ (epiphysis cerebri);
double star, parapinea1 organ {cyclostomes, lacertilian~
or frontal organ {anurans)j single arrow, pineal tract;
double arrow, parietal tract (cyclostomes, lacertilians)
or frontal-organ tract {anurans}. Note diversity in
ultrastructural details of the pinealocytes (1-3)
indicating transmission of outer segment structures and
increasing secretory capacity. For details, see text
(cf. Meiniel, 1980).
ASPECTS OF EVOLUTION OF THE PINEAL ORGAN 17
1974) . Profound evolutionary changes have produced remarkable

variations of the sensory and secretory apparatus of the
pineal organ; at the ultra3tructural and molecular levels
new manifestations of these alterations have been discovered.
During the last two decades a high priority has been

given to the elucidation of the function of the mammalian
pineal gland (cf. Wurtman et al., 1968; Vollrath, 1981).
However, there is no doubt that the pineal organs and complexes
of submammalian vertebrates provide a most important key
for the morphological and physiological analysis of the pineal
gland of mammals including man. The present comparative
report is based on two decades of intense interdisciplinary
research cooperation with Professor Dodt and associates (cf.
Dodt, 1973) and on continuous exchange of thoughts and
information with Professor Collin culminating in a recent
joint report (Collin and Oksche, 1981). Consideration is
gi ven to a number of previous surveys by Oksche (1965, 1970,
1971, 1980) and Collin (1969, 1971, 1977, 1979). In the
present review a replica of these comprehensive treatises
will be avoided; thus emphasis will be placed on general
trends and concepts in comparative pineal research.
PROGRESS IN COMPARATIVE ANALYSIS OF SUBMAMMALIAN PINEAL SYSTEMS
State of Knowledge in 1970

The CIBA Symposium held in London in 1970 (cf.
Wolstenholm and Knight, 1971) marked the end of a decade
of innovative and expanding comparative research in the field.
In the late fifties the structural analysis of the brain
was much helped by the introduction of electron microscopy.
Using this new technique, Steyn and Eakin, both in 1959,
discovered that the parietal eye of lizards is endowed with
true photoreceptor cells resembling the cones of the lateral
eye (cf. Eakin, 1973). This progress was further enhanced
by the first recordings of action potentials from the pineal
complex of anurans, teleosts and lacertilians. In 1961,
Dodt and his associates showed that the pineal 'eye' (frontal
organ) of the frog is a true photoreceptor organ capable
of chromatic ahd achromatic types of response (cf. Dodt,
1973). In 1962, collaborative work of Dodt and Oksche initiated
a series of correlated neurophysiological and ultrastructural
studies focussed on the pineal organs of different teleosts,
amphibians, lacertilians and birds (for references, see Oksche,
1971; Oksche et al., 1971; Dodt, 1973). This work was
followed by extensive comparative investigations of Collin,
1969; see also Collin, 1971). By the use of Nauta-type
methods Paul et al. (1971) succeeded in tracing the pineal
tract to the pretectal region and the tegmentum (the reticular
18 A. OKSCHE
system) of the frog. By means of fluorescence microscopy

(according to Falck-Hillarp) the sympathetic input into the
pineal organ was investigated in a number of species belonging
to different classes of vertebrates (see Wolstenholme and
Knight, 1971; Quay, 1974). Secretory granules were shown
to occur in pinealocytes bearing a rudimentary outer segment
(Collin 1969, 1971; Oksche, 1970, 1971); at the same time
a monoamine fluorophore was demonstrated in pinealocytes of
the sensory type (Owman and RUdeberg, 1970). From these
observations Oksche (1971) concluded that in pineal photoreceptor
organs the input of photic information may be converted into
a neurohormonal mechanism in one and the same sensory cell.
This led to the definition of sensory pinealocytes as photo-
neuroendocrine cells of neuronal origin, a conceptual extension
of the photo neuroendocrine systems as defined by Scharrer
(1964).
Recent Developments (1970 to 1982)
In 1974, three reports contributed substantial new

information toward an understanding of the basic organization
of the pineal sense organs. By means of microspectophotometry
Hartwig and Baumann (1974) showed that the outer segments
in the pineal organ (epiphysis cerebri) of Rana esculenta
contain a rhodopsin-like photopigment 5021 (specific optical
density 0.011-0.016 ~m), whereas the outer segments of the
photoreceptor cells in the frontal organ display a photolabile
substance with an absorption maximum between 560-580 ~m.
Wake et al. (1974) presented a diagram adding more precise
details to the organization of the intrinsic neuronal circuitry
in the frontal organ and the epiphysis of the frog. In addition
to the basically bineuronal chain consisting of receptor cells
and neurons participating in the formation of the pinealofugal
pathways, the pineal complex of anurans contains stellate
acetylcholinesterase-posi ti ve interneurons. The latter may
play an essential role in the generation of the achromatic
or the chromatic types of pineal response. Vigh and Vigh-
Teichmann (1974) came to the conclusion that sensory pinealocytes
of submammalian vertebrates, and even certain cilium-bearing
forms of mammalian pinealocytes, resemble the cerebrospinal
fluid-contacting neurons. This conclusion was further underlined
by the observations (1) that the lumen of most pineal sense
organs is in open communication with the third ventricle,
and (2) that photoneuroendocrine systems of the brain are
concentrated around the third ventricle (Oksche and Hartwig,
1975) .
From the comparative point of view two concepts published

in 1977 deserve particular attention. Collin (1977) gave a
detailed account on the rudimentation of the photoreceptor
cells in vertebrate pineal organs. He emphasized the gradual

transformation of the photoreceptor cell during phylogeny and
established homologies between pineal photoreceptors, rudimentary
photoreceptor cells and secretory pinealocytes. These cells
were claimed to form a single cell line, i. e. 'cells of the
receptor line' (CRL), in contrast to the line of supportive
ependymal elements and secondary neurons. In principle, the
CRL of non-mammalian vertebrates are secretory in function.
However, the rudimentation of the outer segment appears to
be a necessary prerequisite for enhancing full secretory capacity.
Ueck and Wake (1977) classified the pinealocyte as a paraneuron
(Fujita 1977). They emphasized that all forms of pinealocytes
possess a receptive input region, a conductive region and
an output region; thus, the pinealocyte is a recepto-secretory
functional unit. The neuroectodermal orlgln of pinealocytes
has been well established. All criteria characteristic of
a paraneuron appear to hold true for pinealocytes. The boundary
between paraneurons and neurons is, however, a matter for
individual interpretation. Further, it is noteworthy that
about 197"( Collin and Pevet were the first to show that mammalian
pinealocytes and the rudimentary photoreceptor cells of sauropsids
elaborate, in addi tion to the well-known indoleamines, a
proteinaceous secretion. These pioneering studies led to
a series of further investigations (see Collin and Pevet in
Oksche and Pevet, 1981). All three above-mentioned concepts
suggest a relationship of pinealocytes with paraneurons and
also with secretory neurons.
After extensive fluorescence- and electron-microscopic

studies of the pineal organ in Lampetra planeri (Petromyzontidae)
Meiniel (1980) came to the conclusion that the peculiar serotonin-
containing pinealocytes of this lamprey are members of a second
sensory cell line leading from photoreceptor cells to secretory
pinealocytes. Subsequently, this concept was extended to
other vertebrates (Meiniel, see Oksche and Pevet 1981). In
the opinion of Meiniel, only the less conspicuous sensory
elements of the second cell line are endowed with a secretory
capaci ty thus becoming the stem cells of the secretory
pinealocytes.
To prove the existence of two different lines of pineal

receptors in other classes of vertebrates, an improved
indoleamine-fluorescence technique (used without pharmacological
pretreatment) and the light-dependent uptake of tri tiated
deoxyglucose were applied in the analysis of the pineal organ
(epiphysis cerebri) in the frog (Hartwig and Oksche, 1981).
An indoleamine fluorophore was found in the apical and supra-
nuclear positions of sensory pinealocytes; however, cells
displaying very long, birefringent outer segments were free
of this fluorescence. No amine fluorescence was found in
20 A.OKSCHE
the frontal organ, i.e. the distal component of the pineal complex.
After incubation with deoxyglucose in constant darkness (high
spontaneous activity of photoreceptor cells) strongly labeled
pinealocytes bearing long outer segments could be observed.
The pattern of the silver grains in the pineal organ changed
depending on the lighting conditions. Since cyclic changes
in the structure and function of the receptors cannot be excluded,
the existence of two independent receptor-cell lines remains
a possibility. By the use of light- and electron-microscopic
cytochemical techniques melatonin-like immunoreactivity was
demonstrated in cells of the receptor line in the pineal organ
of a teleost, the pike (Falcon et al., 1981). The intensity
of the immunoreaction depended on the phase of the photoperiod.
Remarkably, at the electron-microscopic level, not all cells
of the photoreceptor type appeared to be immunoreacti ve.
The existence of the fluorophore of 5-HT as well as of melatonin-
immunoreactive material in pineal photcreceptor cells sqpports
the concept of a photo neuroendocrine capacity of these elements
(Hartwig and Oksche, 1981; Falcon et al., 1981).
Vigh and Vigh-Teichmann (1981) applied an antiserum against

bovine retinal rods to different pineal systems and were able
to show that the outer segments of the pineal sensory cells
of teleosts, anurans and chelonians give a positive immuno-
reaction. Very recently this reaction was successfully tested
on the pineal organ of Phoxinus phoxinus (Fig. 2), the classical
experimental animal of von Frisch (1911) and Scharrer (1928),
pioneers in the field of photoneuroendocrine research
Vigh-Teichmann et al., 1982). Curiously, the receptors in
the parietal eye and the pineal organ of several lizards were
negative to this antiserum; however, the negative reaction
of the lacertilian retinal receptors with the antiserum used
may indicate a basic difference in the chemical composition
of opsins. On the other hand, the irregular lamellar complexes
in the pineal of the pigeon displayed a positive opsin immuno-
reaction (Vigh and Vigh-Teichmann, 1981). The latter result
is important in suggesting new lines of research into the
function of the avian pineal organ (see below). It is important
to note that the CSF-contacting neurons in different vertebrates,
which had been claimed as potential candidates for the I deep
diencephalic photoreceptors', were completely opsin-negative;
also the occasional 9+0 cilia of mammalian pinealocytes (opossum,
hedgehog) did not display a positive opsin immunoreaction.
In addition, the areas of the ventricular wall described by
Hartwig (see Oksche and Hartwig, 1979) to contain a
microspectrophotometrically detectable photolabile compound
were completely negative to the opsin antiserum. Since micro-
spectrophotometry fails to detect photopigments in pinealocytes
of the receptor line displaying geometrically irregular outer
segments J the opsin immunoreaction will become a very important
• F
"'\
Fig. 2A,B. Pineal organ of Phoxinus phoxinus (Teleostei) A. Opsin-

immunoreactive outer segments (arrow). B. Acetylcholin-
esterase-positive neurons (arrowr:--The other reactive
(dark) structures represent inner segments of the
photoreceptor cells. Bars = 20 ~m. For details, see
text. Unpublished microphotographs; see
Vigh-Teichmann et al. (1982) for methods.
technique in comparative studies of the pineal (as well as other

extraretinal) photoneuroendocrine systems.
Further, progress in demonstrating and mapping central

pinealofugal and pinealopetal pathways by the use of tracers
such as horseradish peroxidase (HRP) has been of great value
in pineal research. In this context, only some of the findings
from our laboratory will be mentioned (Korf and Wagner, 1980,
22 A.OKSCHE
1981; Korf et al., 1982; Korf and M~ller, unpublished results).

They have established clear patterns of pineal afferents and
efferents in a lacertilian and an avian species and shown
that, in addition to the sympathetic input, central pinealopetal
fiber systems exist in several mammalian species (see below).
Comparative Cytology of the Pinealocytes
In comparative terms the pineal organ is composed of

(i) pinealocytes (photoreceptors, rudimentary photoreceptors,
secretory pinealocytes), (ii) ependymal or glial supportive
cells, and (iii) neurons (interneurons, neurons of the pineal
tract). In addition, in the lumen of the pineal organ, which
in sub-mammalian vertebrates communicates with the third ventricle,
free cells, mostly macrophages but probably also supraependymal
elements of neuronal or glial origin, can be observed. In
an open pineal lumen the outer segments of the sensory
pinealocytes are bathed in the Circulating CSF, and there
a release and also an uptake of sUbstances may occur (Oksche
and Hartwig, 1975). This aspect is of importance in connection
with the conspicuous discharge from and renewal of lamellar
systems in the outer segment of pineal photoreceptor cells.
In a number of lower vertebrates, e.g. the frog, indoleamine
fluorophores have been observed within the pineal lumen, seemingly
in connection with a net-like mucopolysaccharide or mucoprotein
matrix.
The following review will concentrate exclusively on cells

belonging to the receptor line (CRL) as defined by Collin
and Oksche (1981).
The cells of the receptor line (CRL) are derived from

sensory, photorecepti ve pinealocytes. The photoreceptor cells
(PC) are gradually transformed into rudimentary photoreceptor
cells (RPC) and, subsequently, into secretory pinealocytes
(Pi). The process of rudimentation began already in anamniotes;
and intermediate forms exist.
The achromatic and chromatic types of electric response

of pineal sense organs are well established (Dodt, 1973;
Hamasaki and Eder,1977). However, the biological significance
of this message conducted to different areas of the brainstem
(see below) is still not understood.
The CRL in non-mammalian vertebrates are secretory in

function. (1) Gradual rudimentation and subsequent disappearance
of the photoreceptor pole, and (2) transformation of the inner
cellular pole from an area involved in neurotransmission into
a vascular contact are important prerequisites of increasing
secretory acti vi ty. It is, as yet, unsolved whether a fully
differentiated PC already possesses a full-range of secretory

capacity. The discrete secretory activity of these cells
is fully manifested in the RPC parallel to the regression
of photosensory structures. However, the PC may display a
higbly active granular endoplasmic reticulum indicating protein
synthesis (Owman and RUdeberg, 1970; Oksche, unpublished
results). Dense-core granules do not belong to the typical
picture of the PC; however, after maintenance in constant
darkness, Omura and Ali (1981) were able to accumulate a
conspicuous number of dense-core vesicles in the PC of a teleost
species. Dense-core vesicles are very abundant in the RPC
of different lacertilians and birds. HIOMT and melatonin
occur in the pineal sense organs (cf. Oksche and Pevet, 1981).
In addition, a secretion of proteinaceous material takes place
in the CRL, even in the PC (see above). With respect to sensory
and secretory functions, it is impossible to draw an exact
borderline between the PC and the RPC. A direct photoreceptive
capacity of the RPC cannot be excluded. The problem of the
presence and nature of membrane receptors in the PC and the
RPC is completely open to discussion. In analogy to the PC,
the RPC may contain synaptic ribbons. However, in the RPC
synaptic ribbons do not mark true synaptic contacts (cf. Coilin
and Oksche, 1981); this pattern of synaptic ribbons already
resembles the arrangement of synaptic ribbons in mammalian
pinealocytes (cf. Vollrath, 1981).
On the basis of the above-mentioned evidence the existence

of photoneuroendocrine cells can be postulated. Gradual evolution
of cellular mechanisms leads from the perception of appropriate
stimuli to the delivery of nervous, neurohormonal and non-
neurohormonal humoral messages (cf. Collin and Oksche, 1981).
This process includes, at the cellular level, a translation
of photic stimuli into a neuroendocrine reaction.
Intrinsic Neuronal Circuitry
In contrast to the retina, the pineal sense organs possess

generally a bineuronal chain. The second neuron displays
a positive acetylcholinesterase reaction and is synaptically
coupled with photoreceptor cells; it is endowed mostly with
a single dichotomously branching process. The axon of these
acetylcholinesterase-positive neurons joins the pineal tract.
Stellate acetylcholinesterase-positive nerve cells, especially
conspicuous in the pineal complex of anurans, can be considered
as interneurons. Unfortunately, the synaptic connections
of these cells, the corresponding neuropil areas, and the
respecti ve neurotransmitters have not been elucidated in detail,
although they appear to be essential for the generation of
the achromatic or the chromatic types of pineal response (cf.
Oksche and Hartwig, 1979). According to Meissl (for references
24 A.OKSCHE
see Collin and Oksche) 1981)) taurine) glutamic acid and glycine
are present in significant concentrations in the pineal organ
of the trout. However) the neurotransmitters of the individual
neurons have as yet not been determined. Serotonin) added
in vitro to the incubation medium) effecte~ a decrease in
the electrical activity of the pineal nerve In the frog) whereas
the application of melatonin was without effect on the activity
of the frontal organ (Donley and Meissl; for references see
Oksche and Hartwig) 1979).
The acetylcholinesterase-posi ti ve neurons of the avian

pineal organ are apparently homologues to the similarly reacting
elements of the pineal sense organs in anamniotes. The intrinsic
circuitry of the nerve cells is) however) less conspicuous
than) e.g.) in anurans and has not yet been deciphered in
greater detail. There is no convincing evidence suggesting
that such acetylcholinesterase-positive nerve cells occur
in the pineal organ of mammals; the acetylcholinesterase-
reactive neurons in the pineal organ of the ferret
Herbert) cf. Wolstenholme and Knight) 1971) are apparently
of habenular origin. The rather scarce) scattered neurons
of most mammalian pineal organs have been claimed to belong
to the autonomic nervous system; the origin of these cells
should, however, be re-examined.
Pinealofugal and Pinealopetal Neuronal Connections
Pineal sense organs communicate with the brain via neural

pathways consisting of myelinated and non-myelinated nerve
fibers (cf. Oksche, 1971; Collin and Oksche, 1981); they
transmit a nervous message to the brain. In phylogenetic
terms the pinealofugal projection shows signs of sequential
regression; at the same time the neuroendocrine communication
gains increasing importance. Pinealopetal nerve fibers of
central origin have been clearly demonstrated in pineal sense
organs by neurophysiological and neuroanatomical methods (cf.
Dodt, 1973; Oksche and Hartwig, 1979). It should be mentioned
that efferent systems also occur in the lateral eye and other
sense organs. Reciprocal neural connections appear to be
characteristic of the pineal sense organs.
In degeneration studies, using methods of the Nauta type,

Paul et al. (1971) had already shown (see Introduction) that
the pineal tract in Rana esculenta projects to the pretectal
area and to the retiCUlar formation of the mesencephalic
tegmentum. The existence of a pineal projection to the deep
mesencephalic gray in the frog was confirmed by neurophysiological
methods (Gaillard et al., 1977; Cadus£eau et al., 1979).
In the rainbow trout, in addition to the above-mentioned

areas, the existence of a habenular projection of the pineal

organ has been shown by means of cobalt chloride iontophoresis
(Hafeez and Zerihun, 1974). Our knowledge of the patterns
of pineal innervation has been greatly extended by tissue
analysis following iontophoretic application of horseradish
peroxidase (HRP). Korf and Wagner (1981) succeeded in showing
that the parietal eye of the lizard, Lacerta sicula projects
not only to the left habenular complex, the pretectal area
and the dorsolateral nucleus of the thalamus, but also to
the paraventricular nucleus. On the other hand, they showed
that the parietal eye receives an input from the same
paraventricular area. A reciprocal innervation is also
characteristic of the pineal organ of the house sparrow, Passer
domesticus (Korf et al., 1982). The pineal tract of this
passerine bird (consisting of approximately 1,000 mostly
unmyelinated nerve fibers) arises from intrinsic acetyl-
cholinesterase-positive neurons, which seem to be homologues
of similar neurons in pineal sense organs. Projections of
this tract were observed in both habenular nuclei and in the
periventricular gray of the hypothalamus. On the other hand,
distinct pinealopetal projections originate in the habenular
complex and in the area of the paraventricular nucleus. Further,
a distinct pinealopetal projection of central origin also
exists in mammals as has been shown in the guinea pig (Korf
and Wagner, 1980) (Fig. 3A,B) and the Mongolian gerbil (Korf
and M~ller, unpublished results). These pinealopetal fibers
originate either in the habenular complex or in the peri ven-
tricular zone adjacent to the paraventricular nucleus. Only
very occasionally, labeled neurons are found in the supra-
chiasmatic nucleus; thus, the existence of a direct connection
between the suprachiasmatic nucleus and the pineal organ has
not been confirmed. The pinealopetal projections of central
nerve fibers in the rat are not very conspicuous; in this
species the sympathetic input is clearly predominant (cf.
Kappers, 1982, this volume). Finally, in the primi ti ve pineal
gland of the hedgehog oxytocin- and vasopressin-immunoreactive
nerve fibers have been demonstrated (NUrnberger and Korf,
1981). (For other references the quoted papers should be
consulted) .
What do the pineal sense organs tell to the brain? It

should be kept in mind that the achromatic pineal systems
may be capable of measuring solar radiation energy and the
chromatic systems - ~he changing spectral composition of daylight
(cf. Dodt and Meissl in Oksche and Pevet, 1981). The neural
connection of pineal sense organs with the reticular formation
of the brainstem, especially via the tegmental projection,
may be involved in phototactic reactions (cf. Oksche and Hartwig,
1979), the control of thermoregulatory behavior (cf. Ralph
et al., 1979), and some unknown activating mechanisms. A
26 A.OKSCHE
direct connection with the neuroendocrine apparatus of the

brainstem is not evident but cannot be excluded. The role
of the neural connections of the pineal organ in the establishment
and maintenance of circadian mechanisms is open to discussion
(cf. Korf et al.) 1982). The suprachiasmatic nucleus of birds
and mammals is a powerful circadian oscillator; significant
variation exists in the coupling of the avian pineal organ
with the secondary self-sustained oscillators (Gwinner) 1978).
The central innervation of the mammalian pineal organ is by
no means rudimentary. The central projection to the pineal
organ might not be involved in circadian mechanisms; it may
serve other functions} e.g. as yet unknown types of neural
feedback} and thus modulate the glandular activity of the
pineal organ. I t should not be overlooked that the pineal
organ is a derivative and integral component of the brain.
Vascular Pattern
In contrast to innervation} vascular patterns of the

pineal organ have not been examined in great detail. In our
experience intravital studies of pineal circulation} whenever
technically possible} are from a functional point of view superior
to all injections of dyes and vascular casts. The knowledge
of the vasculature and microcirculation in pineal sense organs
is very limited. If the pineal organ of lower vertebrates
played an essential role in humoral color-change mechanisms}
the active agent (melatonin) should be released into the
bloodstream either directly or via the CSF. In the frog}
Mautner (1965) studied the arterial supply and venous drainage
of the pineal complex. In addition to the major lateral
connections with venous sinuses} vascular collaterals extend
from the epiphysis cerebri to the choroid plexus of the third
ventricle; however} this link is very discrete and probably
wi thout great functional significance. There is no indication
for a portal vascular system between the epiphysis cerebri
and other neuroendocrine centers in the frog brain. The pathways
of release of indoleamines in the pineal organ of anurans
and other pineal sense organs known to contain indoleamines
are enigmatic. It is an open question whether certain types
of PC} apparently precursors of the RPC} may terminate on
blood vessels instead of forming synapses with secondary neurons.
A direct release of indoleamines into the CSF appears possible
although it is not proved.
In the present context the complex problem of the blood-

brain-barrier cannot be discussed in detail. It is known}
however} that in mammals the pineal parenchyma is outside
the blood-brain-barrier (cf. Vollrath) 1981). However} in
the frog} injection of trypan blue does not lead to a staining
of the pineal complex} in contrast to the heavily stained
B .'
Fig. 3A) B. Labeled nerve fibers of central orlgln (arrow) in the
pineal stalk of the guinea pig. Iontophoretic
application of horseradish peroxidase in the distal
portion of the pineal, organ. Bars = 20~m. For
details, see text. Unpublished microphotographs,
courtesy Dr. H.W. Korf. See Korf and Wagner (1980)
for methods.
choroid plexus (Oksche, unpublished results). Further, there

is no evidence showing that capillaries of the fenestrated
type occur in the pineal organ of anurans. If this can be
proved to be true also for other pineal sense organs, it may
indicate a principal difference in the organization of sensory
and glandular pineal organs.
28 A.OKSCHE
The Avian Pineal Organ
The peculiar comparative position of the avian pineal

organ has been discussed in detail by Oksche and Hartwig (1979),
Collin and Oksche (1981), and Korf et al. (1982). The outer
segments of avian pinealocytes exhibi t distinct signs of
structural regression; this regression coincides wi th a
spectacular increase in the secretory capacity of the pinealocytes.
On the other hand, the isolated avian pineal organ in vitro
displays rhodopsin-like photosensitivity (Deguchi, 1981) and
oscilJ.atory acti vi ty of enzymes involved in melatonin synthesis
(Binkley et al. , 1978; Deguchi, 1980; Takahashi et al.,
1980). According to the usual ultrastructural organization
of the retina, the irregular lamellar whorls of the avian
pinealocytes can hardly be regarded as functional structures.
Retinae displaying a comparable degree of genetic or
experimentally induced alteration of receptor ultrastructure
are incapable of producing an electroretinogram. However,
the lamellar complexes of avian pinealocytes may display a
positive opsin immunoreaction, e.g. in the pigeon (Vigh and
Vigh-Teichmann, 1981). Strangely enough, this immunoreaction
is negative in the morphologically far-better developed outer
segment structures of Passer domesticus (I. Vigh-Teichmann,
personal communication). In electrophysiological studies
conducted in different laboratories the pineal organ of P.
domesticus has not shown a direct response to illumination
(cf. Dodt, 1973). The photic response of the pineal organ
of the pigeon to light stimuli (Semm, 1982, this volume)
differs from the reactivity pattern (on- and off- effect)
of typical pineal sense organs (Semm, personal communication).
The possibility should be discussed that light stimuli perceived
by the avian pineal organ may initiate some kind of chemical
reaction instead of leading to the generation of action
potentials.
To date no structural correlate has been found that would

help to interpret the response of the avian pineal organ to
the forces of the magnetic field (Semm, 1982, this volume).
The avian pineal organ is a self -sustained circadian

pacemaker that acts on a population of secondary self-sustained
oscillators located outside the pineal body, e.g. in the
hypothalamus (Gwinner, 1978). The coupling wi thin this system
shows distinct interspecific differences. The oscillators
of birds can be synchronized by light. In photoperiodic responses
of birds, however, two functions must be considered, time
and photosensitivity, the latter being a periodic (circadian)
function. The problem of the avian circadian and photoneuro-
endocrine systems is very complex due to the fact that they
encompass the deep encephalic photoreceptor(s), the
retino-hypothalamic connection and the pineal organ (cf .
Yokoyama et al., 1978).). Thus the effects of pinealectomy

must be viewed in context with other components of the oscillator
system. In principle, the avian pineal organ is an independent
oscillator; the implantation of a denervated pineal gland
in a pinealectomized bird '(Po domesticus) not only reinstates
the circadian locomotor rhythm but also the donor phase of
this rhythm (Zimmerman and Menaker, 1975, 1979).
CONCLUSIONS
The pineal organ of vertebrates has undergone profound

evolutionary changes which have produced remarkable variations
in its sensory and secretory apparatus. The pineal sense
organs of lower vertebrates are endowed with indoleamine-
containing sensory cells (sensoneuroendocrine cells), which
may be capable of the translation of photic information into
a neuroendocrine response. The concentration of sensory and
neuroendocrine properties in one cell line or even in one
cell may offer functional advantages. The photoneuroendocrine
cells of primitive pineal organs were possibly involved in
the proper timing of biological events. Achromatic pineal
organs might have served (and still serve) as a dosimeter
for solar radiation energy, and the chromatic eye-like components
of pineal complexes as a sensor for discrimination of changes
in the spectral composition of daylight. The target areas
of the pinealofugal neural projections are scattered over
a wide area of the central nervous system including reticulomotor
and neuroendocrine areas of the brainstem. The pineal projection
to the pretectal area displays some overlap with the input
from the lateral eyes. Thus, a fine equilibrium in the retinal
and pineal control of rhythmic motor and endocrine events
seems to have arisen. In parallel with the regression of
their outer segments, pinealocytes of the receptor line appear
to have evolved further a capacity for generating humoral
signals. Humoral agents may be mediated via the bloodstream
and/or the circulating cerebrospinal fluid, and then may be
taken up by specialized membrane receptors of target organ
cells. The above-mentioned mechanisms appear to be invol ved
in color change, endocrine control of reproduction, phototactic
and locomotor reactions, thermoregulatory behavior and, in
a wider sense, in the mechanisms of biological clocks. This
remarkable range of biological effects becomes understandable
when the divergent pattern of the neural projection of pineal
sense organs to extended areas of the brainstem is taken into
account; this type of connecti vi ty may result in triggering
a broad spectrum of different functions. In addition, cells
endowed with melatonin receptors may also show a scattered
pattern of distribution.
It is possible that during an early phase of phylogeny

pineal organs directly or indirectly participated in complex
30 A.OKSCHE
reactions serv~ng adaptation to the environment, and thus

they were enhancing survival. The functional significance
of the pineal parenchyma is emphasized by the discovery, using
advanced techniques, of pineal tissue in several mammalian
species where it was previously thought to be missing (cf.
Hofer et al., 1976). It is also present in several species
of anamniotes claimed to be devoid of a pineal organ; however,
in crocodiles all efforts to demonstrate pineal tissue have
been negative. A possible correlation between the geographical
latitude and the extent of development of the pineal organ
needs further examination. In general, the pineal organs
tend to be prominent in vertebrates at higher latitudes and
smaller at lower latitudes (Ralph, 1975); for example, the
pineals of Antarctic penguins are very large and highly glandular
Piezzi and Gutierrez, 1975). Further, differences between
day- and night-active animals and the special features of
the pineal organ in blind cave-dwelling or burrowing species
deserve particular attention (Pevet, cf. Oksche and Pevet,
1981) .
The photoneuroendocrine capacity is a fundamental factor

which must be considered when developing concepts dealing
with the evolution of the pineal organ. The results and concepts
reported in this paper emphasize the importance of the comparative
structural, physiological and biochemical analysis in pineal
research.
SUMMARY
The pineal organ is a derivative and integral component

of the brain. Phylogenetically it has changed from a 'third
eye' capable of direct achromatic and/or chromatic responses
to light to an endocrine gland influenced by visual stimuli
from the retina. In its phylogenetically ancient form the
pinealocyte resembles a cerebrospinal fluid-contacting neuron
endowed with sensory and secretory properties. Such pinealocytes
have been considered to be photoneuroendocrine cells.
Pinealocytes of the photoreceptor type display in their outer
segments a microspectrophotometrically identifiable photopigment
and an opsin-immunoreactive protein. On the other hand, certain
pinealocytes bearing an outer segment contain microspectro-
fluorimetrically detectable levels 5-HT/5-HTP.
Synthesis, storage and release of indoleamines, and probably

also of peptides and/ or proteins, appear phylogenetically
at an early stage and are fundamental properties of pineal
systems. The capacity of formation of elementary granules
parallels the regression of the geometrically regular outer
segment structures and illustrates that the secretory function
is becoming of increasing importance. The secretory pinealocyte
has been considered to be a paraneuron.
In addition to their neuroendocrine messages acting on

various target organs and systems, the pineal sense organs
are capable of sending neural messages to the brain, e.g.
to the habenular ganglion, pretectal region, tegmental reticular
gray and hypothalamus, via pinealofugal tracts. Pinealopetal
projections have been traced from the brain to the pineal
organ in sub-mammalian and mammalian vertebrates. The avian
pineal organ has retained certain neural elements characteristic
of pineal sense organs.
In evolutionary terms, the pineal organ displays the

capacities of a photoreceptor, biological clock and endocrine
gland. It is a member of the group of circumventricular organs
and a component of the photoneuroendocrine systems. The special
anatomical and physiological features of the pineal organ
enable it to translate photic information into a neuroendocrine
response.
ACKNOWLEDGEMENTS
The investigations of the author cited in this report

were supported by research grants from the Deutsche
Forschungsgemeinschaft (SPp 'Mechanismen biologischer Uhren:
Ok 1/24; 1/25). The author is greatly indebted to Professor
J. Priedkalns (Adelaide) and Dr. H.W. Korf (Giessen) for their
help in preparing the manuscript. Figure 1 was prepared by
Miss K. Michael (Giessen). The secretarial assistance of
Mrs. Julia Brazier (Adelaide) is gratefully acknowledged.
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Mikroskopischen Anatomie des Menschen", Vol. VI/7,
A. Oksche, and L. Vollrath, eds. Springer, Berlin-
Wake, K., Ueck, M., and Oksche, A., 1974, Acetylcholinesterase-
containing nerve cells in the pineal complex and sub-
commissural area of the frogs, Rana ridibunda and Rana
esculenta, Cell Tissue Res., 154:423. --
Wolstenholme, G.E.w.:-and Knight, J., eds., 1971, "The Pineal
Gland", Churchill Livingstone, London-Edinburgh.
Wurtman, R.J., Axelrod, J., and Kelly, D.E., 1968, "The Pineal",
Academic Press, New York.
Yokoyama, K., Oksche, A., Darden, Th. R., and Farner, D.S., 1978,
The sites of encephalic photoreception in photoperiodic
induction of the growth of the testes in the White-crowned
Sparrow, Zonotrichia leucoph!ysgambelii, Cell Tissue Res.,
189:441.
Zimmerman, N.H., and Menaker, M., 1975, Neural connections of
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SCience, 190:477.
Zimmerman, N.H .• and Menaker, M., 1979, The pineal: a pacemaker
within the circadian system of the house sparrow.
Proc. Nat 1. Acad. Sci. USA, 76:999.
COMPARATIVE GROSS AND FINE MORPHOLOGY
OF THE MAMMALIAN PINEAL GLAND
J. Ariens Kappers
Houtweg 13
1251 CS Laren N.H.
The Netherlands
Some knowledge of the structure of an organ is in-

dispensable for the investigation and understanding of
its function. This is also very evident in case of the
mammalian pineal organ the structure of which clearly
points to its endocrine function.
Its classical name, and also its official one now,

is "corpus pineale". This term was given to the structure
by Galen (ca 130 - ca 200 A.n.) who observed that, in man,
the organ is shaped like the cone of a pine tree. In gen-
eral, however, the shape, size and also the location of
the mammalian pineal greatly vary among mammals. Before
dealing with this matter, a short survey of the princi-
ples of pineal embryology is necessary because the onto-
genetic development of the organ elucidates much about
its adult morphology and its relationship to neighbour-
ing structures.
Just like in other vertebrates, the mammalian pineal

organ develops from the neuroepithelial layer which forms
the roof of the dorsal part of the diencephalon, the epi-
thalamus. In fetal stages, this layer, which is situated
between the habenular commissure rostrally and the pos-
terior commissure caudally, evaginates forming a sac-like
structure. In the rat, this first indication of a pineal
anlage is observed between the 12th and the 14ith day of
fetal life (Gardner, 1949; Ariens Kappers, 1960; Voll-
rath, 1981, also for literature). Next, the pineal evag-
ination deepens and its wall becomes thicker by cell pro-
liferation. At days 16 and 17. the wall forllLs infoldings
37
38 J.A.KAPPERS
so that follicles appear. Between the 17th and 18th days

ectodermal mesenchyma grows into the pineal anlage be-
tween the follicles. From this mesenchyma, connective tis-
sue strands and blood vessels originate. The blood ves-
sels are situated close to the parenchymal follicles
the cells of which form perivascular palisades. On day
19 of fetal life, the organ has practically lost its fol-
licular structure now consisting of a dense and solid
cell mass. Between this day and birth the organ grows
rapidly. Perivascular connective tissue forms septa which
separate cell groups, cell cords or islands which show
regional cytological differences. In the marginal lobated
islands, for instance, the nuclei are pale, while in
the central cell islands the nuclei contain more chroma-
tin. Already on the 18th day of fetal life, commissural
nerve fibres penetrate the pineal anlage for about three-
quarters of its length (see Vollrath, 1981, for litera-
tur~. Sympathetic nerve fibres reach the pineal only
postnatally. The rat pineal obtains its adult pattern
of green fluorescence intensity pointing to the presence,
in sympathetic nerve fibres, of an aminergic catechol-
amine at postnatal days 10-11, while the final number of
sympathetic varicose nerve terminals is probably estab-
lished only at day 21 after birth.
In rat, the bulk of the pineal tissue, rapidly grow-

ing in size, gradually shifts in a dorsocaudal direction
finally reaching a superficial position in regard to the
brain. It, however, maintains its connection with the
ependyma of the intercommissural region and some pineal
cells which remain situated at this site by means of a
slender pineal stalk consisting of pineal cells. In this
stalk, nerve fibres are present with which we will deal
in our paper on pineal innervation. In adult rats of
most strains, the bulk of pineal tissue is situated just
ventral to the con£luens sinuum. The great cerebral vein
and the prosencephalic median vein drain into the ros-
tral part of this large venous receptacle. As we will
see later, this topographical relationship of the main
superficial pineal part to the large intracranial veins
is of importance for understanding the drainage o£ the
rat pineal veins.
As has been mentioned, the superficial part of the

pineal gland is connected by a pineal stalk with a much
smaller deep part of pineal tissue which keeps its con-
servative position against the ependyma of the roof of
the third ventricle between the habenular and the pos-
terior commissure. Along with the formation of a pineal
stalk, the pineal recess, which is the original recess
MORPHOLOGY OF MAMMALIAN PINEAL GLAND 39
of the third ventricle into the primordial saccular pine-

al anlage, practically disappears in rat.
In man, the ontogenetic development of the pineal is

somewhat more complicated. Its very first anlage appears
in the beginning of the second month of gestation. From
the neuroepithelial sac, evaginating from the roof of the
third ventricle in the epithalamus, a rostral and a caudal
lobe develop by cell proliferation. Vascular connective
tissue grows in from the periphery of the anlage causing
some lobulation of the pineal parenchyma. Between the
cords of pineal cells, vascularized strands of connective
tissue are now present. A somewhat larger connective tis-
sue septum, originally separating the rostral from the
caudal lobe, completely disappears at day 150 of fetal
life and the two lobes fuse (Htilsemann, 1971). Some struc-
tural differences between the two lobes have been de-
scribed, but not studied in detail (see Vollrath, 1981,
for literature). Before the 60th day of pineal embryonic
development, commissural fibres already penetrate into
the organ, while sympathetic nerve fibres arrive at the
caudal pole of the pineal between day 60 and 82 (Htilse-
mann, 1971), or even somewhat earlier (M~ller, 1976).
Varicosities and nerve terminals of such fibres, frequent-
ly observed at day 150 of gestation, contain clear as
well as dense-core or granulated vesicles.
During its ontogenetic development, the human pineal

keeps its original position close to the ependyma between
the habenular and the posterior commissure, the lamina
intercalaris. The adult organ occupies the depression be-
tween the mesencephalic rostral colliculi. Its stalk is
only very short. Rostrally it is divided into two lamina
which enclose the pineal recess which is still present
in the adult human pineal. The ventral lamina comprises
the posterior commissure and the dorsal lamina the haben-
ular commissure. Most of the organ is covered by tela
chorioidea. The pineal lies in the subarachnoid space
and is, therefore, bathed in subarachnoidal cerebrospinal
fluid. We will deal later with the drainage of the veins
of the human pineal.
As has been mentioned before, the shape, size and

location of the pineal gland greatly vary among mammals.
This has already become clear from the above comparison
of the rat pineal with the human pineal. Many types of
classification have been offered. The most recent one is
that by Vollrath (1981). It is based on the relationship
of the organ to the third ventricle and the shape and
topographical arrangement of the pineal tissue. Accord-
40 J.A.KAPPERS
ing to this classification, a pineal belongs to the prox-

imal type or type A when most of its tissue is situated
close to the third ventricle, that is, as we have seen,
in its embryologically conservative position. When the
pineal is of an elongated type, its length being about
more than twice its largest width, it is designated as
belonging to the proximo-intermedio type, or type AB.
Pineal glands of which the most sUbstantial amount of
glandular tissue lies either superficially or topograph-
ically very closely related to the cerebellum, due to a
considerable ontogenetic shift of the bulk of the struc-
ture in a dorsocaudal direction, belong to the inter-
mediate-distal type or type ABC. For further details of
this very useful classification in which also a more gr
less greater reduction of the three parts, i.e.~ the
proximal, the intermediate and the distal part of the
gland, is taken into account, we refer to Vollrath (1981).
In his book, Vollrath also published tables of the shape,
the dimensions and the weights of the pineal in a great
number of mammals.
Some words may still be said about pineal size. Re-

lating pineal weight either to body weight or to brain
weight (Legait et al., 1975, 1976a,b) it has been shown
that large interspecies variations exist in such rela-
tionships as well as in regard to the. so-called epi-
physial index (see Legait et al., 1976b; Quay, 1980, for
definition and details).
In general, pineal activity is known to be corre-

lated with pineal volume. Variations in pineal volume
have been studied in three species of rodents (Quay,
1956, 1978; Legait et al., 1975). Although pineal weight
is known to show, inter alia~ circadian and seasonal
variations, interspecies variations are much greater. Ap-
parently, therefore, interspecies differences in pineal
weight and volume are also based on genetic differences.
Although the larger size of a pineal gland does not in-
dicate per se that the organ is of greater functional
importance it is nevertheless acceptable to assume that
very large pineals play a more important physiological
role than extremely small ones. As has been stressed sev-
eral times (Ariens Kappers, 1981; Pevet, 1982), the pine-
al can be considered to be principally involved in the
integration of information based on environmental condi-
tions. As was already demonstrated in rodents (Reiter,
1973, 1978; Hoffmann, 1979), this holds especially for
daylength in temperate zones. However, also other envi-
ronmental conditions such as temperature, rainfall, food
supply, etc., depending on the ecological situation in
MORPHOLOGY OF THE MAMMALIAN PINEAL GLAND 41
which the species concerned is living (Pevet, 1979, 1981;

Pevet et al., 1981; Vivien-Roels, 1981), may play an im-
portant role. Evidently, variations in pineal size exist
which are due to the adaptation of the species to geo-
graphical, climatological and meteorological conditions
(Pevet, 1982). Strikingly, animals having a very large
pineal gland such as the sea lion, seal, walrus (Tilney
and Warren, 1919), northern fur seal (Elden et al., 1971),
Weddell seal (Cuello and Tramezzani, 1969), elephant seal
and some Cervidae (Ralph, 1975) are all living under arc-
tic or holarctic conditions ranging to high altitudes and
high latitudes. In contrast, animals such as the anteater,
sloth, armadillo and pangolin (Oksche, 1965; Ralph, 1975;
Quay, 1965) in which pineal cells have been found to be
quite absent or to be present in an extremely small
amount only, and animals such as the rhinoceros, the
elephant dugong (Oksche, 1965; Ralph, 1975; Hill, 1945)
and the red kangaroo which have a small pineal, are all
living under tropical/equatorial conditions. An apparent
latitudinal relationship to pineal size has also been
demonstrated in some rodents.
Next to this it should be remarked that, in general,

small pineals appear to be correlated with a nocturnal
life and largei pineals with a diurnal life of the ani-
mals concerned (Ralph, 1975). Moreover, a correlation
seems to exist between pineal size and the degree of ho-
meothermia. Many heterothermic endotherm animals have a
small pineal, a fact which should perhaps be correlated
with the capacity of pineal function for adaptation to
the thermal environment. Endothermic mammals which live
in a rather harsh thermal environment, such as is the
case in arctic zones, but which nevertheless maintain a
constant and warm body temperature and which have also a
necessarily seasonal-conditioned reproductive pattern.
mostly show a well-developed pineal organ (see Ralph,
1975, for details).
The great variability in pineal size among mammals

is probably associated with the functional capability
of the pineal to adapt them to several very different
environmental conditions. If external conditions are not
very rough as is true for seasonal changes in temperature
in tropical/equatorial zones (DeLany and Happolid, 1979),
the pineal will be relatively small. However, desert life
is difficult for mammals to endure. This could, perhaps,
explain why in some tropical/equatorial species the pin-
eal has been reported to be large and most active. In
the opinion of Pevet (1982, see also for details) it
would be of special importance to investigate the cor-
42 J. A. KAPPERS
relation between pineal size, not with latitude, but with

latitude-related biotopes such as equatorial wet forest,
savanna, desert zones, temperate, arctic and holarctic
zones in combination with zonation in altitude (see also
DeLany and Happolid, 1979, for details), all representing
biotopes in which the influence of environmental condi-
tions on the mammals concerned differs.
In the experimental animal most used, the rat, the

pineal complex shows rather considerable variations de-
pending on the various strains. Basically, it consists of
a proximal or deep part which is situated close to the
third ventricle, a pineal stalk and a distal or super-
ficial part. Especially the pineal stalk may show varia-
tions which are also individually conditioned (Boeckmann,
1980). In Sprague-Dawley rats, for instance, the stalk
may be more or less reduced. If total reduction occurs,
the pineal complex exclusively consists of a deep and a
superficial part. In the albino rat used in the investi-
gations of the present author, the relatively very small
deep part of the pineal was constantly connected by a
slender stalk containing differentiated pinealocytes
with a very well-developed distal or superficial part.
In the hamster, a small but distinct amount of pineal
tissue is situated close to the habenular commissure
forming the "deep pineal", while the bulk of pineal tis-
sue is situated distally and superficially. Occasional-
ly, clusters of pinealocytes could be observed between
these two main parts of the pineal complex (Sheridan and
Reiter, 1970), but often this connection exclusively
consisted of myelinated and non-myelinated nerve fibres
and blood vessels, a true pineal stalk entirely lacking.
Removing for experimental purposes the superficial part
of the pineal complex is relatively easy. It should,
however, be realized that removing this part in animals
having also a "deep pineal" does not lead to a total
pinealectomy. However, according to the results obtained
after such an operation it appears that, at least in the
hamster, removal of the superficial part of the pineal
complex only can practically be considered to be a
total removal of the gland.
In the rabbit pinealectomy is especially difficult

because the long, club-shaped organ consists of a dis-
tinct proximal part, a long stalk and a major distal or
superficial part the terminal end of which is invagi-
nated into the confluens sinuum and either one of the
two transverse sinuses (Smith, 1971; Romijn, 1973).
In many mammals, a conspicuous suprapineaZ- or supra-

habenular recess is present. This is formed by an evagi-

nation of the roof of the third ventricle ontogenetically
arising just rostral to the habenular commissure and ex-
tending in a dorsocaudal direction. The ventral wall of
this recess may cover the deep part of the pineal complex
and part of the pineal stalk, such as in rat and guinea-
pig. If the pineal is exclusively of the deep type, this
recess may cover most of the total gland. Because sub-
arachnoidal tissue is present between the ventral epen-
dymal wall of the recess and the pineal there is no di-
rect contact between this wall and the pineal tissue
proper which is, moreover, covered by a pial envelope and
sometimes by a sheath of glial tissue. Therefore, it can
practically be excluded that, via the ependymal wall of
the suprapineal recess, a direct transport of pineal se-
cretory compounds is possible to the cerebrospinal fluid
present in this recess.
Some findings have, however, provided evidence that,
in mammals, the cerebrospinal fluid may at least serve
as a transport medium for pineal compounds since it has
been found to contain melatonin. The question is how
such compounds may reach the cerebrospinal fluid, either
direcly or indirectly. As we have seen it is not very
reasonable to suppose that pineal compounds can reach the
cerebrospinal fluid via the ependyma of the suprapineal
recess. Interestingly, however, in the pineal recess of
hamster, vole, guinea-pig and Rhesus monkey, an area is
present in which the ependymal lining of this recess
shows interruptions through which the processes of pine-
alocytes penetrate so that the surface of the protruding
cells is in immediate contact with the cerebrospinal
fluid (Hewing, 1978, 1980). This situation reminds of
earlier phylogenetic stages of the pineal and suggests
the possibility of a direct release of pineal compounds
into the cerebrospinal fluid. It is, nevertheless, quite
sure that by far most pinealocytes present in the pineal
complex release their secretory products into the blood
by way of the pineal venous drainage as we will see
later.
By all authors studying this subject it has been

stressed that the blood supply of the mammalian pineal
gland is very large. The minimum rate of rat pineal blood
flow per gram exceeds that of most endocrine organs,
equals that of the neurohypophysis and is surpassed only
by that of the kidney (Goldman and Wurtman, 1964). The
oxygen consumption of pineal tissue is, however, rather
low (Quay, 1962, 1963) and decreases with age. Interest-
ingly, at the onset of light a pronounced vasoconstric-
tion has been observed (Quay, 1972). Removal of the
44 J.A.KAPPERS
pineal sympathetic innervation, known to influence pos-

itively the production of melatonin by the pinealocytes,
especially during the night, reduces the pineal blood
flow by one third (Goldman, 1967). These facts, shortly
mentioned here, already point to the special role of the
pineal blood flow as a vehicle for the transport of pin-
eal secretory products.
Arterial blood reaches the organ either by branches

of the posterior choroidal arteries, which originate from
the posterior cerebral,arteries, or by branches arising
directly from the posterior cerebral arteries. Before
entering the pineal, these very thin arteries run in the
pineal envelope of the organ. In the human, pineal arte-
rioles penetrating the organ follow the connective tis-
sue septa then giving off capillaries. In many other mam-
mals, however, occasional arterioles are present only,
the pineal being directly penetrated by capillaries which
originate from arterioles in the pial pineal covering. In
the organ, the capillaries form an extensive capillary
network which is drained by pineal venules which form
one or more larger veins. In man, having a proximal pin-
eal gland only, a single pineal vein drains into the
great cerebral vein which terminates in the sinus rectus
<Gladstone and Wakeley, 1940), while, according to an-
other author (Bargmann, 1943) venous pineal blood is al-
so drained into the bifurcation of the two internal cere-
bral veins which, more caudally, give rise to the great
cerebral vein. In rodents, in which the distal or super-
ficial part of the pineal complex is very well-developed,
pineal veins drain directly into the great cerebral vein.
From here the blood is transported via the confluens
sinuum and the transverse sinuses into the general circu-
lation. In the Wistar rat, about 12-16 peripheral col-
lecting pineal veins were counted which all drain into
the great cerebral vein. Usually, one central vein was
observed to be present in every pineal gland (Hodde,
1979, 1981).
Because the ultrastructure of pineal capillaries and

their relationship to the pineal parenchyma is of great
importance for understanding pineal function we will
shortly deal with these subjects here although, other-
wise, we will not deal with pineal ultrastructure, a few
topics excepted. In the rat, mouse and ground squirrel
the junctions between adjacent endothelial cells of the
capillaries are variable in shape. Typical tight junc-
tions are absent, but zonulae occludentes have been ob-
served in the chinchilla, while maculae adherentes have
been demonstrated in the cow, sheep and dormouse (see
Vollrath, 1981). Not in all mammalian pineals fenestra-
tions or pores in the capillary endothelium could be ob-

served. In some mammals they are rather scarce, but in
the pineal capillary endothelium of the mouse and the
ground squirrel they occur frequently. The pores are
mostly bridged by a very delicate membranous diaphragm of
about 5 nm in thickness. The pores measure between 50 and
70 nm in diameter.
The cytoplasm of the endothelial cells contains the

organelles usually present such as mitochondria, a Golgi
complex, ribosomes and cisterns of rough endoplasmic re-
ticulum. Occasionally, filaments, micro~ubules and multi-
vesicular bodies are present. A large number of pino-
cytotic vesicles is a regular feature of the endothelial
cells. Their size varies according to the species con-
cerned. The basal lamina of the capillary endothelium
forms the inner lamina of the perivascular spaces which
are a very prominent characteristic in most mammalian
pineals. The outer lamina of the perivascular space is,
in principle, formed by the basal membrane covering the
pineal parenchyma proper. This outer lamina, however, can
be more or less reduced being, therefore, discontinuous.
Extensions of perivascular spaces, which do not contain
a capillary, may penetrate more or less deeply into the
pineal parenchyma separating clusters or cords of pinealo-
cytes. Such spaces have even been supposed to be continu-
ous with intercellular spaces within the pineal paren-
chyma. In this way, a complicated network of communica-
ting spaces is distributed allover the gland.
The width of the perivascular spaces varies among

mammals and so does their content. When the space is
narrow it contains only a few nerve fibres of sympathetic
origin such as has been shown in the squirrel monkey,
cow, sheep and rabbit. In contrast, the perivascular
spaces in cat are wide and contain many nerve fibres. In
rat they are even extremely wide. In them, nerve fibres,
terminals of such non-myelinated nerve fibres, collage-
nous fibres, fibrocytes and the processes of glial cells
have been demonstrated.
The autonomic nerve fibres present in the perivas-

cular spaces may, varying among mammals, enter these
spaces along with the vessels penetrating the pineal
from its outer surface. In that case they may either
terminate within the perivascular spaces or enter, from
here, the pineal parenchyma to end between the pinealo-
cytes and their processes. A number of fibres, however,
directly penetrates into the pineal parenchyma. Some of
such fibres reach, from here, the perivascular spaces
46 J.A.KAPPERS
to terminate in these spaces, while most of them end with-

in the pineal parenchyma proper. Neurotransmitter sub-
stance released from the sympathetic nerve terminals
present in the perivascular spaces can reach the pinealo-
cytes by way of the extensions of these spaces and the in-
tercellular spaces, which, together, form an extensive
and continuous intrapineal canalicular network as men-
tioned above. Probably, neurotransmitter released in the
perivascular spaces can also reach parenchymal cells more
directly via the outer lamina of these spaces which
borders the pineal parenchyma, especially if the outer
lamina is discontinuous. In some mammals, the pineal
parenchyma is separated from the perivascular space not
only by a basal lamina, but also by a thin, often like-
wise discontinuous layer of astrocytic glial processes.
From the foregoing it will be clear that, in many

mammals, a very firm tissue layer separating the peri-
vascular space from the pineal parenchyma which, here,for
the most part consists of the terminals of pinealocyte
processes, is absent. As mentioned, the separation between
the pineal parenchyma and the perivascular space is
formed only by an often discontinuous basal lamina and!
or a thin glial sheath which may likewise be discontinu-
ous. It can, therefore, not wonder that in some mammals
the terminal endings of the pinealocyte processes occa-
sionally herniate or bulge directly into the perivascular
spaces. In the cat such bulging endings are covered by
an extremely thin glial barrier and the basal lamina.
In the Rhesus monkey, the terminals of the pinealocyte
processes penetrating deeply into the perivascular spaces
are exclusively covered by the basal lamina, while, in
the rat, the terminals of such processes bulging into
the perivascular spaces are often completely devoid of
either a glial sheath or a basal lamina (Wartenberg,
1968). Pinealocyte processes directly terminating in
perivascular spaces have also been observed in the dog,
hamster, guinea-pig, mouse and gopher (see Vollrath,
1981, for literature). Evidently, a close topographical
relationship exists between the terminals of pinealocyte
processes - in which at least part of the pineal secre-
tory products accumulate and from which these are re-
leased - and the perivascular spaces. This situation in
combination with the fact that in many mammals the endo-
thelium of the capillary wall is fenestrated points
morphologically already to the possibility that pineal
secretory products can easily be taken up by the blood
and illustrates the endocrine function of the mammalian
pineal organ.
A feature much discussed especially in clinical cir-

cles and shortly to be mentioned here is the presence, in
the pineal, of brain sand~ corpora arenacea or acervuli~
all synonyms for the same type of concretions. As such
concretions are opaque in X-ray photographs of the human
brain they form a point of orientation for the radiolo-
gist. It has long been maintained that the occurrence of
brain sand would in some way be negatively related with
the secretory activity of the organ, especially since, in
man, they are more numerous in old age. Such concretions
are not exclusively present in the pineal of Primates in-
cluding man as was thought in earlier days, but they may
also occur in other mammals such as, for instance, in the
rat. Their number generally greatly varies as does their
shape, histological appearance and topography. In prin-
ciple, corpora arenacea have been observed in glial and
stromal compartments. In them, the presence of hydroxy-
hepatite or carbonate apatite, iron, magnesium and phos-
phorus have been demonstrated. It should be noted that
such concretions have not only been found in the pineal
gland, but also in the habenulae and in the leptomenin-
geal tissue adjacent to the pineal. From the rather ex-
tensive literature (see Vollrath, 1981) it can be con-
cluded that both the origin and the formation of these
acervuli and their functional meaning, if any, are far
from clear. Formerly it has often been held that the
presence of brain sand would point to an atrophy and
functional degeneration of the gland. It is, however, now
known that this is not the case, It was, for instance,
demonstrated that the metabolic activity of the human
pineal does not alter in old age in which, generally,
more acervuli are present (Wurtman et al., 1964) and
that there is no correlation between the degree of pine-
al calcification and either the overall histological
structure of the gland (Cassano et al., 1961) or pineal
enzyme levels including those enzymes which are of cru-
cial importance for the production of pineal indole-
amines which are generally considered to play a most im-
portant role in pineal function (Otani et al., 1968).
Pineal cysts are also a rather regular feature. They

may already be found at birth. In some cases it is quite
apparent that such cysts can be considered pinched off
parts of the normally occurring pineal recess of the
third ventricle because, in such cases, the wall of the
cysts consists of ependymal and pineal parenchymal cells.
In other cases, however, cysts are clearly present in
glial patches which may point to a more or less patholog-
ical process such as likewise occurs in other parts of
the central nervous system. Apparently, such cysts are
48 J. A. KAPPERS
of no consequence for a normal pineal function. As this

paper deals with the normal mammalian pineal gland, pine-
al pathology will not be dealt with.
In some mammals, such as the rat, the presence of

striated muscle fibres has been observed in the pineal.
Ultramicroscopically, such fibres show embryonic features
and either or not a close topographical relationship to
blood vessels. Their origin and functional significance,
if any, is quite unknown. Most probably they originate
from the mesenchyma invading, in embryonic stages, the
neuroepithelial anlage of the organ.
In most mammals, pineal connective tissue is scarce.

In man, connective tissue septa and strands may separate
islands, cords, rosettes or follicles of pinealocytes.
The septa often contain blood vessels and nerve fibre
bundles. Mast cells have also been observed to be present
in small numbers, while pigment-containing melanophores
could be demonstrated in the pineal stroma in a number of
mammalian species. It is most questionable whether mel-
anophores are involved in pineal function.
As will more elaborately be dealt with in the paper

on pineal innervation, in some few mammals nerve cells
were noted to be present within the pineal parenchyma.
They are certainly not a constant feature of the mamma-
lian pineal. Abundant nerve fibre bundles and single
nerve fibres are, however, constantly present. They are
of different origin as will be mentioned in our second
lecture.
Proceeding now to the histology and cytology of the

pineal parenchyma proper it can be stated that its major
component is the pinealocyte 3 sometimes termed pineocyte.
In the rat, pinealocytes make up about 82% of the total
pineal cell content (Wallace et al., 1969). The pinealo-
cyte is a polymorphous cell element especially in the
human pineal in which unipolar, bipolar and multipolar
pinealocytes have been demonstrated by means of the sil-
ver carbonate staining technique. In most mammals, how-
ever, the pinealocyte consists of a cell body and a
variable number of cell processes which vary in length
and width. The terminal endings of the pinealocyte pro-
cesses are often club-shaped. Ultramicroscopically, they
are sometimes difficult to distinguish from nerve termi-
nals, the more so because they may likewise contain gran-
ular vesicles. The ter'minal endings of pinealocyte pro-
cesses are situated either within the parenchyma between
adjacent pinealocyte perikarya or processes, or in close
vicinity to perivascular spaces from which they are then

separated by a thin basal lamina, a thin glial sheath or
both, the glial sheath and/or the basal lamina being of-
ten discontinuous as has been mentioned before. As has
also been described, in some mammals the endings of pine-
alocyte processes bulge into the perivascular spaces.
As to the phylogenetic and ontogenetic origin of the

mammalian pinealocyte the following may be remarked. It
has been maintained that, phylogenetically, the mammalian
pinealocyte is the final stage of a so-called sensory
cell line. Earlier stages in this line are the true photo-
sensory cells or photoreceptor cells and the secretory
rudimentary photoreceptor cells which are present in the
pineal organ of non-mammalian vertebrates (Collin, 1971).
Rather recently it could be demonstrated that, also onto-
genetically, pinealocytes show signs of such an origin.
In this regard, the presence of centrioles and cilia in
mammalian pinealocytes is of special interest.
If at all present, centrioles may show a number of

rather unusual features. Apparently, they may transform
into microtubular sheaves or bouquets (Wolfe, 1965j Lin,
1969, 1970, 1972). Probably, such a transformation does
not point to their degeneration, but to the formation of
structures which recall outer segments or cilia as pres-
ent in the above-mentioned phylogenetic precursor cells
of pinealocytes, such as cilia of the 9 + 0 pattern
(Vollrath, 1981). No centrioles are present in the adult
rat pinealocyte, but they still occur in the pinealocytes
of fetal (Clabough, 1973) and of newborn (Zimmerman and
Tso, 1975) rats. Here they display the usual fine struc-
ture becoming elongated between 15 days and 6 weeks of
age (see Vollrath, 1981, for further details). Abortive
remnants of a ciliary apparatus, such as a Ciliary root-
let in continuity with a centriole, have even been de-
scribed in pinealocytes of the adult guinea-pig (Lin,
1972) and of the adult hedgehog (Pevet and Saboureau,
1973) .
True cilia occur in a number of very different cells

of the adult mammalian organism, such as in some ganglion
cells. Their presence in adult mammalian pinealocytes
attracted special attention because they have also been
considered to po~nt to the phylogenetic as well as the
ontogenetic origin of pinealocytes from their precursor
cells of the sensory cell line. In rat pinealocytes, the
presence of cilia, like that of centrioles, has been
noted in fetal (Clabough, 1970, 1973) and in early post-
natal stages (Zimmerman and Tso, 1975) of development.
50 J. A. KAPPERS
In pinealocytes of the adult rat cilia are, however, very

rare. In the pinealocytes of most other mammals the pres-
ence of clilia shows great variability. In the pinealo-
cytes of the noctule bat (Pevet et al., 1977), mole-rat
(Pevet et al., 1976) and mole (Pevet, 1974) cilia are,
however, frequently observed. Like in the phylogenetic
precursor cells of pinealocytes they are of the 9 + 0
type. In the adult mole each pinealocyte has been report-
ed to contain a cilium (Pevet and Collin, 1976). Struc-
tures reminiscent of rudimentary outer segments such as
are present in the secretory rudimentary photoreceptor
cells in the pineal organ of lizards and birds, have been
demonstrated in pinealocytes of the noctule bat (Pevet et
al., 1977), while even concentric lamellar structures
resembling outer segmental discs were observed in close
vicinity to cilia (Pevet and Collin, 1976; Pevet et al.,
1977). The findings mentioned clearly indicate that, in-
deed, the mammalian pinealocyte is derived not only phy-
logenetically, but also ontogenetically from precursor
cells of the so-called sensory cell line. At least in
some mammals such an origin has been demonstrated during
the ontogenetic development of the pinealocyte.
Mammalian pinealocytes not seldomly contain more

than one nucleus showing one or more nucleoli. The nuclei
are polymorphic. During the times light microscopy was
exclusively available for pineal investigation, much has
been speculated about the true nature of the so-called
"Kernkugeln" or nuclear pellets which present themselves
as nuclear inclusions. Electron microscopical research
has, however, clearly demonstrated that these are, in
fact, parts of the cell cytoplasm which are invaginated
into the lobulated nuclei. Pinealocyte nucleoli are
prominent, but contain little RNA. Nuclear size shows
small variations according to the site of the pinealo-
cytes either in proximal or distal regions of the gland,
while differences in size have also been described in
the so-called cortical and medullar region of the organ.
Nuclear size also varies with the age of the animal, the
seasons, the functional state of the gonads and, in
general, with the activity of the pineal gland. Such
changes are, however, small and do not offer very re-
liable indications as the functional state of the pine-
alocyte is concerned, at least not without very stringent
statistical investigations. The circadian changes in size
shown by pinealocytes can be expected to be related with
the well-known circadian changes in pineal gland secre-
tory activity as stated by cytochemical and pharmacolog-
ical research.
In the fully adult mammalian pineal the pinealocytes

show very little mitotic activity. In the rat it has, how-
ever, been demonstrated that the mean number of mitoses
follows a circadian rhythmicity showing a peak at midday
(Renzoni and Quay, 1964; Quay and Renzoni, 1966).
On morphological grounds, different types of pine-

alocytes have been distinguished. The most frequent dis-
tinction made is that between so-called light and dark
pinealocytes. By some authors it has, however, been
claimed that such a distinction is merely based on different
functional states of the same cell type (see Vollrath,
1981). Recently, however, two different populations of
pinealocytes have been described on the ground of their
different ultrastructure and possible fUnction (Pevet,
1977a, 1979). Pinea10cytes of population I, representing
the majority of pinealocytes, contain a well-developed
Golgi apparatus producing granular vesicles, while pine-
alocytes of population II are characterized by the pres-
ence of accumulations of material or by the formation of
vacuoles containing flocculent material of moderate elec-
tron density, both in the cisterns of the endoplasmic re-
ticulum which is more abundant in these cells. In the
mole, this type of pinea10cyte shows a rather rough en-
doplasmic reticulum comprising lozenge-shaped paracrys-
tal1ine structures (Pevet, 1974, 1976, 1977a, 1979; Pevet
and Smith, 1975) which are of a proteic nature disappear-
ing after pronase treatment (Pevet, 1977b). Formation of
vacuoles by the endoplasmic reticulum has, under natural
conditions, been observed in the hedgehog (Pevet and Sa-
boureau, 1973; Pevet, 1976) and the noctu1e bat (Pevet
et al., 1977) and,under experiment~l conditions, in the
hedgehog (Pevet and Saboureau, 1974) and rat (Karasek et
a1., 1976; Karasek and Marek, 1978). Such material has
been considered a special type of secretory product, in
some way related to the antigonadotropic activity of the
pineal gland (Pevet, 1977bj Pevet and Ariens Kappers,
1977a,b). In the mole, the paracrysta11ine structures
have been demonstrated to increase in number in males
during the period of high sexual activity and in females
during oestrus, gestation and lactation (Pevet and Smith,
1975). These findings are, at least, suggestive of some
relationship between their presence, the functional state
of this type of pinealocyte and the functional state of
the sex organs. In castrated rats, the endoplasmic re-
ticulum in such pinealocytes has been shown to produce
vacuoles containing moderately dense material which is
released into intercellular spaces (Karasek et al.,
1976) Similar vacuoles the origin of which could, how-
ever, not be established exactly, have been demonstrated
52 J.A.KAPPERS
in hedgehog pinealocytes at the beginning of the physio-

logical phase of quiescence (Pevet and Saboureau, 1973)
and also when such a quiescence was experimentally in-
duced (Pevet and Saboureau, 1974). Not in all mammalian
pineals two populations of pinealocytes showing clearly
the different characteristics mentioned can be distin-
guished. In such cases, in which the organ shows one
population of pinealocytes only, a single pinealocyte may
combine the cytological and functional properties of cells
belonging to the two different populations to be found in
other mammals. Further investigations on this subject are
certainly necessary and could prove to be most fruitful
for the undertanding of pineal function.
In this necessarily short survey of the comparative

gross anatomy of the mammalian pineal organ, its devel-
opment, microscopic anatomy and cytology we cannot deal
with all of the structures present in pinealocytes. We
will, however, shortly mention the presence of lipid
droplets in such cells. Pinealocytes contain varying
amounts of such droplets and their size, location in the
cell and shape are also variable. MUch has been specu-
lated about their possible functional significance. The
chemistry and histochemistry of pineal lipids has been
thoroughly reviewed by Quay (1974). No lipid compounds
have been identified which are characteristic of pinea-
locytes only. Probably, pineal lipids are primarily in-
volved in the general cell metabolism and in membrane
structure and function of pinealocytes, not playing any
role as hormones or hormone precursors. It is, however,
of some interest that pineal lipid content in the rat
demonstrates a circadian rhythmicity showing a peak at
midday and being lowest at midnight (Quay, 1965, 1974).
Moreover, pineal lipid content is in some way correlated
with the activity of the gonadal axis. In female rat it
was observed to be lowest just before the start of ovu-
lation and maximal at the time of dioestrus (Zweens,
1965). Pineal lipid content proved also to be age-depen-
dent. In the human it increases with age (Quay, 1957),
but it was shown to decrease in female rats between 8
and 15! weeks of age (Zweens, 1965). Under different
experimental conditions either an increase or a decrease
in the mammalian pineal lipid content has been observed
(see Table 16 in Vollrath, 1981). From the above find-
ings the conclusion can be drawn that the lipid content
of pinealocytes is somehow correlated with their func-
tion, but that the exact role played by these lipids is
still enigmatic.
Finally we will turn to another constituent of the

mammalian pineal parenchyma, the gZiaZ ceZZ which has al-

so been termed interstitial cell. The number of pineal
glial cells greatly varies among mammals. In the pineal
parenchyma of the rat their number amounts to about 12%
of the total number of pineal cell elements (Wallace et
al., 1969), while in cat and monkey the ratio of glial
cell perikarya to pinealocyte perikarya is less than
1 : 10 (Wartenberg, 1968). In the rat pineal gland the
glial cells differ in some aspects from the classical
astrocytes, more especially because they have some ultra-
structural features in common with the so-called dark
pinealocytes. This is the reason why, by some authors,
they have been designated by the more neutral term in-
terstitial cells. Recently, however, light microscopical
studies in which antibodies against characteristic glial
marker proteins were used, suggested that the "intersti-
tial cells" in the rat pineal are, indeed, astrocytes
(M¢ller et al., 1978).
In general, mammalian pineals which are superficial-

ly situated contain but few glial cells and glial pro-
cesses, whereas deep pineals mostly show an abundance of
astrocytic perikarya and processes (HUlsemann, 1967;
Sheridan and Reiter, 1973). Astrocytes in the pineal may
be of either the fibrous or the cytoplasmic type, just
like in the brain. In the pineal, however, fibrous
astrocytes apparently outnumber protoplasmic astrocytes.
In the human pineal, which is deeply situated, glial
fibres being the processes of fibrous astrocytes are
very abundant (HUlsemann, 1967). The glial fibres form
an intricate network which surrounds groups of pinealo-
cytes. This network is specially dense in the periphery
of the organ where glial patches have occasionally been
observed. In other primates, regional differences in the
amount of glial fibres and their arrangement have also
been described (HUlsemann, 1967; Eckhardt, 1972; Merker,
1968). Not all glial fibres necessarily originate from
within the pineal organ proper (see Vollrath, 1981, for
literature). In the rhesus monkey, glial fibres reach
the pineal via its short stalk. Here such fibres evident-
ly originate from the sub ependymal layer of the third
ventricle (Eckhardt, 1972).
In non-primates the amount of glial fibres shows

great interspecies variations. In the pineal of some
mammals even no glial fibres could be demonstrated at
all, while in that of others they are relatively scarce
(see Vollrath, 1981, for literature). The localization
of glial fibres within the pineal also varies. They
may be distributed in the marginal zone of the organ,
54 J. A. KAPPERS
in its centre, close to blood vessels and in connective

tissue septa.
As protoplasmic astrocytes are concerned, little in-

formation is available from light microscopic investiga-
tions. As has, however, been mentioned before a glial
barrier may separate pinealocytes, especially the termi-
nals of their processes, from the perivascular spaces as
is the case in cat and monkey (Wartenberg, 1968). This
barrier is formed by cytoplasmic glial cells and their
processes. About the exact function of pineal glial cells
nothing is known that fundamentally differs from what we
know about the function of astrocytic glia in general.
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Wurtman, R.J., Axelrod, J., and Barchas, J.D., 1964, Age
and enzyme activity in the human pineal, J. clin.
Endocrinol., 24:299.
Zimmerman, B.L., and Tso, M.O.M., 1975, Morphologic evi-
dence of photoreceptor differentiation of pinealo-
cytes in the neonatal rat, J. Cell BioI., 66:60.
Zweens, J., 1965, Alterations of the pineal lipid content
in the rat under hormonal influences, Progr. Brain
Res., 10:540.
PERSPECTIVES OF COMPARATIVE ANATOMY
Lutz Vollrath
Department of Anatomy
Johannes Gutenberg-University
Mainz
INTRODUCTION
As the precise role of the pineal gland has been established

in a few mammalian species only, every effort should be made to
elucidate the function of this potentially important neuroendocrine
organ in as many species as possible. One way in which the morpho-
logist can contribute to reach this goal is to carry out comparati-
ve studies. Comparative anatomical studies with respect to the pi-
neal gland can be, and should be, carried out at different struct-
ural levels: (a) At the gross anatomical level; (b) at the tissue
level; (c) at the cellular level, and (d) at the level of cell or-
ganelles.
GROSS ANATOMY
From a comparative point of view, the pineal gland is a most

interesting organ. The morphologist is particularly impressed by
the great structural diversity of the organ at the gross anatomical
level, though it should be noted that due to the small size micros-
copic methods may have to be used to examine the organ and its topo-
graphic relationships. The present contribution restricts itself to
the situation in mammals, which alone give enough food for thought.
An often discussed point is whether a pineal gland is
regularly present or not (Bargmann, 1943; Oksche, 1965; Ralph, 1975).
From a recent survey (Vollrath, 1981), it can be seen that the or-
gan has been clearly demonstrated in a large number of mammalian
species. Therefore, the species are of special interest which lack
a pineal organ or have very small, atrophic pineals. One Order of
61
62 L. VOLLRATH
mammals in which the pineal is apparently not regularly present is

the Xenarthra. Whereas Bargmann (1943) mentioned an absence of the
pineal in Dasypus novemcinctus. D. villosus and Bradypus and Ralph
(1981) in D. novemcinctus, Hofer et alo (1976) found diffuse pineal
tissue arranged around the intercommissural recess in the latter
species. The absence of pineal tissue has been established also for
some marsupials, i.e. Petaurus breviceps and P. norfolcensis (John-
son, 1977).
An atrophic pineal gland has recently been described for the

equatorial African species Heterocephalus glaber, measuring as little
as 0.002135 mm 3 and representing the smallest rodent pineal gland
in absolute size reported so far (Quay, 1981). This finding is of
particular importance as it conforms to the trend noted by Ralph
(1975) and Quay (1980a) that rodents whose geographic distributions
are centred in lower latitudes have relatively small pineal glands,
whereas rodents at higher latitudes are characterized by greater
pineal volume. In view of this situation it would appear wise to car-
ry out comparative anatomical and comparative physiological studies,
concentrating on species inhabiting lower latitudes. in particular
as their pineal glands reveal ultrastructural peculiarities (Pevet
and Kuyper, 1978; Pevet and Yadav, 1980).
Moreover. attempts should be made to breed small equatorial

rodents in the laboratory to have a sufficient amount of experimen-
tal animals available for systematic studies. In my view, it would
be highly desirable to select rodents characterized by very small,
yet non-atrophic pineal glands which could be easily quantitatively
surveyed with respect to a perhaps mosaic-like structure of the pa-
renchyma (Vollrath, 1979), direction and course of pinealocyte pro-
cesses, distribution of peripheral sympathetic as well as central
commissural fibres and ultrastructural peculiarities.
Apart from the size of the pineal gland, comparative anatomical

studies have to take into consideration the form and the position
of the pineal complex. A classification has been proposed (Vollrath,
1979, 1981) which enables researchers to indicate with a few letters
the type of pineal gland present. According to this classification.
an ABC pineal gland is a dumbbell-shaped organ in which the proximal
part (area A) lies in the immediate vicinity of the third ventricle
and the distal part (area C) just beneath the skull, in close rela-
tion to the cerebellum; the B indicates that the intermediate area
of the pineal complex (area B) is distinctly reduced in size when
compared with areas A and C. The respective letter is omitted when
the area it stands for is missing.
The usefulness of this classification has yet to be demon-

strated, but one advantage is that researchers are beginning to
look very carefully at the arrangement of pineal tissue and are
COMPARATIVE ANATOMY OF THE MAMMALIAN PINEAL GLAND 63
beginning to realize the distinct inters train differences that exist.

These are particularly noticeable in rats. In the albino rats stud-
ied by Gregorek et al. (1977), a distal pineal was regularly present
whereas a proximal or deep pineal was always lacking. By contrast,
in the 106 Sprague-Dawley rats studied by Boeckmann (1980) a deep
pineal gland was always present. The Wistar rats investigated by
Korf and M~ller (unpublished observations) resembled those described
by Gregorek et al. (1977). In view of these diverse findings it may
be wise to breed a specific strain of albino rats characterized by
a uniform pineal complex. Another interesting approach might be to
select extreme forms and to examine what effect cross-breeding has
on the pineal complex. In this context it is interesting to note
that according to Gardner (1953) the superficial pineal of the hood-
ed rat is not connected to the epithalamus. This poses the question
as to how the central innervation (Buijs and Pevet, 1980; Korf and
Wagner, 1980; Schneider et al., 1981) of the pineal gland is ef-
fected.
The classification referred to above is also a useful basis

for studying whether or not closely related mammalian species have
similar types of pineal complexes. It is premature to answer this
question as an insufficient number of mammalian species has been
investigated in this respect. Nevertheless, some interesting facts
emerge. The Primates, Chiroptera, Tupaiodea, Insectivora, Marsupi-
alia and Monotremata studied so far are mainly characterized by
proximal or Type A pineals; in Artiodactyla and Perissodactyla there
appears to be the tendency of the pineal complex to extend into a
posterior direction, forming what can be classified as a Type AB
pineal; a similar trend is noticeable in fissipede carnivores and
especially the pinnipede carnivores, which have Type ABC pineals.
The greatest variability is found in rodents, ranging from a Type
A pineal (Glis glis) to an aC pineal (Mesocricetus auratus) (for
further details, see Vollrath, 1981). This structural diversity of
the pineal complex clearly calls for an explanation, but before
this goal can be achieved the pineal complexes of many more mamma-
lian species have to be analysed. In this context it is interesting
to note that the latitudinal trend in relative pineal size observed
in rodents (Quay, I 980a) is not seen in Insectivora, Microchiroptera,
Artiodactyla, Prosimii and Anthropoidea (Quay, I 980b) •
HISTOLOGY
Recent systematic comparative studies of the pineal gland at

the tissue level are lacking. Among the many interesting points to
be studied, three aspects are particularly important. One is the
arrangement of pinealocytes within the parenchyma. Often it has been
observed that the pinealocytes are not randomly arranged, but form
cords, clusters or follicles (cf. Vollrath, 1981). A follicular ar-
64 L. VOLLRATH
rangement of pinealocytes is of interest as these cells, in certain

respects, appear to resemble photoreceptors more closely than ran-
domly arranged pinealocytes. Fig. I of the detailed paper by Wolfe
(1965) on the rat pineal gland shows an abundance of mitochondria
in the juxta-luminal areas, resembling the ellipsoid-regions of in-
ner segments of photoreceptors. Whether these photoreceptor-like
pinealocytes differ in function from "ordinary" pinealocytes remains
to be elucidated. An interesting point is that pinealocytes arranged
in follicles, in some species, appear to be restricted to certain
areas of the pineal gland. In sheep, they were found to be restricted
to the dorsal tip of the gland (Lang, 1959). In Pirbright White guinea-
pigs follicles are conspicuous, however, the proximal area and in
the distal area a small zone facing the colliculi are regularly de-
void of these structures (Jung and Vollrath, 1982). Interestingly,
the follicles were more prominent in continuously illuminated guinea-
pigs than in animals kept under LD 12:12.
A second important point is the characterization and the inves-

tigation of the distribution of the cells immunoreactive for argi-
nine vasotocin-, a-MSH-, LHRH- and somatostatin-like compounds (Pe-
vet et al., 1980) as well as their relationships to interstitial or
glial cells. Should these cells turn out to be a special category
of pinealocytes, then ultrastructural studies would be particularly
important in order to identify the different types of pinealocytes.
Thirdly, the innervation invites comparative studies, in par-

ticular as the central innervation reaching the pineal gland via
the commissures is becoming more and more important. Recent studies
using the horse-radish peroxidase technique have shown in a variety
of mammalian species that central fibres from diverse sources inner-
vate the gland and, particularly important, not just the proximal
part of the pineal complex but also the distal parts (Korf and
Wagner, 1980; Nurnberger and Korf, 1981; Korf an M~ller, 1982).
Further studies have to elucidate the main centres of the brain that
regularly send nerve fibres to the pineal gland via the commissures
and to clarify their functional significance. An interesting ques-
tion is whether or not the central fibres and the peripheral sympa-
thetic nerves supply different areas of the parenchyma. Electron
microscopic studies have revealed well developed neuropil areas
within the pineal parenchyma characterized by synapses between
axonic and dendritic profiles (Schneider et a1., 1981). As it is
not yet clear whether the perikarya of these neurons lie outside
the pineal, a search for small intrapineal nerve cells is necessary
in order to assess the functional significance of the nerve fibres
leaving, or reaching, the pineal gland and to understand the intra-
pineal neuronal circuitry in mammals.
Finally quantitative studies on both types of innervation,

central commissural and peripheral sympathetic, are of interest. An
important first step in this direction has been taken by Karasek et
al. (1982a) who assessed the number of nerve endings per unit area
in a variety of mammalian species and found highly significant in-
terspecies differences. Moreover, an interesting inverse correlation
between adrenergic nerve fibre endings and pineal noradrenaline con-
centrations on one hand and synaptic ribbons on the other was noted.
Although we still do not know the function of the synaptic ribbons
in the mammalian pineal gland, these studies provide an impetus for
carrying out further studies on possible functional interrelation-
ships between synaptic ribbons and sympathetic nerve fibres.
CYTOLOGY
Comparative studies at the cellular level are also needed, as

such basic questions as to whether pinealocytes are monopolar, bi-
polar and/or multipolar cells and whether particular forms of pine-
alocytes can be attributed to a particular type of pineal organ have
not been answered. However, this kind of study should be preceeded
by detailed investigations of individual species, as differences
may exist in the different regions of the pineal complex. An impor-
tant problem is to assess the length of the neurite-like pinealocyte
processes and to examine to what extent they form bundles and run
in a particular direction within the parenchyma.
The uneven distribution of cell organelles, especially mito-

chondria, in some pinealocytes invites studies as to the presence
or absence of polarized pinealocytes in different species, a ques-
tion that is particularly relevant with respect to pinealocytes ar-
ranged in follicles. As reviewed in detail (Vollrath, 1981), pinealo-
cyte mitochondria exhibit a number of unusual features that appear
to make comparative studies worthwhile Besides giant mitochondria
which have been found in some species, the mitochondrial crests
show great variation: In addition to shelf-like cristae, tubular
and tubulovesicular cristae have been described. In view of the
fact that mitochondria with tubular cristae are typical of steroid-
producing cells a systematic search for such mitochondria on a com-
parative basis may be useful. Finally, it should be recalled that
Clabough (1971) noted in the hamster that small mitochondria with
few cristae were exclusively present in what was termed PI pinealo-
cytes, while larger mitochondria with a plexiform array of crests
in a dense matrix were typical of P~ pinealocytes. As the problem
of different types of pinealocytes ~s still unsettled, every attempt
should be made to solve it.
Comparative studies are also needed with respect to the synap-

tic ribbons and spherules, especially on a quantitative basis and
under experimental conditions. As clearly shown by Karasek et al.
(1982a) synaptic ribbons exhibit prominent interspecies differences
in amount, being especially abundant in ground squirrel and chip-
munk. According to personal experience, the ribbons are least
frequent in the pineal gland of the laboratory mouse. The inverse
66 L. VOLLRATH
correlation between ribbons and noradrenergic nerve terminals de-

scribed by Karasek et al. (1982a) indicates that stimulating results
can be obtained when comparative studies are carried out on a quanti-
tative basis. Experimentally the question could be answered whether
the striking increase in number of synaptic ribbons in animals ex-
posed to continuous illumination (Lues, 1971; Vollrath and Huss,
1973; Roux et al., 1977) which is in contrast to the low number of
ribbons during the photophase of the 24 hour cycle, is generally
present or not.
Comparative studies may also provide useful information with

respect to the topographical relationships of synaptic ribbons.
Whereas in the guinea-pig pineal 93.1 % of the ribbon fields bor-
dering the plasmalemma faced adjacent pinealocytes, in the vole,
Microtus agrestis, the majority of ribbons lay opposite glial cells
and only about 15-20 % faced clearly identified pinealocytes (He-
wing, 1981). These results clearly indicate that the concept that
the synaptic ribbons may be involved in a functional coupling of ad-
jacent pinealocytes as proposed for the guinea-pig (Vollrath and
Huss, 1973; Vollrath, 1973) does not appear to be generally valid.
Occasionally "paired" ribbon fields (Vollrath and Huss, 1973)

are present in the pineal gland, especially after exposure to con-
tinuous illumination (Vollrath and Huss, 1973; Vollrath, 1973).
These structures in which ribbons lie directly opposite one another,
yet in different pinealocytes, are functionally even more enigmatic
than the ordinary synaptic ribbons. Comparative studies by Karasek
et al. (1982a) have clearly indicated that the number of "paired"
ribbon fields differs in various species.
Finally, comparative studies are required with respect to the

synaptic spherules (see Vollrath, this volume). As in the guinea-
pig pineal these structures may be peculiar to diurnal pinealocytes
of the proximal pineal gland, it would be interesting to know
whether the spherules are regularly present in the different species
and whether they are confined to a particular type of pinealocyte.
Currently, studies are in progress in my laboratory to answer this
question.
Another component of the pinealocyte that will greatly benefit

from quantitative comparative studies is the dense-core vesicles
(DCVls). Their precise functional significance is not known, though
quantitative studies have shown that they are more numerous during
photophase than scotophase, which speaks against an assumption that
they contain melatonin. Comparative studies in a number of mammalian
species have supported the visual impression that the DCVls are
quite variable in amount and are predominantly present in pinealo-
cyte processes in some species (Karasek et al., 1982b). Again, what
this means in terms of function remains to be elucidated.
PINEALOCYTE-SPECIFIC (1) ORGANELLES
A potentially important component of the mammalian pinealocyte is

what has been called microtubular sheaves (Wolfe, 1965; Lin, 1969,
1970, 1972). These structures are transformed centrioles. The trans-
formation involves elongation of the centriole, apposition of fine-
granular, electron-dense material at the outer surface of the
centriole and lateral tilting of their microtubules. giving rise to
microtubular sheaves. The sheaves are interesting from a comparative
point of view, as they are present in rat (Wolfe, 1965; Arstila,
1967), guinea-pig (Lin, 1972), gerbil (Welsh and Reiter, 1978) and
hamster (Bucana et al., 1974) but are apparently absent in mouse
and monkey (Lin, 1970). In the rat apparently all the centrioles
transform into microtubular sheaves between 15 days and 6 weeks of
age (Lin, 1969, 1970), as in adult rats typical centrioles are en-
tirely lacking (Wolfe, 1965; Lin, 1970). Comparative studies should
be carried out using morphometric techniques to assess (i) the re-
lative amounts of centrioles and microtubular sheaves in different
species, (ii) the length of the microtubular sheaves under different
physiological and experimental conditions, (iii) their topographical
relationships and (iv) to find out whether their frequency can be
correlated with other structural features of the pineal gland in a
meaningful manner. Such a comparative approach may provide useful
hints as to the functional significance of the microtubular sheaves,
in particular as it has been suggested that the formation of the
sheaves may represent an abortive attempt of the pinealocyte to
produce outer segments (Vollrath, 1981) which in photoreceptors are
linked to the inner segment by 9 x 2 + 0 cilia.
CONCLUSIONS
The present survey has shown that comparative studies are an

important tool to study specific aspects of the mammalian pineal
gland. These studies may not solve the pineal problem, but would at
least provide incentives for further investigations which may even-
tually lead to a fuller understanding of some of the inherent
problems and especially of the structural and functional complexity
of the pineal system.
REFERENCES
Arstila, A. U., Electron microscopic studies on the structure

and histochemistry of the pineal gland of the rat.
Neuroendocrinology, 2 Suppl.: 1 (1967)
Bargmann, W., 1943, Die Epiphysis cerebri. In: Hdb. mikrosk. Anat.
Mensch •
W. v. Mollendorff, ed., Springer, Berlin. VI,4: 309
Bucana, C. D., Nadakavukaren, M. J. and Frehn, J. L., 1974.
Novel features of hamster pinealocyte ultrastructure.
Tissue Cell 6: 85
68 L. VOLLRATH
Clabough, J. W., 1971, Ultrastructural features of the pineal

gland in normal and light deprived golden hamsters.
Z. Zellforsch 114: 151.
Boeckmann, D., 1980, Morphological investigation of the deep
pineal of the rat.
Cell Tiss. Res. 210: 283.
Buijs, R. M. and Pevet, P., 1980, Vasopressin- and oxytocin-
containing fibres in the pineal gland and subcommissural
organ of the rat.
Gardner, J. H., 1953, Innervation of pineal gland in hooded rat.
J. compo Neurol. 99: 319.
Gregorek, J. C., Seibel, H. R. and Reiter, R. J., 1977,
The pineal complex and its relationship to other epithalamic
structures.
Acta Anat. 99: 425.
Hewing, M., 1981, Topographical relationships of synaptic ribbons
in the pineal system of the vole (Microtus agrestis).
Anat. Embryol. 162: 313.
Hofer, H. 0., Merker, G. and Oksche, A., 1976, Atypische Formen
des Pinealorgans der Saugetiere.
Verhandl. Anat. Ges. 70: 97.
Johnson, J. I. Jr., 1977,Central Nervous System of Marsupials.
In: The Biology of Marsupials.
D. Hunsaker II, ed., Academic Press, New York, San Francisco,
London, 157.
Jung, D. and Vollrath, L., 1982, Structural dissimilarities
in different regions of the pineal gland of Pirbright White
guinea-pigs.
J. Neural Transmiss.(in press).
Karasek, M., King, T. S., Brokaw, J., Hansen, J. T., Petterborg, L.J.
and Reiter, R. J., 1982a, Inverse correlation between "synaptic"
ribbon number and the density of adrenergic nerve endings
in the pineal gland of various mammals.
J. Compo Neurol. (in press)
Karasek, M., King, T. S., Petterborg, L. J., Hansen, J. T.,
Bartke, A. and Reiter, R. J., 1982b, Dense-core vesicles in
the mammalian pinealocyte and their relation to secretory
processes.
TSEM Journal 13: 13.
Korf, H.-W. and M~ller, M., 1982, Zentrale Innervation der Epiphyse
bei verschiedenen Saugetierarten.
77. Verso Anat. Ges. 1982: 28. Abstract.
Korf, H.-W. and Wagner, U., 1980, Evidence for a nervous connection
between the brain and the pineal organ in the guinea-pig.
Lang, K., 1959, Anatomische und histologische Untersuchungen
der Epiphysis cerebri von Rind und Schaf.
Thesis, Miinchen
Lin, H.-S., 1969, A special morphological expression of

centriolar formation.
Anat. Rec. 163: 313.
Lin, H.-S., 1970, The fine structure and transformation of
centrioles in the rat pinealocyte.
Cytobios 2: 128.
Lin, H.-S., 1972, Transformation of centrioles in pinealocytes
of adult guinea-pigs.
J. Neurocytol. 1: 61.
Lues, G., 1971, Die Feinstruktur der ZirbeldrUse normaler,
trachtiger und experimentell beeinfluBter Meerschweinchen.
Z.Zellforsch. Mikrosk. Anat. 114: 38.
NUrnberger, F., Korf, H.-W., 1981, Oxytocin- and vasopressin-
immunoreactive nerve fibers in the pineal gland of the
hedgehog, Erinaceus europaeus L.
Oksche, A., 1965, Survey of the development and comparative
morphology of the pineal organ.
Progr. Brain Res. 10: 3.
Pevet, P. and Kuyper, M.A., 1978, The ultrastructure of
pinealocytes in the golden mole (Amblysomus hottentotus)
with special reference to the granular vesicles.
Cell Tiss. Res. 191: 39
Pevet, p. and Yadav, M., 1980, The pineal gland of equatorial
mammals. I. The pinealocytes of the Malaysian rat (Rattus
sabanus).
Pevet, P., Ebels, I., Swaab, D. F., Mud, M. T. and Arimura, A.,
1980, Presence of AVT-, a-MSH-, LHRH- and somatostatin-
like compounds in the rat pineal gland and their relation-
ship with the UM05R pineal fraction. An immunocytochemical
study.
Quay, W. B., 1980a, Greater pineal volume at higher latitudes
in rodentia: exponential relationship and its biological
interpretation.
Gen. Compo Endocrinol. 41: 340.
Quay, W. B., 1980b, Pineal volume and latitudinal and
chronobiological characteristics: Comparison of five
mammalian orders.
Am. Zool. 20: 898.
Quay, W. B., 1981, Pineal atrophy and other neuroendocrine
and circumventricular features of the naked mole-rat,
Heterocephafus glaber (RUppel), a fossorial, equatorial
rodent.
J. Neural Transm. 52: 107.
Ralph, C. L., 1975, The pineal organ and geographical
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Int. J. Biometeorol. 19: 289.
70 L. VOLLRATH
Ralph, C. L., 1981, Melatonin production by extra-pineal tissue.

In: Melatonin: Current Status and Perspectives.
N. Birau, W. Schloot, eds., Pergamon Press, Oxford.
Advances in the Biosciences 29: 35.
Roux, M., Richoux, J. P. and Cordonnier, J. L., 1977, Influence
de la photoperiode sur l'ultrastructure de l'epiphyse avant
et pendant la phase genitale saisonniere chez la femelle
du lerot (Eliomys quercinus).
Schneider, T., Semm, P. and Vollrath, L., 1981, Ultrastructural
observations on the central innervation of the guinea-pig
pineal gland.
Vollrath, L., 1973, Synaptic ribbons of a mammalian pineal
gland. Circadian changes.
Z. Zellforsch. Mikrosk. Anat. 145: 171.
Vollrath, L. and Huss, H., 1973, The synaptic ribbons of the
guinea-pig pineal gland under normal and experimental
conditions.
Vollrath, L., 1979, Comparative morphology of the vertebrate
pineal complex.
Vollrath, L., 1981, The Pineal Organ.
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VI/7. Springer, Berlin-Heidelberg-New York.
Welsh, M. G. and Reiter, R. J., 1978, The pineal gland of the
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Wolfe, D. E., 1965, The epiphyseal cell: an electron-microscopic
study of its intercellular relationships and intracellular
morphology in the pineal body of the albino rat.
THE USE OF ELECTRON MICROSCOPY AND STEREOLOGY IN THE STUDY
Lutz Vollrath
Johannes Gutenberg-University
Mainz
INTRODUCTION
Now that transmission electron microscopy (TEM) has been used

for many years to study the pineal gland it is timely to evaluate
what progress has been achieved by using this technique and to spe-
culate which aspects of pineal research may benefit most by its ap-
plication in the future.
THE BASIC ULTRASTRUCTURE OF THE PINEALOCYTE
One of the main achievements of TEM is the clear demonstration

that the functionally important intrinsic cells of the pineal organ
are photoreceptor cells in lower vertebrates, closely resembling
the cones of the retina. The intrinsic cells of the mammalian pineal
gland, the pinealocytes, have some structural features in common
with photoreceptor cells, i.e. presence of synaptic ribbons, ellip-
soid-like aggregations of mitochondria, but on the whole it would
appear that they represent a cell type sui generis, the ultrastruc-
ture of which reveals little about its "profession". As extensively
reviewed (Vollrath, 198Ia), the mammalian pinealocyte is a neuron-
like cell consisting of a cell body, or perikaryon, and cytoplasmic
processes.
Little is known from TEM studies about the number and the
length of the processes that emanate from the perikaryon. This kind
of information could be very useful as it seems possible that the
pinealocytes, by way of special structures ("synaptic" ribbons and
spherules, vesicle-crowned rodlets), are functionally interrelated
(Hopsu and Arstila, 1965; Vollrath and Huss, 1973; Vollrath, 1973)
71
72 L.VOLLRATH
and form intricate neuronal circuits. Pictures obtained in human

pineals (Hortega, 1922) showing pinealocyte perikarya with long or
short cytoplasmic processes suggest. that different types of pinealo-
cytes may be present, resembling the Golgi Type I and GeIgy Type II
neurons. Interestingly, silver impregnation studies in sheep, cow
(Lang, 1959), dog, cat (Zach, 1960) and horse (FaSbender, 1962) have
have revealed monopolar pinealocytes in the periphery of the
pineal gland, whereas pinealocytes with 2-4 processes occupy
the centre of the gland. Systematic ultrastructural studies as to
the number of processes leaving a pinealocyte perikaryon are lacking.
In one study, in which serial sections were used, it was found that
cat pinealocytes have only one (or perhaps two) processes (Warten-
berg, 1968). In view of the structural complexity of mammalian pi-
nealocytes, reconstructions of a number of cells from serial sections
would be highly desirable.
The ultrastructure of the pinealocytic cytoplasm has been exten-

sively reviewed (Pevet, 1979, 1981a; Vollrath, 198Ia). Here it is
relevant to note that in my opinion the pinealocytes of different
mammalian species appear basically similar and resemble neurons in
many respects. Clearly defined sub-types of pinealocytes such as
observed in the mole, !al~a europaea (Pevet and Collin, 1976),
have not yet been conv1nc1ngly described in detail for other mam-
malian species. The distinction made by Pevet (1977, 1979, 1981a)
based mainly on the presence or absence of small granular vesicles
is debatable, as in my experience these granules are so rare in many
species, and in some strains of animals even lacking, as to use them
as distinct markers of a specific sub-type of pinealocyte. Never-
theless, the search for different types of pinealocytes should con-
tinue, for a number of reasons (see below).
Among the various cytoplasmic components of mammalian pinealo-

cytes, mitochondria, microtubules and microfilaments often stand
out due to their concentrations in certain cell regions. Peculiar
cell organelles of the mammalian pinealocyte, the so-called "synap-
tic" ribbons and the microtubular sheaves, the latter representing
centriole derived structures, have attracted, and no doubt will con-
tinue to attract, special attention.
QUANTITATIVE ELECTRON MICROSCOPY
Now that the basic ultrastructure of the mammalian pinealocyte

and the pineal gland as a whole is known, the question arises as
to how further progress can be achieved. As the ultrastructure of
the pinealocyte is such that its function cannot be deduced at first
sight, if at all, special approaches have to be used to unravel
its mysteries. One such approach is quantitative electron microsco-
py. What has been achieved with this technique in the past and what
can be attained in the future?
ELECTRON MICROSCOPY AND STEREOLOGY 73
QUANTITATIVE CHANGES OF SYNAPTIC RIBBONS AND SPHERULES
In what was probably the first quantitative ultrastructural

approach to the mammalian pineal gland the functionally obscure
"synaptic" ribbons were investigated. As synaptic ribbons are present
also in the photoreceptor cells of the pineal in lower vertebrates,
they were regarded as "possible reminders of the original neurosen-
sory function" of the pinealocyte (Kappers, 1971). Experimental
studies in guinea-pigs, involving exposure to continuous illumina-
tion or continuous darkness for several weeks revealed that the
ribbons cannot be regarded as functionless, phylogenetic relics
(Vollrath and Huss, 1973). Under constant light, the ribbons showed
a striking increase in size and number as well as changes in shape.
These results then led to the question as to whether synaptic rib-
bons show numerical changes also under physiological conditions. In
an attempt to answer this question the number of ribbons was counted
in guinea-pigs over a period of 24 h and it was found that the
amount of ribbons decreases during the day and increases during the
night (Vollrath, 1973). These day/night differences have been con-
firmed for other species such as rat (Kurumado and Mori, 1977; King
and Dougherty, 1980; Karasek and Vollrath, 1982), baboon (Theron et
al., 1979, 1981) and goldfish (McNulty, 1981). As these results
are in complete agreement with the day/night differences in mela-
tonin formation, one may question whether this kind of quantitative
approach to the pineal problem leads to new insight. In my opinion
- it does. First, the numerical changes of synaptic ribbons under
physiological and experimental conditions led to the concept that
the structures in question may functionally couple adjacent pinealo-
cytes and that the pinealocytes are clearly neuronal or neuroendo-
crine in nature (Vollrath and Huss, 1973; Vollrath, 1973). This
concept in turn triggered studies into the electrophysiological na-
ture of mammalian pinealocytes carried out in conjunction with Dr.
Semm, now at the Zoological Institute in Frankfurt/M., which have
led to a better understanding of mammalian pinealocyte function
(see below). Finally, right now we are witnessing that quantitative
studies dealing with pineal "synaptic" structures may lead to an
interesting new concept.
The synaptic structures I am referring to are the so-called

synaptic spherules (cf. Vollrath, 1981a), also known under the
terms rosette-like structures (rabbit, Leonhardt, 1967; Romijn,
1973, 1975) and vesicle-crowned balls (guinea-pig, Lues, 1971) in
the pineal, and synaptic body in the organ of Corti (Ades and Eng-
strom, 1974; Baird, 1974) and the vestibular organ (Wersall and
Bagger-Sjoback, 1974).
That these structures may be functionally different from

synaptic ribbons has been suggested by the findings that in preg-
nant guinea-pigs spherules show a strong increase in number whereas
ribbons increase less conspicuously (Lues, 1971). In rabbits,
74 L. VOLLRATH
sympathectomy of the pineal gland resulted first in an increase in

the number of spherules, followed by that of the ribbons (Romijn,
1975). Recently, the day/night changes of ribbons and spherules
were investigated in the rat pineal gland in vivo and in vitro
(Karasek and Vollrath, 1982). It was found that in vivo both ribbons
and spherules show corresponding changes in number, i.e. low numbers
at daytime and high numbers at night. In vitro, the day/night chan-
ges did not occur, but interestingly after 3 h in culture the sphe-
rules had increased considerably in number, again suggesting that
spherules and ribbons may differ in their response.
That the spherules are of great potential importance occurred

to us when we studied them in the guinea-pig. In this species the
pineal gland is a dumbbell-shaped, Type ABC or ABC organ (Vollrath,
1979, 198Ia). Quantitative assessment revealed that ribbons and
spherules may differ in their distribution within the parenchyma
and may differ in their rhythmicity (Vollrath, Schultz and McMillan,
unpublished observations). In all three pineal regions investigated
(proximal = A, intermediate = B, distal = C) the ribbons were equal-
ly abundant and showed the expected behaviour, i.e. small numbers
during daytime and high numbers at night. However, when looking
at the spherules it is striking to note that during day time they
are more abundant in the proximal area than in the intermediate
and distal regions and that at night time they exhibit low numbers
in all three pineal regions. This indicates that ribbons and spheru-
les, in the proximal region, show an inverse behaviour. What could
this mean in terms of function? Three aspects have to be considered.
First, as in the majority of instances, ribbons and spherules are
not found in one and the same pinealocyte profile. different popu-
lations of pinealocytes could exist. characterized by ribbons and
spherules respectively. Second, it may mean that there are differen-
ces in the circadian rhythmicity of the pinealocytes. one group of
pinealocytes being diurnal and the other nocturnal. In this context
it is interesting that electrophysiological studies carried out in
guinea-pigs also point into the direction of different rhythmicity
(Semm and Vollrath. 1980): One group of cells exhibited"an electri-
cal activity corresponding to the melatonin forming activity of the
pineal gland, i.e. low activity during daytime and high activity at
night. A second category of pineal cells showed a inverse electri-
cal behaviour: High activity during daytime and low activity at
night. In view of the morphological results obtained it is tempting
to speculate that the day-active pinealocytes correspond to the
cells that contain synaptic spherules whereas the night - active
cells are identical with the ribbon-containing pinealocytes. Pinealo-
cyte profiles exhibiting both spherules and ribbons could correspond
to those cells which did not exhibit circadian rhythmicity electro-
physiologically (Semm and Vollrath, 1980). More detailed studies
have to be carried out to prove or to disprove this concept. The
third implication of the morphological findings is that the differ-
ent regions of the guinea-pig pineal complex contain different types

of pinealocytes in different amounts and hence may exhibit function-
al heterogeneity. In this context the question arises as to which
role the central commissural fibres play, that are prominent in
the proximal pineal region (cf. Schneider et al., 1981).
Finally, morphometric data are of value also with respect to

the length of synaptic ribbons. In the pineal gland of the goldfish
it was found that synaptic ribbons are short during the day and al-
most twice as long during night (McNulty, 1981).
QUANTITATIVE DATA ON DENSE-CORE VESICLES
In view of the fact that the mammalian pinealocyte is an en-

docrine cell it is logical to search for morphological correlates
of its secretory products. According to Pevet (1977, 1979, 198Ia,b)
flocculent material in cisterns of the endoplasmic reticulum or in
vacuoles represents one type of secretory product and dense-core
vesicles (DCV's) another. It should be noted though that in commonly
used experimental animals the two types of secretory processes are
not so conspicuous as to solve the inherent problems at first sight.
It is therefore relevant to enquire what kind of information can
be gained fron quantitative studies. Quantitative studies so far
concern only the DCV's, which are known to be highly variable in
number in pinealocytes of different mammalian species (cf. Pevet,
198Ib). Recently, hard quantitative data have been obtained with
respect to the number of DCV's present within a clearly defined
unit area of pinealocyte parenchyma and equally important, with
respect to their location within the pinealocyte (Karasek et al.,
1982a). The following sequence (from lowest to highest numbers) was
obtained: Fox, cat, Richardson's ground squirrel, Sprague- Dawley
rat, Eastern chipmunk, Cotton rat, Djungarian hamster, white-footed
mouse and laboratory mouse, a clear tendency being present for the
DCV's in some species to accumulate in pinealocytic processes. No
systematic studies have been carried out yet to determine whether
the amount of DCV's present can be correlated with the serotonin
and melatonin levels of the pineal gland.
In different species data are available with respect to the

24 h rhythmicity of DCV's. In the rabbit it has been shown that in
the vicinity of the Golgi apparatus DCV's exhibit a distinct in-
crease in number between 7 a.m. and noon, followed by a prominent
decline coming to a halt at midnight; in the terminals of pinealo-
cytic processes DCV's show a peak 7 hrs later suggesting a trans-
port of the DCV's from the perikaryon to the processes (Romijn et
al., 1976). In the laboratory mouse (Benson and Krasovich, 1977;
Kachi, 1979), Mongolian gerbil (Welsh et al., 1979a) and Syrian
hamster (Krasovich and Benson, 1982) the numbers of DCV's also
increase during the day and decrease at night. In view of the con-
76 L.VOLLRATH
sistency of the results obtained in different mammalian species it

can be concluded that the DCV's appear to be intimately related to
circadian events in the pineal gland. Their numerical increase
during the day speaks against the supposition that 'they contain me-
latonin; they may contain serotonin, as their qualitative behaviour
parallels the circadian rhythmicity of serotonin (cf. Vollrath,
198Ib). This conclusion is in agreement with the cytochemical find-
ings by Lu and Lin (1979) and Juillard and Collin (1980). Accord-
ing to another view (Collin, 1981), they main contain a specific
protein termed pinealin~ Whatever the DCV's contain, it is clear
that it is a substance that is present in larger amounts during the
day than at night.
STEREOLOGICAL METHODS
In the above studies the quantitative methods applied were

mostly rather simple. Pineal tissue covering a specified number of
holes of electron microscopic supporting grids was scanned and all
the structures in question were counted. This rather crude method
is applicable only when the structures to be quantitated are rela-
tively rare, as is the case with synaptic ribbons and ncv's. Among
the different morphometric techniques available, stereo logical
methods appear to have the greatest scope for future quantitative
investigations of the pineal gland.
Stereology is a special type of morphometric analysis based

on "a geometric analysis of structures and textures; it includes
methods that allow direct derivation of metric properties of struc-
tures from two-dimensional sections on the basis of geometric-sta-
tistical reasoning" (Weibel, 1969). It is an important tool to
correlate structure and function. Technical details may be obtained
from the publications by Weibel (1969, 1979, 1980) and others.
In the field of pineal research, stereo logical methods have

so far been applied with three aims: (i) to obtain data on the
volume fractions of the different pineal components; (ii) to mo-
nitor circadian changes, and (iii) to study seasonal rhythmicity.
COMPOSITION OF PINEAL TISSUE
In one stereo logical study in which the data were obtained by

means of the point counting method, the pineal glands of 10 adult
male Wistar rats, 14 weeks old, were analysed (Krstic, 1977). In a
first step the material was studied at the light microscopic level.
The data obtained are given in Table I.It should be noted that the
data stem from immersion fixed material. Hence, the volume fraction
of capillaries may differ when perfusion-fixed pineals are investi-
gated. The presence of two types of cell, dark and light, is also
most likely due to immersion fixation.
Table
Morphometric analysis at the light microscopic
level. Rat pineal gland.
From Krstic, 1977.
Capillaries 5.8 + 0.15 %a

Pericapillary spaces and 13.1 + 0.22 %
pineal canaliculi
Dark cells 2.2 + 0.09 %
Light cells 78.8 + 0.27 %
aexpressed as % volume fraction mean + SEM
The stereological analysis of individual rat pinealocytes at

the electron microscopic level is shown in Table 2. These kinds of
data are of value for comparative studies only.
Table 2
Stereological analysis of "light" pinealocytes

of rats.
From Krstic, 1977.
Nucleus 22.3 + O. I %a
Mitochondria 6. I + 0.06 %
Golgi apparatus 0.56 + 0.02 %
Endoplasmic reticulum, 5.37 + 0.07 %
rough and smooth
Lipid droplets 1.19 + 0.03 %
Lamellar bodies O. I + 0.008%
Cytoplasmic matrix 64.38+0.13 %
a
expressed as % volume fraction, mean + SEM
When a structure-function correlation at the organ level is intended,

a more sophisticated approach is necessary, taking into consider-
ation all the structural components of the pineal gland, i.e., blood
78 L. VOLLRATH
vessels, perivascular spaces, nerve fibres, connective tissue if

present, pinealocytes, interstitial cells and last, but not least,
the intercellular canaliculi. These 0.2 - 0.4~ wide canaliculi
which extend from the perivascular spaces and appear to enmesh in-
dividual pinealocytes (Quay, 1973, 1974; Krstic, 1975) are espe-
cially important as their width changes over a period of 24 h, being
wide during the day and small at night. Moreover, as pinealocytes
may have a follicle-like arrangement, the central lumen or remnants
of it have to be taken into consideration.
Finally, it should be noted that a stereological analysis of

the pineal gland would be incomplete without dealing with pinealo-
cyte perikarya and processes separately.
STEREOLOGICAL STUDIES WITH RESPECT TO CIRCADIAN RHYTHMICITY
One promising application of stereology is the study of cir-

cadian rhythmicity. In one such investigation a careful analysis
of the various components of gerbil pinealocytes was carried out
(Welsh et al., 1979a). It was found that most of the parameters
studied were smallest between 7 a.m. and 1 p.m., followed by an
increase continuing into the dark phase. From these results and
those on melatonin synthesis it looks as though the morphological
parameters increase in anticipation of the increased melatonin
formation in scotophase. Further studies have to show whether the
changes observed in the gerbil principally apply to other species
as well. In view of the well established melatonin rhythmicity
of the pineal gland, the pineal gland appears to be an ideal object
to correlate structure and function.
One morphological parameter, the volume fraction of microtu-

buIes, has been studied in two species. In the gerbil, microtubule
volume fraction increased from 7 a.m. to 7 p.m., followed by a
slight decrease at night (Welsh et al., 1979a). In the baboon,
Papio ursinus, the cytoplasmic content of microtubules was small
during photophase and high during scotophase (Theron et al., 1981).
These divergent results show how important it is to carry out
detailed stereological studies in a variety of species before ge-
neralizations with respect to circadi~n rhythmicity are made.
Recently, the conspicuous nucleoli of rat pinealocytes have

been investigated with respect to 24-hour-rhythmicity (Lew et al.,
1982). Four parameters were studied: (i) Total nucleolar volume,
(ii) granular and (iii) fibrillar components as well as (iv) clear
areas. According to these studies the nucleolus is largest 1 h
after the onset of light and smallest towards the end of the dark
period (LD 12:12). the amplitude of the daily change being + 15 %.
The granular component exhibited a daily amplitude of + 29 ~. the
maxima and minima occurring at the same time as the peaks and
troughs of total nucleolar volume. In view of the many unresolved

questions with respect to the structure and function of the nucleolus,
studies dealing with the prominent nucleoli of the pinealocytes
may yield important information.
STEREOLOGICAL STUDIES WITH RESPECT TO CIRCANNUAL RHYTHMS
Stereological analyses are also an important tool to study cir-

cannual rhythmicity of the pineal gland. At the light microscopic
level seasonal changes of the pineal gland have been clearly estab-
lished (cf. Vollrath, 198Ia,b). In one ultrastructural study using
stereology it was found that in the ground squirrel the volume den-
sities of mitochondria and lipid droplets increased from a minimum
in winter to a maximum in the fall, whereas the surface density
of endoplasmic reticulum showed an inverse behaviour. The means for
number of Golgi bodies per test area of cytoplasm were small in
winter and consistently high in spring, summer and fall. DCV's were
highly variable in amount, showing a distinct depression in spring
only (McNulty and Dombrowski, 1980).
Before systematic stereo logical studies on an annual basis

are carried out, in my view a number of points should be taken into
consideration. First, it is desirable to perform a 24-hour study
in the species under question, with two aims: One is to establish
that 24-hour rhythmicityofaparticular parameter is clearly de-
monstrable and to clarify the order of magnitude of the daily
changes; only when these data are available, the annual data can
be seen in the right perspective. Moreover, proper sampling tech-
niques and appropriate statistical analyses are important prere-
quisites to exclude, or to confirm, a mosaic-like structure of the
pineal parenchyma (cf. Vollrath, 1979) and to cope with this situa-
tion accordingly. As usually the sampling is done in different sec-
tions and in different regions of the gland, an appropriate sta-
tistical test should be used, such as a nested or hierarchic ana-
lysis of variance. Secondly, it should be taken into consideration
that infradian rhythms, i.e., rhythms with periods longer than 28 h,
may be present in the pineal gland (Vollrath et al., 1975, 1981;
Welker et al., 1982), which may give tha false impression of annual
rhythmicity. Moreover, ultradian rhythms may complicate the picture,
the superimposition of ultradian and circadian rhythms resulting
in beat frequencies and an attenuation of the daily amplitudes
(Wallen and Yochim, 1974). Thirdly, as seasonal studies are by na-
ture long-term investigations it should be made sure that the ani-
mal supply from the dealer is carefully controlled, i.e., that the
dealers do not buy extra animals from other dealers, change the
diet etc. As a last point it should be mentioned that the stereolo-
gical methods applied give valid results only when the proper
methods of randomization are observed.
80 L.VOLLRATH
APPLICATION OF STEREOLOGY IN EXPERIMENTALLY-AFFECTED ANIMALS
It should be noted that stereological studies, especially

when carried out on a 24-hour- or an annual basis, are very labor-
ious, time-consuming, and expensive. These disadvantages are of
lesser concern in investigations whose aim it is to study the effects
of a special treatment on the pineal gland.
In one study in which the function of the pineal gland was

severely impaired by sympathectomy (Welsh et al., 1979b, gerbils)
a clear decrease was noted with respect to total pinealocyte size,
smooth and rough endoplasmic reticulum, free cytoplasm, mitochondria
and presumptive secretory vesicles, whereas synaptic ribbons in-
creased in volume. This study has also revealed the important point
that sham-operation is not without effect on the pinealocytes. Hence,
in addition to the experimental and sham-operated animals, untreated
controls should be included in the studies to obtain a complete
picture.
CONCLUSIONS
The present survey has shown that quantitative studies at the

ultrastructural level are an important tool to investigate the struc-
ture and function of the pineal gland. In contrast to other organs,
stereological analyses have been surprisingly little used to unravel
the structural and functional complexity of the mammalian pineal
gland.
In my view, undoubtedly a new era of pineal ultrastructural

research will begin when the approach so often used in the past is
abandoned, viz. to carry out painstaking experiments and subsequently
to be content with purely descriptive and often meaningless and de-
batable semiquantitative results which cannot be subjected to rigor-
ous statistical analyses.
The application of stereological techniques to all the compo-

nents of the mammalian gland will be of great value, especially
when they are carried out on a comparative basis. Then we will be
able to make definite statements about a number of important points:
The nerve fibre density in different species and in different
regions of the pineal complex. The volume fraction of the blood
vessels in the pineal gland can be compared with other organs. The
length, width, course and volume fraction of pinealocyte processes
can be objectively assessed and attempts can be made to relate
these findings to the structure of the pineal complex. Is it just
a coincidence that the small, roughly sperical rat pineal gland
contains pinealocytes with few and short processes whereas the elon-
gate, dumbbell-shaped guinea-pig pineal gland has relatively long
processes forming bundles which not rarely run in preferred dire-
tions?
Last, but not least, hard quantitative morphological data pro-

vide an impetus for correlations which may eventually lead to new
concepts. One such study is that by Karasek et al. (1982b) in which
an inverse correlation between synaptic ribbon number and the den-
sity of adrenergic nerve endings was found in the pineal gland of
various manunals.
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ELECTRON MICROSCOPY AND STEREO LOGY 85
Wartenberg, H., 1968, The mammalian pineal organ: Electron

microscopic studies on the fine structure of pinealocytes,
glial cells and on the perivascular compartment.
Weibel, E. R., 1969, Stereological principles for morphometry
in electron microscopic cytology.
Int. Rev. Cytol. 26: 235.
Weibel, E. R., 1979, Stereological methods. Vol. 1: Practical
methods for biological morphometry.
Academic Press, London- New York-Toronto.
Weibel, E. R., 1980, Stereological methods. Vol. 2: Theoretical
foundations.
Academic Press, London-New York-Toronto
Welker, H. A., SchUhle, A. and Vollrath, L., 1982, Infradi an
rhythms of serotonin and serotonin-N-acetyltransferase
in the pineal gland of male rats.
J. interdiscipl. Cycle Res. (in press).
Welsh, M. G., Cameron, I. L. and Reiter, R. J., 1979a, The pineal
gland of the gerbil, Meriones unguiculatus. II. Morpho-
metric analysis over a 24-hour period.
Welsh, M. G., Hansen, J. T. and Reiter, R. J., 1979b, The pineal
gland of the gerbil, Meriones unguiculatus. III. Morpho-
metric analysis and fluorescence histochemistry in the intact
and sympathetically denervated pineal gland.
Wersall, J. and Bagger-Sj5back, D., 1974, Morphology of the
vestibular sense organ. In: H. H. Kornhuber, ed.
Handbook of Sensory Physiology, Vol. VI,I, p. 123.
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Zentralbl. Veterinarmed. 7: 273.
INNERVATION OF THE VERTEBRATE PINEAL ORGAN
J. Ariens Kappers
Houtweg 13
1251 CS Laren N.H.
The Netherlands
In the present survey we will deal with the innerva-

tion of the pineal organ proper only, not with that of
other parietal organs such as the parapineal organ, the
frontal organ of anuran amphibians, and the parietal eye
of lizards which have been termed "accessory pineal or-
gans" .
Phylogenetically, the pineal develops from a direct-

ly photoreceptive, mainly sensory organ into an indirect-
ly photosensitive secretory organ by which also other ex-
ternal as well as internal stimuli are integrated. The
latter is often termed a neuroendocrine gland, not be-
cause its secretory cells which release their products
into the blood would be true nerve cells, but because
these cells are of neural origin. Evidently, the function-
al changes which the organ phylogenetically shows will
not only be reflected by changes in the structure of the
cells by which it is composed, but also by changes in
its innervation.
INNERVATION OF THE NON-MAMMALIAN PINEAL
Before going into more detail, some fundamental

structural and functional principles should be mentioned
first. Especially in more primitive non-mammalian verte-
brates such as fish and amphibians, the pineal can pri-
marily be considered a directly photoreceptive organ
conveying photic stimuli to brain centres along a neural
pathway. In principle, this pathway consists of two types
of cells, photoreceptive neurosensory cells and nerve
87
88 J. A. KAPPERS
cells. Embryologically, both cell types originate from

the same neuroepithelial anlage. In the adult stage both
are situated in the pineal epithelium or pineal paren-
chyma. Sensory nerve cells may, in part, also be situated
in the pineal stalk which connects the pineal organ prop-
er with the brain.
Neurosensory cells in general combine two different

functions. In the first place they are directly sensory
in being susceptible to stimuli originating in the en-
vironment of the organism. In addition they are capable
of "translating" or transducing such stimuli into a neu-
ral impulse. This means that the external stimulus causes
an action potential in the very same cell by which this
stimulus is received. Such neurosensory cells show a
polar surface which is structurally specialized for the
reception of specific external stimuli and a basal pro-
cess which is comparable to an axon. The polar surface
of photosensitive neurosensory cells or photoreceptor
cells is characterized by an outer segment which, as has
been shown in a previous lecture, consists of a more or
less complicated lamellar structure. The terminal ending
of the basal process of any neurosensory cell conveyes
the neural impulse, generated in it by the external
stimulus, by means of a synaptic contact to the dendrites,
or more directly to the soma of a true nerve cell
changing the spontaneous activity of this nerve cell.
The axon of the sensory nerve cell then conducts the ef-
fect of the neural impulse, originally generated in the
neurosensory cell by external stimuli, to brain centres.
In case of the lateral eyes of all vertebrates as well
as in the pineal organ of many non-mammals, such sensory
nerve cells are situated in the sensory organ proper,
while, for instance, the neurosensory cells present in
the olfactory epithelium convey their action potential
to nerve cells which are situated in the brain, i.e.~
in the olfactory bulbs.
As has been mentioned in our earlier lecture, the

pineal organ of all vertebrates develops from the epi-
thalamic neuroepithelial layer which forms the roof of
the third ventricle between the habenular and the poste-
rior commissure. During its ontogenetic development this
layer bulges out in a dorsal direction forming the pri-
mary pineal anlage. In the lowest vertebrates this pine-
al anlage then grows along a shorter or longer distance
in a rostral direction forming at its most rostral end
a pineal vesicle which remains in connection with the
epithalamus by means of a primary hollow pineal stalk,
differing in length. The lumen of this stalk is in open
INNERVATION OF THE VERTEBRATE PINEAL ORGAN 89
communication with the third ventricle. In some lower

vertebrates the pineal end-vesicle keeps its lumen, while
the walls of the pineal stalk may fuse so that its lumen
disappears and the stalk becomes solid and the open com-
munication between the pineal lumen and the third ventri-
cle does not exist any longer.
Part of the cells of the neuroepithelium from which

the pineal organ proper originates differentiates into
photoreceptor cells, sensory cells and, in some lower
vertebrates, intercalated nerve cells, while other cell
elements differentiate into supporting or interstitial
cells. Being non-neural in function, this latter cell
type will not be discussed here. As has been mentioned
before, sensory nerve cells may also be present in the
pineal stalk, not exclusively in the pineal organ proper.
The axons of the sensory nerve cells grow in the direc-
tion of the brain via the walls of the pineal stalk
forming together the pineal tract. From the foregoing it
is evident that the direction of the neural impulse,
generated in the neurosensory photoreceptor cells by
photic stimuli and conveyed by sensory nerve cells is to
the brain. Therefore, this type of pineal innervation is
termed afferent or pinealofugal innervation.
By a number of different investigations it has been

demonstrated that, most probably, secretory cells are
already present in the pineal organ of fishes and amphib-
ians. In principle, in such lower vertebrates three cell
types could be secretory in function: (1) photoreceptor
cells having a well-developed fully functional outer
segment and conveying a neural impulse to sensory pineal
neurons may show, next to their neurosensory function
also a secretory function, (2) part of the photoreceptor
cells which show degeneration of their outer segment have
lost their neural function if their basal process is not
in synaptic contact with pineal sensory neurons, now ex-
clusively exerting a secretory function, (3) ontogenet-
ically prospective photoreceptor cells do not develop an
outer segment at all, but differentiate directly into
purely secretory elements. In the pineal organ of the
cyclostome Petromyzon planeri pinealocyte-like secretory
cells have even been described (Meiniel, 1980). It can-
not be entirely excluded that all of these three possi-
bilities are, indeed, realized, even in rather primitive
vertebrates. More investigations are, however, necessary,
but many studies confirmed that the pineal organ of fish
and amphibians is already involved in the production of
secretory compounds, indoleamines and probably also pep-
tides (see Ariens Kappers, 1981, for literature and dis-
90 J. A. KAPPERS
cussion) It might be possible that, in such lower ver-

tebrates, photic stimuli are not exclusively transduced
into a neural, but also into a secretory response. It
should, however, be stressed that some problems are still
to be solved, especially as the virtual secretory response
to photic stimuli of pinealocyte-like cells is concerned,
which, next to the so-called secretory rudimentary photo-
receptor cells (Collin, 1971) are the most likely candi-
dates for a secretory function.
In analogy to the conditions present in mammalian

pineals the question can be raised whether the secretory
production in such primitive non-mammalian vertebrates
is neurally regulated at least in so far as such a pro-
duction would not be exclusively regulated by direct
photic stimuli. As we will mention later, the presence of
such a pinealopetal or efferent pineal regulating inner-
vation is doubtful in fish, while it is still not very
well proved to exist in amphibians.
In fish the histochemical technique for demonstrating

acetylcholinesterase has given the best results for ob-
serving the presence of pineal sensory nerve cells in the
pineal epithelium. Their number, which varies greatly
among the different species, is smaller than that of the
photoreceptor cells. This means that a number of photo-
receptive neurosensory cells convey their neural impulse
to the same sensory nerve cell. In some fish, nerve cells
are more densely packed in the stalk region than in the
pineal end-vesicle in which such cells are also not
equally distributed. Multipolar as w~ll as pseudo-uni-
polar sensory nerve cells have been distinguished, the
latter type being the most common. Synaptic contacts of
a special type between the basal process of photorecep-
tor cells and the dendrites of the intrapineal sensory
nerve cells have been described, but very little is
known about the intrapineal connectivity perhaps exist-
ing between such nerve cells and, when present, inter-
calated nerve cells.
As has been mentioned before, the axons of the sen-

sory nerve cells form the afferent or pinealofugal pineal
tract. This tract contains mostly non-myelinated nerve
fibres. In fish their number may vary from 120 to more
than 2.000. Their diameter is also variable. In some
pineal tract fibres dense-core or granular vesicles meas-
uring 50 - 140 nm in diameter have been observed. Their
functional significance is, so far, unknown. In two
species of fish indications for the presence ofaxo-
axonal contacts between pineal tract fibres have been

demonstrated.
With the cobalt chloride iontophoresis technique,

the central projections in the brain of pineal tract fi-
bres have been studied in detail (Hafeez and Zerihun,
1974). In SaLmo such fibres could be traced to the para-
pineal organ, lateral habenular nucleus, pretectal area,
di- and mesencephalic periventricular grey, dorsomedial
and dorsolateral thalamic nuclei, nucleus of Darksche-
witsch, cells of origin of the medial longitudinal
fasciculus and the dorsal tegmentum which suggests that
the afferent neural pathway from the pineal influences
the function of many important brain centres. Interest-
ingly, no fibres running to the subcommissural organ,
optic tectum and interpeduncular nucleus could be ob-
served in SaLmo. According to the experience of a number
of authors using less sophisticated methods, in most fish
pineal tract fibres could not be followed beyond the
level of the posterior commissure. In Phoxinus~ the pine-
al tract was found to be connected with the right habenu-
lar ganglion (Bhargava, 1973). Recently, some pineal
tract fibres of the teleost Puntius sophore were observed
to bifurcate after reaching the brain. Some branches en-
ter the optic tectum and the subcommissural organ, while
others enter the ependymal lining of the third ventricle
just ventral to the posterior commissure, a few fibres
even projecting into the ventricle (Sathyanesan and
Sastry, 1982).
Electrophysiological recordings from the fish pineal

end-vesicle and the pineal stalk provided clear evidence
that the pineal is light-receptive (see Dodt, 1973, for
a review). In cyclostomes and fish, light inhibits the
spontaneous electrical activity of the organ. Two dif-
ferent responses to light could be distinguished. Usual-
ly, an achromatic response is obtained, the effect of
stimulation solely depending on luminance and not on
wavelength. A few cells, however, show a chromatic re-
sponse being inhibited by light of short wavelength and
excited by exposure to longer wavelengths.
In fish no clear evidence exists about the presence

of pinealopetal or efferent nerve fibres. Using the
Falck-Hillarp technique, some catecholaminergic green
fluorescent fibres were observed in the peripheral menin-
geal tissue of the pike (Esox Lucius)~ but not within the
pineal parenchyma proper (Owman and RUdeberg, 1970). As
to the granular vesicles observed in some pineal tract
92 J.A.KAPPERS
fibres it is not known whether these are present in

either afferent or efferent nerve fibres. The exact na-
ture of their content is also still enigmatic.
In the pineal of amphibians 3 well-developed true

photoreceptor cells as well as secretory rudimentary pho-
toreceptor cells have been described in detail by many
authors (see Vollrath, 1981, for a review). In the rudi-
mentary photoreceptor cells, the outer segments show
signs of degeneration. It is, however, questionable
whether such cells represent true and permanently rudi-
mentary photoreceptor cells or true photosensory elements
the outer segments of which are involved in a process of
more or less cyclic break-down and renewal. It has, how-
ever, been supposed that a true phylogenetically progres-
sive degenerative process of the directly photoreceptive
capability of pineal photoreceptor cells in favour of
their secretory capability sets in clearly in amphibians.
The basal process of secretory rudimentary photoreceptor
cells is no longer in synaptic contact with pineal sen-
sory neurons, but ends on the limiting membrane of the
pineal epithelium.
The basal process of true amphibian photoreceptor

cells is in axo~dendritic or axo-somatic synaptic contact
with intrapineal sensory nerve cells. Three types of
such synaptic connections have been distinguished: (1)
ribbon synapses, the ribbons having also been termed
vesicle-crowned rodlets, (2) convential synapses, and
(3) synapses characterized by the presence of subsurface
cisterns. Ribbon synapses are rather typical of verte-
brate photoreceptor cells in general. The axo-dendritic
synapses have been subdivided in two main types
(Bayrhuber, 1972). In type I, 2-5 dendrites lie opposite
a synaptic ribbon, while, in type II, 1-6 ribbons or
vesicle-crowned rodlets lie opposite a single dendrite.
Usually, the dendrites are not invaginated into the
photoreceptor cells.
Conventional synapses lacking ribbons have been ob-

served less frequently. They may connect intercalated
neurons, also termed interneurons (Bayrhuber, 1972; Korf,
1976). Synapses showing subsurface cisterns have been
observed in the pineal of Rana pipiens. They have been
held to be involved in efferent transmission of feed-
back mechanisms (Kelly and Smith, 1964).
For the investigation of different types of ampbib-

ian pineal nerve cells and their relationships best re-
sults have been obtained by applying the acetylcholin-
esterase reaction after Karnovsky and Roots (Wake et al.

1974; Ueck, 1971). In Rana ridibunda and Rana esculenta
the pineal organ contains large and small multipolar
nerve cells, pseudo-unipolar cells and a few bipolar
ones. It has been suggested that the pseudo-unipolar
cells give rise to the pineal tract fibres, whereas the
multipolar nerve cells would be interneurons. However,
the wiring of the pineal nervous circuitry and its func-
tional significance is a most complicated matter which
has still to be elucidated.
A system ofaxons running through the pineal lumen

has also been observed (Vigh-Teichmann et al., 1973).
The fibres are synaptically connected with the plasmo-
lemma of supporting pineal cells. They show some resem-
blance with cerebro.pinal fluid-contacting axons in
other parts of the central nervous system present in
other classes of vertebrates. In the amphibian pineal,
these non-myelinated axons contain dense-core vesicles,
but their origin and function are unknown.
The amphibian pineal tract consists of both myeli-

nated and non-myelinated nerve fibres, the number of the
latter prevailing. Part of the fibres in this tract
originate in the frontal organ, present in anuran amphib-
ians, which will not be dealt with here. In addition to
undoubtedly pinealofugal nerve fibres, the pineal tract
of anurans also contains some possibly aminergic fibres
showing dense-core vesicles measuring 85 - 95 nm in dia-
meter (Oksche and Vaupel-von Harnack, 1965). Similar
fibres were observed in the peripheral regions of the
organ (Ueck, 1968). Green fluorescent nerve fibres being
evidently aminergic have, however, been mostly observed
in the peripineal leptomeningeal tissue. According to
some authors (Ueck, 1968) such fibres would be exclusive-
ly confined to blood vessels, but others (Iturizza, 1967)
have pointed out that they do not show special relation-
ships with vessels and are particularly abundant in the
ventral region of the pineal. Their pinealopetal nature
would be indicated by a complete disappearance of fluor-
escence in the pineal and an accumulation of fluorescent
material in the proximal stump of the nervous pathway
after transsection of the posterior commissure. Although
observations by other authors also indicate the possible
presence of an efferent pinealopetal innervation (Kelly
and Van de Kamer, 1960; Kelly and Smith, 1964; Paul,
1972) the origin of the fibres involved and their exact
course and function still need to be established. Theo-
retically the regulation of secretory processes in the
amphibian pineal by pinealopetal nerve fibres is attrac-
94 J.A.KAPPERS
tive because it is improbable that the secretory func-

tion of the secretory rudimentary photoreceptor cells is
directly regulated by photic stimuli.
The amphibian pineal tract leaves the organ at its

caudal border and comes in close contact with the base
of the commissural organ giving off some fibres which,
however, do not originate in the pineal organ but in the
frontal organ. The majority of the tract fibres runs
further caudalward and penetrates the posterior commis-
sure. Fibres originating in the pineal organ proper reach
the pretectal region and the periventricular grey, while
both the pineal and the frontal organ may also influence
the function of the habenular nuclei and the dorsal thal-
amus.
Electrophysiological studies of the amphibian pineal

organ show an exclusively achromatic response to light
proving that the pineal is, indeed, directly photosensi-
tive (see Vollrath, 1981, for the extensive literature
on this subject).
In reptiles the pineal organ is characterized by a

progressive reduction of its photoreceptive sensory func-
tion and a progressive development of its secretory ca-
pability. This is also reflected by the afferent inner-
vation of the reptilian pineal. Considering the scarcely
present fully differentiated true photoreceptor cells in
most reptiles, it is obvious that the number of pineal
sensory nerve cells is also very rare (Lacerta: Ariens
Kappers, 1976). The relatively few afferent or pinealo-
fugal nerve fibres originating from these nerve cells
and forming the pineal tract are, for the most part,
myelinated. They run to the habenular and posterior com-
missure, and, more caudally, to the region of the sub-
commissural organ and the periventricular fibre layer.
Their exact function is, so far, unknown.
The basal process of the rare well-developed photo-

receptor cells forms synaptic connections with a number
of dendrites of a sensory nerve cell, while a number of
terminals of basal processes of several photosensory
cells may also form synaptic connections with a single
dendrite of a sensory nerve cell. The synapses are of the
ribbon or vesicle-crowned rodlet type.
Efferent or pinealopetal non-myelinated monoaminer-

gic nerve fibres containing dense-core vesicles have
often been described in reptilian pineal organs. They
are nor adrenergic as is shown by their green fluorescence
and the increase of their granular vesicular content af-

ter the administration of false sympathetic transmitters
such as 5-hydroxydopamine and 5-hydroxydopa. Moreover,
the administration of tritiated hydroxytryptamine fol-
lowed by electron microscopic autoradiography and a num-
ber of pharmacological tests point to the presence of
both noradrenaline and serotonin in these fibres. Like
in the mammalian pineal, the presence of serotonin in
such noradrenergic fibres is very likely due to their up-
take of this compound which is produced by the pineal
secretory parenchymal cells.
The exact origin of the autonomic pineal efferent

fibres is not known. In the snake Natrix they largely
derive from a caudal and dorsal meningeal nerve bundle
reminding of the conary nerves in mammals. Most probably,
these peripheral noradrenergic nerve fibres originate in
the superior part of the sympathetic trunks to regulate
the function of the pineal organ which, in snakes, is
exclusively secretory. In the pineal, the fluorescent
fibres are primarily situated in the perivascular spaces
accompanying the blood vessels which penetrate into the
organ from the periphery. Some fibres, however, apparent-
ly innervate parenchymal lobules (Quay et al., 1968). In
the lizard, such fibres are not in true synaptic con-
tact with the secretory rUdimentary photoreceptor cells
(Wartenberg and Baumgarten, 1969). Some fibres lie free-
ly in the pineal lumen sometimes in close topographical
association with the degenerated outer segments of such
cells. The functional significance of the peripheral
pinealopetal autonomic innervation has, in reptiles, not
been experimentally established. However, as has already
been mentioned, it can be assumed that it regulates the
secretory function of the organ.
In birds~ the afferent pineal innervation is still

more reduced than in reptiles, snakes excepted in which
no such innervation is present at all, due to the phylo-
genetically continuing regression of the neural direct-
ly photoreceptive function of the organ. Rather large
interspecies differences do, however, exist regarding
the development of pinealofugal innervation. Even by
means of the Karnovsky-Roots histochemical technique
for the demonstration of acetylcholinesterase, sensory
nerve cells cannot easily be demonstrated (Ueck and
Kobayashi, 1972a,b). In the Japanese quail, nerve cell
bodies were not observed at all, while they are rare in
the dove, but more abundant in the chicken. In the house
sparrow and the canary, sensory nerve cell bodies and
their processes are also present.
96 J.A.KAPPERS
Avian afferent pineal innervation has been most ex-

tensively studied in the house sparrow. Mostly, the sen-
sory nerve cells are bipolar, occasionally, however,
pseudo-unipolar (Ueck and Kobayashi, 1972a). Their axons
form small nerve bundles which run in the pineal stalk.
The presence of such a pineal tract has also been demon-
strated in other birds. Most often its fibres are non-
myelinated and always non-fluorescent suggesting their
afferent function.
In the basal process of the photoreceptor cells

which, for the most part, are secretory rUdimentary photo-
receptor cells, synaptic ribbons have been demonstrated
(Bischoff, 1969; Ueck, 1970a,b), but there is little
evidence for the presence of functional synapses between
such photoreceptor cells and the sensory neurons. It is
also typical of the much reduced afferent pineal inner-
vation that, for instance in the sparrow, several hun-
dreds of rudimentary photoreceptor cells face a single
nerve cell only. In accordance with the evident pre-
ponderant secretory function of the avian pineal, its
pinealofugal or afferent innervation is far from well-
developed and does not appear to play an important func-
tional role.
Practically nothing is known about the sites of

ending of the afferent nerve fibres. In some birds they
could be followed into the posterior commissure, while
some reach the habenular commissure or seemingly disap-
pear in the intercommissural region.
In agreement with the more important secretory

function of the avian pineal, the pinealopetal innerva-
tion by peripheral efferent autonomic nerve fibres is by
far better developed than the pinealofugal afferent one.
As has been demonstrated using several techniques, most
efferent fibres are noradrenergic. It has been supposed
that, in the chicken, a noradrenergic as well as a cho-
linergic efferent pineal innervation may be present
(Wight and MacKenzie, 1970). Most probably, however,
just like in mammals some sympathetic noradrenergic
nerve fibres also demonstrate a cholinergic reactivity
(Ueck and Kobayashi, 1972a). In some species, the sympa-
thetic fibres show, next to a green fluorescence, also a
yellow fluorescence due to the presence of serotonin
which has evidently been taken up by such fibres from the
secretory parenchymal cells as is also the case in rep-
tiles and mammals.
The sympathetic origin of the avian pinealopetal

peripheral nerve fibres has been clearly established. The

green fluorescence disappears after sympathetic ganglion-
ectomy (Hedlund and Nalbandov, 1969). In the pineal, the
number as well as the location of such fibres vary con-
siderably among birds. Their location may be exclusively
perivascular, both perivascular and parenchymal, or en-
tirely parenchymal (see Vollrath, 1981, for literature)
The terminals of the noradrenerg~c fibres are not in
direct contact with pineal parenchymal cells. As usual,
such fibres which are non-myelinated contain dense-core
as well as electron-lucent vesicles.
In a large number of avian species, cell staining

with chrome-alum haematoxylin phloxine or aldehyde-fuchsin
are embedded in the pineal parenchyma close to the haben-
ular and posterior commissure (Oksche et al., 1972). Sup-
posedly they are extrahypothalamic neurosecretory ele-
ments (Quay and Renzoni, 1963, 1966). Futher investiga-
tion of such cells and their processes would be of much
interest because of the recent observations on the pres-
ence of extrahypothalamic neurosecretory nerve fibres in
the mammalian pineal gland (see later).
In respect to the afferent innervation morphologi-

cally observed in some avian pineals it cannot completely
be ruled out that, in such cases, the organ is directly
photosensitive and influences the function of brain cen-
tres. In most species, however, the number of true photo-
receptor cells is small, the synaptic connections between
the basal process of photoreceptor cells, if present, and
of the secretory rudimentary photoreceptor cells have not
been thoroughly studied, and the number of pinealofugal
nerve fibres is relatively small. Pertaining to this
afferent pineal innervation, electrophysiological in-
vestigations did not give unequivocal results (see Voll-
rath, 1981). In some studies, light stimuli did not
exert any influence on spontaneous electrical pineal ac-
tivity, while, in others, light flashes resulted in a
marked inhibition of electrical discharges. This was,
however, only the case if the optic nerves were intact
which would speak against a direct pineal photoreceptiv-
ity, at least in the species concerned, the quail.
Curiously, light has been shown to directly effect

the chemical composition of explanted avian pineal cells.
In cultured chick pineal, darkness stimulated the produc-
tion of N-acetyl transferase (Wainwright and Wainwright,
1978), the crucial enzyme involved in the production of
melatonin. In birds, the problem whether the pineal is
directly photosensitive or not is complicated by the fact
98 J.A.KAPPERS
that there is evidence of the presence of extraretinal as

well as of extrapineal photoreceptivity (see Menaker and
Oksche, 1974).
INNERVATION OF THE MAMMALIAN PINEAL
In mammals, the pineal is a purely secretory endo-

crine organ. The pineal-specific cells, the secretory
pinealocytes which are phylogenetically as well as, at
least clearly in some mammals, ontogenetically derived
from precursor cells of the sensory cell-line, do not
transduce direct photic stimuli into a neural impulse
which is conveyed to brain centres via sensory pineal
neurons. In the adult mammalian pineal, sensory neurons
are lacking with a possible exception of one species only
(see later). However, light or its absence, darkness,
still plays amost important role in the function of the
mammalian pineal. The secretory function of the pinealo-
cytes is, besides a number of other factors (Ariens Kap-
pers, 1979), regulated by efferent pinealopetal nerve
fibre systems which convey photic, but also external
stimuli of other types such as olfactory and acoustic
stimuli, to the organ. We will now deal with the systems
of efferent mammalian pineal innervation.
An orthosympathetic innervation of the pineal gland

was suggested to be present in mice as early as 1904 by
the father of neuroanatomy, Ramon y Cajal, and subsequent-
ly by some few other authors. It was then described in
man and monkey by Le Gros Clark in 1940 and finally
demonstrated and experimentally confirmed in rat by supe-
rior cervical ganglionectomy by the present author
(Ariens Kappers, 1960, also for earlier literature) who
erroneously held this efferent type of pineal innervation
to be the exclusive one. In the sixties, mammalian pineal
orthosympathetic innervation has drawn special attention
because mainly American authors proved that this neural
pathway conveys indirect photic stimuli to the organ
regulating the secretory function of pinealocytes,
especially as the metabolism of indoleamines is con-
cerned. As has been demonstrated by autoradiography, ad-
ministration of false transmitters, chemical sympathec-
tomy, fluorescence histochemistry, electron microscopy
and pharmacological experiments, the neurotransmitter
substance in the sympathetic fibres is noradrenaline.
Noradrenaline, when released from the fibre terminals
and probabiy also from preterminal varicosities, is
taken up by S-adrenergic receptors present in the cell
membrane of pinealocytes. Via the adenyl cyclase-cAMP
INNERVAliON OF THE VERTEBRATE PINEAL ORGAN 99
system, noradrenaline then stimulates the production of

melatonin in these cells.
As was observed in some reptiles and birds, the ter-

minal part of the sympathetic nerve fibres in the pineal
of a number of mammals contains serotonin which is pro-
duced by pinealocytes and taken up and stored by the nerve
fibres. The presence of octopamine has also been demon-
strated (see Vollrath, 1981, for literature). In the pine-
al parenchyma, the sympathetic fibre terminal endings do
not show true synaptic connections with pinealocytes,
ending freely. In general, the fibres are incompletely
surrounded by lemmocyte cytoplasm (Bondareff, 1965; Arstila,
1967; Ariens Kappers, 1969), while the basal lamina usual-
ly covering nerve fibre bundles is also discontinuous
(Ariens Kappers, 1969). Most probably, the noradrenaline
released from intraparenchymal sympathetic nerve fibres
affects the function of the pinealocytes by way of dif-
fusion. The same may be true for noradrenaline released :f;rom
the terminals of perivascular sympathetic nerve fibres,
the more so as the perivascular spaces show outpocketings
penetrating into the parenchyma. As has been mentioned in
our first lecture, it has, moreover, been demonstrated by
light- (Quay, 1974) and by electron microscopy (Krstic,
1975) that the pinealocytes are surrounded by a system of
continuous fine channels which enhance the diffusion of
compounds. Together with the extensions of the perivas-
cular'spaces, these channels form an extensive canalicu-
lar network.
The orthosympathetic nerve fibres are postganglionic,

originating from the perikarya of the superior cervical
ganglia. Having entered the skull with other fibres of
the same origin they reach the pineal gland along two
somewhat different pathways. In rat, most fibres run to
the organ in two bilateral symmetrically nervi conarii
which course to the dorsocaudal pole of the pineal. A num-
ber of fibres also enter the organ by way of the peri-
vascular spaces of pial vessels which penetrate into the
gland (Ariens Kappers, 1960). After superior cervical
ganglionectomy all sympathetic pineal fibres disappear
as has been shown by silver staining (A~iens Kappers,
1960), while, after transection of the nervi conarii by
far most of the green fluorescent noradrenergic autonomic
fibres in the organ are absent (Owman, 1964). In some
species, such as in the cat and bat, the two symmetrical
conary nerves fuse before reaching the pineal (Kenny,
1965). This is also the case in man (Le Gros Clark, 1940)
and monkey (Kenny, 1961). The fibres in the conary nerves
distribute directly in the pineal parenchyma.
100 J. A. KAPPERS
The way in which sympathetic nerve fibres enter the

organ varies among mammals as does their intrapineal dis-
tribution. In some mammals, sympathetic fibres are prac-
tically exclusively situated in the perivascular spaces,
while in others they are found exclusively in the pineal
parenchyma proper. As has been mentioned, a perivascular
and a parenchymal distribution also occur (see Vollrath,
1981, for literature). Moreover, perivascular nerve
fibres sometimes enter the pineal parenchyma, while
parenchymal fibres reach the perivascular spaces. In
species in which the pineal complex is constituted of a
superficial and a deep part, autonomic nerve fibres were
observed to leave the superficial part to run uninter-
ruptedly through the pineal stalk to the deep part of the
gland and, in some mammals, even to the habenular nuclear
complex.
In the terminals of the sympathetic fibres as well

as in their preterminal varicosities small and large
dense-core vesicles measuring 40 - 60 and 60 - 120 nm, re-
spectively, are present. Spherical and flat electron-
lucent vesicles also oocur. By a number of different
techniques it has been shown that the more abundant small
granular vesicles and the flat clear vesicles are in-
volved in the storage and release of noradrenaline, the
flat clear vesicles representing depleted granular vesi-
cles. The spherical electron-lucent vesicles may be
cholinergic in nature constituting a cholinergic link in
postganglionic transmission (Eranko et al., 1970; Macha-
do and Lemos, 1971; Vollrath, 1981, also for the exten-
sive literature on this subject). It has been suggested
that, functionally, the small dense-core or granular
vesicles are basically similar to the larger ones.
After the onset of darkness, the number of granular

vesicles strikingly decreases, while the number of flat
electron-lucent vesicles increases (Matsushima and Ito,
1972; Matsushima et al., 1979). This agrees with the
concept that noradrenaline, the amount of which in-
creases during darkness (Wurtman et al., 1967), is soon
released at that time of the day. As has been mentioned,
noradrenaline release induces an increased production of
melatonin by the pinealocytes during darkness (Wurtmann
et al., 1968).
Many experiments have shown that information about

environmental conditions, especially changes in photic
input in the lateral eyes, is conveyed to the pineal
gland by way of its peripheral orthosympathetic innerva-
tion. The central neural pathway involved has been thor-
oughly studied by Moore (1978a,b) who will speak himself

about this subject during this course. Therefore, I will
only shortly indicate this pathway here. The ganglion
cell layer of the retina conveys photic stimuli bilateral-
ly to the suprachiasmatic nuclei. These hypothalamic nu-
clei project caudal ward into the peri ventricular hypothal-
amus and the ventral tuberal area, while neurons of this
latter area project to the lateral hypothalamic nucleus.
Neurons of this nucleus have efferent connections, run-
ning in the medial forebrain bundle, with the sympathetic
intermediolateral cell column in the upper thoracic part
of the spinal cord. From this part of the cell column,
preganglionic orthosympathetic fibres run to the superior
cervical ganglion. The central neural pathway from the
lateral eyes to the cervical sympathetic fibres has been
confirmed by electrophysiological experiments (Brooks et
al., 1975; Dafny and McClung, 1975; McClung and Dafny,
1975). Furthermore, it has been shown that effects of
light on the pineal are not mediated by any of the endo-
crine organs (Fiske et al., 1962) and that light does not
exert any direct influence on the function of cultured
pinealocytes.
As was first shown in the rat (Ariens Kappers, 1960)

and later confirmed in rat and other mammals by several
authors, nerve fibres originating from both the habenular
and posterior commissure course in the pineal stalk. How-
ever, most of them form hairpin loops joining again their
commissure of origin, especially the habenular, at the
contralateral side. Some fibres, however, were found to
run not only in the pineal stalk, but also to reach the
pineal organ proper not returning to their commissure
of origin. Because of a number of reasons, such fibres
were first regarded to be aberrant commissural fibres
going astray (Ariens Kappers, 1960, 1965). However, re-
cent findings fully confirmed the concept held by much
earlier authors (see Ariens Kappers, 1960) that the
habenulo-pineal complex is a functional neural entity.
As was earlier observed (Schapiro and Salas, 1971), even
after bilateral superior cervical ganglionectomy light
stimuli are still able to influence rat pineal function
while, more recently, a number of neuroanatomical and
electrophysiological investigations proved the presence
of a central epithalamo-pineal neural connection~ espe-
cially between the habenular region and the mammalian
pineal gland (see Ueck, 1979; Guerillot et al., 1979;
Semm et al., 1981; Vollrath, 1981). Some of the pathways
involved are pinealofugal and either catecholaminergic or
acetylcholinergic, while others are clearly pinealopetal
(see Ariens Kappers, 1981, and Vollrath, 1981, for lit-
102 J.A.KAPPERS
erature). The most important results obtained can be sum-

marized as follows. In rat, nerve fibres from the habenu-
lar nuclei, but probably also stria medullaris fibres,
run via the pineal stalk to the pineal gland. By record-
ing average photic responses from the rat pineal in light
and dark adaptation, it was observed that in the dark-
adapted state responses exhibited a higher amplitude
(Dafny, 1980). It has been assumed that photic stimuli
are transmitted to the pineal along two separate routes:
(1) a fast one via the central habenulo-pineal pathway,
and (2) a slower one via the peripheral sympathetic inner-
vation of the pineal. It was also found (Semm and Voll-
rath, 1979) that the guinea-pig pineal contains two dif-
ferent populations of spontaneously active pinealocytes.
The cells of the first population respond to olfactory,
acoustic and short-term photic stimuli which evidently
reach the gland via the central habenulo-pineal neural
pathway. After sympathectomy the spontaneous activity of
such cells in the pineal is diminished, but not quite
suppressed. The cells of the second category do not re-
spond to short-term sensory stimulation. Assumedly, mel-
atonin production happens in pinealocytes showing an in-
creased electrical activity during the night and a de-
creased activity during the day. These cells cease firing
after removal of the peripheral sympathetic pineal in~er
vat ion which is known to enhance melatonin production
during the night. Because, moreover, the activity of some
pinealocytes is proved to be stimulated by light and in-
hibited by darkness it was, furthermore, hypothesized
that a positive feedback mechanism regulates melatonin
synthesis which is induced by darkness and terminated by
light. The activity of the two different populations of
pinealocytes has been supposed to depend on the central
and the peripheral innervation of the pineal, respective-
ly.
In the guinea-pig, nerve fibres coursing from the

habenular nuclei to the pineal have also been demonstrat-
ed, while fibres joining the striae medullares and run-
ning in the habenula reach the gland somewhat more dor-
sally (Semm and Vollrath, 1979). On the base of electro-
physiological findings it was suggested that the habenu-
lar nuclei, probably the lateral ones, modify pineal
gland activity, while, vice versa, the pineal influences
single unit activity in the habenular nuclei. In exactly
which way the latter possibility is realized is still
open to further research. It is known that, in the fer-
ret, intramural pineal cholinergic nerve cells send fi-
bres to the habenular region, while from this region
nerve fibres run to this pineal ganglion (Trueman and
Herbert, 1970; Herbert, 1971; David and Herbert, 1973;

David et al., 1973). To our knowledge in no other adult
mammal such a cholinergic pinealo-habenular connection
has, so far, been observed.
In some mammalian fetuses, however, morphological

research suggested the presence of a pinealofugal nerve
fibre bundle closely associated with clusters of nerve
cells (M_ller, 1979, a~so for literature). It has been
held that this bundle may represent a vestigial homologue
of the non-mammalian pineal tract which, perhaps, per-
sists in adult mammals (Ueck, 1979) having been incorpo-
rated in the pineal parenchyma and the pineal stalk (M_l-
ler et al., 1975). As has been mentioned, however, the
ferret is the only mammal in which, in the adult state,
pineal sensory neurons have at present been observed.
The investigations shortly surveyed clearly estab-

lished the presence of a central pineaopetal innervation
via the habenular region. They, furthermore, suggest that
(1) other sensory stimuli than just photic ones may regu-
late pinealocyte activity via this pathway, and that
(2) photic stimuli of a different duration influence the
activity of different pinealocyte populations along two
different pinealopetal neural pathways, the peripheral
sympathetic and the central habenulo-pineal pathways.
Irregularly occurring intrapineal nerve cells have

been observed in several mammals including primates. The
distinct intramural pineal ganglion in the ferret has
already been mentioned. Its cells are acetylcholinergic
and probably of a sensory nature. Intramural cholinergic
nerve cells of a quite different nature are present in
the rabbit pineal. They have been considered postgangli-
onic parasympathetic neurons. The parasympathetic pineaZ.
innervation has, so far, only been investigated in the
macaque monkey (Kenny, 1961) and, more extensively, in the
rabbit (Romijn, 1972, 1973a,b, 1975a,b). In the rabbit,
preganglionic parasympathetic nerve fibres probably
originate from the superior salivatory nuclei in the me-
dulla oblongata. Some of such fibres run along with the
facial nerves, the greater petrosal nerves and the conary
nerves to the pineal gland in which they establish axo-
somatic and axo-dendritic synapses with intramural pineal
postganglionic parasympathetic neurons, the number of
which is about 40. Postganglionic cholinergic parasympa-
thetic nerve fibres originating from these cells inner-
vate the pinealocytes. Other parasympathetic pregangli-
onic fibres, likewise originating from the superior sali-
vatory nuclei, do, however, synapse with postganglionic
104 J.A.KAPPERS
parasympathetic cells present in the greater petrosal

nerves. The axons of these cells run to the pineal via the
nervi conarii to distribute directly in the pineal paren-
chyma. In the rat pineal, cholinergic nerve terminals ex-
clusively situated in the organ and presumably making true
synaptic contacts with pinealocytes have also been de-
scribed (Wood, 1973). However, bilateral stereotaxic coag-
ulation of rabbit facial nerves caused only minor changes
in the ultrastructure of pinealocytes (Romijn, 1975a),
while the administration of parasympatholytic drugs showed
comparable results (Romijn, 1976). By some authors such a
parasympathetic pineal innervation has, however, not been
accepted on biochemical grounds, choline acetyl transfer-
ase being barely or not at all detectable in the pineal.
The very little activity measured could, in fact, be due
to the presence of carnitine acetyl transferase (LaBella
and Shin, 1968; Schrier and Klein, 1974). So far it would
appear that there is little evidence that the parasympa-
thetic autonomic system exerts any important effect on
mammalian pineal function.
Extramural pineal nerve cells forming distinct gan-

glia which are probably functionally associated with the
organ have occasionally been observed (see Bargmann,
1943). Two of such ganglia showing a somewhat different
topography have been distinguished. After their discover-
ers they have been named parietal or Warburg's ganglion
and Pastori's ganglion. By one author (Kenny, 1965) a
small Warburg's ganglion was observed in a young adult
human between the suprapineal recess and the dorsal sur-
face of the gland. In older individuals it could, however,
not be found. By other authors (M~llgaard and M~ller,
1973) a Warburg's ganglion was demonstrated in human
fetuses on the dorsal surface of the great cerebral vein
which lies in between the ganglion and the pineal, while
a ganglion of Pas tori was present at the caudal pole of
the gland, ventral to the great cerebral vein. It has
been suggested that both extramural pineal ganglia are
parasympathetic (Kenny, 1965).
Neurosecretory nerve fibres are also present in the

mammalian pineal gland. They stain with chrome-alum haem-
atoxylin aldehyde fuchsin or alcian blue. Such extra-
hypothalamic neurosecretory fibres have been demonstrated
to occur in small amounts inthe pineal of the hedgehog
(Bargmann, 1964; Hartmann, 1957; Suomalainen, 1960; Ok-
sche, 1965), the Rhesus monkey (Lukaszyk and Reiter, 1974,
1975) and the Mongolian gerbil (Japha et al., 1974). Re-
cently, vasopressin- and oxytocin-containing nerve fibres
were demonstrated in the area of the subcommissural organ
and in the pineal of the rat (Buijs et al., 1978; Buijs

and Pevet, 1980). By radioimmunoassay, vasopressin, oxy-
tocin, neurophysin I and neurophysin II were also quanti-
tatively determined in the bovine pineal (Pevet et al.,
1980). The ratio of vasopressin to oxytocin was similar
to that of neurophysin I to neurophysin II. This suggests
that the neurophysins present in the pineal are, indeed,
the carrier proteins of the two peptidergic hormones
rather than of arginine-vasotocine, the pineal presence
of which was earlier supposed (see Dogterom et al.,
1979) .
Such extrahypothalamic neurosecretory fibres were

also specifically demonstrated in the rat pineal by the
unlabelled antibody-enzyme technique (Buijs and Pevet,
1980). They run via the subcommissural organ or the ha-
benular commissure into the pineal stalk to end in the
rostral part of the organ proper. It has been speculated
that they are possibly involved in the regulation of
water balance. In the guinea-pig pineal, probably iden-
tical nerve fibres have been observed using the horse-
Tadish peroxydase method (Korf and Wagner, 1980). Their
origin from the region of the neurosecretory paraventri-
cular hypothalamic nucleus suggests their peptidergic
nature. From here, the fibres course via the habenular
or the posterior commissure and the pineal stalk to the
caudal part of the pineal gland. Further investigations
concerning the functional significance of such neuro-
secretory nerve fibres in the pineal organ are neces-
sary.
In concluding this survey it appeared that, in par-

allel with the change in function which can be observed
in the pineal organ during its phylogenetic development,
pineal innervation also changes from a practically pure-
ly pinealofugal or afferent innervation into a pinealo-
petal or efferent innervation, a more or less gradual
process which is fully realized in mammals. The periph-
eral sympathetic pineal innervation is certainly of the
utmost importance for pineal function. Besides, however,
many investigations have now shown that central efferent
nerve fibres of very different origins convey sensory
input and also special chemical input to the organ influ-
encing its function.
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112 J. A. KAPPERS
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THE DIFFERENT CLASSES OF PROTEIC AND PEPTIDIC
SUBSTANCES PRESENT IN THE PINEAL GLAND
1 2
Paul Pevet '
1) The Netherlands Institute for Brain Research

Amsterdam, and 2) Dept. of Anatomy and Embryol-
ogy, University of Amsterdam, The Netherlands
It is now well known that the pineal gland is principally

involved in long-term adaptation of Iunctions such as re-
production to environmental conditions. The mechanism by
which the pineal acts on the gonadal axis is, however, not
yet known. Recently it has been hypothesized that the
5-methoxyindo1es (melatonin, 5-methoxytryptamine, 5-meth-
oxytryptophan, 5-methoxytryptophol and 5-methoxyindole-3-
acetic acid) synthesized by the pineal gland as well as by
the retina, Harderian gland and intestine would be im-
plicated in a system by which the pineal and some other
organs such as the brain would be able to perceive, to
differentiate and to integrate environmental information
such as photoperiod, temperature, food, etc. In response,
the pineal, then, would synthesize and release proteic/
peptidic hormone(s) which would act on the reproductive
axis (Pevet et al.,198Ib; Pevet and Haldar-Misra, 1982).
Considering this hypothesis, the study of the pineal ac-
tive proteic/peptidergic fractions - the discovery of
which dates back as far as the 1920's - appears to be of
fundamental interest. According to the literature, the
pineal appears to contain a large number of proteic or
peptidic compounds, some of them being identified or par-
tially characterized, others not (see table 1). However,
although present in the pineal, these various compounds
can be of one or more of the following origins:
1) synthesis in organs other than the pineal, but found
there because they are present in cell elements such as
nerve axons which connect the gland to different brain
structures;
2) synthesis in organs other than the pineal, with a
113
114 P. PEVET
Table 1. Proteins, peptides and proteic/peptidic fractions reported

to be present in the pineal of mammals
Arg: arginine, Gly: glycine, Glu: glutamine, Leu: leucine,

LHRH: luteinizing hormone releasing hormone, a-MSH: a-
melanocyte stimulating hormone, MIF: a-MSH-release inhib-
itor factor, MW: molecular weight, PAG: pineal antigonado-
trophin, PPIF: pineal prolactin inhibiting factor, PPRF:
pineal prolactin releasing factor, Pro: proline, TRH:
thyrotropin releasing hormone, TSL: threonylserinyllysine,
Tyr: tyrosine, VIP: vasoactive intestinal peptide.
Name/terminology Species References
I IDENTIFIED PEPTIDES AND PROTEINS

A) Neurohypophysial principles
Neurophysins cow, hamster, Reinharz et al.,1981*,
mouse, man, Weindl & Sofroniew, 1982
pigeon', rat,
sheep
Oxytocin cow, hamster, P~vet et al., 1981a~

hedgehog, mouse, Gee1en et a1., 1981;
~ rat, sheep Nurnberger & Korf,1981
Vasopressin cow, hamster, P~vet et a1., 1981a,*

hedgehog, mouse, Geelen et al., 1981"
man, rat, Nurnberger & Korf,
rabb it, sheep 1981
Vasotocin** cat, chicken, Pavel, 1979,

cow, eel, Vaughan, 1981,*
gerbil, guinea Vivien-Roe1s et al.,
pig, hamster, 1979, 1981*
lizard, man,k.
mouse,quai1,
rat, sheep,
snake, trout
B) Other hypothalamic hormones

LHRH*** cow, pig, P~vet, *
1982a, P~vet et
rat, sheep a1., 1981a, King &
Millar, 1981
MIF rat Kastin et a1., 1980

PROTEIC AND PEPTIDIC SUBSTANCES IN PINEAL GLAND 115
Table 1 (continued)
TRH*** cow, frog, pig, Pevet, 1982a*

sheep, rat
Somatostatin *** rat Pelletier et al., 1975
C) Various other peptides

ACTH (1-39) rat Vuolteenaho & Leppal-
uoto, 1981
Angiotensin I rat Coculescu et al., 1977
Angiotensin II rat Changaris et al., 1979
a-Endorphins rat Rossier et al., 1977;
Vuolteenaho et al.,
1980
Enkephalin rat Rossier et al., 1979
a-MSH rat Pevet et al., 1982a*
Substance P cat, human Powel et al., 1977
VIP cat, pig, Uddman et al., 1980;
rabbit
TSL cow Orts et al., 1980
II PARTIALLY IDENTIFIED PEPTIDES AND/OR PROTEINS
E5 (MW ~ 2000, peptide of 14 cow Neac~u, 1971, 1972

amino-acids. Ring with an S-S
bridge. Lateral chain of 5
amino-acids with Pro-Arg-
Gly(NH2) for the N-terminal
amino-acids)
p-Glu- X -Leu-Arg-Pro-Gly(NH2) sheep Millar et al., 1981
Pineal in (MW ::: 10.000,
cow Milcou et al., 1963a
14 amino acids)
X-Pro-Arg-Gly(NH2) cow, sheep, rat Pevet et al., 1981c
X-Tyr-Pro-Leu-Gly(NH2) rat Kastin et al., 1981
(continued)
116 P.PEVET
Table 1 (continued)
III NON IDENTIFIED PROTEIC OR PEPTIDIC FRACTIONS****
Anestrine cow Bianchi & Osima, 1960

Altschule's pineal extract cow Altschule, 1957
Anovulin cow Chazov et al., 1972
Crinofizin cow Milcu et al., 1976
Epiglandol cow Hofstatter, 1914
Epiphysan cow Engel, 1935;
Hofstatter, 1938
Epifizhormon/epiphysormone cow Parhon et al., 1940
Epithalmin cow Dil 'man & Anisimov ,1975
Extracts IIa, IlIa cow Ota et al., 1975
Fraction F2 (MW < 1500) sheep Ebels et al., 1965;
Moszkowska et a1.,1974
Fraction F 3 (MW < 700 when sheep Moszkowska et al.,1973
purified)
Fractions Al and A3 cow Vaughan et al., 1974;
Blask et al., 1976
Fraction II I (MW '" 500-1000) cow Thieblot et al., 1979
PAG (MW '" 500-1000) cow, human Benson et al., 1972;
Matthews et al., 1971
Peptide II F (lJW '" 3000) cow Rudman et al., 1970
PPRF cow Blask et al., 1976
PPIF cow Blask et al., 1976
PP7,2 sheep Noteborn et al., 1982
UM05R (MW '" 500-100) cow, sheep Ebels et al., 1973
XM300R PP7,2 sheep Noteborn et al., 1982
* Review article to which we refer for details and references.

** Its presence is doubtful in mammalian species (see text).
*** The large amount found by some authors is doubtful (see text).
**** Many fractions isolated from the pineal and termed 'pineal ex-
tracts' have not been quoted in this table.
secondary uptake by the gland from the general circula-

tion;
3) synthesis in the pineal by specific pineal cells.
Such a classification of the proteic/peptidergic com-
pounds into three different classes is very important to
consider. Indeed, although all of them can be isolated
from the gland, only the proteic/peptidic compounds of
class three can be considered as true pineal hormones.
The present review deals with the results obtained
the last few years in studies concerned with this prob-
lem, using different techniques such as electron micro-
scopy, ultracytochemistry, immunocytochemistry, radio-
immunoassay and bioassays.
I. PEPTIDES AND PROTEINS OF THE FIRST CLASS
Complex nervous connections between the central

nervous system and the pineal gland are known to exist in
vertebrates (see the articles by Ariens Kappers and
Moore, this volume). Thus, neurotransmitters such as for
example noradrenaline and acetylcholine (at least in some
species) are present in nerve endings located in the pin-
eal and consequently can be isolated from pineal ex-
tracts. However, they are not and have never been con-
sidered as pineal factors. Similarly, since it is known
that in the brain neuropeptides may act as neurotrans-
mitters (Buijs et al., 1982), the existence of peptider-
gic nerve fibres might explain the presence of neuro-
peptides in the pineal. Vasopressin (AVP) and oxytocin
(OT) have been detected in the pineal gland of numerous
mammalian species (see table 2) but at a relatively low
concentration. Their presence has been demonstrated - by
the use of the unlabeled antibody enzyme method - to be
due, at least in the rat (Buijs and Pevet, 1980) and the
cow (Pevet, unpublished data), to the presence in the
pineal of AVP- or OT-containing fibres. In the rat these
fibres are extrahypothalamic fibres, originating - at
least partly - from the magnocellular AVP- and OT-pro-
ducing nuclei which run via the subcommissural organ and
the deep pineal (if present) or via the habenular com-
missure into the pineal stalk, terminating in the ros-
tral part of the pineal (Buijs and Pevet, 1980). This
work has recently been confirmed in the hedgehog by
NUrnberger and Korf (1981). These authors indeed have
observed that the perikarya giving rise to these AVP- or
OT-containing fibres were located in the dorsal part of
the paraventricular nucleus, the fibres entering the
pineal also via the habenular commissure. The extrahypo-
thalamic AVP- or OT-containing fibres explain also the
presence of the two neurophysins described in the pineal
00
Table 2. Neurohypophysial Hormone Concentrations in the Mammalian Pineal

G = gland; n.d. = not detectable; Pr = protein, WT = wet tissue
Species AVP OT References
Cow '" 1.6 pg/mg WT '" 0.75 pg/mg WT Fisher and Fernstrom, 1981
3.7 ~ 0.7 pg/mg WT 3.7 ~ 1.7 pg/mg WT Dogterom et al., 1980
407 + 66 pg/G 238 ~ 41 pg/G Fernstrom and Fisher, 1980
Hamster Trace Trace Pevet et al., 1981a
Human 306.4 + 27.7 pg/G 386 + 42.1 pg/G Geelen et al., 1981
Mouse Trace Trace Pevet et al., 1981a
Rabbit 0.032 + 0.008 pg/ugPr Negro-Vilar et al., 1980

Rat 0.186 + 0.019 pg/~Pr Negro-Vilar et al., 1980
(Sprague-Dawley '" 1.99 pg/mg WT '" 2.46 pg/mg WT Fisher and Fernstrom, 1981
(Wistar) 2.7 + 0.8 pg/mg WT 1.3 + 0.8 pg/mg WT Dogterom et al., 1980
(Brattleboro) n.d. 8.7 + 3.7 pg/mg WT Dogterom et al., 1980
Sheep 19.74 + 2.3 pg/mg WT 35.3 + 7.1 pg/mg WT Reinharz et al., 1981
"'1J
"'1J
m
<
m
-i
of different mammalian species (details in Reinharz et

al., 1981). Neurophysins, indeed, are cystein-rich pro-
teins which in the pineal, like in the neurohypophysis,
are associated with the neurohypophysial hormones (Pe-
vet et al., 1980c).
Vasotocin (AVT) is one of the neurohypophysial hor-

mones in the nonmammalian vertebrate species. For some
authors (review in Pavel, 1979), AVT would be also a hor-
mone present in and synthesized by the pineal gland of
mammals. This conclusion now appears to be very improb-
able (see details in P~vet, 1981b, 1982a,b; P~vet et al.,
1981a, and in the next subheading), but AVT is really
present in the pineal of some nonmammalian vertebrate
species (table 3). Vivien-Roels et al. (1981) have sug-
gested that this presence could be due - in analogy to
mammals - to the presence in the pineal of extrahypo-
thalamic fibres containing the neurohypophysial hormone
(here AVT). Such an interpretation which, to date, has
not yet been proved, seems very likely because Weindl
and Sofroniew (1982) have detected by means of immuno-
cytochemistry neurophysin~containing nervous fibres in
the pineal of the pigeon.
The existence of other peptidergic fibres in the

pineal explains also the presence of numerous other
neuropeptides. For example, Uddman et al. (1980) and
M~ller et al. (1981) have demonstrated that the VIP im-
munocytochemically detected in the pineal of the cat,
pig and rabbit, was exclusively found in nerve fibres
running in the pineal and especially in the pineal
stalk. Following Hansen and Karasek (personal communica-
tion) the substance-P detected by radioimmunoassay, for
example in the human pineal (300-500 pg/mg tissue; Pow-
el et al., 1977), would be also located exclusively in
nerve fibres and endings, at least in the cat.
However, these results cannot be generalized to all

neuropeptides. For some of them indeed, the problem ap-
pears to be more complicated. For example, as will be ex-
plained under the next subheadings, the determination of
the exact origin of the LHRH-immunoreactivity found in
the pineal is a difficult problem. However, the pres-
ence in the pineal of some extrahypothalamic LHRH-con-
taining fibres, although not yet clearly demonstrated,
is very probable. Indeed, Barry (1979), in the female
squirrel monkey, has described an LHRH-reactive peri-
karyon in the habenular commissure or in the rostral
part of the pineal, a perikaryon which might send fibres
into the pineal. Similarly, although not any immunocyto-
Table 3. Vasotocin Concentration in the Pineal of Nonmammalian Vertebrates N
o
G = gland; Pr = protein
Species AVT References
Birds
Chicken 0.180 ~ 0.04 pg/~g Pr Negro-Vilar et al., 1980
'" 9.1 pg/G. Fisher and Fernstrom, 1981
31 ~ 5 pg/G Vivien-Roels et a1., 1981
312 ~ 39 pg/G Fernstrom et al., 1980
Quail 44 + 21 pg/G Vivien-Roels et al., 1981
Reptiles
Lizard (Lacerta agilis) 210 + 74 pg/G Vivien-Roe Is et al., 1981
Snake (Natrix tessala) 245 ~ 89 pg/G Vivien-Roels et al., 1981
Tortoise (Testudo hermanni) 181 ~ 118 pg/G Vivien-Roels et a1., 1981
Amphibians
Frog (Rana esculenta) 324 ~ 27 pg/G Vivien-Roels et al., 1981
'" 8.5 pg/G Fisher and Fernstrom, 1981
Fishes
Salmo gairdneri 58 ~ 15.7 pg/G Vi vien-Roels et al., 1979; Holder et al. , 1979
124 ~ 17 pg/G Vivien-Roe1s et al., 1981
Sa1mo gairdneri Richardson 30.7 + 7.2 pg/G Holder et al., 1979; Vivien-Roels et al. , 1979
Salmo trutta morpha fario 34.8 ~ 9.1 pg/G Holder et a1., 1979; Vivien-Roels et al . , 1979 :-0
'1J
Sa1velinus frontalis 24. 0 ~ 6.7 pg/G Holder et al., 1979; Vivien-Roels et al. , 1979 m
Anguilla anguilla 32.8 ~ 6.9 pg/G Holder et a1., 1979; Vivien-Roels et al. , 1979 <
m
--!
chemical study has confirmed the radio immunological ob-

servations of White et al. (1974) and of Jackson et al.
(1977) and though, at the contrary, Youngblood et al.
(1979) have chemically demonstrated that the immunore-
activity observed in bovine and sheep pineal was due to
an unknown compound different from the genuine peptide,
it is not possible to exclude completely the presence of
some TRH-containing fibres in the pineal.
Concerning somatostatin, Pelletier et al. (1975)

have noted that in the rat the reaction was only present
close to the capillaries. Thus, here also, although it
is known that some of the observations are due to cross
reaction of the antibody used with an unknown compound
(see after), the presence of some somatostatin-containing
fibres is a possibility.
a-MSH is known as a pituitary hormone but is also

produced by the nervous system (see review in Swaab,
1982). Consequently we have also to examine the possi-
bility that the a-MSH found in the pineal would be also
a neurotransmitter present in nerve fibres or endings. To
date, however, not any information permits to support
this idea (see after for the problem of the presence of
a-MSH in the pineal).
II. PEPTIDES AND PROTEINS OF THE SECOND CLASS
The pineal gland is an endocrine organ capable of

synthesizing and releasing material which can affect
numerous organs and systems and which, in turn, is in-
fluenced by a variety of stimuli and signals. Especially
the pineal, like all endocrine organs, appears to be un-
der feed-back control by target tissues (see review in
Ariens Kappers, 1978). This peculiar aspect of pineal
physiology is important to consider here. Indeed, a
system feedback operating between the hypothalamus or
the hypophysis and the pineal would implicate that hypo-
thalamic or hypophysial hormones are transported via the
general circulation into the pineal. Looking now at the
list of identified proteins and peptides found in the
pineal (table 1), it appears that some of them are hypo-
thalamic and hypophysial principles. Consequently, the
possibility that the presence of such hormones within
the pineal would be due to an accumulation of circula-
ting hormones has to be taken into consideration.
This aspect of the pineal peptide/protein problem

has not been deeply investigated. Clear results, how-
ever, have been obtained with two hypothalamic hormones.
122 P.PEVET
After intravenous injection of labelled MIF, Redding et

al. (1973) have observed that the radioactive hormone
was especially concentrated in the pituitary and the pin-
eal. A similar observation was made after injection of
labelled LHRH by Redding and Schally (1973), Admundson
and Wheatons (1979) and Trentini et al. (1980). The last
mentioned group noted also that the pineal gland accumu-
lated 3H-LHRH in an amount significantly higher than the
pituitary gland. These experiments which demonstrated
that the pineal, at least in rat, is able to concentrate
in a notable amount circulating MIF and LHRH, can easily
explain the presence in the pineal of these hypothalamic
hormones. Such a phenomenon of accumulation, however,
does not permit to explain all results published in the
literature and especially the finding that the pineal
would contain and synthesize a very large amount of
'hypothalamic hormones' (White et al., 1974). Cross re-
actions of the antibodies used with unidentified com-
pounds could be also responsible of some of these obser-
vations (see next sub-heading). Moreover, as explained
before, LHRH for example is probably also present in ex-
trahypothalamic nervous LHRH-containing fibres.
The physiological significance of such a phenomenon

of accumulation of circulating hormones is not known but
at least it demonstrates that a feed back system operates
between the hypothalamus and the pineal. Moreover, this
raises the question of the possibility of a similar
mechanism for other hormones such as hypophysial ones,
a phenomenon which would explain the high amount of a-
MSH detected in the pineal by some authors (Olivier and
Porter, 1978; Vaudry et al., 1978; O'Donohue et al.,
1980). As after hypophysectomy the amount of immunore-
active a-MSH does not change (Vaudry et al.,. 1978; O'Don-
ohue et al., 1980), this presence of a-MSH in the pineal
cannot be attributed to an accumulation of circulating
hypophysial hormone. It is very probable that these re-
sults are due to a cross reaction of the used antibodies
with an unknown compound (see below). However, as in in-
tact as well as pinealectomized rat, exogenously admin-
istered H 3 -a-MSH was demonstrated to be also accumulated
in the pineal (Kastin et al., 1976), the presence of a
small amount of accumulated a-MSH, TRH or other peptidic
of proteic hormones is probable and at least cannot be
excluded. Interestingly, Vuolteenaho and Leppaluoto
(1981) who have detected ACTH (1-39) in the rat pineal
(42-166 fmol/gland), concluded also that this ACTH in
fact originated from the pituitary.
III. PEPTIDES AND PROTEINS OF THE THIRD CLASS
Processes involved in the synthesis and release of

proteic/peptidic substances have been identified at the
ultrastructural level (see below). The existence of pro-
teins or peptides of the third group thus cannot be dis-
carded. To date, however, none of these substances have
been structurally identified and by consequence their
studies are always indirect ones.
A. Fluorescent Histochemical Studies
The pinealocytes of the hedgehog (Pevet, 1976), the

mole (Pevet, 1976; Pevet et al., 1976), the rabbit (Smith,
1972; Smith et al., 1972a,b) and the rat (Pevet et al.,
1975; Smith et al., 1975) contain a yellow autofluores-
cent sUbstance which was demonstrated to be, in the rab-
bit, a protein containing much tryptophan (Smith, 1972).
A comparison of the fluorescent histochemical observa-
tions with the ultrastructural ones in the mole (Pevet,
1974, 1976; Pevet et al., 1976) suggests that this auto-
fluorescent material could be identical with the protein-
aceous material (or with part of it) which is located in
the cisterns of the rough endoplasmic reticulum (the
'ependymal-like secretory process', see below). This
opinion is supported by the fact that in rat after cas-
tration or injection of gonadotropins, both experiments
which provoke an increase in number of pinealocytes con-
taining autofluorescent material (Pevet, 1976; Pevet et
al., 1975; Smith et al., 1975),an increase in number of
vacuoles characteristic of the ependymal-like secretory
process has been described (Karasek and Marek, 1978;
Karasek et al., 1976a,b). This, however, remains to be
demonstrated.
B. Immunocytochemical studies
(1) LHRH Immunoreactivity
In 1974 White et a1., using a radioimmunoassay,

demonstrated the presence of a very large amount of LHRH
in the pineal gland of the cow, pig and sheep and con-
cluded that this gland might be considered as a supple-
mental source of hypothalamic releasing hormone. This
result was the center of a large controversy. Some au-
thors denied its presence in the ovine pineal (Carson
et al., 1977) while others (Duraiswani et al., 1976;
Gradwell et al., 1976; Joseph, 1976; Millar and Tobler,
124 P.PEVET
1981; Wheaton, 1980), although confirming the presence

of detectable amounts of LHRH, noted that the LHRH-
immunoreactivity found in the pineal of cow, rat and
sheep was considerably lower than that in the hypothala-
mus. New results obtained in the last few years by dif-
ferent teams, however, now permit to have a better idea
on this LHRH problem. The low LHRH-immunoreactivity ob-
served in the pineal is in fact due, as was demonstrated
in the sheep by a combination of immunological and chem-
ical techniques by King and Millar (1981), to the pres-
ence of the genuine peptid~. This presence is probably
due, as explained before (subheadings I and II), to the
existence in the pineal of LHRH-containing extrahypothal-
amic fibr~s and/or to a phenomenon of accumulation in the
pineal of circulating LHRH. Immunocytochemically, how-
ever, an intense positive reaction has also been obtained
in the rat pineal gland (Pevet et al., 1980b). In this
study, three different antibodies against LHRH were used.
All stained equally well both rat hypothalamic tissue and
synthetic LHRH-~ontaining agarose beads. In the pineal,
however, only one gives positive results (see details in
Pevet et al., 1980b). This difference between the anti-
body potencies in the rat pineal, the rat hypothalamus
and the synthetic LHRH-containing agarose beads suggests
strongly that the intense reaction in the pineal is due
to the presence of a compound different from LHRH but
recognized by the antibody. This cross reaction of some
anti-LHRH with an unknown compound, a LHRH-like compound,
could explain the high LHRH immunoreactivity found in
the pineal by White et al. (1974). Indeed, if we use the
same antibody which has given us a positive reaction in
immunocytochemical studies, in a radioimmunoassay system
(Arimura, personal communication), a large amount of im-
munoreactive LHRH similar to that described by White et
al. (1974) is detected in the pineal. The presence of
such a LHRH-like compound has recently been confirmed
immunocytochemically by Piekut and King (1981) and
chemically by Miller et al. (1981), who isolated from
bovine pineal a substance recognized by different anti-
bodies against LHRH but structurally different from the
hypothalamic LHRH.
However, to date the nature of the immunocytochem-

ically stained LHRH-like compound which, as demonstrated
by means of organ culture (Pevet et al., 1980a), is syn-
thesized in the pineal itself, has not been determined
and more specially, we don't know whether this LHRH-like
compound is identical to the peptide immunologically re-
lated to LHRH detected by Millar et al. (1981) in the
bovine pineal.
(2) a-MSH~ Somatostatin and TRH Immunoreactivity
a-MSH is a hormone synthesized primarily in the in-

termediate lobe of the pituitary and also in the central
nervous system (for details see Swaab, 1982). A large a-
MSH immuno- and bioactivity has also been detected in the
rat pineal by different authors who assume that this im-
muno- and bioreactivity is caused by the presence of a-
MSH (O'Donohue et al., 1979, 1980; Oliver and Porter,
1978; Vaudry et al., 1978). Moreover, as hypophysectomy
does not deplete pineal a-MSH content, O'Donohue et al.
(1980) concluded that very likely this a-MSH is synthe-
sized by the pineal itself. Using antibodies against a-
MSH, cells have been immunocytochemically stained in the
rat pineal (Pevet and Swaab, 1979; Pevet et al., 1980b).
After 6 days culture in an in vitro system with synthetic
medium, some cells in the pineal explant are immunocyto-
chemically stained (Pevet et al., 1980b). This means that
the immunocytochemical result is not due to the accumula-
tion of circulating a-MSH (see subheading II) but to the
synthesis by the pineal of an a-MSH immunoreactive com-
pound. This immunocytochemical observation which confirms
the observation made by the earlier cited authors does
not permit to conclude with them that a-MSH is present in
the pineal gland. Indeed, the three antibodies against
a-MSH used in this study, although they stained equally
well both a-MSH containing agarose beads and cells of
the intermediate lobe of the rat pituitary, have quite
different staining properties in the pineal (see details
in Pevet and Swaab, 1979). These differences in antibody
potencies permit the conclusion that the a-MSH immuno-
reactivity observed in the rat pineal is due to the
presence of an a-MSH-like compound different from pitui-
tary a-MSH, a conclusion which had already been made on
the basis of bioassays by Rudman and Scott (1975).
Somatostatin has been immunologically detected in

the rat pineal gland (Pelletier et al., 1975). However,
this result has been later contested by Berolowitz et al.
(1978). Probably, as suggested by the immunocytochemical
study of Pevet et al. (1980b) in which only one of the
seven antibodies against somatostatin tested gives a
positive reaction in the pineal while all of them react
in the different control tests, this discrepancy is due
to the fact that it is a somatostatin-like compound dif-
ferent from the genuine peptide which is present in the
pineal. This is probably also the case for the TRH im-
munoreactivity detected by White et al. (1974) in the
pineal of cow, pig and sheep. Indeed, Youngblood et al.
(1979) have chemically demonstrated that the immunore-
126 P. PEVET
activity observed in sheep bovine pineal was due to an

unknown compound different from TRH.
(3) The Vasotocin Controversy
In 1963 Milcou, Pavel and Neacsu discovered in ex-

tracts of bovine pineal glands a peptide with pressor and
oxytocic activities. Although "unfortunately no synthetic
arginine vasotocin was available as standard", they in-
dicated that the chromatographic characteristics of this
peptide were similar to those of vasotocin and concluded
that the pineal peptide was "arginine vasotocin, or a
hormone with similar structure". This result was immedi-
ately checked by Ebels et al. (1965) who were not able to
confirm the presence of AVT in the pineal of sheep and
cow. One year later, however, Pavel and Petrescu (1966),
after a comparative study of the antigonadotrophic ef-
fects of the purified peptide and of synthetic AVT, con-
cluded again that the peptide was identical with AVT.
This result once more was contested by Neac~u (1972) who,
interestingly, was co-author with Pavel of the original
1963 paper. This author, after a chemical analysis, con-
cluded that the pineal peptide (termed by him "fraction
E5") was different from AVT and was composed of 14 amino-
acids. The last-mentioned study was never considered,
probably because two years earlier Cheesman (1970) and
Cheesman and Farris (1970) had isolated and structurally
identified AVT from bovine pineal extracts. This work
of Cheesman - which to date has not yet been confirmed -
was considered as the definite proof of the existence of
pineal AVT. The vasotocin controversy was thus provision-
ally stopped and in the next few years Pavel and his
group, still using the bioassay approach, "demonstrated"
that the pineal gland of all mammals so far examined, in-
cluding man, contained AVT (or lysine vasotocin, in
pigs); that AVT was synthesized by the ependymal cells
located in the pineal recess and by the cells of the sub-
commissural organ (SCO), and that AVT was released into
the cerebrospinal fluid (CSF) (see details and refer-
ences in Pavel, 1978). At the same time, the physiolog-
ical properties of AVT were extensively investigated by
the physiologists (see review in Vaughan, 1981, and
Vaughan and Blask, 1978) and the results obtained were
such that at the end of the seventies - and although in
the same period AVT was also considered as a neurohor-
mone of the fetus implicated in the maintenance of its
own intra-uterine water balance (Vizsolyi and Perks,
1976) - practically no pinealogist questioned the pres-
ence of AVT in the mammalian pineal gland. It is inter-
esting to note here that it is probably this general

consensus which is at the origin of the new revival of
the vasotocin controversy. Indeed, the acceptation of the
concept that AVT is a pinealantigonadotrophic agent has
led to intensified efforts to measure the AVT content in
tissues and body fluids. The bioassays being particularly
tedious techniques, especially for large numbers of
samples, radioimmunoassays were developed. Different as-
says had been made already in the mid-seventies, but none
of them was really specific for AVT, the antibodies used
being antibodies against AVP or aT (Rosenbloom and Fisher,
1975; Legros et al., 1976) .The first radioimmunoassay for
AVT using an AVT-antiserum was developed by Dogterom et
al. (1978, 1979, 1980). Using this radioimmunoassay,
these authors were not able to find an AVT immunoreactiv-
ity in the pineal, subcommissural organ or fetal neuro-
hypophysis of any of the mammalian species studied and
they concluded that the AVT biological activity found by
some authors (see above) was probably due to a compound
different from AVT itself. Negro-Villar et al. (1979) who
had also developed a specific radioimmunoassay for AVT,
were also unable to detect AVT immunoreactivity in the
pineal of the rat and rabbit and they strongly questioned
the role of AVT as a pineal hormone in mammals. These ob-
servations were quickly contested by Fernstrom et al.
(1980) who, having also developed a radioimmunoassay
specific for AVT, concluded that AVT is present in the
mammalian pineal. One year later, however, the last-men-
tioned authors, having refined their technique~ re-
evaluated their results and concluded that AVT is prob-
ably not present in the mammalian pineal (Fisher and
Fernstrom, 1981). At the same time Bowie also reconsider-
ed her original immunocytochemical work which had led
her to accept the presence of AVT in the rat pineal
(Bowie and Herbert, 1976) and concluded that this result
was due to a cross reaction of the antibody used with an
albumin-like substance (Bowie, 1980).
It thus appears that the long standing vasotocin

controversy has now, at least apparently, been solved,
the more so since when the physiological effects of AVT
are considered, it is possible to note, with Vaughan,
1981), that " ... where tested, AVP (which is really pres-
ent in the pineal) has many of the same effects as AVT".
It should be realized, however, that these negative re-
sults obtained with radioimmunoassay do not contest the
reality of the presence of an AVT-biological activity,
a presence which might be due to either vasopressin and
oxytocin, two peptides present in the pineal of mammals
(see subheading I) and which, taken together, would re-
128 P.PEVET
act in different bioassays or to a compound different

from AVT itself but related to it, an idea already sug-
gested by Neacsu (1972) and which could eventually explain
the present "inexplicable" chemical results of Cheesman
and Farris (1970).
Using the antibody against AVT raised by Fernstrom,

a positive staining similar to that described by Bowie
and Herbert (1976) has been obtained immunocytochemically
in the rat pineal (Pevet et al., 1980b). Moreover, the
relative number of immunostained cells in the pineal of
3 week old rats appears to be larger than in that of
adult rats. These immunocytochemical results which have
to be related to the observation that, as compared to
adult rats, a higher AVT biological activity is found in
the pineal of young rats (Pavel et al., 1975), and to
the positive radio immunological results obtained in the
rat pineal by Fernstrom et al. (1980), indicate clearly
the presence of an AVT immuno- and bioactivity. During
this immunocytochemical study four antibodies which all
stained equally well tissue of avian neurohypophysis, of
a turtle hypothalamus and synthetic AVT-containing agar-
ose beads, were used. Of these antibodies only that
raised by Fernstrom gave positive results. This differ-
ence between the antibody potencies in the rat pineal,
the avian neurohypophysis, the tortoise hypothalamus and
in the synthetic AVT-containing agarose beads has per-
mitted the conclusion that the reaction obtained in the
pineal was due to the presence of an AVT-like compound
different from AVT itself to which the antibody of Fern-
strom would cross-react. Interestingly, this interpreta-
tion is now confirmed by Fisher and Fernstrom (1981) who,
after reconsidering their first observation (Fernstrom
et al., 1980), concluded that their results were probably
due to antiserum nonspecificity. This interpretation is
also supported by the fact that using our radioimmuno-
assay for AVT, vasotocin has never been detected in
the mammalian pineal while, using the bioassay developed
by Holder and Guerne (1976), a biological activity sim-
ilar to that of AVT has been found in all species studied
(Pevet, 1982a; Holder et al., 1982). It agrees also with
the conclusion raised by Neacsu in 1972 that the biolog-
ical activity of AVT in the m~mmalian pineal gland is
due to a compound different from AVT itself.
Some preliminary results have permitted us to sug-

gest that this compound could be a peptide possessing
the same Pro-Arg-Gly(NH2) tripeptidic carboxy terminal
end as vasotocin (P~vet et a1., 1979, 1980a,c; P~vet,
1981b, 1982a).
(4) Perspectives
Looking at the results published in the literature

it appears that very often a close correlation exists be-
tween results obtained with the bioassays, the radioimmu-
noassays and the immunocytochemical studies. For example,
as explained before, using either the bioassay technique
or immumocytochemistry, a higher pineal "AVT-like" con-
tent is found in the young rat than in the adult one.
Positive immunocytochemical results were obtained with
antibodies which give also positive result in a radio-
immunoassay system. It would thus be tempting to "logi-
cally" conclude that the compound responsible for a given
biological activity is the compound which is recognized
by the corresponding antibody and consequently to decide
to use these antibodies for further isolation and puri-
fication of the compounds concerned.
The results obtained in immunocytochemistry, how-

ever, oblige us to be careful with such a conclusion. In-
deed, surprisingly, similar staining patterns were ob-
tained with all antibodies tested, anti-angiotensin-I
(Changaris et al., 1979), anti-AVT (Bowie and Herbert,
1976; Pevet et al., 1980b), anti-a-MSH (Pevet et al.,
1980b), anti-LHRH (Pevet et al., 1980b; Piekut and
Knigge, 1981), anti-somatostatin (Pelletier et al., 1975;
Pevet et al., 1980b), anti-UM05R (Pevet et al., 1980b).
Moreover, by means of serial section it has been demon-
strated in the rat that different antibodies stained the
same pineal elements near the perivascular space (Pevet
et al., 1980b). It appears thus that the a-MSH-like com-
pound, the LHRH-like compound, the somatostatin-like
compound and the AVT-like compound detected immumocyto-
chemically are all present in the same structure. More
exactly, as it was also demonstrated that the staining
potentialities of all antibodies were affected after
adsorption to heterologous peptides, it seems that all
antibodies react with a unique compound, possibly a pro-
tein or a protein complex. The nature of this compound
of which, as demonstrated by means of in vitro tech-
niques, the synthesis by the pineal is controlled by
sympathetic innervation (Pevet, unpublished results)
"could be a pineal endocrine principle, an enzyme, or
any structural protein" (Pevet et al., 1980b). Recently
Rix et al. (1981) using different antibodies against
angiotensin, have obtained a staining pattern similar
to that already described. Examining adjacent semithin
and ultrathin sections by immunocytochemistry and elec-
tronmicroscopy respectively, they have moreover demon-
strated that the staining reaction was located in the
130 P.PEVET
flocculent material present in the extracellular perivas-

cular compartment. Although in our own study we have been
able to observe some stained cell bodies (for example
fig. 3 in Pevet et al., 1980b), it is very probable that
the observations of Rix et al. (1981) are also applicable,
at least partly, to our own results. The nature of the
flocculent material, its origin and especially its re-
sponsibility in a possible false positive immunoreaction
have thus to be determined now before pursuing this ap-
proach of the pineal peptide problem.
B. ULtrastructuraL Aspect
The observations reported in this part concern ex-

clusively the major cell type of the pineal: the pinealo-
cyte or its phylogenetic homologues when non-mammalian
vertebrates are concerned. More than one way of produc-
tion and release of proteinaceous substances is taking
place in these cells. Indeed, two processes involved in
the synthesis and release of pineal proteic/peptidic
agents have been identified at the ultrastructural level
and are characterized either by the formation of granular
vesicles (GV) by the Golgi saccules, or by the formation
of vacuoles containing flocculent material of moderate
electron density by the cisterns of the rough endoplasmic
reticulum (RER), the so-called ependymal-like secretory
process. For an exact definition of these two processes
and details on the demonstration of their proteic nature
as well as on their possible relationships, we refer to
previous review articles (Pevet, 1979; 1981a,b; 1982a,b)
The identification of these secretory processes

permits now by a qualitative and quantitative electron
microscopic analysis of the different cell organelles,
to study, although their chemical nature has not yet
been determined, the regulatory mechanisms of the pineal
protein/peptide production and especially to identify
the substances (hormones, neurotransmitters, etc.) in-
volved in the control of their production.
(1) The influence of noradrenaLine on the processes

of protein/peptide secretion
The idea that the activity of the pineal gland is

controlled by the sympathetic innervation via its neuro-
transmitter release is now well accepted. This opinion,
however, is based essentially if not exclusively on bio-
chemical and physiological results concerning indole-
amines, especially melatonin. Information on a possible
effect of the sympathetic innervation on the processes

of protein/peptide secretion is very rare.
In in vitro experiments after application of nor-

adrenaline (NA) to the medium, an increase in the forma-
tion of GV by the Golgi apparatus has been demonstrated
in the pinealocytes of the hamster, rabbit and rat (Kara-
sek, 1974; Romijn and Gelsema, 1976; Haldar-Misra and
Pevet, 1982a). NA appears thus to stimulate the formation
of GV at least in these three species. This observation
is confirmed by different other results. In rat, for ex-
ample, pineal tissue grafted under the kidney capsule,
a situation in which the pineal is completely denervated,
a drastic decrease in number of GV was observed (Aguado
et al., 1977). Moreover, after pineal denervation by
superior cervical ganglionectomy, Romijn (1975) and Lin
et al. (1975) described a decrease in number of GV in
the pinealocytes of the rabbit and the hamster respective-
ly. This observation however is contested by Sheridan
(1975) who observed in the hamster under apparently the
same experimental conditions as well as after destruction
of the noradrenergic innervation by 6-hydroxydopamine
injections an increase in number of GV. To date there is
no explanation for this discrepancy.
In in vitro experiments, after application of NA to

the medium, a decrease in the number of GV is observed in
mouse pinealocytes (Haldar-Misra and Pevet, 1982a). Thus,
contrary to the observations in the hamster, rabbit and
rat (see above), NA appears in the mouse to inhibit the
process of protein secretion characterized by the forma-
tion of GV. Other findings in the mouse, demonstrating a
direct correlation between a high NA content in the pin-
eal and a low number of GV in the pinealocytes or, on
the contrary, between a low pineal NA concentration and
a large number of GV, support this interpretation. Ben-
son and Krasovich (1977), for example, have observed a
circadian rhythm in the number of GV in ~he ending pro-
cess of the mouse pinealocytes characterized by a high
number during the light phase and a low number during
the dark phase. The low number of GV is thus directly
related to the nocturnal discharge of NA from the
sympathetic nerve terminals. Moreover, Krasovich and
Benson (1979) have also observed an increase in number
of GV after pharmacological blockage of NA synthesis.
All these results indicate clearly that NA has a

clear influence on the process of protein/peptide secre-
tion characterized by the formation of GV. This effect,
however, appears to be species specific (for more de-
tails see Haldar-Misra and Pevet, 1982a) and this could
132 P.PEVET
possibly explain some contradictory observations reported

in the literature when the relationship light/dark perio-
dicity and pineal function is considered.
Concerning the ependymal-like secretory process the

observations are very scarce. Haldar-Misra and Pevet
(1982a) have observed in the cultured pineal of rat that
the addition of NA to the medium led to the formation of
an extended complex of cisterns of the RER and of vacuoles
containing flocculent material originating from the cis-
terns. This result, however, needs a more detailed anal-
ysis.
From the above data it can be concluded that NA

strongly influences the processes of protein/peptide se-
cretion in the pinealocytes. This, therefore, emphasizes
again the importance of the sympathetic innervation for
the activity of the pineal (see review in Ariens Kappers,
1981). However, as NA induces the formation of melatonin
which, in turn, can also act on the process of protein
secretion (see below), it should be realized that we are
facing a very complex system of regulation.
(2) The infZuence of hormones of the sexuaZ axis on

the process of protein/peptide secretion
The proteic/peptidic agents synthesized via the pro-

cesses studied at present are believed to act on the
resproductive axis (see Introduction). Consequently, as
the function of the pineal, like that of every endocrine
organ, depends o~ feedback systems (see details in Ariens
Kappers, 1978), an effect of the hormones of the sexual
axis is very probable. Many studies in the literature
deal with effects of sexual and hypophysial hormones on
the pineal (see review in Vollrath, 1981) but surprising-
ly few concern specifically an effect on the processes of
protein/peptide secretion.
After castration Clementi et al. (1965) and Karasek

et al. (1976a) observed an increase in number of GV in
the rat pinealocytes. According to the last-mentioned
authors, the increase was due to the plasmatic elevation
of gonadotropins induced by gonadectomy. This interpreta-
tion was later demonstrated to be true. Administration
of gonadotropins to rats provoked indeed similar changes
either in vivo (Karasek and Marek, 1978) or in vitro
(Karasek et al., 1978). A similar gonadotrophin-induced
increase in number of GV in the pineal of snakes and
lizards has also been demonstrated by Petit (1976) and
Vivien (1965). Karasek et al. (1982b) have also observed
a very large increase in number of GV (100%) in the pin-

ealocytes of the female rat at days 21 and 22 of pregnan-
cy. They related this increase to the hormonal changes
which occur during the late stage of gestation. All these
results led Karasek to suggest the existence of a feed-
back mechanism between the pineal gland and the hypoph-
ysis: "This mechanism may consist in enhancement of the
synthetic activity of the pineal gland by gonadotropins
causing a subsequent increased elaboration of pineal anti-
gonadotropic factors" (Karasek, 1979). Yet, this concept
is supported by new findings by these authors. Indeed,
Karasek et al. (1982a) observed that when compared to
phenotypically normal mice, the pinealocytes of the snell
dwarf mouse (a mutant with hereditary hypopituitarism)
contain a very low number of GV but that this number was
increased after a pituitary graft.
Considering other hormones of the gonadal axis and

especially those like LHRH, oestrogen, progesterone and
testosterone which are known to be taken up by the pin-
eal (see details and references in Vollrath, 1981), rel-
evant studies are even more rare. It appears, however,
that LHRH does not act directly on this process of pro-
tein secretion. Indeed, in in vitro experiments Karasek
et al. (1978) were not able to observe any effect of this
peptide. After administration of oestradiol, morphologi-
cal features of increased pinealocyte activity have been
also observed (Karasek and Marek, 1980).
The activity of the ependymal-like secretory process

seems also to be especially influenced by the endocrine
activity of the gonadal axis. In the mole a parallelism
between this synthetic activity of the pinealocytes and
the hormonal production of the gonadal axis has been es-
tablished (Pevet and Smith, 1975). Formation of accumu-
lations of proteinaceous material in the pinealocytes of
the garden dormouse has been observed during the natural
period of anoestrus (winter, during hibernation) and also
during experimentally induced anoestrus (Roux et al.,
1977, 1981). In the hedgehog, vacuoles containing floc-
culent material have been described in the cisterns of
the RER, both at the beginning of the period of sexual
quiescence (Pevet, 1976; Pevet and Saboureau, 1973) and
at the beginning of the period of an experimentally pro-
voked decrease of sexual activity (Pevet and Saboureau,
1974). After castration (Karasek et al., 1976a,b) or after
administration of gonadotropic hormones (Karasek and
Marek, 1978) an increase in the number of vacuoles con-
taining flocculent material is observed in the rat pin-
ealocytes. Very interesting, in the last days of preg-
134 P.PEVET
nancy when, as noted before, a strong increase in the

number of GV was observed, a decrease in the number of
these vacuoles was described (Karasek et al., 1982b). Ac-
cording to Karasek these results could be explained, as
demonstrated by a study of the effect of prolactin on
rat pinealocytes (Karasek et al., personal communication),
by the fact that the ependymal-like secretory process is
implicated in the regulation of the prolactin secretion
and that characterized by the formation of GV in the re-
gulation of gonadotropins.
(3) Relationships between indoleamines and the pro-

cesses of protein/peptide secretion
The pinealocyte in mammals or the rudimentary photo-

receptor cell - its phylogenetic homologue - in birds and
reptiles is engaged in both the metabolism of indoleamines
and the production of proteinaceous substances (see details
in Collin, 1979; Collin and Oksche, 1981; Pevet, 1981b,c,
1982b), a situation which permitted Ueck and Wake (1977,
1979) to assert that these cells belong to the paraneurons
according to the definition of Fujita (1979). Consequent-
ly, as in other paraneurons, a physiological relationship
between indoleamines and proteins has to be taken into
consideration. The intracellular relationship of indole-
amines with protein synthesis in pineal cells has been
essentially studied in non-mammalian species, the results
concerning mammals being yet fragmentary. However, these
studies have clearly shown that in the hamster, lizard,
mouse and parakeet, serotonin coexists with a proteinace-
ous compound in the GV, an observation which recalls the
APUD concept of Pearse (1969) and which would indicate
that serotonin could change membrane permeability and
thus allow or control the release of peptidergic hormones.
All these results have been reviewed recently in detail
by Collin (1979), Collin and Oksche (1981) and Pevet
(1981b,c, 1982b), and we refer to these articles for more
details.
In the pineal gland, however, the problem of the re-

lationship of indoleamine with protein presents another
aspect which, in our opinion, is also of great physiolog-
ical importance. It is known indeed that the pineal gland
synthesizes and releases indoleamines but also concen-
trates circulating indoleamines, at least serotonin and
melatonin (Wurtman et al., 1964). Consequently, the in-
doleamines released from the pineal and probably also
from the retina, Harderian gland, intestin, etc. (details
and references in Pevet et al., 1980a) may reach (intact?
modified?) the pineal via the general circulation and then

act on the pineal secretory activities. Unfortunately,
only very few data related to this problem have been re-
ported in the literature. Injections of serotonin during
the period of sexual activity in the hedgehog provokes,
in addition to an inhibition of the sexual activity,
the formation in the pinealocytes of vacuoles containing
flocculent material (Pevet and Saboureau, 1974) character-
istic of the so-called ependymal-like secretory process
(Pevet, 1976). Melatonin, chronically administered via
implantation of melatonin-containing beeswax pellets
causes in hamster pinealocytes an increase in the number
of GV. A single injection of 25 ~g of melatonin induces
a decrease in number of GV in the ending of the mouse
pinealocyte processes. In contrast, melatonin administered
in daily intraperitoneal doses of 50 ~g over a period of
5 days increases markedly the number of GV at least dur-
ing the light period of a light/dark cycle (Benson and
Krasovich, 1977). Pinealocytes of melatonin-treated rats
are characterized by a prominent Golgi apparatus and
especially by an increased number of micro tubules (Freire
and Cardinali, 1975), structures which are known to be
implicated in secretory processes in a variety of cells.
In in vitro experiments melatonin as well as other

5-methoxyindoles such as 5-methoxytryptophan, 5-methoxy-
tryptamine, 5-methoxytryptophol, induce in mouse pinealo-
cytes, depending of the time of the treatment and of the
presence or absence of NA, an increase or a decrease in
the number of GV (Haldar-Misra en Pevet, 1982b,c). 5-Meth-
oxyindole-3-acetic acid, however, provokes always inde-
pendent of the experimental conditions, an increase in
GV number (Haldar-Misra et al., 1982c).
These ultrastructural results demonstrate clearly

that indoleamines, especially 5-methoxyindoles, partici-
pate in the regulation of the activity of the processes
of protein/peptide secretion. These results are probably
of great importance when the physiology of the pineal is
concerned. However, this does not mean that all indole-
amines are working at the same time. We know for example
that in hamsters kept under laboratory conditions, 5-meth-
oxytryptophol is synthesized by the pineal during the
light phase (Pevet et al., 1980a) while melatonin in
hamsters as well as in other species is produced during
the dark period (Klein et al., 1981). Moreover, we also
know that in hamsters kept under natural environmental
conditions the synthesis of 5-methoxytryptophan by the
pineal, retina and Harderian gland is high in winter and
very low in summer, an opposite evolution being observed
136 P.PEVET
for melatonin and 5-methoxytryptamine, at least in the

pineal (see details in Balemans et al., 1982).
IV. CONCLUSION
From the experiments described in this review it is

now clear that a lot of proteic or peptidic sUbstances
isolated from various pineal extracts and known to be very
active in some bioassays, cannot be considered as pineal
factors. They are indeed not synthesized by the pineal it-
self (peptides and proteins of classes I and II).
The problem of structural identification of one or

more true pineal proteic or peptidic factors (hormones?)
remains as yet unsolved. However, the ultrastructural
identification of processes of protein/peptide secretion
in specific pineal cells and the observation that the
activity of these processes is regulated by the sympathet-
ic innervation as well as by the 5-methoxyindo1es, demon-
strate clearly the reality of the existence of such pro-
teic/peptidic pineal factors, a reality which should not
be doubted anymore.
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Acknowledgement: The author wishes to thank Miss J.Sels

for her skillful secretarial aid.
PTERIDINES IN THE PINEAL AND EFFECTS OF THESE SUBSTANCES ON THE
INDOLE METABOLISM OF THIS ORGAN*
I. Ebels, M.G.M. Balemans**, J. van Benthem**, H.P.J.M.

Noteborn, and A. de Moree
Department of Organic Chemistry and **Zoological

Laboratory, State University of Utrecht
The Netherlands
pteridines are very light sensitive substances. The first com-

pounds of this class were isolated as a yellow pigment from the wings
of the brimstone butterfly by Wieland and Schopf (1925) and as the
white pigment from the wings of the cabbage butterfly by Schopf and
Wieland (1926). These pigments were named xanthopterin and leuco-
pterin indicating the colour and source of the compounds. The real
chemical structure of these substances was not solved until 1940 by
Purrmann. The structure of a third insect pigment, isoxanthopterin
was also elucidated by Purrmann in 1941. The well-known structure
folic acid, and its derivatives which play a key role in metabolism,
contains also a pteridine ring. For a survey of the most important
literature in the pteridine field till 1969, see Blakly's "Bio-
chemistry of folic acid and related pteridines".
In mammals three aromatic amino acid monooxygenases: phenylala-

nine-, tyrosine-hydroxylase and tryptophan-hydroxylase require a
reduced pterin as cofactor (Nagatsu et al., 1981). It is generally
thought that tetrahydrobiopterin (BH4) is the natural factor for
these mammalian monooxygenases. These three hydroxylases play an
important role in the biosynthesis of biogenic monoamines from
aromatic amino acids, i.e.:
(i) the catecholamines dopamine, norepinephrine, adrenaline, and
(ii) the indole amine serotonin.
In this respect BH4 is also very important for the biosynthesis of
melatonin and other methoxyindoles in the pineal.
*Our research is supported by grants of the Netherlands Organisation

for the Advancement of Pure Research (ZWO) through the Foundation for
Biological Research (BION) no. 14.70.03 and through the Foundation
for Medical Research (FUNGO) no. 13-35-33.
151
1 52 I. EBELS ET AL.
In this paper we will give a survey of:

I. The isolation of pteridines from sheep pineals
II. The influence of synthetic pteridines and that of low molecular
weight pineal substances, present in fractions which show a
pteridine-like fluorescence, on indole-metabolism of pineals
III. Some general remarks
I. THE ISOLATION OF PTERIDlNES FROM SHEEP PINEALS
I.a. The Isolation of 6-L-Erythrobiopterin from an Aqueous Extract

of Sheep Pineals
It was in 1973, that we first observed, that when an aqueous

extract of sheep pineals was separated on Sephadex G-25 and Sephadex
G-IO columns, besides the well-known fluorescence like indoles have,
another high fluorescent peak was present, which could not be detec-
ted in a comparable aqueous extract of sheep cerebral cortex. This
high peak showed an excitation maximum (e) at 360 nm and a fluores-
cence maximum (f) at about 440 nm. After ultrafiltration of the low
molecular weight Sephadex G-25 fraction F2 - F3 of a pineal extract
through different Amicon ultrafiltration membranes, it was found that
the substance with these special fluorescence characteristics was
located in the UM05F-fraction, which contains substances with a
molecular weight (MW) < 500. ~Vhen this UM05F sheep pineal fraction
was further separated on a Sephadex G-10 column (142 x 1 cm), the
substance with the above mentioned fluorescence was eluted between
about 150 and 250 ml from that column. For the extraction and sepa-
ration see Scheme 1 and Ebels (1979).
When these Sephadex G-10 fractions were tested in a bioassay by

Moszkowska et al. (1976) using anterior pituitaries of male rats in
vitro, an inhibition of the gonadotropic activity of these pituitaries
was observed. In paper electrophoresis and paper chromatography exper-
iments, the substance with special fluorescence characteristics, like
pteridines, and the inhibiting activity in the bioassay in vitro, was
always found in the same fraction. After preparative paper chromato-
graphy, elution of the special fluorescent band and lyophilization
of the eluate, trimethylsilylation was carried out. Gas liquid chro-
matography (GLC) gave a peak with the same retention time as a syn-
thetic preparation of 6-biopterin. With GLC-mass-spectrometry (GC-
MS) a spectrum was obtained, identical with that of 6-biopterin.
Thin layer chromatography and the activity in a Chrithidia

fasciculata-test revealed that we had isolated 6-L-erythrobiopterin
(6-L-B), Fig. 1. from sheep pineals. For details see the experiments
of Van der Have-Kirchberg et al. (1977). Since the time we knew that
6-L-B was present in our extracts, we have carried out all our experi-
ment.s under dim red light-conditions: A > 600 nm, because pteridines
are very light sensitive substances. Moreover, some of the oxidation
products, such as pterin-6-aldehyde and pterin-6-carboxylic acid, can
PTERIDINES IN THE PINEAL AND INDOLE METABOLISM 153
SCHEME 1. OUTLINE OF THE METHODS USED IN PART I.a.
aqueous pineal extract
I
Sephadex G-25
(F2 + F3)
I
ultrafiltration through UM2
/
UM2 filtrate
~
UM2 residue
ultrafiltration
through UM05
UM05 filtrate UM05 residue
Sephadex G-10
paper electrophoresis
I
paper chromatography
I ______
trimethylsilylation thin layer chromatography
I
gas liquid chromatography
and mass spectrometry
154 I. EBELS ET AL.
o
N H H
H- N) I: ~ C - C- CH3
H2N
AN .J
N
I
. OH
I
OH
Figure 1. 6-L-erythrobiopterin
A-:Jr
o
N H H
H-N +:-C-C-CHl
I I I
H2N N ~ H H OH OH
7,8 - dihydrobiopterin
H-N :Jr
o H
~ I
N
N
~ H H
H
H
I
H
~ C-C-CH3
I
OH OH
5,6,7,8 tetrahydrobiopterin
Figure 2. Reduced 6-L-erythrobiopterins
also influence metabolic processes, related to gonadal growth. For

details see Ebels (1981) and Ebels, another chapter in this book.
The experiments of Fukushima and Nixon, (1979, 1980) have suppor-

ted our results. These authors observed, using high performance liquid
chromatography, that the pineal of the rat contains more biopterin
than seven other organs tested and also much more than several other
regions of the rat brain per gram wet tissue. By using an oxidation
method, the authors concluded from their results, that biopterin is
present in rat pineals in the reduced form, mostly as BH4, the cofac-
tor for aromatic amino acid monooxygenases (Fig. 2).
Levine et al. (1979) studied the tryptophanhydroxylase-, tyro-

sinehydroxylase- and cofactor-activity, expressed as BH4, in different
regions of the rat brain. These authors detected the highest amount
of cofactor-activity in the pineal.
Kapatos et al. (1981) observed that rat pineal glands in organ

culture synthesize and release biopterin and are able to maintain
concentrations of biopterin, occurring in vivo, for up to 54 hours in
vitro. These authors found also that the intracellular biopterin con-
tent is reduced 50 percent by treatment with L-norepinephrine. This
is, according to these authors, an indication that biopterin levels
are regulated by an adrenergic cyclic adenosine monophosphate-depen-
dent mechanism. The observed decline in tissue biopterin content, pro-
duced mainly by inhibition of the biosynthesis, is not associated with
either an increase in biopterin release or a shift in the reduction
state of biopterin. Kapatos et al. (1981) postulate that the decline
in biopterin might be related to the increase in the activity of N-
acetyl transferase. These authors hope that a further study of the
adrenergic cyclic AMP-induced inhibition of pineal biopterin biosyn-
thesis may lead to the discovery of a new function of biopterin in
the pineal gland and possibly to a new role for this compound in
other tissues.
Ebels (1979, 1981, and another chapter in this book), has des-
cribed, that different pterins show antigonadotropic activity in in
vitro and in vivo experiments. Therefore, we have undertaken experi-
ments with the aim to study whether pteridines are present in dif-
ferent pineal fract~, prepared by a Bensinger-extraction method,
which show antigonadotropic activity.
I.b. Isolation of Neopterin, pterin-6-Carboxylic Acid and Biopterin

from Sheep Pineal Fractions, Prepared with a Bensinger-Extrac-
tion Method
Bensinger et al. (1973) published in abstract-form a method for

aqueous acetone extraction of bovine pineals and the further removal
of fats and indoleamines by chloroform/methanol extraction. A compen-
satory ovarian hypertrophy (COH)-inhibiting substance was subsequently
extracted into isobutanol and further purified by gelfiltration on
Sephadex G-15 and thin layer chromatography. Although the details of
the method and all the results were not published in detail, the
authors stated that the active principle was probably a small, non-
tryptophan containing peptide.
Blask et al. (1976) observed, using the same method, that two
partially purified bovine pineal fractions, apparently free of indole-
amines, inhibited prolactin release from anterior pituitaries in vitro.
Ebels et al. (1978, 1979) compared an acetic acid extraction

method and the Bensinger-extraction method. From these experiments was
156 I. EBELS ET AL.
concluded that although both extracts were active in inhibiting

COH, after unilateral ovariectomy, different Rf-values for the active
paper chromatographic regions were obtained. Moreover, these authors
could detect more COH-inhibiting activity when the Bensinger extrac-
tion method was used than with aqueous and acetic acid extraction
methods, used earlier by the same group. Therefore, we started experi-
ments to study the presence of pteridines·which can have antigonado-
tropic activity, in pineal fractions prepared by the Bensinger extrac-
tion method.
The different fractions were first prepared according to Scheme

2. The isobutanol extract of sheep pineals was separated on a Sephadex
G-ls COlillilll, and a sheep cerebral cortex isobutanol extract was sepa-
rated in the same way. Absorbance-measurements of the Sephadex G-ls
fractions at 280 nm are given in Fig. 3.
- - pineaL extract
1.0 - - - - cortex extract
0.8
0.6
E
c
C)
CD
N
"
:.
I I
....>- 0.1,
~
: I
:\
,",
c'"
/i\! \~ \ Vli \
"" I ,
CII ' I ' I •
""0
o 0.2
....a.u
D
: . \}J. ~
---~: '----"'-" ' .... _--
tube 50 ~OO 150 200
fraction FO F1 F2 3 FI, F5 F6 F7 F8 F9 F10
ml 126 212 257 306 1,32 522 617 729 833 923
365
Figure 3. Comparison of the 280 nm absorbance-elution-profiles of the

isobutanol extracts of sheep pineals and sheep cerebral
cortex after gelfiltration on Sephadex G-ls columns (85 x
2.5 em), equilibrated and eluted with 0.2 N acetic acid.
SCHEME 2. OUTLINE OF THE HETHODS USED IN PART I. b.
sheep pineal glands

extraction with
acetone/water (3:1 v/v)
acetone extract residue

after evaporation
of the acetone,
extraction with
chloroform
methanol 2:1 v/v
chloroform methanol water layer

extract
after evaporation of
chloroform/methanol
extraction with isobutanol
water layer isobutanol extract
ultrafiltration gelfiltration over

through PM10R Sephadex G-1S
Sephadex G-1S fractions
FO -4 FlO
PM10 residue (PM10R) PM10 filtrate
ultrafiltration
through UM2
UM2 residue (UM2R) UM2 filtrate
ultrafiltration
through UMOS
UMOS residue UMOS filtrate

(UMOSR) (UMOSF)
158 I. EBELS ET AL.
In fig. 4a and 4b the fluorescence measurements are presented.

In both extracts several peaks with excitation and fluorescence maxi-
ma like indoles, pteridines and flavines could be detected. The UV-
absorbance results are comparable to those obtained by Ebels et al.
(1979) for bovine pineal extracts. It can be concluded that the
curves of Fig. 4a and 4b give more detailed information about the
different compounds, present in the extrac"ts than the curves in Fig.
3. On this Sephadex G-15 column dextran blue 2000 was eluted from 115~
225 ml and Cl- from 325 ~ 420 mi.
In the pineal isobutanol extract Sephadex G-15 fraction F5,

eluted between 384 and 465 ml a high peak is present with a pteri-
dine-like fluorescence. This fraction is further studied with paper
chromatography and after elution of the bright blue fluorescent band
and lyophilization, this fraction was trimethylsilylated and studied
with GLC and GC-MS as described before in part I.a for biopterin.
Table 1 shows some Rf-values of the isolated pineal compound and

some synthetic pteridines.
Table 2 gives the retention time of the isolated compound and

some pteridines after silylation.
From Table 1 and 2 can be concluded that the Rf-value in paper

chromatography and the retention time in GLC-experiments is corres-
ponding to those of synthetic 6-D-neopterin.
Fig. 5 gives the mass spectrum of the isolated pteridine-like

compound of the Sephadex G-15, F5-fraction, which is identical to the
spectrum of 6-neopterin (Fig. 6) and in agreement with the spectrum
published by Lloyd et al. (1971).
The UM05R-fraction of the water layer, which is obtained after

the isobutanol extraction (see Scheme 2) is also studied, while Ebels
et al. (1979) detected in a comparable bovine pineal fraction COH-
inhibiting activity. This fraction was studied with the same tech-
niques as described before. Table 3 gives the Rf-values of a pineal
UM05 residue (UM05R) and some synthetic pteridines.
Table 4 shows the retention time in GLC-experiments after

trimethylsilylation of the paper chromatography fraction Rf = 24
(Table 3, UM05R).
From the GLC experiments can be concluded that in the UM05R a

substance is present with about the same retention time as 6-neo-
pterin, while the Rf-value of neopterin is more than twice that of
the bright yellow band (Rf = 24) in paper chromatography. The mass
spectrum which was obtained with GC-MS was the same as given in
Fig. 5.
Pineal extract
450
1100
400 e 1 e 1
280/350 1 295/350
350
• 1 • 1
300 350/450 285/350
~
250
c
~ , pH •
.6 200 • 1 :- 2,5456 9 10 " 11 11 n~
ec e 1 450/530
301380
• 1
~ 150 • 1460/530 e 1
~ 55/405 370/530
~
100
• 1
~
a 50
2951340
~
~
O+---~~~~~~~~~~~~,--r-,,-~--~~~~~-p--~~~~~~
lube
fraction
ml
FO
117 599 735
210
F9+-FlO
883 953
240
+
1083
F11
340
1575 1647 17R2

430
F14
490
-------j
2205
Figure 4.a. Separation of a sheep pineal isobutanol-extract by gel-

filtration on Sephadex G-15 (85 x 2.5 em), equilibated
and eluted with 0.2 N acetic acid.
e: excitation; f: fluorescence
Cortex -extract
150
::- 125
.
';;;
:£
c:
100
e f
2851360
~
c:
e
..
oil
(;
75 e
460/530
f
:~,:tft~
~ 50
'"
.?:
;:; 25
~
0 i
--------11
tube 30 60 270 370
fraction FO---f1 F2 - 1 - - - - - - - F7
ml 261 297 391,5 589,5 724,5 868,5 972 1665
Figure 4.b. Separation of a sheep cerebral cortex isobutanol-extract

by gelfiltration on Sephadex G-15 (85 x 2.5 em), equi-
librated and eluted with 0.2 N acetic acid.
e: excitation; f: fluorescence
160 I. EBELS ET AL.
Table 1.
Rf-values (xI02) of the main spot with a pteridine-like
fluorescence of the sheep pineal Sephadex G-15, F5 fraction
(Fig 4a) and of some synthetic pteridines, in two solvent-
systems.
Solvents
Compound or fraction B-A-W E-W
pterin-6-carboxylic 12 72
acid
6-D-neopterin 13 60
6-L-erythrobiopterin 28 65
Sephadex G-15, F5.

(eluted between 384 15 64
and 465 ml)
B-A-W n-butanol-acetic acid-water, 4:1:1
E-W ethanol-water, 1:3
Table 2.
Retention time in minutes in gas liquid chromatography of the main

fluorescent compound of a sheep pineal, Sephadex G-15, F5 and some
synthetic pteridines on a Dexsil-column (2.10 m x 2 mm).
Compound Retention time (min)
6-D-erythroneopterin 20.98
6-L-er~throbiopterin 16.44
7-L-erythrobiopterin 14.34
pterin-6-carboxylic acid 18.76
Sephadex G-15, F5-fraction
eluted from 384 ~ 465 ml 7.40, 16.72, 21.08
(Fig. 4a)
paper chromatography band 20.52
(Rf, 64) see Table I.
73 0/0
1000
20.0
?:
'iii
c 147 205 337
<I>
:£ 0 0
100 150 200 250 300 350
.,.
50 400 mle
1000
409 x5.0 20.0
;- 613
?: 598
'iii 481
c
<I>
:£ 0 0
450 500 550 600 650 700 750 800 mle
Figure 5. Mass spectrum of 6-neopterin, isolated from a sheep pineal

Sephadex G-15, F5-fraction (see Fig. 4.a), after trimethyl-
silylation.
o
N H
H-N : ]I( ~CHOH-CHOH-C~
)-::,. ..J OH
H2N N N
Figure 6. 6-neopterin
162 I. EBELS ET AL.
Table 3.
2
Rf-values (xlO ) of compounds, present in a sheep pineal UM05-residue
(UM05R) and of some synthetic pteridines.
Compound or fraction Rf-value (xI02) ~n

ethanol-water I: 3
isoxanthopterin 31
pterin-6-aldehyde 34
xanthopterin 52
6-D-erythroneopterin 59
6-L-erythrobiopterin 65
pterin-6-carboxylic acid 72
folic acid 78
riboflavin 22
UM05R 23, 45, 65, 73, 81, 86
UM05R* 44, 74, 82
UM05R** 24
* This UM05R fraction was irradiated with normal laboratory light during
about 3 hours.
** The bright yellow fluorescent band (rf, 23) was eluted, lyophilized
and re-chromatographed.
Table 4.
Retention time in minutes in gas liquid chromatography of an UM05R, paper

chromatographic band (Rf, 24, see Table 3) and of some synthetic pteri-
dines, on a Dexsil-column (2.00 m x 2 mm).
Compound Retention time (min)
leucopterin 11.32
pterin 5.60
6-D-erythroneopterin 31.44
pterin-6-carboxylic acid 26.62
UM05R-paper chromatographic fraction 3.28, 31.68

(Rf, 24)
Table 5.
The relative intensities (RI) of the excitation (e) and fluorescence (f)
maxima of the isobutanol- and water-layers of different ultrafiltration-
fractions corresponding to 5 grams of sheep pineals
Fraction isobutanol-layer water-layer

elf in nm R.I. elf in nm R.I.
UM2R MW > 1000 300/350 43 290/345 200

and < 10.000
350/415 22 350/430 64
350/450 12
370/520 37 380/520 60
450/520 58 450/520 92
UM05R MW > DOO 300/350 17 290/345 16

and < 1000
340/390 12
UM05F MW < 500 300/350 470 300/355 400

355/445 185 355/455 360
450/525 88 450/525 67
164 I. EBELS ET AL.
From these experiments can be concluded, that neopterin can be

isolated from UM05R and from the Sephadex G-15, F5-fraction, two
fractions prepared with a Bensinger-extraction method. In UM05R,
neopterin is probably bound. From the experiments we carried out till
now, we have the impression that we can detect neopterin in the
isobutanol-extract, when higher molecular weight material is extrac-
ted into the isobutanol (see Fig. 4a, F1). When that peak is absent,
then we could isolate neopterin from the water layer. Therefore, we
have the impression that neopterin can be bound to higher molecular
weight material.
Another pteridine could be isolated from a Sephadex G-l5 fraction,

obtained when the column was eluted with NH 4 0H after the acetic acid
elution. This compound has an Rf-value of about 60 in the solvent
ethanol:water 1:3. Fig. 7 gives the mass spectrum, which was obtained
after trimethylsilylation. This spectrum is identical to that of syn-
thetic pterin-6-carboxylic acid (Fig. 8) and in good agreement with
the spectrum published by Lloyd et al. (1971). From that pineal frac-
tion was obtained another mass spectrum after trimethylsilylation
which can be explained by a structure like "biopterin-glucopyranose-
phosphate". All the main peaks of a biopterin mass spectrum were found
and those of a-D-glycopyranose-6-phosphate (see Zinbo and Sherman,
1970) were observed. This is again an indication of a pterin which
can most probably be bound to other compounds.
As it was observed, using the extraction- and separation-methods,

presented in Scheme 2, that a fluorescence, like pteridines have, could
be detected in the isobutanol- and the water layer, we have changed
our last separation-steps in order to remove first the substances with
MW > 500, present in the water layer, after the chloroform/methanol
extraction, by ultrafiltration and to carry out subsequently the iso-
butanol-extraction on the UM05F-fraction (MW < 500), with the aim to
separate "free" and "bounded" pteridines.
The different pineal fractions were now prepared according to

Scheme 3. The isobutanol extracts and water layers were compared and
their excitation (e) and fluorescence (f) maxima were determined. The
results are summarized in Table 5.
In the UM2R water layer the compounds with an indole-like fluor-

escence were located, while in UM05F these fluorescence characteris-
tics are present as well in the water layer as in the isobutanol frac-
tion. In UM05R only little fluorescence is found in both layers.
In the UM05F-fraction compounds with an indole-, a pteridine-

and a flavine-like fluorescence could be located in the water- and
in the isobutanol-Iayer (see also Ebels, 1981).
The elution profiles after gel filtration of the UM05F-water layer

and the UM05F-isobutanol layer we published before (Ebels, 1981). In
1000 73 .,.
I 100
o~~~~~~--~~~~~~--~.-~--~o
50 100 150 200 mle
1000 408 423
306
O~~--~--~~--~~~--~~---.-=~~-LO
250 300 350 400 mle
Figure 7. Mass spectrum of pterin-6-carboxylic acid, isolated from

an UM05 Sephadex G-15 fraction, (see Scheme 2), after tri-
methylsilylation.
Figure 8. Pterin-6-carboxylic acid.

166 I. EBELS ET AL.
SCHEME 3. OUTLINE OF THE METHODS USED IN PART 1. b.
sheep pineal glands
iextraction with acetone/water 3:1
)
residue acetone extract
after evaporation of the acetone,
extraction with chloroform/methanol
water layer chloroform/methanol extract

after evaporation of the
chloroform/methanol,
ultrafiltration through
PM10
PM10 residue (PM10R) PMI0 filtrate
extraction with
isobutanol
ultrafiltration
throught UM2
PM10R PM10R
water layer isobutanol layer
UM2 residue (UM2R) UM2 filtrate
extraction with
isobutanol
1
see next page
UM2R UM2R
water layer isobutanol layer
SCHEME 3. OUTLINE OF THE METHODS USED IN PART I.b. (continued)
UM2 filtrate
ultrafiltration through
UM05
UM05 residue UM05 filtrate

(UM05R) (UM05F)
extraction with extraction with
isobutanol isobutanol
UM05R UMOSR
\later layer isobutanol layer
UMOSF water layer UMOSF isobutanol layer
gelfiltration over gelfitration over

Sephadex G-1S sephadex G-1S
Sephadex G-1S Sephadex G-1S

fractions WFO ~ WFll fractions FO ~ F14
HPLC; HPLC;
GLC; GLC;
GC-MS GC-MS
168 I. EBELS ET AL.
the water layer the different fluorescence peaks were about twice as
high as in the isobutanol layer. In the isobutanol layer however,
one very high peak with a pteridine-like fluorescence could be detec-
ted, eluted between 347 and 408 ml (F4), and for the water layer a
comparable fluorescent peak was eluted between 303 and 358 ml (WF2).
In the Sephadex G-15 fraction F2 (eluted from 202 ~ 230 ml) of the
isobutanol layer a fluorescence could be observed with elf = 350 nml
430 nm. In that Sephadex G-15 fraction applied to a high performance
liquid chromatography column (HPLC) a peak was observed with a reten-
tion time identical to that of pterin-6-carboxylic acid; a Partisil
PXS 10/25 SCX; col. no lE (Whatman Inc.) was used.
The main peaks obtained in HPLC-experiments are summarized in

Table 6. The isobutanol-Sephadex G-15 fraction F4 was further studied
with GLC and GC-MS after trimethylsilylation, and a spectrum identical
with that of 6-biopterin was obtained.
Although the retention time of the highest peaks in HPLC-experi-

ments were comparable to those of respectively pterin-6-carboxylic
acid and 6-biopterin, only from the isobutanol Sephadex G-15 fraction
F4, a mass spectrum, identical to that of synthetic biopterin was
obtained, besides the mass spectra of three other compounds with
molecular weight peaks M/e of 309; 282; 541. From the water layer
Sephadex G-15 WF2-fraction no mass spectrum of biopterin was obtained,
but from that fraction the mass spectra of four other compounds were
determined with M/e = 304; 367; 533; 628. All these mass spectra
could not be identified till now.
With these results it is again clearly demonstrated, that HPLC

is d very nice analytical method, but that the final step for struc-
ture elucidation of compounds must always be a GC-MS-experiment! From
all these experiments can be concluded that in different ultrafiltra-
tion fractions of the water layer and in several Sephadex G-15 frac-
tions of sheep pineals, prepared according to a Bensinger-extraction
method, neopterin, pterin-6-carboxylic acid and biopterin could be
isolated and identified. Moreover, in the fractions with the highest
pteridine fluorescence several other compounds could be detected, but
the structure of those is till now unknown. Thus several fractions
are still mixtures of compounds! As comparable bovine pineal fractions
showed COH-inhibitory activity and pteridines can have an antigonado-
tropic activity in in vitro and in vivo experiments (Ebels, 1979,
1981, and another chapter of this book) it will be worthwhile to
study, whether the pteridines detected in the above described pineal
fractions can be responsible for the observed antigonadotropic
effects.
II. THE INFLUENCE OF SOME SYNTHETIC PTERIDlNES AND LOW MOLECULAR

WEIGHT PINEAL FRACTIONS ON INDOLE METABOLISM OF PINEALS
Cremer-Bartels and Hollwich (1978) were the first authors who

~
m
::0
o
Z
m
CJl
Z
Table 6. --i
:::c
m
HPLC-retention times of the main peaks with a pteridine-like fluorescence, in some Sepha- ::!!
z
dex G-15 sub-fractions of UM05F (isobutanol- and water-layer) m
»
r
»
z
Isobutanol-layer water-layer
o
co-chromato- retention co-chromato-
Z
fraction retention fraction o
time in min. graphically iden- time in min. graphically iden- o
r
tical with tical with m
~
m
--i
»
OJ
or
F2 1.3 pterin-6-car- WF4 1.5 pterin-6-car-
boxylic acid boxylic acid Vi
~
F4 6.S 6-biopterin 6.4 6-biopterin
Ol
CD
170 I. EBELS ET AL.
indicated an influence of pteridines (triaminophenylpteridine or

triamterene) on hydroxyindole-O-methyltransferase (HIOMT), the enzyme
responsible for all O-methylating processes in indole metabolism in
the pineal and the retina (Balemans, 1979). A decrease of HIOMT-acti-
vity was observed when low concentrations of the substrate N-acetyl-
serotonin were tested. These authors postulate that naturally occuring
pteridines might be involved in the regulation of HIOMT in dependence
of light.
Cremer-Bartels (1979) showed the effect of two synthetic pteri-

dines: triamterene and xanthopterin on multiple forms of HIOMT in
the retina and the pineal gland. These authors, using flat-bed-gel-
electrofocusing technique, found several HIm·iT enzymes, which differ
in the pH of the isoelectric point. The pattern of the concentrations
of the enzymes differed in dependence on light adaptation. The block-
ing effects of synthetic pteridines varied depending on light adap-
tation and species. Moreover, it was observed that retina extracts
can inhibit the activity of purified pineal HIOMT.
Cremer-Bartels and Ebels (1980) have shown that pterin-6-aldehyde,

one of the photolytic products of folic acid, can decrease the acti-
vities of bovine pineal HIOMT, purified by flat-bed-gelelectrofocus-
ing, with isoesectric points at pH 5.0, 5.2 and 5.7. These authors
suggest that pteridines may be involved in non-retinal effects of
light on pineal melatonin biosynthesis, besides the retinal pathway.
Ebels and Cremer-Bartels (1982) observed that low molecular weight

substances in partially purified pineal fractions, which show a pter-
idine-like fluorescence, can also influence the activity of HIOMT,
isolated from avian pineals and retina.
The results described demonstrated that this mechanism of inhi-

biting HIOMT activity seems to be independent of the species and the
organ. A blocking effect was found in this in vitro system, whether
retinal or pineal HIOMT-preparations were used.
In other experiments, with dialyzed and non-dialyzed bovine

pineal extracts, was shown that low molecular weight substances, pro-
bably accompanying the high molecular-weight HIOMT-enzyme preparation,
can suppress the activity of the enzyme. It is however, not known
at this moment if these "suppressor" molecules are pteridines. How-
ever, it must be considered that in the experiments, described above,
only the activity of HIOMT at one point in a 24 h period was tested.
In the experiments described in the following part, the influence
of pteridines on the capacity of methylation in pineals is determined
every 2 or every 4 hours during a 24 h period.
Balemans (1979, 1981); Balemans et al. (1980) studied the influ-

ence of three synthetic pterins (2-amino, 4-oxo-pteridines) on the
diurnal fluctuations of HIOMT activity in the isolated pineal gland

of 42-day old male Wi star rats, during the month of October. The
method used to determine the HIOMT-activity of the pineal (Balemans
et al., 1978) permits the separate determination of the synthesis of
5-methoxyindoles. The results of these experiments showed that reduced
neopterin stimulated the methylation of the substance 5-hydroxytrypto-
ph an (5-HTP) 5-hydroxytryptamin (5-HT) and 5-hydroxyindole-3-acetic
acid (5-HIAA), during the night, HIOMT-action on the combination
N-acetylserotonin with 5-hydroxytryptophol (N-Ac-5-HT/5-HTL) was
shifted to a later moment in the dark period.
Pterin-6-aldehyde stimulated HIOMT-action on 5-HT during the

day-time, HIOMT action on the substrates 5-HTP, 5-HIAA and N-Ac-5-
HT/5-HTL was shifted towards an earlier period.
, Isoxanthopterin did not exert any influence on diurnal variation

in the HIOMT-activities studied in this period of the year. From
these experiments was concluded that reduced neopterin and pterin-6-
aldehyde can influence the activity and the circadian rhythmicity of
5-methoxyindole synthesis. In later experiments has been shown that
the above described, circadian rhythmicity of HIOMT activity could
only be influenced by pteridines, when the maximal HIOMT-activity
was measured at 24.00 o'clock (in June and in the period from half
September till the beginning of October). There was no or only a
small influence detectable, when the maximal HIOMT-activity was deter-
mined at 04.00 o'clock (in April and in the last half of October) .
These alterations in sensitivity of HIOMT-activity, also striking in
the month of December and January (Ebels et al., in preparation, see
Fig. 9a and 9b) may be of physiological importance. For details see
also Ebels (1981).
Recently, Balemans et al. (in press) studied changes in the cir-

cadian rhythmicity of HIOMT activity in the synthesis of 5-methoxy-
indoles in the pineal gland of 28 day old male Wistar rats, exposed
to white, red and green light. The results of these experiments showed
that red light, like pterin-6-aldehyde shifts the peak of activity of
HIOMT to the light period, whereas green light, like reduced neop-
terin, shifts that peak of activity to the dark period. In juvenile
rats (Miline, 1949) and in birds (Benoit, 1964, 1972) red and green
light show opposite effects on gonadal growth, red light showing a
stimulatory and green light an inhibitory effect. From these experi-
ments (Balemans et al., in press) it appears that red and green light
show also opposite effects on HIOMT circadian activity. Therefore,
Balemans et al. concluded that it seems likely that a relationship
exists between light of different wavelenghts, pterins, indolemetabo-
lism and gonadal development.
The above described experiments show two different approaches

to study the influence of pteridines on the activity of HIOMT. In
the first part only one point of the 24 h period and in the second
172 I. EBELS ET AL.
dpm/pineal
4000
I
December
3000
2000
. I
"
/\
./
" J
...... "{
.'
./
" /
1000
in hours
08.00 12.00 16.00 20.00 24.00 04.00
Figure 9a: The day/night rhythm of the HIOMT-activity in synthesizing
melatonin/S-methoxytryptophol (---) 1 and the influence of
pterin-6-aldehyde ( .... ) and reduced neopterin (---) on this
enzyme activity, in pineals of 2B-day old male Wistar rats
in vitro, in December.
dpm/pineal
4000 January
3000
j
/t\. · · I
. \
\
.....
".
'. ;'
:',
:i \
\......,
\
".
---",:. '1"'''~' .'. '. I/','/

"./"
2000
,
. . ,,' ' ..-{
1000
time in hours
OB.OO 12.00 16.00 20.00 24.00 04.00
Figure 9b: gives the curves of comparable experiments carried out in
January.
part the influence of pteridines on the circadian rhythmicity of the

enzyme activity was studied. In the last part i t is observed that
pterin-6-aldelhye can shift the peaks of activity, this means that
in that model it cannot be said if j_t is stimulatory or inhibitory.
Therefore, by testing only one point the then observed inhibition
can only be concluded for the time examined.
From the above described results has been concluded that pteri-
dines can influence the HIOMT activity of the pineal. At the other
side the biosynthesis of pteridines can be influenced by indoles.
Katoh et al. (1982) described that rat brain sepiapterin reduc-

tase, which is involved in the biosynthesis of tetrahydrobiopterin,
was directly inhibited by a catecholamine, L-norepinephrine and by
the indoleamines; serotonin, N-acetylserotonin and melatonin. These
authors postulate that there might be a regulatory system in the for-
mation of norepinephrine or serotonin in the brain by the amount of
melatonin.
Much more work has to be carried out before we will really under-
stand how the biosynthesis of pteridines and that of indoles are
related.
SOME GENERAL REHARKS
The following diseases show a disorder of pteridine metabolism:

Schaub et al. (1978) described a patient with atypical phenylketo-
nuria, due to defective dihydrobiopterin biosynthesis. The same group
have published several cases; see also Ebels (1981). Nagatsu et al.
(1981) found that in the striatum of Parkinsonian human brain both
biopterin concentrations and tyrosine hydroxylase activity was great-
ly reduced compared to age-matched control brains. Moreover, the same
group observed that the dopamine beta-hydroxylase in the cerebro-
spinal fluid of Parkinsonian patients was greatly decreased as com~
pared with that of control patients (Nagatsu et al., 1982). Ziegler
and Kokolis (1979) found very high levels of BH4 in all tumour
patients; see also Ebels (1981). Recently Ziegler et al. (1982a,b)
published the presence of a glycoprotein in tumour-bearing patients',
which binds pterins. The authors discuss the possibility that this
glycoprotein may cause the disorder in pteridine-metabolism in these
patients. Whether this effect can be influenced by pineal substances
remains to be studied. From the work of Lapin (1974, 1976, 1978,
1979) we know that the pineal can influence neoplastic growth, see
also Lapin and Ebels (1981); Lapin and Frowein (1981).
Kapatos and Kaufman (1981) observed that peripherally adminis-

tered reduced pterins do enter the brain. Tnese authors concluded
from their experiments, that the content of tetrahydrobiopterin (BH4)
in rat brain was doubled by peripherally administered BH4, the natu-
ral L-stereoisomer being more effective than the unnatural D-configu-
174 I. EBELS ET AL.
ration. The model pteridine, 6-methyltetrahydropterin was ten times

more efficient than BH4, crossing the bloodbrain barrier. These auth-
ors suggest that administration of tetrahydropterins might provide
a pharmacological technique for increasing the biosynthesis of cate-
cholamines and serotonin in the brain and periphery, and could per-
haps be useful in the treatment of disorders resulting from deficits
in biogenic amines biosynthesis.
Levine et al. (1981) concluded from their experiments, that the

hydroxylase cofactor BH4, and its biosynthetic system are localized
in dopaminergic nerve terminals in the striatum. This conclusion was
based on the nearly equivalent loss of tyrosinehydroxylase and BH4
and its initial biosynthetic enzyme, guanosine triphosphate cyclo-
hydrolase, after injection of 6-hydroxydopamine into the substantia
nigra. These authors found moreover, that the phosphorylated form
of tyrosine hydroxylase is saturated with cofactor and therefore
optimally active. Conversely, the nonphosphorylated enzyme would be
relatively inactive and would not contribute significantly to the
endogenous synthesis of catecholamines. It will be very interesting
to carry out comparable experiments with pineal tissue in the future,
in order to locate the high amount of cofactor BH4, the hydroxylases
and its biosynthetic system.
Different methods are used for the determination of pteridines,

see Ebels (1981). The most sensitive and specific method seems to be
the method reported by Nagatsu et al. (1981). Using a specific anti-
serum against 6-L-erythrobiopterin, a radioimmunoassay (RIA) has been
developed to measure the biopterin concentrations in urine, serum,
cerebrospinal fluid and tissues. The limit of sensitivity was 0.5 p
mol/tube. For details see also Ebels (1981). This RIA may be of great
importance for a better understanding and the significance of the
high amount of pineal biopterin for different metabolic processes,
especially those in which catecholamines and indoles play an impor-
tant role.
SUMMARY
1. 6-L-erythrobiopterin was isolated and identified from sheep pin-

eals by means of rather simple and mild extraction and separation
methods, from a low molecular weight fraction MW < 500.
2. A second pterin neopterin was isolated and identified, from two
different fractions, prepared with a Bensinger isobutanol extrac-
tion method. This pterin can probably be present in sheep pineal
fractions as a complex which is to date still unidentified.
3. Other substances in pineal fractions, displaying a pteridine-
like fluorescence were detected; they are isolated but not yet
identified.
4. Pterin-6-carboxylic acid was isolated and identified from an
UM05R fraction, MW > 500 and < 1000, indicating that a bound
pterin may be present in that fraction.
5. pteridines can influence the indole metabolism of rat pineals in

vitro, and probably reverse.
6. Pterin-6-aldehyde and low molecular weight sheep pineal fractions,
displaying a pteridine-like fluorescence, can decrease the activi-
ty of isolated HIOMT.
7. A disorder of pteridine metabolism is found in atypical phenyl-
ketonuria, Parkinson disease and in neoplastic growth.
8. The radioimmunoassay for 6-L-erythrobiopterin will be of great
importance for pterin-metabolism in general and hopefully for
pineal-biopterin metabolism particularly.
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176 I. EBELS ET AL.
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PEPTIDIC AND PROTEIC SUBSTANCES ISOLATED FROM PINEALS AND THEIR
RELATION TO THE HYPOPHYSIAL-HYFOTHALAMIC-GONADAL AXIS
I. Ebels*
Department of Organic Chemistry

State University of Utrecht
The Netherlands
At the ultrastructural level, processes implicated in the syn-

thesis and release of peptidic/proteic compounds have been clearly
identified in pineal specific cells (Collin, 1979, 1981; Collin and
Oksche, 1981; Pevet, 1979, 1981a,b, 1982a,b). Thus the synthesis
and release of peptidic/proteic substances cannot be discarded. The
isolation, purification and characterization of this class of pineal
compounds hav~ however presented a formidable challenge, since extrac-
tion methods for peptide purification have failed to yield large
quantities of pure compounds, sufficient for structure elucidation.
Moreover, a simple and specific bioassay, indispensable for the iso-
lation of biologically active compounds has not been available till
now. Different observations however support the presence of pineal
peptidic and proteic substances showing a definite biological acti-
vity.
The research group of Thieblot has worked since about 1950 on

the isolation and purification of (a) pineal peptidic/proteic sub-
stance(s). For a survey of this work see Thieblot and Thieblot
(1981) .
Milcu et al. (1963) isolated a proteic product from the pineal

with hypoglycemic properties that they termed "pinealin".
Reiss et al. (1963a,b) found two dramatically opposed actions

of different fractions of aqueous extracts of pineal glands on the
*Our research is supported by grants of The Netherlands Organization

for the Advancement of Pure Research (ZWO) through the Foundation
for Medical Research (FUNGO) No. 13-35-33 and for the Foundation
for Biological Research (BION) No. 14.70.03.
179
180 I. EBELS
gonadal system. The supernatant of an aqueous extract of pineals,

deproteinized by trichloroacetic acid (TCA) to a final concentration
of 3.3%, freed from TCA by repeated ether extraction, and lyophilized
was used. This fraction contained a substance inhibiting gonadotropin
activity, while the TCA precipitate after being dried by repeated
washing with acetone, contained a stimulatory principle. While study-
ing different pineal gland fractions, one fraction reduced whereas
another increased the phosphorus turnover of the pituitary. The same
authors observed that the inhibitory pineal extract stimulated the
32p incorporation in the brain and decreases considerably that of the
liver. One of the most interesting aspects of these investigations
was the observation of the opposite roles played by the pineal and
pituitary in their metabolic actions on the brain. When these authors
extracted rat cerebrum and cerebellum, freeze dried cattle brain, rat
muscles, spleen, liver and kidney, the above described effects could
not be observed with a comparable fraction obtained from these tis-
sues. Therefore it seems that the observed effects are rather speci-
fic for the pineal.
Neac 7u (1972) isolated a peptidic fraction from bovine pineals,

which he termed the E5-fraction. This fractions blocks spermato-
genesis in gonadotropin-treated male frogs and acts on pituitary LH
and prolactin levels in intact rats (Vaughan et al., 1982).
Ota et ale (1975) observed an inhibition of ovulation induced

with pregnant mare serum (PMS) and human chorionic gonadotropin (HCG)
by a melatonin-free extract of bovine pineal powder. The authors hold
that perhaps two substances which possess anti-ovulatory properties
are present in the pineal: one having a molecular weight (MW) of more
than 100.000 and one with a MW less than 10.000.
Slama-Scemama et ale (1979) have shown that high- and lower

molecular weight ovine pineal fractions, prepared under dim red light
conditions stimulated the gonadotropic releasing activity of the
medial basal hypothalamus. For details see Ebels and Benson (1978)
and Benson and Ebels (1981).
Several studies indicate also the presence of some of the hypo-

physial and hypothalamic hormones in the pineal, detected in general
with radioimmunological methods. These compounds, however, can accor-
ding to Pevet (see this book) originate:
- from other organs than the pineal because these compounds are
present in cells such as nerves, which connect the pineal with
other brain structures.
- from other organs than the pineal, with a secondary uptake by the
gland from the general circulation
- from the pineal, synthesized by specific cells in the gland.
Only compounds of the last group can be considered as true pineal

"hormones".
PEPTIDIC AND PROTEIC SUBSTANCES FROM PINEALS 181
The present paper will give a survey of the results, obtained

in the last decades with different pineal fractions. Especially
effects of different partially purified pineal fractions on the repro-
ductive system will be described. The following subjects will be
discussed.
I. Experiments in vitro, using a bioassay or/and radioimmunoassay
1.1. Experiments using anterior pituitaries, and a bioassay with
immature female mice
1.2. Experiments using anterior pituitaries and a radioimmunoassay
(RIA) for the detection of prolactin
1.3. Experiments with rat and mice hypothalami, using a bioassay
with immature female mice
II. Experiments in vivo, using hemicastrated and castrated animals
II.l.Effects of bovine and ovine pineal extracts on compensatory
ovarian hypertrophy (COH)
II.2.The isolation of the tripeptide threonylseryllysine from bovine
pineals using the COH-test
III. Effects of a partially purified bovine pineal fraction E5
IV. Comparison of ovine peptidic/proteic pineal fractions with a
E5-fraction, isolated from bovine pineals
I. EXPERIMENTS IN VITRO, USING A BIOASSAY OR/AND RADIOIMMUNOASSAY
1.1. Experiments Using Anterior Pituitaries, and a Bioassay with

Immature Female Mice
It has been known for many years that either fresh pineal glands
of rats and sheep, or acetone-dried sheep pineal powder, can dimin-
ish or inhibit the secretion of anterior hypophyses of adult male
rats in vitro (Moszkowska, 1956, 1958, 1964 1965; Moszkowska and
Heersche~2). When an extract of sheep pineal powder was gelfil-
trated on a Sephadex G-25 column, two subfractions were obtained
which demonstrated opposite effects on the follicle stimulating
activity of anterior hypophysis of the male rat in vitro. The low
molecular weight Sephadex G-25 fraction F2 increased the follicle
stimulating activity. The subfraction Sephadex G-25 F3, could dimin-
ish the follicle stimulating activity of the anterior hypophysis
(Ebels et al., 1965). In further purification steps only a slight
anti gonadotropic activity was found in one experiment, while in
other experiments no activity was observed at all.
Different regions of sheep hypothalamic tissue, cerebral cortex

and cerebellum were extracted and separated with the same methods
as employed in the experiments with sheep pineal bodies. In these
experiments no antigonadotropic activity was found in the Sephadex
G-25 F3-fraction of an extract of the median eminence region of
sheep hypothalami. Significant stimulation was observed with the
Sephadex G-25 F3-fraction of sheep cerebral cortex extracts and
neither stimulatory nor inhibitory activity was found in the low
molecular weight Sephadex G-25 F3 of cerebellum.
182 I. EBELS
In other experiments was shown that the anti gonadotropic

activity of sheep pineal fraction F3 differed from the activity of
synthetic melatonin, s-methoxytryptophol,serotonin and arginine
vasotocin (Moszkowska and Ebels, 1968, 1971). The conclusion was drawn
that the antigonadotropic activity of sheep pineal Sephadex G-2s F3
seemed to be fairly specific for the pineal body, but that it is
unstable under the experimental conditions used at that time (Ebels
et al., 1970; Moszkowska and Ebels, 1971).
Therefore, simple and milder extraction and separation methods

were developed (Balemans et al., 1970; Ebels et al·., 1972a,b; Zurburg
and Ebels, 1975; Ebels and Horwitz-Bresser, 1976). With these methods
a substance showing special fluorescence characteristics could be
isolated from a low molecular weight Sephadex G-2s fraction. This
substance showed an excitation maximum at 360 nm and a fluorescence
maximum at 440 nm, and could not be detected in similar amounts of
sheep cerebral cortex treated in the same way (Zurburg and Ebels,
1974) •
It has been shown in later experiments that the substance(s)

with that special fluorescence, was associated with an antigonado-
tropic activity in vitro, using anterior hypophysis of male rats.
After ultrafiltration through Amicon membranes, it was observed that
the anti gonadotropic activity and the special fluorescence was loca-
ted in the UMOsF-fraction (MW < 500). After gel filtration of UMOsF
on Sephadex G-10 this fluorescent compound and the anti gonadotropic
activity was found in the same fraction. From that Sephadex G-10
fraction 6-L-erythrobiopterin was isolated and identified using subse-
quently preparative paper-electrophoresis and paper-chromatography,
gasliquid-chromatography (Ge) after trimethylsilylation and GC-mass-
spectrometry.
It has been shown in later experiments that isolated and syn-

thetic 6-L-erythrobiopterin preparations can diminish the gonado-
tropic activity of anterior hypophyses of male Wi star rats in vitro
and moreover decrease radioimmunologically detectable plasma LH-
levels of immature pinealectomized and hemicastrated male rats in
vivo, when measured 24 h after a single injection (for details see
Ebels, 1981).
From the work of Fukushima and Nixon (1979, 1980) we know that
the pineal gland of rats contain much more biopterin than in seven
other organs and several different regions of the brain. Moreover,
these authors stated that biopterin in the pineal is mostly present
in the reduced form: tetrahydrobobiopterin. This compound is the
natural cofactor of tryptophan hydroxylase, phenylalanine- and
tyrosine hydroxylase. We have observed later on that reduced biop-
terin is a very potent inhibitor of the gonadotropic activity of
anterior pituitaries of male rats in vitro. Therefore, we believe
that 6-L-erythrobiopterin in the reduced form is (partly) responsible
for the antigonadotropic activity, found in low molecular weight

sheep pineal fraction in in vitro and in vivo experiments (Ebels,
1981). pteridines are very unstable, light sensitive compounds
which may explain that pineal fractions may lose their antigonado-
tropic activity during isolation procedures. For details see Ebels
(1979, 1981) and another chapter on pteridines in this book.
1.2. Experiments In Vitro Using Anterior Pituitaries and a RIA for

the Detection of Prolactin
Bartke et al. (1975) induced pineal mediated gonadal regression

in male hamsters by exposure to "short days" and observed that early
recrudescence was observed after treatment with exogenous prolactin.
Blask et al. (1976) have demonstrated that hemi-pituitaries,

incubated with a crude acidic extract of bovine pineals after neutral-
izing to pH 7.4 with NaOH, released significantly more prolactin into
the incubation medium, than the corresponding control hemi-pitui-
taries, incubated with rat cortical extracts. It was also shown that,
when incubated with bovine pineal extracts, hemi-pituitaries released
significantly more prolactin into the medium than their corresponding
halves, treated with rat hypothalamic extract.
On the other hand, bovine pineal fractions, prepared according

to Bensinger et al. (1973), which show the most potent inhibition of
compensatory ovarian hypertrophy (COH) in mice, further separated by
thin-layer c'hromatography, gave two sub fractions , which could inhibit
the release of prolactin from hemi-pituitaries. From these results
is concluded that crude bovine pineal extracts contain prolactin-
releasing activity (PRF) and prolactin-inhibiting activity (PIF).
These PRF and PIF activities can both act directly on the anterior
pituitary in vitro. These results were confirmed and extended by
Chang et al. (1979), using other extraction and separation methods.
Dilute acetic acid extracts of bovine pineals were gelfiltrated on
Sephadex G-2s and the low molecular weight Sephadex G-2s fractions
were separated further by ultrafiltration, using the Amicon membranes
PM10, UM2 and UMOs. These last fractions were incubated with rat
hemi-pituitaries and the effects on prolactin secretior. determined.
The UMOsF-fraction (MW < 500) shows inhibition of prolactin release
in this system. The PM10R-fraction (MW > 10.000) could stimulate the
release of prolactin in the same system.
However, the UMOsR-fraction (MW > 500 and < 1000) did not show
any activity in this in vitro-system, but could reduce plasma prolac-
tin in vivo in urethane-anesthesized rats. Larsen and Benson (1979)
using high performance liquid chromatography as a final step, obtain-
ed the same results with a more purified prolactin-inhibiting frac-
tion from bovine pineals.
184 I. EBELS
From these experiments can be concluded that pineal extracts

contain PRF- and PIF-activities, acting directly on the pituitary,
but that there is also a partially purified low molecular weight
pineal fraction, which can decrease the plasma prolactin levels,
by possibly stimulating the secretion of a hypothalamic prolactin
inhibiting factor.
The results of Matthews et al. (1978), Bex et al. (1978) Reiter

and Ferguson (1979), and Benson and Matthews (1980), in which the
transplantation of pituitaries beneath the kidney capsules either
partially or totally prevented gonadal atrophy in light-deprived
hamsters, suggest a possible role for prolactin and pineal prolactin
regulating factors in pineal mediated gonadal atrophy. Therefore,
the pineal regulation of prolactin secretion may be more important
than was thought in previous years. See also Leadem and Blask (1982).
Benson and Matthews (1980) concluded from the results of their

and other's experiments, that the pineal may secrete a prolactin
regulating substance. Such a substance could act by either regulating
the synthesis and/or release of hypothalamic prolactin releasing
factors or prolactin release inhibiting factors such as dopamine.
Recently, Martens et al. (1981) showed in a microsuperfusion-system
of neurointermediate lobes of Xenopus laevis, that dopamine can
inhibit the release of all newly synthesized peptides except the pre-
cursor, leading to an enhanced level of these peptides remaining in
the lobe. Cyclic AMP stimulated the release of all peptides, except
the precursor, leading to reduced levels of lobe peptides. A compar-
able mechanism may exist in the hypothalamus for gonadotropic releas-
ing substances regulated by pineal substances. Benson et al. (in
press) have found more experimental data to support the last hypo-
thesis. See also Benson and Ebels (1981).
I.3. Experiments In Vitro with Rat and Mice Hypothalami Using a

Bioassay with Immature Female Mice
In 1973, Moszkowska et al. showed the modulation of gonado-

tropin-releasing factor content in the rat hypothalamus by pineal
factors in vitro and in vivo. The same group demonstrated an effect
on the hypothalamus in vitro by addition of sheep pineal fractions.
Those fractions were prepared by gelfiltration on Sephadex G-25 of
an aqueous pineal extract and subsequent ultrafiltration through
the Amicon membranes UM2 and UM05 of the low molecular weight
Sephadex G-25 fractions (see Citharel et al., 1973). Mice hypo-
thalami incubated with an UM2R-fraction (MW > 1000) showed a highly
significant decrease in the secretion of hypophysiotropic activity
in vitro as compared to the controls. When the same fraction was
tested with anterior hypophyses of male mice, no effect on the
gonadotropin was observed. Therefore it was concluded that this
UM2R-fraction has an influence on the hypothalamus. Hypothalami
incubated with the sheep pineal fraction UM05R (MW > 500 and < 1000)
showed increased hypophysiotropic activity. No significant influence

on the anterior hypophyses alone were observed with UM05R-fractions
(Citharel et al., 1973; Ebels et al., 1975).
All these experiments were carried out under normal laboratory

light conditions. However, in later experiments, using an isobutanol
extraction method (Ebels et al., 1979) and ultrafiltrating the
remaining water layer through Amicon membranes PMI0, UM2 and UMOS,
the PM10R-fraction (MW > 10.000) and UM2R-fraction (MW > 1000 and
< 10.000) showed a stimulating effect on the hypophysiotropic acti-
vity on the median eminence of the hypothalamus. Further ultrafil-
tration of this PM10R-fraction on the filters XM100 and PM30, and
testing of those residues have shown that XMI00R (MW > 100.000) and
PM30R (MW > 30.000 and < 100.000) contain the stimulating activity.
Nearly no activity was left in the PMIOR fraction (MW < 30.000 and
> 10.000) of the PM30 filtrate. All these experiments were carried
out in red light (A > 600 mm) and as much as possible in darkness.
This was for us the first time that we found biologically active
high molecular weight pineal fractions. Later on, comparable results
were obtained with ultrafiltrated fractions, obtained by direct
ultrafiltration of aqueous ovine pineal extracts. It may be that the
experimental lighting conditions, used in the last years to protect
the light-sensitive pteridines in our pineal extracts during the
isolation procedure, preserve high molecular weight complexes which
increase the gonadotropic-releaseing activity of male rat medial
basal hypothalami. For details, see Slama et al. (1979). It may be
that the pineal fractions XM100R and PM30R contain active complexes,
which can be destroyed by normal light. This supposition is supported
by the results of Frowein and Lapin (1979) who have compared the
effects of sham-pinealectomy in newborn and adult rats performed
under white and red light. These authors observed that melatonin
biosynthesis is altered by hitting the pineal with white light
in situ during sham-operation. The melatonin synthesis was reduced
irreversibly for at least 2 months. It is left unaltered when surgery
is performed under red light.
Deguchi (1981) observed that direct illumination of cultured

chicken pineals suppressed the night-time increase of N-acetyltrans-
ferase activity. The sensitivity curve of the enzyme activity for
light of different wavelengths resembled the absorption spectrum of
rhodopsin. In the near future more attention will be paid to these
high molecular weight complexes.
II. EXPERIMENTS IN VIVO, USING HEMI-CASTRATED AND CASTRATED ANIMALS
11.1. Effects of Pineal Extracts on Compensatory Ovarian Hypertrophy

(COH)
Moszkowska (1951) first showed that aqueous pineal extracts

can inhibit (COH) and the formation of corpora lute a in guinea pigs
186 I. EBELS
after unilateral ovariectomy (UO). Later i t was shown that daily

administration of a pineal extract (Reiss et al., 1963a) or of mela-
tonin (Sorrentino, 1968), blocked COH after UO in the rat. Especial-
ly the research group of Benson and co-workers have carried out
numerous experiments in this field. The results of the earlier work
of this group are summarized in a review by Benson and Orts (1972)
in which the hypothesis was postulated that pineal antigonadotropic
substances act by altering the sensitivity of the hypothalamo-hypo-
physial axis to the feedback effects of gonadal steroids. A support
for this hypothesis was found in experiments, in which i t was shown
that fractions of aqueous extracts of bovine pineal glands, partially
purified to exclude melatonin, inhibited the post-castration rise
in LH in orchidectomized rats. In later studies it was shown by the
same group that similar LH-inhibitory properties can be observed in
aqueous extracts of rat pineals. The media of hemi-pituitaries incu-
bated with cerebral cortex and cerebellar, did not demonstrate simi-
lar anti gonadotropic activity. Inactivation of the anti-COH substance
in rat pineal incubation media and bovine pineal extracts, was pro-
duced by proteolytic enzymes (Matthews and Benson, 1973). These
authors postulated already at that time that the non-melatonin pineal
anti gonadotropin (PAG) is a small peptide in the molecular weight
range of 500 - 1000, based on ultrafiltration experiments.
From studies carried out with ovine pineal glands, using simple
and mild extraction methods, it could be concluded that the ovine
UM05-residue, (MW > 500 and < 1000) contains (a) substance(s) which
inhibited COH and reduced ventral prostate weights in mice; the same
fraction retarded vaginal opening in young female mice. These studies
showed clearly that (an) antigonadotropic substance(s) could be
separated from melatonin, since this indoleamine, as well as 5-
methoxytryptophol and 5-hydroxytryptophol are eluted from Sephadex
G-25 and Sephadex G-l0 columns after the fraction in which COH-
inhibiting activity is localized.
Similarly prepared melatonin-free fractions, obtained from

extracts of bovine pineal glands delayed vaginal opening time, reduce
the incidence of estrous vaginal smears in rats and reduce serum
LH-levels in long term orchidectomized rats.
Benson et aL (1976a,b) studied also the effects of antigonado-

tropic fractions derived from bovine pineal extracts on fertility
and ovulation. A gonadal site of action was ruled out by Benson et
al. (1976a) and the effects on ovulation and fertility were postu-
lated to be the result of inhibition of LH-secretion. In other
studies the anti gonadotropin PAG was localized to pineal parenchyma
and vasotocin-like activity to the pineal stalk by the same group.
In later years Benson et al. attempted final purification of bovine
PAG. Throughout these studies it was learned that the sulfur-contain·
ing amino acid taurine was closely associated with the COH-inhibit-
ing factor. Further studies showed that the COH-inhibiting substance
could be separated from taurine, hypotaurine and reduced and oxidized

glutathione, by paper-chromatography (see also Benson and Ebels,
1981) .
In 1973 Bensinger et al. described in an abstract a method for

aqueous-acetone extraction of bovine pineal and the further removal
of fats and indoleamines by chloroform/methanol extraction. Although
the details of the methods and all the results were not published in
detail the authors stated that the active principle was probably a
small, non-tryptophan-containing peptide. We have extracted also
bovine pineals according to the method of Bensinger et al. (1973) and
confirmed the presence of COH-inhibiting activity (Ebels et al.,
1979). However, further separation by gel filtration and paper-
chromatography revealed different Rf-values for the inhibiting activ-
ity than the COH-inhibiting substances obtained from acetic and
aqueous extracts of ovine and bovine pineals. In later studies the
anti gonadotropic substance(s) obtained by both acid and isobutanol
extraction could be localized using high pressure liquid chromato-
graphy. Since the amount of residue obtained are extremely small, a
large amount of pineals will be needed to obtain enough pure material
for structural determination by mass-spectrometry. For more details
of the many experiments carried out by Benson et al., see the reviews
written in recent years by Ebels and Benson (1979) and by Benson and
Ebels (1981).
11.2. The Isolation of the Tripeptide Threonylseryllysine from Bovine

Pineal Glands
Orts et al. (1978) isolated from bovine pineals a tripeptide,

using glacial acetic acid extracts, separated by Sephadex gelfiltra-
tion, ultrafiltration and paper-electrophoresis. The antigonadotropic
activity was followed through the purification steps using the
compensatory ovarian hypertrophy test with female mice. The sequence
was determined to be threonylseryllysine (Thr-Ser-Lys). The tripep-
tide was synthesized by Larsen et al. (1979) and the biological
effects were studied. Studies on mice and rats have shown that Thr-
Ser-Lys causes a weak anti gonadotropic activity (Vaughan et al.,
(1980).
Although the effects on prolactin observed with Thr-Ser-Lys are

consistent with those of the pineal prolactin regulating substance,
partially purified by Larsen and Benson (1979), the elution times,
using high performance liquid chromatography, are different, which
implicate different chemical structures.
This tripeptide is of a smaller molecular weight than most

pineal antigonadotropic factors prepared by the group of Benson et al.
in the last years, suggesting that the use of glacial acetic acid as
Orts et al. did, may have effectively cleaved this tripeptide from a
larger parent molecule.
188 I. EBELS
III. EFFECTS OF A PARTIALLY PURIFIED BOVINE PINEAL FRACTION E5
Milcu et ale (1963) have published the biological and chromato-

graphic characterization of a polypeptide with pressor and oxytocic
activities, isolated from bovine pineal glands. These authors con-
cluded from the results of their experiments that their pineal poly-
peptide was neither arginine- or lysine vasopressin, nor oxytocin,
but that the biological and chromatographic characteristics are
siDilar to those of arginine vasotocin or a compound with similar
structure. Ebels et ale (1965) attempted to isolate a peptide with
pressor and oxytocic activities from sheep and bovine acetone desic-
cated pineal bodies by gelfiltration and column chromatography. These
authors could not confirm the occurrence of arginine vasotocin in
the pineal organ. Moszkowska and Ebels (1968) studied the antigonado-
tropic action of synthetic arginine vasotocin. These authors concluded
from their experiments that arginine vasotocin can act on the gonads
or on the gonadotropic hormones and not on the secretion of the
anterior hypophysis in vitro, as was observed in in vitro experiments
with acetone-dried sheep pineal powder and with a:3ephadex G-25 F3
fraction (Moszkowska, 1965; Ebels et al., 1965).
Neac~u (1972) used a bovine peptidic/proteic fraction E5, pre-

pared according to the method published by Milcu et ale (1963) and
proposed a composition for the peptide E5, having a molecular weight
of about 2000. Under the experimental conditions this author used, the
"polypeptide" E5 can inhibit, when injected prior to human chorionic
gonadotropin, the activity of this hormone on spermatogenesis in male
frogs. It was concluded that E5 influenced rather LH than FSH.
In another publication Neacsu (1972) postulated that the E5-

peptide has a larger ring than oxytocin and vasopressin and a larger
side chain of five amino acids but also ending by pro-Arg-Gly(NH 2 ) as
AVT and AVP, although no experimental details of methods and results
were presented for this statement.
Since the time Cheesman (1970), Cheesman and Fariss (1970) pub-
lished the isolation and identification of AVT from bovine pineals,
a large number of publications appeared in which the effects of syn-
thetic AVT were described. For a survey of this literature see:
Vaughan and Blask (1978); Pavel (1979); Vaughan (1981) and Reiter
(1980, 1981).
Recently Vaughan et ale (1982) tested again an E5-fraction pre-

pared by Dr. Neac 7u, Bucharest, Romania. These authors concluded from
their experiments that: pituitary levels of prolactin were signifi-
cantly diminished after five subcuteneous injections of 5 ~g of this
partially purified bovine pineal peptide fraction E5, in both intact
and castrated rats. E5 treatment did not significantly affect the
castration-induced changes in plasma LH, FSH or pituitary LH, but did
partially block the pituitary fall in FSH in castrated animals.
Pevet et al. (1980) using radioimmunological methods, concluded

from their experiments, that the pineal does not contain AVT, but an
AVT-like peptide, which may be responsible for the anti gonadotropic
activity, observed with the bovine ES-fraction. See also Pevet (1979,
1981b and this book) .
It can be concluded that the presence of AVT or and AVT-like

peptide in the mammalian pineal is still debated. Vaughan (1981) wrote
in one of her reviews: "that after 2 decades of research can be con-
cluded that the nonapeptide AVT directly or indirectly affects the
reproductive system of all classes of vertebrates, but that there is
considerable controversy whether an antigonadotropic peptide of the
pineal is AVP, AVT, oxytocin, ES or another unidentified peptide".
Although different biological effects are obtained with bovine

"ES-fractions", it is not proven that this fraction is a pure peptide,
because a description with details of the exact experimental condi-
tions used for the preparation of the ES "peptide" from bovine pineals
is still lacking. Therefore, in the next part of this paper we will
describe a comparative study, in which different peptidic/proteic
ovine pineal fractions were compared with a bovine ES-fraction, pre-
pared by Dr. Neac?u, Bucharest, Romania.
IV. COMPARISON OF SOME PEPTIDIC AND PROTEIC PINEAL FRACTIONS WITH A

BOVINE ES FRACTION
The bovine pineal ES-fraction was prepared by Dr. C. Neac9u,

sent to Dr. P. Pevet, Netherlands Institute for Brain Research,
Amsterdam and there stored at -80°C until use in our institute in
Utrecht. The ovine pineals were collected by Ersco, San Mateo, Calif-
ornia in the winter months, shipped on dry ice and stored at -20°C
until use.
Using rather simple and mild extraction and separation methods,

three ovine pineal fractions: XM300R, PP7.2' and PP7.2S were prepared
according to Scheme 1. From the results of the experiments can be
concluded that these sheep pineal fractions and the bovine pineal
ES-fraction contain peptidic and proteic substances as measured with
the "micro-Lowry" method. However, the proteic content of the frac-
tions is between 60 and 70% for the ovine pineal fractions and below
40% for the bovine ES-fraction, using serum albumin as a standard,
and the same method under the same experimental conditions. This may
mean that the above mentioned fractions contain besides their proteic
part, another structure. At this moment we have no other explanation
for this fact. All the above mentioned fractions show excitation and
emission maxima just like indoles show. These fluorescence character-
istics are sensitive to normal laboratory light and most stable in
red light (A > 600nm).
190 I. EBELS
SCHEME 1. OUTLINE OF THE PREPARATION OF DIFFERENT OVINE PINEAL

FRACTIONS.
sheep pineals
extraction
with H2 0 (3x)
supernatant residue (1)
~..
ultrafiltration extraction with
through XM300 O.2M HCl
~---~
residue (XM300R) filtrate residue (2) supernatant

ultra- adjusted at
filtration pH 7.2; two
through volumes of
XM100 ethanol 96%
added
residue (XM100R) filtrate
supernatant protein (PP7.2)
extraction extraction
with O.2M HCl with 10mM TrisHCl
pH 8.2
residue (4) residue (3) supernatant
supernatant UHOS]
lyophilization
lYOPhiliZationl protein
(PP7.2S)
adjusted at
protein
pH 7.2; two
(PP7.2 I )
volumes of
ethanol 96% added
supernatant protein (XM300R-PP7.2)

Several years ago, Ariens Kappers et al. (1974) mentioned that

in rabbit and rat pineals protein(s) are probably present which con-
tain much tryptophan. In gel-electrophoresis experiments the trypto-
phan-rich protein band showed also a specific yellow autofluorescence
(for details see also Smith and Ariens Kappers, 1975).
In the near future special attention will be paid to the indole-

like fluorescence in our fractions, since it has been shown that
tryptophan is very important for the biological activity of somato-
statin (Hirst et al., 1980) and of pancreozymin (Rajh et al, 1980).
From the staining pattern obtained with Coomassie blue of the

bovine pineal fraction E5 and the ovine pineal fraction PP7.2 using
disc gel-electrophoresis, it can be concluded that a similarity exists
between the patterns of the bovine pineal fraction E5 and the ovine
fraction PP7.2S. However, using sodium-dodecyl sulphate slab gel-
electrophoresis, unexpected high molecular weight proteic bands were
located in the gel. This justifies the conclusion that the bovine
pineal fraction E5 contains proteinaceous material, which was not
detected with the disc gel-electrophoresis method.
When the ovine fraction XM300R - PP7 . 2 and PP7. 2S were treated
in the same way a number of proteic/peptidic bands were also visible.
The protein bands can be divided into 5 major groups:> 94K; > 68K;
> 43K; > 30K and> 21K.
Data from HPLC experiments in combination with those of gel-

electrophoresis experiments suggest however that polypeptidic sub-
stances of a lower molecular weight are also present in the bovine E5-
and the ovine PP7.2-fractions. In a RIA developped by Dr. A. Reinharz,
Geneva, Switzerland, use was made of an antibody against the carboxy
terminal tripeptide AVP or AVT. The ovine fractions xM300R-PP7.2
and PP7.2 are recognized by this antibody. The dilution curve is
parallel to the E5 and to the standard (here AVP). This implies that
(a) peptide(s) present in all these fractions are most probably ter-
minating by Pro-Arg-Gly(NH 2 ). It thus appears that (a) peptide(s)
ending by the same carboxy terminal end as AVP or AVT is present in
the E5-fraction obtained from bovine pineals as well as in the ovine
pineal fractions XM300R-PP7.2 and PP7.2 (for details of these experi-
ments see Noteborn et al., (in press» .
The present findings do not allow to conclude that the blockage

of spermatogenesis in gonadotropin-treated male frogs; the inhibition
of uterine weights and 32 p incorporation in gonadotropin-treated
female mice (Neac 7u, 1972); the activity on pituitary levels of LH
and prolactin in intact rats (Vaughan et al., 1980, 1982) and the
high activity in compensatory ovarian hypertrophy test (unpublished
results) present in the bovine pineal fraction E5, are due to pineal
peptidic antigonadotropic "hormones" (Pevet et al., 1981a,b) , because
the bovine pineal fraction E5 contains also a large amount of proteic
192 I. EBELS
compounds MW > 14.300 daltons), which could be responsible for bio-

logical activities. It remains, however, possible that the same pep-
tide is present in the bovine pineal fraction ES and the ovine pineal
fractions studied because it is difficult to detect a similarity in
the bands < 14.3K due to diffusion in this front region in our slab
gel-electrophoresis experiments. It has been demonstrated that the
ovine pineal fractions share several chemical and immunological
properties with the bovine pineal fraction ES. However, the struc-
ture of the compound(s) and their biological activity remain to be
elucidated.
SUMMARY
1. The antigonadotropic activity in vitro of a low molecular weight

(MW < 500) ovine pineal fraction, using male rat anterior pituit-
aries and a bioassay, is most probably caused by reduced 6-L-
erythrobiopterin.
2. High and lower molecular weight ovine pineal fractions, prepared
under red light conditions, can increase the gonadotropin releas-
ing activity of male rat medial basal hypothalamus in vitro.
3. A low molecular weight pineal fraction (MW > 500 and:< 1000) con-
tains a substance, which inhibit compensatory ovarian hypertrophy,
reduces ventral prostate weights in mice, retards vaginal opening
in young female mice and can decrease plasma LH-levels.
4. A tripeptide Thr-Ser-Lys with antigonadotropic capability is iso-
lated from bovine pineals and is synthesized later on.
5. Crude and partially purified bovine pineal fractions contain pro-
lactin regulating substances in vitro.
6. A low molecular weight bovine pineal fraction (l~ > 500 and <
1000) can decrease plasma prolactin levels in vivo.
The pineal regulation of prolactin secretion may therefore be
more important than was thought in previous years.
7. Comparison of some ovine peptidic/proteic pineal fractions with a
bovine pineal fraction ES has learned that several chemical and
immunological properties are shared. The results obtained with an
antibody against the carboxy terminal tripeptide of AVP or AVT
indicates that a peptide(s) ending by Pro-Arg-Gly(NH 2 ) is probab-
ly present in the bovine ES fraction, as well as in some ovine
pineal fractions.
8. Although the presence of peptidic/proteic substances in biological-
ly active pineal fractions cannot be discarded, the chemical
nature of the biological active substances remain to be eluci-
dated.
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gonadectomy, p-CPA and pineal extracts on the rat parvocel-
lular neurosecretory hypothalamic system; a fluorescence
histochemical investigation, Brain Res., 86:353.
Sorrentino, S. jr., 1968, Antigonadotropic effects of melatonin in
intact and unilaterally ovariectomized rats, Anat. Rec.,
160:432. - - --
Thieblot, L., and Thieblot, Ph., 1981, "La Gland Pineale," Physio-
logie et Clinique, S.A. Maloine, ed., Paris.
Vaughan, M.K., 1981, Arginine vasotocin and vertebrate reproduction,
in: "The Pineal Gland," vol. II, Reproductive Effects, R.J.
Reiter, ed., CRC Press, Boca Raton, Florida.
Vaughan, M.K., and Blask, D.E., 1978, Arginine vasotocin - A search
for its function in mammals, in: "The Pineal and Reproduction,"
Progress in Reproductive Biology, Vol. 4., R.J. Reiter, ed.,
S. Karger, Basel.
Vaughan, M.K., Johnson, L.Y., Pevet, P., Neacsu, C., and Reiter, R.J.,
1980, Effect of a polypeptide (E5) ext~acted from bovine pineal
glands on plasma and pituitary levels of luteinizing hormone
(LH) and prolactin in normal and castrated adult male rats.
Tenth Ann. Mtg., Soc. Neurosci., 16:457.
198 I. EBELS
Vaughan, M.K., Richardson, B.A., Johnson, L.Y., Reiter, R.J., Pevet,

P., and Neac 7u, C., 1982, Ef~ects of a bovine pineal peptidic
fraction (E5) on plasma and pituitary levels of LH, FSH and
prolactin, Experientia, in press.
Zurburg, W., and Ebels, I., 1974, Separation of pineal extracts by
gel filtration. I. Isolation from sheep pineals of a substance
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35:117.
Zurburg, W., and Ebels, I., 1975, Separation of pineal extracts by
gel filtration. II. Identification and isolation of two
indoles from sheep pineal glands, J. Neural Transm., 36:59.
MOLECULAR ASPECTS OF NEUROENDOCRINE INTEGRATIVE PROCESSES
IN THE PINEAL GLAND *
** *** **
D.P.Cardinali, Monica N.Ritt~~ Maria I.Vacas,
P.R.Lowenstein,*** P.V.Gejman, * C.Gonzalez Solve~
and Elba Pereyra
Centro de Estudios Farmacologicos y de Principios
Naturales (CEFAPRIN), Serrano 665/669, 1414 Buenos
Aires, Argentina
INTRODUCTION
The mammalian pineal gland fulfills the criteria of a
"neuroendocrine" transducer~ It translates a neural language
provided by norepinephrine (NE) released at the synaptic bio-
phase to a hormone language,melatonin and perhaps endocrine
active peptides. The pinealocytes are also "endocrine-endo-
crine" transducers inasmuch as they convert an endocrine lan-
guage, e.g. estradiol attaining the gland via the general ci~
culation, to a different endocrine signal like melatonin. Ad-
ditionally "endocrine-neural" ~ransducer events occur in the
pineal gland, as revealed by the significant modifications of
the activity of the innerv~ting sympathetic pathway after sev-
eral hormone treatments. 1 ,
NEUROENDOCRINE TRANSDUCTION
Historically the quality and quantity of ambient illumina-
tion have been pointed out as the major environmental signal
affecting the pineal gland. Light information is relayed
neurally by the retino-hypothalamic tract and it is integrat-
* Supported by grant nQ 6638 from Consejo Nacional de Inves-

tigaciones Cientificas y Tecnicas (CONICET), Argentina.
** Established Investigator, CONICET.
*** Research Fellow, CONICET.
199
200 D. P. CARDINALI ET AL.
ed with circadian information in the suprachiasmatic nucleus

of the anterior hypothalamus (Fig. 1). Caudal projections from
the suprachiasmatic nucleus connect via a multisynaptic path-
way with the superior cervical ganglia (SCG).3 Axons from the
SCG innervate the pineal gland where they release NE in the
vicinity of pineal cells. It is now accepted generally that
darkness stimulates the release of NE from these nerves. 4 ,5
The biochemical mechanisms by which NE released from pineal

nerve endings accelerates melatonin synthesis have been ana-
lyzed in detail using organ cultures (Fig. 1). The catechol-
amine acts on5-adrenoceptors of the pineal cells and stimu-
lates the activity of aden~l cyclase which results in an in-
creased synthesis of cAMP.5-8 In the subsequent induction of
enzymes participating in melatonin synthesis e.g. serotonin
N-acetyltransferase (SNAT) and hydroxyindole-O-methyl trans-
ferase (HIOMT), both RNA and protein are synthesized within
the pinealocytes. 9 ,10 Presynaptic5-adrenoceptors modulating
NE release are presumably present in pineal nerve endings. 11
There is also information on the existence ofo(-adrenergic

effects of N~ in the pineal gland. These include in viv0 12 and
in vitr0 13 ,1 modification of SNAT activity and stimulation of
phosphatidylinositol turnover. Either by acting directly on
pineal nerve endings or through the modification of a hypo-
thetical trans-synaptic feedback signal'~2-adenoceptor ago-
nistsand antagonists modify NE release from pineal nerves. 16,17
Postsynapticoe-adrenoceptors that increased significantly in
number following s~mpathetic denervation of the pineal gland
were described by H-dihydroergocryptine binding in rat pineal
membranes. 16
Most neurotransmitters act via membrane receptors to in-

duce a variety of second messengers. For one receptor sub-
class the transducing event depends upon the activation of
adenyl cyclase to produce a single output signal: cAMP. The
link between the.5 -adrenoceptor-adenyl cyclase system and
melatonin secretion in the pineal gland has extensively been
reviewed. 8 ,18 The second major receptor sub-class is more
ubiquitous in that it gives rise to various output signals
including calcium, cyclic nucleotides and several arachidonic
acid metabolites like prostaglandins (PGs), thromboxanes, and
leukotrienes. Possibly all these signals are intimately relat-
ed to the hydrolysis of phosphatidylinositol. 19 ,20,21
The arachidonic acid metabolites seem to perform a variety

of functions depending on the cell considered. In some cases
they subserve the role of second messengers within the cells;
in other cases they diffuse away from the cell and thus act
as aotacoids to modulate the surrounding cells. 21 This dual
NEUROENDOCRINE INTEGRATIVE PROCESSES IN PINEAL GLAND 201
+-~ +--LlGHT
..10-----,-+ PlQlein Synlhesls
Kynurenamine
PlNEALOCYTE
5HT
~-------------------5HT
Fig. 1 Diagram representing the subcellular mechanisms under-

lying neuroendocrine-transducing events in the mamma-
lian pineal. TP: tryptophan; HTP: 5-hydroxytryptophan;
5HT: serotonin; NAS: N-acetylserotonin; HTOH: 5-hy-
droxytryptophol; HIAA: 5-hydroxyindoleacetic acid;
MTOH: 5-methoxytryptophol; MIAA: 5-methoxyindoleacetic
acid.
role is of particular importance in the adrenergic neuroef-

fector junction, where PGs play both pre- and postsynaptic
functions; for example, besides influencing the cellular re-
sponses to the neurotransmitter, the PGE2 released from vascu-
lar smooth muscle functions as a negative feedback signal to
depress transmitter release from the nerve endings. 22
In 1976 Szabo and Friedhoff 23 reported that the injection
of 10-50 mg/kg of the PG synthesis inhibitor indomethacin (Id)
to adult male rats for 4-5 days decreased by 65-95% the activ-
ity of pineal SNAT when measured at the 4th h of dark during
daily photoperiod. Essentially similar results were obtained
by using smaller amounts of Id 24 • A single injection of 5
mg/kg to rats at the end of the light phase of daily photo-
period brought about a significant, 60%-depression of the
dark-induced activation of pineal SNAT,and diminished signif-
202 D. P. CARDINALI ET Al.
icantly pineal melatonin levels by 27%. The treatment also

decreased HIoMT activity by 60%. Although no attempt was made
to characterize whether the depressed melatonin synthesis and
levels after Id were due to a true impairment of pineal NE-
driven phenomena or merely to a shift in the peak levels of
melatonin at night, our results argued in favor of a modulato-
ry role of PGs at any of the several anatomical stages of the
retinal-pineal pathway (Fig. 1). Obviously the location(s) of
the effect (pineal and/or extrapineal) could not be determined
from those experiments.
In the pineal gland monoamine oxidase (MAO) type A activit~
but not MAO type 8 activity, was found to depend on intact sym-
pathetic nerves. 25 ,26 This observation led to the conclusion
that the former is located in the nerve endings (or needs ab-
solutely of their integrity) whereas the latter is located in
the pinealocytes. Id treatment of rats increased by 55% pineal
MAO type A and decreased by 37% MAO type 8; ~tneal catechol-
o-methyl transferase was not affected by Id. These observa-
tions suggested that the drug enhanced by acting on pineal or
extrapineal sites, the intraneuronal metabolism of NE.
Providing that all Id pineal effects can be attributed

to inhibition of PG synthesis, the above discussed results su~
gested the occurrence of pre- and/or postsynaptic effects of
PGs (or of some of the products of lipoxygenase e.g. leuko-
trienes, whose synthesis may increase after cyclooxygenase
inhibition))9-21 However besides cyclooxygenase, Id alters at
submicromolar amounts several other relevant enzyme activities
like phospholipase, phosphodiesterase or cAMP-dependent pro-
tein kinase. 27 ,28 Therefore as it is usual when only a single
experimental approach is used to answer a posed question, the
foregoing results were beset by the vagaries of unspecified
site(s) and uncertain mechanisms of action of the drug used.
The following experiments were carried out in vitro in
order to supersede some of these drawbacks. Specifically they
sought to fulfill the following criteria to attribute a role
for PGs in NE-drivenmelatonin biosynthesis: (a) NE should re-
lease physiologically significant amounts of PGs at the con-
centrations that increase melatonin production in vitro; (b)
incubation of NE with various PG synthesis inhibitors should
impair or block NE-induced melatonin release; (c) physiolog-
ical concentrations of PGs should increase melatonin synthe-
sis in vitro, as well as counteract the inhibition of mela-
tonin release brought about by cyclooxygenase inhibitor; (d)
specific recognition sites for PGs should be identified in
pineal subcellular fractions.
The effects of NE on PG release by pineal glands in vitro

were examined in bovine and rat explants. By applying specific

bioassays for PGE-and PGF- like materials, we observed that
the basal release of PGs by bovine pineals in vitro was in-
creased by 2-3-fold after adding 1-100 uM NE to the medium. 29
Obviously these experiments were not addressed to the question
on the site of origin (pre- or postsynaptic) of the PGs re-
leased by NEj however in view of the very small pOBulation of
sympathetic nerve endings in bovine pineal gland,3 the pos-
sibility that NE could release PGs from the nerve varicosities
rather than from the pinealocytes was considered remote.·
More recently we undertook a series of experiments aiming

to assess the adrenoceptor involved and site of origin of
PGE2 released by NE from rat pineal explants. Pineal glands
incubated with 10 or 100 uM NE released 51%-and 415% more
radioimmunoassayable PGE2 to the medium than in the absence
of NE. Phentolamine (10 uM) fully prevented NE effect at both
concentration of the transmitter, whereas propranolol failed
to affect it significantly. After pineal sympathetic denerva-
tion by superior cervical ganglionectomy (SCGx) the release of
PGE 2 evoked by NE was significantly higher than in sham-operat-
ed controls, an effect also abolished by ~-adrenoceptor (but
not ~-adrenoceptor) blocking agents (results submitted for
publication). Thus the dose dependent, NE-stimulated PG re-
lease in rat pineal gland appears both to be mediated by a
postsynaptic ~-adrenoceptor and to originate at a postsynap-
tic site. The increased ~-adrenergic response to NE in de-
nervated rat pineals correlated well with the priorly report,d
increase in the number of~ -adrenoceptor sites after SCGx. 1
In mammals certain membrane phospholipids like phosphatidy~

inositol, phosphatidylcholine and phosphatidylethanolamine are
the cellular repositotles of arachidonic acid, the precursor
molecule for PGs, thromboxanes and leukotrienes. 20 ,21 ThE
number 2 position of phosphat idyl inositol is higly enriched
in arachidonic acid, and it would not be unreasonable to pos-
tulate that the acceleration of pineal phosphat idyl inositol
turnover brought about by NE via ~-adrenoceptors leads ulti-
mately to the liberation of this rate-limiting precursor for
the synthesis of PGs and other products of cyclo-oxygenase
and lipoxygenase. Moreover in those cases where the specifici-
ty of the receptor mechanism has been examined, it is clear
that the PGs are released by the same receptor sub-class that
gives rise to the phosphat idyl inositol response and that gates
calcium. 19
In order to examine the possible occurrence of pineal PG
receptors.radioligand binding studies were carried out in
900 g supernatants of bovine glands by usinQ a charcoal sus-
pension to separate free from boxd ligand. 29
3H-PGF2~ and 3H-PGE2 high affinity binding to pineal protein

components was augmented significantly by increasing calcium
concentrating in medium and was decreased after preparation
of homogenates in buffer not containing Id (a finding which
suggests that the endogenous PGs formed during the homogeniza-
tion and incubation procedure occupy the available pineal
binding sites)29. Pineal PG binding, was maximal in the
27,000 g pellet, where mitochondria, plasma membrane fragmen~,
lysosomes and large microsomal particles precipitate.
Analysis of the binding data by Scatchard plots revealed
the presence of single populations of binding sites for PGE2
and PGF2~' with Kd of about 1-2 nM. Comparison of the rela-
tive affinities of PGE1, PGE2 and PGF2Q(suggested that both
sites were sep~rate entities, and that the PGE 2 binding required
the 9-keto moiety while the PGF2~binding requlred the 9
hydroxyl group.29 Therefore, not only NE releases PGs from rat
and bovine pineal glands via post-synaptic ox. -adrenoceptors
but also the released PGs become bound to specific calcium-de-
pendent recognition sites, probably in pineal membranes.
The next series of experiments sought the answer two ques-
tions: (a) do PGs affect pineal SNAT activity and melatonin
accumulation and release in pineal organ cultures? (b) does
blockade of PG synthesis alter the NE-evoked melatonin release
from pineal explants ?
So that the effects of PGs on SNAT activity and melatonin

accumulation and release could be examined pineal glands from
male rats were incubated for 6 h in TC 199 medium. 31 ,32 Among
the several PGs tested only PGE2 increased the enzyme activity
at all studied doses (1-1000 nM) the stimulation curve being
bell-shaped with a maximum at 100 nM.31 PGE1 increased pineal
SNAT only at the highest studied dose while PGF2~was devoid
of effect. Nanomolar concentrations of PGE2 increased mela-
tonin accumulation and release to the medium. 32 Differing
from results on SNAT the maximal effect of PGE2 was detect-
able on both pineal and medium concentrations of melatonin at
1 nM PGE2, with gradual decreases at greater concentrations.
Therefore actual secretion of melatonin may not accompany
always the changes in SNAT activity.
To disclose a pre- or postsynaptic site of action of PGE2,
pineal glands from rats subjected to SCGx or sham operation:
7 days earlier were ~ncubated with PGE2 and the activity of
SNAT was determined. 1 PGE2 caused a significantly greater
stimulation of SNAT in pineal glands from SCGx than from con-
trol rats. These findings indicated that the stimulatory ef-
fects of PGE2 on melatonin synthesis is predominantly post-
synaptic, and that the pineal supersensitivity that develops
~ INDO 100 I'M

fi'·;··'}·]ASA 100 I'M
:IE E:::':;:;) MEF 100 I'M
::> 35
a
IJJ
:IE c
0
IJJ += 30
:r:: a
t- .a
:::J
0
u 25
t- .50
..c:
en ~ 20
IJJ
« "ii
IJJ •
.
..J c
IJJ ·ii
a::: .....
z E 10
Z a
~
~..J
0
0
c
a 5
c
IJJ
:IE
0
HE 10 I'M + + + + + + +
PGE2 10 nM + + +
Fig. 2 Effect of indomethacin, acetylsalicylic acid and
mefenamic acid on NE- and PGE-induced melatonin re-
lease by rat pineals in organ culture. Shown are
the mean ± SEM (n= 6). Controls differed from all
the remaining groups (p (0.01, ANOVA). For experi-
mental details see ref. 33.
after SCGx is not restricted to the adrenoceptor-mediated

mechanisms. However, differing from the adrenergic supersen-
sitivity phenomenon after SCGx in rat pineal,12 supersensitivi-
ty to PGE 2 is accompanied by an increase in the maximal res-
ponse to the agonist without any shift in the dose-response
curve. 31
The effect of 10 uM NE on melatonin release to the medium

is shown in Fig. 2. Exposure to the adrenergic transmitter
elicited a 13- to 20-fold increase in melatonin release. Ad-
dition of Id, acetylsalicylic acid or mefenamic acid (100 uM)
brought about a si~nificant impairment of melato.nin release
stimulation by NE. 2 However in spite of that at the inhibitor
concentration used PG synthesis is totally blocked (as assessed
by measuring radioimmunoassayable PGE 2 in medium, unpublished
results) a residual 4-6- fold stimulation of melatonin release
was observed. In every case the addition of 10 uM NE and 10 nM
PGE 2 after the PGs synthesis inhibitor resulted in a stimula-
tion of melatonin release that was indistinguishable from that
found with NE alone (Fig. 2).
These results strongly suggest that PGE2 is a physiological

intermediary in NE-controlled melatonin biosynthesis in the
pineal gland. Additionally they suggest that the PG link in
melatonin biosynthetic mechanisms appears to be complementary
rather than necessary inasmuch as supramaximal amounts of
cyclo-oxygenase inhibitors failed to prevent fully melatonin
33
release. A similar observation has been made by oth s in
studies or NE-driven renin release in renal cortex.
The molecular mechanisms through which PGs exert their ef-
fects in the brain remain undefined. PGs appear to be involved
with the regulation of adenyl cyclase- cAMP system either direc-
tly through a receptor-mediated activation of adenyl cycl~se
or indirectly through PG-mediated effects on NE release. 2 ,34,35
In a series of in vitro experiments we assessed the effects of
different PGs on pineal cAMP accumulation and its binding to
intracellular receptors. 31 'A dose-response experiment for the
effects of PGE 2 , PGE 1 , PGF2~ , 15-keto PGF2~ and PGI2 indi-
catedthat only PGE2 increased significantly the cAMP concentra-
tion in doses between 10 and 1000 nM. This effect of PGE2 on
cAMP was also observed in the pineal glands of rats subjected
to SCGx (unpublished results), a finding which sugges~the post-
synaptic nature of the event. Incubation of pineal explants
with 50 nM PGE2 decreased the unoccupied cAMP-binding sites in
pineal homogenates while the total number of binding sites re-
mained unaltered. 31 Since a direct link between the adenyl
cyclase-cAMP system and SNAT induction has been demonstrated
6,8,13,18, it seems feasible that the two observed effects of
PGE 2 ,i.e. increased cAMP and melatonin synthesis, are causally
related.
It has been well established that in addition to the nega-
tive feedback control exerted by NE itself a number of sub-
stances (e.g. PGs) released at the neuroeffector junction may
play a role in modulating adrenergic neurotransmission. 22
PGE 2 locally mobilized by sympathetic nerve stimulation may
counteract further release of NE by a negative feedback mech-
anism. Such an effect is exerted by an action on stimulus-se-
cretion coupling and more specifically on the availability of
calcium. 35
In order to examine the presynaptic effect of PGE2 in the
rat pineal gland we studied the release of NE from pineal
sympathetic nerves, after labelling the endogenous NE stores
with 3H_NE.32 Only 100 nM PGE2 was able to impair significan-
tly NE release in this preparation. Neither PGF2~ nor PGE 1
exerted, at concentrations up to 100 nM, any effect on the
potassium-elicited 3H- NE release from the pineal glands in
vitro.
Therefore the PGE Z braking effect on NE release also ap-

pears to take place ln the pineal neuroeffector junction. It
should be stressed however that concentrations of PGE2 100
times greater than those re~uired to enhance melatonin secre-
tion were needed to affect H-NE release. Interestingly enough
these are the PGE 2 concentrations attained in incubation medium
after exposing bovine and rat pineal explants to NE.29 Thus an
attractive hypothesis concerning the role of PGE2 at the pineal
neuroeffector junction is that at low concentrations (i.e. 1-10
uM, close to the Kd of PG receptor sites 29 ) PGE 2 is an intra-
cellular intermediary (or messenger) involved in NE stimula-
tion of melatonin secretion. Increasing amounts of PGE Z mobi-
lized towards the synaptic cleft would effectively inhlbit NE
release from nerve endings, therefore exerting a negative
feedback on the whole system. Perhaps the simultaneous impair-
ment of dark-induced melatonin synthesis and stimulation of
MAO type A activity (an alleged pineal presynaptic parameter)
reported by us in Id-treated rats,24 were due to a dual effect
of the drug on pre- and postsynaptic compartments by removing
an inhibitory signal for the former and a stimulatory one for
the latter (Fig. 1).
Another postsynaptic signal that may play a role in regulat-

ing NE release from pineal nerve endings is angiotensin II.
Components of the renin-angiotensin system are present in the
pineal gland. Renin 36 and angiotensin 1 3 "/ and 11 38 are detec-
table in intact pineals and cultured pinealocytes, the pineal
being the richest source of renin in the brain. Angiotensin
II is known to increase adrenergic varicosities 39 and seroto-
nin content 40 in the pineal gland. Proteolytic enzymes play
an important role in the production of angiotensin II. Renin
converts renin substrate to angiotensin I (a decapeptide),
and a "converting enzyme" converts angiotensin I to angioten-
sin II (an octapeptide). Angiotensin converting enzyme also
degrades peptides like bradykinin, insulin and enkephalin. 41
Angiotensin converting enzyme was detectable in rat pineal
gland and exhibited a circadian rhythm in activity with maxi-
mum· at the end of the light phase of daily photoperiod. 42 SCGx
or exposure to light for 6 days increased enzyme activity and
obliterated morning-evening differences whereas injection of
the 6-agonist isoproterenol depressed the high levels observed
in SCGx rats. 42 The significant increase in converting enzyme
activity after SCGx strongly suggested that angiotensin II pro-
duction may increase. Indeed in a preliminar·· set of experi-
ments we observed an increase in immunohi~£cich~mically detect-
able angiotensin II after SCGx. 43 Conversely angiotensin I
exhibited a negative correlatien with converting enzyme acti-
vity during the diurnal cycle. 2
The pineal renin-angiotensin syst~m could be relevant for

loc~l control of organ's blood Flow~ It may also facilitate
the release, inhibit the uptake and accelerate the biosynthe-
sis of catecholamines in pineal adrenergic nerves. Lastly it
could have also an endocrine role, inasmuch as the pineal
gland appears to participate in the regulation of hydration. 45
ENDOCRINE-ENDOCRINE TRANSDUCTION
Although environmental lighting is the major input control-

ling pineal activity, hormones secreted by endocrine organs
whose activity is modulated directly or indt6ectly by the pi-
neal also affect the function of the gland. Hormone effects
on the pineal are exerted either directly or through functional
changes in the neural input to the gland. Experiments carried
out in animals injected with labeled steroids, or on subcel-
lular fractions incubated with different radioactive hormones
indicate that the pineal gland of various species (rat, sheep,
cow, rhesus monkey) exhibits protein components which bind
the hormone with high affinity and specifically. To date re-
ceptors for estradiol,47 testosterone,47,48 5oe-dihydrotestos-
terone (DHT),48 progesterone,49 prolactin 2 and melatonin 50 have
been detected in pineal subcellular fractions. Autoradiographic
studies indicated also nuclear binding sites for estradiol and
androgens in rat pineals. 51 Enzymatic activities that convert
testosterone to estrogens,52 as well as progesterone 53,54 or
testosterone 48 to 5 0<.. -reduced metabolites are present in rat
pineal homogenates. Several in vivo and in vitro studies in-
dicate that 8 number of pineal constituents and enzymatic
activities are changed by hormone treatment in mammals. In
both female humans and rats available data support the conclu-
sion that melatonin synthesis and release are depressed at
the time of mid-cycle LH peak (for references see ref. 46).
It is generally accepted that after transversing the plasma

membrane of the cell steroid hormones bind with high affinity
and specificity to receptor proteins and that due to this
interaction an alteration of the receptor occurs, rendering
the hormone-receptor complex significantly more nucleotropic. 55
The translocation of complexes to the nucleus is a direct
consequence of this interaction. As first showed by Jensen and
co-workers for the uterus 56 , deplenishment of cytosol estrogen
receptor (ER) is followed by an active mechanism of R replen-
ishment, partly sensitive to protein synthesis inhibitors (for
references see ref. 55). We have recently examined several as-
pects of this depletion-replenishment cycle of ER in the pineal
gland. The effect of a single s.c. injection of 0.1-20 ug of
estradiol 3 h earlier of cytosol ER and nuclear ER complex is
shown in Fig. 3. A 2 ug-dose resulted in ER values close to
those reported at proestrus during the estrous cycle. 58 Infant
DOSE OF ESTRADIOL <I'g)

Fig. 3 Effect of estradiol (0.1-20 ug, 3 h earlier) on
pineal ER in spayed rats. The concentration of
cytosol ER 57 and nuclear ER complex 58 was estimated
from single point binding assays of pooled pineal
glands (17-28 mg). Shown are the means ± SEM (n= 4
in each group).
rats exhibited about half cytosol ER content as compared to

adult spayed rats, while spayed 20-day-old rats showed inter-
mediate levels (Fig. 4). In all 3 groups estradiol treatment
depressed cytoplasmic ER for up to 18 h, and a significant
increase was found 30 h after injection. These observations
are in general agreement with the results obtained when the
depletion-replenishment pattern of hypothalamic and adeno-
hypophysial ER ~as examined in castrated rats. 59 ,60
The observation that SCGx decreased both cytoplasmic and
nuclea.r estrogen and androgen receptors 47 ,61 and inhibited
hormone-induced increase of pineal protein synthesis 61 provid-
ed the first evidence that the activity of pineal sympathetic
nerves controls gland's sensi tivi ty to hormones. NE injection
restored pineal cytosol estrogen and androgen receptor sites
in SCGx rats; this effect was mediated by a 8 -adrenoceptor
and changes in RNA synthesis. 47 ,61 We have further examined
the interaction of neurotransmitters and ER by studying the
effect of adrenergic drugs on the cycle of depletion-re-
plenishment of cytosol ER. SCGx caused 1 week later a signif-
icant impairment of estradiol induced depletion of cytosol ER
as well as of its nuclear accumulation and replenishment (Fig.5).

Treatment with isoproterenol (1 mg/Kg, 19 and 3 h before, and
simultaneously with estradiol) restored-cytosol ER levels, but
either impaired or blocked the depletion and translocation to
the nucleus induced by estradiol. Blockage of ~ -adrenoceptors
by propranolol (20 mg/Kg) inhibited partially the estrogen-
induced depletion of cytosol ER. Thus although the sympathetic
input was needed to keep the cycle of receptor depletion-re-
plenishment intact, repetitive injection of the ~ -agonist
isoproterenol failed to restore this parameter in SCGx rats.
Perhaps another nerve-originated input besides NE keeps pineal
ER kinetics normal, or the schedule of injection of isoprote-
renol failed to reproduce the normal interaction of endogenous
NE with its pineal 3-adrenoceptors. An answer to this query
was given by the examination of ER kinetics and estradiol bi-
ological effects in pineals of SCGx rats killed 8-18 h after
surgery. During this time period,that elapses for about 10 h~
adrenergic varicosit~s degenerate and the NE released exerts
post-synaptic effects that are indistinguishable from those
observed after stimulation of the sympathetic nerves. 52 In
the rat pineal,degeneration of sympathetic nerves, as measured
18
ELAPSED AFTER 2 I.l9 ESTRADIOL S.C.
Fig. 4 Effect of estradiol on pineal cytosol ER in intact

infant rats~or spayed 20- or 90-day -old rats.
Other details as in Fig. 3.
SCG.
~~ 40~------------~--~~1 40~--------------------'
~ I ~~Prol/pranolol9 1 SCG•• lsoprot
~~
o j1
1 o-9-cr-
1 1
Y- -Y
~ ~
/ /r---r- ------1------ 1
u:=
o 20
I
o
J
OLO.L-~3---,6~------:-12':-------~18~0 0 0 3 6 12
o
18
HOJRS ELAPSED AFTER 2 f.I9 ESTRADIOL S.C.
Fig. 5 Effect of estradiol on pineal ER in spayed rats sub-

jected to SCGx 7 days earlier, and/or isoproterenol
(1 mg/Kg) or propranolol (20 mg/Kg). Drugs were
given as 3 s.c. injections 19 and 3 h before, and
simultaneously with estradiol. Other details as in
Fig. 3.
by the decay of organ's NE content or by the increase in pineal

melatonin levels (unpublished results) starts at the 8-10 h
after SCGx. Translocation of cytosol ER complexes to the nu-
cleus was maximal in estradiol-treated rats 12 h after SCGx,
while it was impaired prior or after the expected nerve ending
degeneration period. Propranolol injection (but not phentola-
mine) prevented the effect of degeneration. Essentially similar
results were obtained when estradiol effects on pineal melato-
nin synthesis was examined at the same time intervals after
SCGx (results submitted for publication). Therefore these data
strongly support the conclusion that the continuous, physiolo-
gical interaction of NE with its receptors is needed to modu-
late pineal ER kinetics.
Another physiological example of neuronal modulation of

pineal responsiveness to circulating hormone signals was given
by studies at different times of the day in rats. In male rats
pineal response to androgens (as well as to other hormones,
e.g. ACTH.63) depends largely on th~ time of day when the

hormone is injected. The extent of stimulation of pineal pro-
tein synthesis brought about by testosterone 64 parallels the
daily rhythm in pineal NE content and turnover 4 and pineal
testosterone receptors 65 in that all these reach maximal
values at night. At this time pineal testosterone aromatiza-
tion was high whereas testosterone 5~-reduction was low. 2
Recently time-dependent effects of testosterone 0g7pineal
electrical activit y66 and cAMP phosphodiesterases have been
reported.
In vitro addition of NE or cAMP mimicked the effect of
night-time on pineal testosterone metabolism; they also de-
pressed progesterone 5 oL-reduction. During the degeneration
of nerve terminals after SCGx an increase in testosterone
arometization and a decrease in 5 eX. -reduction was found
(results submitted for publication). Collectively~the afore-
mentioned results indicate that the over~ll effect of testos-
terone on the pinealocytes depends on the level of activity
of the innervating sympathetic neurons through regulation of
hormone binding sites and metabolism in target cells.
A B
CONTROUED
STEROID-INDUCED INCREASE '"
STPCI'D RECEPnII NEURAL ACTIVITY.
£STRADfOL..
TESTOSTERONE
IELF-IIOOULATION OF STERQD "fECT BY eN_HG NEURAL

INPUT.
Fig. 6. A hypothetic self-modulatory mechanism for estra-

diol and testosterone effects in pineal gland.
i:c ~Co
t 1~0 ~'"
'"
E
"-
1 200
-0
E
UJ
UJ
a:
LJ..
"- 80
Cl 1 2 ] 4 5
Z
:::l PH-QNBJ oM
0
CIl 60
S
z Kd, 1.92 oM
0 ~O
± Kd,2.08 oM
veh.
" 20 lH
50 100 150 200 250 300

[ 3H-ONB] BOUND ( fmollmg prot.)
Fig. 7 Effect of LH (NIH-S-19) (100 ug, 3 and

18 h after orchidectomy) on quinuclydinil
benzylate (QNB) binding in rat SCG; receptor
studies were carriEd out by conventional
techniques 74 • Differences in Bmax are signif-
icant (p <0.05, analysis of covariance).
ENDOCRINE-NEURAL TRANSDUCTION
Several hormones affect NE turnover in pineal sympathetic

nerves. Estradiol or testosterone treatment of castrated rats
increased pineal NE turnover rate,68 whereas LH or FSH injec-
tion depressed it. 69 Changes in intra- and extraneural metabo-
lism of NE in the pineal gland may explain in part those ef-
fects. 26 ,69 Prolactin treatment increased pineal and SCG tyro-
sine hydroxylase activity in rats. 7o Since estradiol or testos-
terone treatment augments NE release from pineal nerve ending,68
and inasmuch as NE is needed absolutely for the steroid hormone
to affect pineal function (see above) a self-modulatory mecha-
nism for hormone action can be hypothesized (Fig. 6).
Some hormones also affect the post-synapsis at the pinealo-
cyte level;for example testosterone injection decreased sig-
nificantly ~-adrenoceptor density in rat pineal gland, as
well as in MBH and cerebral cortex. 71 The inhibitory effect
of testosterone on pineal ~ -adrenoceptors were not mimicked
by estradiol, LH or FSH administration to castrated rats. 71 ,72
It occurred even in the absence of intact sympathetic nerve
terminals indicating its intrinsic post-synaptic nature.
214 D, p, CARDINALI ET AL.
Sex steroid and gonadotropin injection affect NE turnover

and several enzymatic activities related to NE synthesis and
metabolism in rat SCG.26,61,69,70,73. For estradio1 73 and
testosterone 70 specific receptor sites occur in SCG. Other
relevant metabolic activities and constituents of SCG are
also affected by sex steroids, gonadotropins, prolactin and
corticosteroids (for references see ref. 70). For example
LH treatment of castrated rats decreased muscarinic receptor
binding in SCG (Fig. 7), an effect observed also after incuba-
tion of SCG slices with LH in vitro. These data strongly sug-
gest that neuroendocrine integrative processes take place in
the SCG. Such observations can be of physiological importance
in view of the several neuroendocrine efferent pathways of
SCG. 4~, 70, 75-'/7
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THE RESPONSES OF MELATONIN RHYTHMS TO ENVIRONMENTAL LIGHTING
R.J. Wurtman, M.H. Deng and P. Ronsheim

Laboratory of Neuroendocrine Regulation
Department of Nutrition and Food Science
Massachusetts Institute of Technology
Cambridge, MA 02139
Two very powerful generalizations characterize present con-

ceptions of normal pineal function (1-3): In almost all species
examined thus far in laboratory situations, the pineal secretes
more melatonin at nighttime than during the day, and melatonin
secretion is suppressed when the animals are placed in a lighted
environment. Since nighttime for humans coincides with the daily
dark period, investigators have often tacitly assumed that the
second generalization explains the first, - that is, that the cir-
cadian increases in melatonin secretion occurs nocturnally because
the absence of light terminates the retina-mediated suppression of
pineal sympathetic activity, thereby increasing the organ's stimu-
lation by norepinephrine.
This formulation - that melatonin secretion occurs noctur-
nally because nighttime coincides with the absence of sunlight -
overlooks the fact that many nocturnally-active species probably
spend most or all of their lives in light intensities below those
reportedly needed to suppress the pineal. Daytime is passed in
dark burrows within the earth, while nighttime is passed under the
light of the moon and the stars. Thus, full moonlight provides,
on the earth's surface, about 0.05 ~watts/cm2 (4,5), while the
light intensity that, in a laboratory situation, causes a 50%
inhibition of serotonin N-acetyltransferase (6) in rats [or a
reduction in pineal melatonin in hamsters (7)] is about 0.5 ~W/cm2,
or tenfold greater. In neither situation would light intensity be
expected to be sufficient, by itself, to inhibit pineal sym-
pathetic activity and melatonin synthesis. Moreover, when rats
are exposed to "dim light" (0.1-0.3 ~watts/cm2) for twelve hours
daily and then to "darkness" (less than 0.01 ~watts/cm2) or room
221
222 R. J. WURTMAN ET AL.
light (greater than 45 ~watts/cm2) for the other twelve hours, the
dim light is neither inhibitory nor stimulatory to melatonin
synthesis; rather, it is associated with either high or low rates
of melatonin secretion, depending on the intensity present during
the rest of the day (5). If albino rats live under light inten-
sities less than or equal to 0.05 ~watts/cm2, their propensity to
secrete melatonin nocturnally clearly cannot be explained by
postulating that their lighting environment cyclically inhibits
pineal sympathetic outflow. How then are we to understand the
nocturnal entrainment of melatonin secretion among nocturnal
burrowing animals, and how can we relate their melatonin rhythms
to natural ambient lighting?
Any analysis of these relationships requires consideration of
those parameters of light which influence its biological con-
sequences. They include, among other things, its spectrum, its
intensity, and its timing.
The spectrum of sunlight at the earthls surface resembles
that emitted by a theoretical IIblack bodyll heated to 5500 oK, as
filtered through the atmospherels ozone layer. It is continuous
between 290 and 700 nanometers, and thus includes some mid-range
ultraviolet, more long-wave ultraviolet, and major, nearly-equal
proportions (in terms of percents of total energy) of visible
irradiations. The effects of various visible and ultraviolet wave
lengths on the ratls pineal have been examined by assessing their
relative potencies in suppressing HIOMT activity. These potencies
parallel their abilities to activate rhodopsin: yellow-green is
most effective, blue and yellow less so, and red and ultraviolet
not at all (8). This pattern isnlt surprising, given the abundant
evidence that more than 95% of the photoreceptive units in the
ratls retina are rod-type cells. One might anticipate that the
action spectrum for the photic suppression of melatonin secretion
from the humanls pineal will differ considerably from the rat IS,
since human retinas contain several types of photoreceptive cells
and several photopigments. (Parenthetically, it might be noted
that exposing people and laboratory animals to light spectra that
differ considerably from the sunls constitutes an experiment which
investigators mayor may not realize that they are performing: It
would seem wiser to standardize the light spectrum utilized for
photobiologic studies, and to use one that resembles as closely as
possible the spectrum present on the earthls surface.)
The int~nsities of light under which humans and other animals
normally live can vary over 8 or more log units, depending on how
members of each species choose to pass their time: One pressing
task for the student of pineal function involves relating each
species l circadian and circannual patterns of melatonin secretion
to the light intensities that it normally receives. Humans spend
portions of each day within four intensity ranges (Figure .1):
RESPONSES OF MELATONIN RHYTHMS TO LIGHTING 223
104
I HUMAN
THRESHOLD
104
;:;- 10 2
E
.....
u
'"
c;
I 10 2
~
::t..
:: 10° 10°
.....
en
:z
RAT
w THRESHOLD
.....
~
~
~ 10-2 10-2 - '
Q
...J
10 10· 4
~ 0600 1800
TIME OF DAY
Figure 1. Ambient light intensities under various conditions, and

to which a nocturnal, burrowin rodent
mi ht be ex osed on a tical da. Daylight occurs
for 12 hrs, from 0600 to 1800. The threshold light
intensities required in the laboratory to produce
significant suppression of melatonin synthesis (albino
rats, ref. 6) or secretion (human, ref. 8) are taken
from the Literature.
Working out of doors in daytime they are exposed to 2,000 to

20,000 vwatts/cm 2 , or even more; indoor lighting usually provides
50-500 ~watts/cm2; the night light of the moon and stars varies
between 0.001 ~watts/cm2 and, at full moonlight, 0.05 ~watts/cm2;
and the light of a "dark" environment is probably below 0.001
~watts/cm2 or below. The light intensity needed to suppress noc-
turnal melatonin secretion from the normal human's pineal is about
1,000 ~watts/cm2, or greater (9); this is well below that present
out of doors in the daytime, but greater than that usually pro-
vided indoors. As discussed above, two of these ranges are above
the intensities needed in the laboratory to suppress the rat's
pineal (daytime outdoor light and interior light), and two are
below (nighttime outdoor light and "darkness").
If albino rats are placed in a cage that allows them to
choose between being exposed to ambient laboratory light or to the
darkness of an artificial burrow (less than 0.01 ~watts/cm2), and
if the ambient light intensity is in the usual indoor range (66
~watts/cm2) for twelve hours and in the nocturnal moonlight range
(0.03 ~watts/cm2) for the other twelve hours, we find that the
animals spend almost all of the "daylight" period in the dark
burrow, and all of the "nighttime" in the outer cage (4,10).
Since the light intensity at nighttime is greater than that In the
burrow (0.03 vs. 0.01 ~watts/cm2), nighttime actually coincides
with the animals's daily light period. This schedule probably
simulates the animal's schedule in the wild state, where rats are
nocturnally active under the light of the moon and stars. It
contrasts, of course, with the situation for humans - who are
active under sunlight or indoor light, and inactive during the
dark period, sleeping in darkness indoors. Melatonin secretion
from the rat's pineal is now also maximal during the animal's
light period. The generalization that peak melatonin secretion
coincides with nighttime is thus preserved, but at the apparent
expense of the generalization that it coincides with the daily
dark period.
If an additional constraint is now imposed on the animals
such that, once they enter the burrow each day, they are required
to remain there until just after the onset of "moonlight," the
amplitude of the pineal melatonin rhythm diminishes markedly,
suggesting that either it has become free-running - and its peaks
and valleys have been missed - or that it has in fact disappeared.
Now animals spend their 24-hour days exposed alternately to two
light intensities which are both below that shown experimentally
to suppress melatonin's synthesis and secretion. If the rats are
again allowed to leave the burrow at intervals, then the melatonin
rhythm recurs and its peak is re-entrained to nighttime. Such
sampling of the outside world need only be very brief, - a minute
or two - a finding compatible with the evidence (11) that
similarly-brief light pulses are adequate to suppress melatonin
RESPONSES OF MELATONIN RHYTHMS TO LIGHTING 225
synthesis for hours, if delivered at times of day appropriate for

the species. (Thus the importance of the timing of light exposure.)
Perhaps one stimulus that keeps the albino rat's melatonin
rhythm entrained to the daily light cycle - and causes its secre-
tion of melatonin to be maximal during nighttime - is these brief,
daytime exposures to bursts of intense (for the albino rat) ambient
light. This formulation allows the two seemingly-contrary general-
izations to coexist (Figure 1). Melatonin secretion is again
both nocturnal and suppressed by light; its entrainment to night-
time - the albino rat's daily light period - occurs because the
animal is sporadically exposed to intense light during the day.
(Of course the formulation ignores the fact that other sensory
inputs, besides those generated by light, affect behavior and
pineal function.) It-will be interesting to see if similar expla-
nations, relative to how each species actually chooses to live,
can be adduced for the patterns of melatonin secretion found in
other mammals besides albino rats and people. [That these pat-
terns might exhibit great variety is suggested by the finding that
melatonin is secreted nocturnally when sheep are exposed to long
days (and short nights), but barely at all when the animals are in
long nights (12).J In any case, an appreciation of light's photo-
biologically-important parameters, and of how species behave in
relation to normal variations in their lighting environment, will
probably be very important in unravelling the ways that mammals
use their pineals.
ACKNOWLEDGEMENTS
These studies were supported by a grant from the National
Institutes of Health (HD-11722). The experiments on animals with
access to burrows were conceptualized and conducted by Or. Harry
Lynch.
REFERENCES
1. Wurtman, R.J., J. Axelrod, and D.E. Kelly, "The Pineal,"
Academic Press, New York, 1968.
2. Wurtman, R.J. and M. Moskowitz, Medical Progress: The Pineal
Organ. N. ~. ~. Med. 296:1329-1333 (1977);
296:138J-l~6-{197rr:-
3. Waldhauser, F. and R.J. Wurtman, The secretion and actions of
melatonin, in: "Biochemical Actions of Hormones," G.
Litwack, ed., vol. 10, Academic Press, New York (in
press) •
4. Lynch, H.J., R.J. Wurtman, and P. Ronsheim, Activity and

melatonin rhythms among rats with recourse to dark
burrows, in: "The Pineal and Its Hormones," R.J. Reiter,
ed., Alan R. Liss, New York (1982).
5. Lynch, H.J., R.W. Rivest, P.M. Ronsheim, and R.J. Wurtman,
Light intensity and the control of melatonin secretion in
rats, Neuroendocrinology 33:181-185 (1981).
6. Minneman, K.P., H. Lynch, and R.J. Wurtman, Relationship
between environmental light intensity and retina-mediated
suppression of rat pineal serotonin-N-acetyltransferase,
Life Sci. 15:1791-1796 (1974).
7. Brainard~.C., B.A. Richardson, L.J. Pettibone, and R.J.
Russel, The effect of different light intensities on
pineal melatonin content, Brain Res. 233:75-81 (1982).
8. Cardinali, D.P., F. Larin, and R.J.Wurtman, Action spectra
for effects of light on hydroxyindole-O-methyl transferase
in rat pineal, retina and harderian gland, Endocrinology
91:877-886 (1972).
9. Lewy, A.J., T.A. Wehr, F.K. Goodwin, D.A. Newsome, and S.P.
Markey, Light suppresses melatonin secretion in humans,
Science 210:1267-1269 (1980).
10. Lynch, H.J., M.H. Deng, P. Ronsheim, and R.J. Wurtman, Daily
rhythms in activity and melatonin secretion among rats in
a naturalistic environment, Abstract, Soc. Neurosci.
Minneapolis, Minn., November, 1982. ----
11. Illnerova, H., J. Vanecek, L. Wetterberg, and J. Saaf, Effect
of one minute exposure to light at night on rat pineal
serotonin-N-acetyltransferase and melatonin, J. Neurochem.
32:673 (1978). -
12. Lincoln, G.A., O.F.X. Lameida, H. Klandorf, and R.A.
Cunningham, Hourly fluctuations in the blood levels of
melatonin, prolactin, luteinizing hormone, follicle-
stimulating hormone, testosterone, tri-iodothyronine,
thyroxine, and cortisol, in rats under artificial photo-
periods, and the effects of cranial sympathectomy, J.
Endocrinol. 92:237-250 (1982). -
THE ROLE OF LIGHT AND AGE IN DETERMINING MELATONIN
PRODUCTION IN THE PINEAL GLAND
Russel J. Reiter
The University of Texas
Health Science Center at San Antonio
San Antonio, TX 78284
INTRODUCTION
Roughly two decades ago it was discovered that the photo-

periodic environment to which animals are exposed determines the
structure (Quay, 1956) and metabolism (Quay, 1963; Wurtman et
al., 1963) of the mammalian pineal gland. Shortly thereafter, it
was uncovered that the ability of the pineal to influence the
reproductive system also depended on the photoperiod (Czyba et
al., 1964; Hoffman and Reiter, 1965). This series of experiments
provided not only an impetus for research on the pineal gland but
also a renewed interest in the impact of light and darkness on
the organism. In the intervening years the enthusiasm was
translated into investigations that have definitively shown that
the daily light:dark cycle provides the primary regulatory
influence on the pineal gland. When the role of the photoperiod
is discussed relative to the pineal, typically, indoleamine
synthesis is considered (Cardinali, 1981) along with the hormonal
effects of the pineal gland on the neuroendocrine-reproductive
axis (Reiter, 1980). It is the purpose of the present survey to
summarize the influence of light and darkness on indole
metabolism within the pineal gland. Additionally, the resume
will consider age of the organism as a factor in determining
pineal melatonin production.
227
228 R. J. REITER
IMPACT OF LIGHT AND DARKNESS ON MELATONIN
Neural connections between eyes and pineal
In many non-mammalian vertebrates the pineal responds

directly to light rays which reach the pineal gland after
penetrating the skulL In mammals, however, it is light and
darkness perceived by the lateral eyes which is critical in
determining serotonin metabolism within the pineal gland.
Because of this indirect action of photoperiod on the pineal,
mammals have had to evolve a system of cummunicating information
about the photoperiod, which is detected by the eyes, to the
pineal gland. There were at least two means available whereby
the retinas could signal the pineal gland. The retinas could
either send a hormonal signal to the gland via the blood vascular
system or it could transfer the information to the pineal over a
neural route i during evolution mammals opted for the latter
means. It is now known that photic information perceived by the
Pineal gland
J
Post·ganglionic
I sympathetic neuron
Superior
cervical ganglion
Upper
thoracic
cord
'" Pre-ganglionic
Intermediolateral ~ sympathetic
cell column
neuron
Fig. 1. Schematic representation of the neural connections

between the eyes and the pineal gland as they are
currently believed to exist in mammals. Information
about the light:dark cycle reaches the pineal primarily
via the peripheral sympathetic nervous system. Other
fibers from the brain probably enter the pineal stalk
and eventually terminate within the gland. SCN =
suprachiasmatic nuclei.
LIGHT AND AGE IN MELATONIN PRODUCTION 229
retinas is transduced into a neural message which is subsequently

transferred over a complex series of neurons to reach the pineal
gland (Fig. 1). The neural signal reaches the hypothalamus by
means ofaxons of ganglion cells which terminate in the
suprachiasmatic nuclei (SCN); in the rat, fibers from a given
retina end primarily, although not exclusively, in the
contralateral SCN. Axons of SCN neurons theoretically project to
the medial basal hypothalamus where another synapse is made and
then the neural message is transferred to the lateral
hypothalamic area (Moore, 1978). From this location long
descending axons presumably carry the transduced photoperiodic
message to the intermediolateral cell column of the upper
thoracic cord; this cell column is composed of preganglionic
sympathetic neurons whose axons emerge in the ventral roots of
the thoracic spinal nerves. They pass up the sympathetic trunk
to eventually make a terminal synapse in the superior cervical
ganglia; these ganglia lie in the carotid sheath at the division
of the common carotid into the internal and external carotid
arteries. Postganglionic axons follow blood vessels into the
skull and eventually, in the company of the arterial supply to
the gland, the nerves arise in the vicinity of the pineal.
Before they penetrate the gland they form one or two rather
distinct nerve bundles, the nervi conarii (Kappers, 1960). After
penetrating the capsule of the gland the nerve bundles break up
into smaller fascicles which ramify among the parenchymal
elements; they terminate primarily in the pericapillary spaces
but occasionally endings are also found among parenchymal cells
(Matsushima et al., 1981).
The sympathetic nerve endings which terminate within the

pineal gland reportedly release norepinephrine (NE) especially
during the dark phase of the light:dark cycle. The catecholamine
subsequently acts on ~-adrenergic receptors on the pinealocyte
membrane (Zatz, 1981). NE as a neurotransmitter within the
pineal has been proven in only a small number of animals, e.g.,
whereas it stimulates the metabolism of serotonin to melatonin in
the rat pineal (Zatz, 1981) this has not been shown to be the
case for the Syrian hamster (Lipton et al., 1982). The released
neurotransmitter, be it NE or some other compound, interacts with
S-receptors which are linked to an adenylate cyclase-cyclic AMP
system (Klein et al., 1981). Via this mechanism the conversion
of serotonin to melatonin is promoted (Cardinali, 1981). The two
pineal indices which are most frequently measured are the
increased acetylation of serotonin by the enzyme N-acetyl-
transferase (NAT) and the tissue levels of melatonin. Both these
parameters increase during the daily dark period; the increases
are often parallel. Once produced melatonin seems not to be
stored within the gland but rather it is rapidly released.
Hence, blood levels of melatonin are closely related to the
230 R. J. REITER
production of the indole in the pineal gland (Wilkinson et al.,

1981).
Under either artificial or natural photoperiodic conditions

the daily period of light is associated with low levels of·pineal
NAT activity (Klein et al., 1981) and melatonin content (Lynch,
1971; Rollag et al., 1980a) and depressed titers of circulating
melatonin (Wilkinson et al., 1977).
Light:dark cycle and patterns of melatonin production
Sometime during the daily dark period, pineal melatonin

production rises and, as a consequence, blood levels of the
constituent also increase (Panke et al., 1978; Brown et al.,
1981). However, the pattern of the nocturnal pineal production
of melatonin varies among species (Fig. 2). For example, when
Syrian hamsters are kept under 14 hours of light and 10 hours of
darkness (LD 14:10) daily, the first 4-6 hours of dark exposure
is associated with essentially no rise in pineal melatonin
content; thereafter, pineal melatonin levels increase rapidly to
Pattern of
Melatonin Production Description Exomples
peak late
in dark period
_..!/"Il •i.••. •
.
peak near
middle of
dark period
I
albino rat
Richardson's ground squirrel
13-lined ground squirrel
eastern chipmunk
I
·.
•.•.•.•.
· •••.•.....•..•..••.•.••.•••.•..•..••.•.•..••.•..•.••.••
.VA·
Turkish hamster
white-foated mouse
prolonged peak
r during majority
of dark period
"Cotton rot
djungarion hamster
Time
Fig. 2. Patterns of nocturnal melatonin production in rodents

toat have been studied to date. Whereas some species
tend to exhibit a brief rise in pineal melatonin
levels, in others elevated melatonin levels may be
measured throughout the dark period.
reach a peak roughly 8 hours after darkness onset (Panke et al.,

1979). Conversely, in the albino rat kept under the same photo-
periodic conditions peak pineal melatonin levels are usually
reached as soon as 4-5 hours after darkness onset with a gradual
sustained drop thereafter (Johnson et al., 1982). In the
Richardson's ground squirrel (Spermophilus richardsonii) , a
diurnally active rodent, pineal melatonin levels rise quickly
after darkness onset and begin to drop soon thereafter (Reiter et
aL, 1981b). Finally, in a number of species melatonin levels
rise rapidly during darkness and remain elevated during the
majority of the dark period; examples include the Djungarian
hamster (Phodopus sungorus) (Goldman et aL, 1981), the cotton
rat (Sigmodon hispidus) (Matthews et al., 1982), and the white-
footed mouse (Peromyscus leucopus) (Petterborg et al., 1981). If
the daily period of darkness to which these species are exposed
is increased by 4 hours, i.e., to LD 10:14, melatonin levels
remain elevated virtually throughout the daily dark period. In
these species i t appears as if the pineal gland may actually
measure the length of the daily dark period, at least within the
limits in which ~he system has been tested. In species such as
the Syrian hamster where a single short term peak of melatonin
occurs, prolonging the daily dark period seems not to greatly
alter the duration or the timing of the nocturnal melatonin peak
(Tamarkin et al., 1979; Reiter, 1981).
How these different patterns of melatonin relate to the

ability of the pineal to inhibit reproduction remains unknown.
In cotton rats, white-footed mice, and Djungarian hamsters it may
be the increased and prolonged rise in melatonin which initiates
gonadal involution when these species are placed under short day
conditions (Petterborg et al., 1981). In the Syrian hamster, the
mechanisms may be different inasmuch as the quantity of melatonin
produced by the pineal gland of hamsters kept under artificially
regulated long or short days varies rather minimally, yet under
reduced photoperiods (less than 12.5 hours of light daily)
gonadal regression ensues (Elliott, 1976). Perhaps in this
species there are two rhythms, i.e., the melatonin rhythm and a
rhythm in the sensitivity of the neuroendocrine-reproductive
system to melatonin (Fig. 3). Under long day conditions these
rhythms are out of phase and melatonin, although synthesized and
secreted, does not inhibit reproduction. On the other hand,
under short day conditions peak melatonin levels coincide with an
increased sensitivity of the animal to the indole; as a result,
reproductive collapse follows. This is an example of an internal
coincidence model. Assuming there are, in fact, two rhythms (the
melatonin cycle and the sensitivity fluctuation), what mechanisms
are brought into play to bring them in and out of phase with each
other remains unknown. Finally, under natural photoperiodic and
temperature conditions the actual amount of melatonin produced
wi thin the pineal may vary seasonally (Brainard et al., 1983).
232 R. J. REITER
Cause of
Gonadal
[..,-~
Long Days Short Days Atrophy
coinciding
with
sensitivity
n
c:
0
:l
1 period
e
-0
a.. prolongotion
c:
·c of melatonin
.E
[1
peak
1
0
"ii
::'i!
"0
Gl
c:
a:: increased
height
of melatonin
1 [1 peok
Time
Fig. 3. Theoretical means whereby melatonin may induce its

endocrine influences. As seen in the top panel, in
some species there may be two rhythms, i.e., the
melatonin rhythm and the cycle of sensitivity to
melatonin (cross-hatched bar) Only when the melatonin
peak coincides with the increased sensitivity is the
indole amine capable of exerting its effects.
Alternatively, perhaps the amount of melatonin produced
[due to a prolongation of the melatonin peak (middle
panels) or to an increased height of the melatonin peak
(bottom panels)] determine its action.
Influence of intensity on melatonin production
Animals seem to vary greatly in reference to the sensitivity

of their pineal gland to light. In nocturnally active rats,
Syrian hamsters and Djungarian hamsters born in captivity and
raised under laboratory photoperiods, prolongation of normal
artificial room light (intensity of 1000 lux or about 172 ~W/cm2
and much lower intensities as well) into the night prevents the
normal nocturnal rise in pineal melatonin levels (Panke et al.,
1979; Tamarkin et al., 1979). Likewise, the abrupt exposure of
these animals to light at night when pineal melatonin levels are
high leads to a precipitous decline in the melatonin content of
the pineal gland (Rollag et al., 1980b; Brainard et al., 1982b).
In the case of the Syrian hamster, the intensity of light

required to cause such a decline is very low, on the order of 1
lux {about 0.186 ~W/cm2.
There are some other species, however, which seem to be less

sensitive to light in terms of the inhibition of nocturnal
melatonin levels. For example, in the human it has been shown
that the intensity of light at night required to inhibit serum
melatonin titers (Vaughan et aL, 1976; Lewy et aL, 1980) is
greater than 500 lux (about 86 ~W/cm2); this is much higher than
the intensity which suppresses pineal melatonin production in the
Syrian hamster pineal gland (Brainard et al., 1982b). When these
two species are compared it is important to keep in mind that the
human is usually diurnally active whereas the hamster is a
nocturnal species. This could account for differential response
to light. With this is mind, we examined pineal melatonin levels
in two other diurnally active species, the Richardson I s ground
squirrel (Hurlbut et aL ,1982; Reiter et aL, 1982a) and the
Eastern chipmunk (Tamias striatus) (Reiter et al., 1982b; 1982c),
after their exposure to light during the normal dark period. As
with the human, in neither species did normal room light suppress
the rise in pineal melatonin normally associated with darkness
(Fig. 4). Thus, these diurnal species responded in a manner
similar to the human, i.e., they were considerably less sensitive
to light than the nocturnal species previously investigated
(Panke et al., 1979; Tamarkin et al., 1979; Rollag et al., 1980b;
Brainard et al., 1982b). Do these differences really relate to
the fact that the human, the Richardson I s ground squirrel, and
the Eastern chipmunk are diurnal while the other species examined
are nocturnal? There is another difference between these two
groups of animals. The diurnally active species were exposed to
sun light during at least a portion of the life history while the
rat and hamster were born in captivity and were raised under
artificial lighting of the laboratory. Sun light has a much
higher intensity (up to 100,000 lux) than do most artificial
lights currently in use in animal rooms (approximately 1000-1500
lUx). If the intensity of light to which animals are exposed
during the day in fact determines what they interpret as darkness
(Rivest et al., 1981; Lynch et al., 1981) then the results sum-
marized above may not relate to whether the animals are diurnal
or nocturnal but rather to their previous lighting history.
Perhaps higher intensity light during the day, i.e., sun light,
makes them insensitive to relatively lower intensity room light
at night. Conversely, animals that are exposed to artificial
room light during the day are much more sensitive to light at
night. If this latter interpretation has validity, then perhaps
raising rats or Syrian hamsters under natural sun light would
also render them insensitive to room light at night. We have
evidence that the diurnally active 13-lined ground squirrel
(Spermophilus tridecemlineatus) , born in captivity and exposed
234 R. J. REITER
Richardson's Ground Squirrel

~ 20 oLD 14:(0
"t>
oLL
~
>-., 16
~.S
.:l.a
+=E
12
ti~
Z>.
5i li 8
.5 ~
a.z 4
Time
Fig. 4. Pineal N-acetyltransferase activity (top panel) and

melatonin content (bottom panel) in the Richardson IS
ground squirrel. Animals were either kept in 10 hours
of darkness (LD 14: 10) during the night they were
killed or they were kept in continual room light (LL)
(approximately 4,000 lux or 925 ~W/cm2). Although
light exposure at night slightly depressed the rhythms
in NAT and melatonin they were not prevented by this
treatment procedure.
throughout its life to artificial room light, responds to low

intensity light at night in the same manner as does the rat and
hamster (Reiter et al., unpublished observations).
Influence of wavelength on melatonin production
Besides light intensity, it is of interest to determine what

wavelengths of light are most important in influencing pineal
melatonin levels. These interactions have been only sparingly

investigated. Recently, we used the Syrian hamster to examine
which wavelengths of visible light are most efficient in
depressing nocturnal pineal melatonin (Brainard and Reiter,
1982). During peak nocturnal melatonin production groups of
hamsters were exposed to light sources with one half peak band
widths of 339-371 nm (near ultraviOlet), 435-500 nm (blue),
515-550 nm (green), 558-636 nm (yellow), and 635-770 nm (red).
Animals were exposed to each color at an intensty of either 0.928
(about 5.4 lux) or 0.200 ~W/cm2 (about 1.16 lUX). At the 0.928
~W/cm2 intensity, the blue, green and yellow light sources led to
a 70-80% reduction of pineal melatonin within 20 minutes; the
near ultraviolet light caused a 40% drop while the red light
source was ineffective in reducing pineal melatonin in the Syrian
hamster. At o. 200 ~W/ cm 2 , the blue and green light caused a
70-90% depression of pineal melatonin while near ultraviolet,
yellow and red lights did not depress pineal melatonin levels
during the 20 minute exposure period. Thus, at least in the
Syrian hamster visible light of the blue and green wavelengths of
the spectrum seem most efficient in suppressing pineal melatonin
when the animals are abruptly exposed to these light sources at
night.
To attempt to distinguish which wavelength of visible light,

that is, blue or green, was most effective another experiment was
performed in which animals were exposed to these wavelengths at
the following intensities: 0.186, 0.074 or 0.019 ~W/cm2. Again,
the animals were killed after 20 minutes exposure and pineal
melatonin levels were estimated (Brainard et al., 1982a). At
each of the intensities tested, blue light caused a 20-25%
greater depression of pineal melatonin than did the green light,
thus indicating that blue wavelengths of visible light are the
most efficient wavelengths of those tested to date in depressing
pineal melatonin levels in laboratory born Syrian hamsters.
IMPACT OF AGE ON MELATONIN
Although it has been assumed for years, with little

substantive evidence that pineal function diminishes with age,
only recently has specific information on pineal melatonin pro-
duction in aging animals been reported. The circadian rhythm in
pineal melatonin levels in 2-month-old and 18-month-old male and
female hamsters were compared (Reiter et al., 1980b). During the
daily light period the pineal gland of both young and old
hamsters contained equivalent amounts of melatonin (roughly 100
236 R. J. REITER
pg/gland). During the 10 hour period of darkness pineal

melatonin levels in the 2-month-old male and female hamsters rose
to 835 and 887 pg/ gland, respectively. In both sexes, peak
melatonin levels were achieved at 0400 hours, 8 hours after
darkness onset. By comparison, in the old animals peak melatonin
levels reached only about 200 pg/gland. This marked reduction in
pineal melatonin levels in aging Syrian hamsters could be related
to a number of factors, the most likely of which may be the
reduced sensitivity of the ~-adrenergic receptors on the
pinealocyte membrane to the sympathetic neurotransmitter within
the gland (Greenberg and Weiss; Reiter et al., 1982d). Besides
pineal levels of the indole, plasma titers of immunoreactive
melatonin are also diminished in aging Syrian hamsters (Reiter et
al., 1982e) (Fig. 5).
In a detailed study using albino rats, Reiter and colleagues

(1981a) also found a marked reduction in pineal melatonin pro-
duction during the aging process. In this study both pineal NAT
activity levels and immunoreactive melatonin concentrations were
compared in 2, 12 and 29-month-old female rats killed either
during the day or during darkness. Whereas pineal NAT values
varied little among the three age groups, melatonin values
differed greatly. Although daytime values of pineal melatonin
Time
Fig. 5. Schematic diagram of pineal and plasma levels of

immunoreactive melatonin in young and old rodents.
Perhaps both daytime and nighttime levels of melatonin
are reduced in old animals. It may be important that,
although reduced, weak rhythms may still persist.
did not vary among the three groups, at night the 29-month-old
rats exhibited a weak rise in pineal melatonin levels compared to
that of the 2-month-old animals; the 12-month-old rats had night-
time melatonin levels between those of the other two groups.
These findings suggest that there may be a gradual (as opposed to
an abrupt) reduction in the production of pineal melatonin as
these animals age.
Finally, in Mongolian gerbils the pineal production of

melatonin also appears to be altered as animals become older
(Reiter et al., 1980a; King et al., 1982). Of special interest
in this species is that the pineal gland becomes heavily
calcified and the nocturnal levels of melatonin, even in young
animals, are much lower than in other rodents studied to date.
Perhaps the weak melatonin rhythm in the pineal of this species
is related to the high degree of pineal calcification.
CONCLUDING REMARKS
The patterns of nocturnal melatontn production vary con-

siderably even among closely related rodent species. Whether
these different patterns or alterations of these patterns during
different light:dark cycles determine the action of melatonin in
a given species remains to be determined. Both the intensity and
the wavelength of light seem important in influencing pineal
melatonin levels. The importance of light intensity in altering
nocturnal pineal melatonin concentrations may be related to the
activity patterns of the animals, i.e., whether they are
nocturnal or diurnal, or to their previous lighting history.
This remains an area of interest for future pineal research.
Also, the role of different wavelengths of light in determining
pineal melatonin synthesis will undoubtedly receive continued
investigation in the future. Finally, it appears at least in
rodents, that the age of the animal may be important in
determining the ability of the pineal to produce one of its
hormonal constituents, melatonin.
REFERENCES
Brainard, G. C., and Reiter R. J., 1982, The influence of

different light spectra on pineal melatonin content in the
Syrian hamster, Abstr. Endocrine Mtg., in press.
Brainard, G. C., Richardson, B. A., King, T. S., and Reiter, R.
J., 1982a, The influence of low irradiances of blue and
green light on pineal melatonin content in the Syrian
hamster, Abstr. Neurosci. Mtg., in press.
238 R. J. REITER
Brainard, G. C., Richardson, B. A., Petterborg, L. J., and

Reiter, R. J., 1982b, The effect of different light
intensities on pineal melatonin content, Brain Res., 233:
75.
Brainard, G. C., Petterborg, L. J., Richardson, B. A., and
Reiter, R. J., 1983, Pineal melatonin in Syrian hamsters:
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laboratory and natural conditions, Neuroendocrinology, in
press.
Brown, G., Grota, L., and Niles, L., 1981, Melatonin: Origin,
control of circadian rhythm and site of action, in:
"Melatonin - Current Status and Perspectives", N. Birau and
W. Schloot, eds., Pergamon, New York.
Cardinali, D. P., 1981, Melatonin. A mammalian pineal hormone,
Endocr. Rev., 2: 327.
Czyba, J. C. ,Girod, C., and Durand, N., 1964, Sur I' antagonisme
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Morgan, W. W., Vaughan, M. K., Johnson, L. Y., and
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Craft, C. M., 1982e, Reduced pineal and plasma melatonin
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Rivest, R. W., Lynch, H. J., Ronsheim, P. M., and Wurtman, R. J.,
1981, Effect of light intensity on regulation of melatonin
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Rollag, M. D., Panke, E. S., and Reiter, R. J., 1980a, Pineal
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and Reiter, R. J., 1980b, Quantification of daily melatonin
synthesis in hamster pineal gland, Endocrinology, 106: 231.
Tamarkin, L., Reppert, S. M., and Klein, D. C., 1979. Regulation
of pineal melatonin in the Syrian hamster, Endocrinology,
104: 385.
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Wilson, K. M., Sandock, K. J., Vaughan, M. K., Kaslow, S.
H., and Reiter, R. J., 1976, Nocturnal elevation of plasma
melatonin and urinary 5-hydroxyindole acetic acid in young
men: Attempts at modification by brief changes in
environmental lighting and sleep and by autonomic drugs, ~.
Clin. Endocr. Metab., 42: 752.
Wilkinson, M., Arendt, J. Bradtke, J., de Ziegler, D., 1977,
I
Determination of a dark-induced increase of pineal N-

acetyl transferase activity and simultaneous radioimmunoassay
of melatonin in pineal, serum and pituitary tissue of the
male rat, J. Endocr., 72: 243.
Wurtman, R. J.,-Axelrod, J., and Phillips, L. S., 1963, Melatonin
synthesis in the pineal gland: Control by light, Science,
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Zatz, M., 1981, Pharmacology of the pineal gland, in: "The Pineal
Gland, Vol. I, Anatomy and Biochemistry", R. J. Reiter, ed.,
CRC Press, Boca Raton.
DIFFERENTIAL REGULATION OF THE 24 HOUR PATTERN
OF SERUM MELATONIN AND N-ACETYLSEROTONIN
G.M. Brown, L.J. Grota*, L. Harvey,

H.W. Tsui, and S.F. Pang**
Department of Neurosciences
McMaster University, Hamilton, Ontario
*Department of Psychiatry, University of Rochester
**Department of Physiology, University of Hong Kong
INTRODUCTION
While conducting a series of investigations with the object-
ive of developing a highly specific radioimmunoassay for melatonin
(MEL), we developed two assay systems one capable of measuring
both MEL and N-acetylserotonin (NAS) activities, the other capable
of measuring melatonin specifically. Results of investigations
using these assays provided strong evidence that the 2 substances
have different 24 hour patterns in serum. We have therefore under-
taken the development of a highly specific radioimmunoassay for
NAS and have used it to conduct a more detailed examination of this
issue. Current findings indicate that dissociation of the 24
hour pattern of serum MEL and NAS occurs under conditions of short
photoperiod. These findings suggest that different mechanisms may
be involved in regulation of circulating NAS as compared to MEL.
DEVELOPMENT OF MANNICH ANTISERA TO MEL AND NAS

In 1974, we reported on production of antiserum capable of
binding NAS and MEL equally and with high affinity, but discrimin-
ating against a large number of other indoleamines (Grota and
Brown, 1974). The antigen used in these investigations was N-acetyl-
serotonin coupled to bovine serum albumin uSing formaldehyde con-
densation (Mannich reaction). Subsequently, using MEL coupled to
bovine serum albumin in the same manner, we succeeded in obtaining
specific antisera which discriminated MEL from NAS and a wide
variety of other indoles (Pang et al., 1977).
243
244 G. M. BROWN ET AL.
The two types of antisera appeared to differ primarily in

their ability to discriminate between substitutions on the indole
molecule in or around the five position. We teasoned that the site
of conjugation for the NAS conjugate must be adjacent to the 5
position of the indole ring so that antibodies stimulated by the
conjugate would not discriminate between groups in this region
although they would discern changes at the amino end-of the mol-
ecule. Our findings on cross-reactivity of the NAS conjugate
were subsequently confirmed by Kennaway and co-workers (1977).
Since the MEL conjugate stimulated antisera which showed discrim-
ination at both the five and the amino positions, we inferred that
this conjugate was coupled at a site remote from both of those
positions. We have recently investigated this antigen further
on the grounds that it might serve as a useful antisera for other
indoleamines.
STUDIES ON MEL MANNICH ANTIGEN

Two approaches were used to determine the site of conjugation
of MEL-M-8SA. In one approach we reacted model amines with for-
maldehyde and MEL, isolated the intermediates and chemically
identified the reaction products. We found that carbon 2 was the
most likely site of methylene bridge formation for the hapten
MEL, based on nmr analysis of reaction products with glycine-ethyl-
ester or piperdine (Grota et al., 1981). In the second approach
we utilized cross-reactivity of antisera stimulated by the con-
jugate. The analysis showed that the presence of 5-methoxy, 6-
unsubstituted, unsubstituted indole nitrogen, and N-acetyl(side
chain) were all necessary for the binding of indolealkylamine to
the antisera. See (Pang et al., 1977) for details. This approach
was consistent with the chemical analysis which indicated that
the methylene bridge coupling melatonin to BSA occurred at carbon
2 of the indole nucleus. Consistent with these observations are
the data of Besselievre et al., (1980) and Wolinsky and Sundeen
(1979) which show respectively that analogues of MEL with alkyl
substitutions at the indole nitrogen do not form conjugates with
protein in the formaldehyde reaction and that formaldehyde coup-
ling of the indole to prolactin is unstable if it occurs at the
indole nitrogen. Taking these data together, it appears that mel-
atonin reacts with formaldehyde at the indole nitrogen initially
but this binding forms a substitution which is somewhat unstable
and the eventual stable bond is formed at Carbon 2 (Fig. 1).
One implication of our data is that the methylene bridge
between hapten and the free amino groups on the protein occurs at
different sites depending on the substitutions on the indole hap-
tens. If a 5-hydroxy substitution is present, formaldehyde con-
jugation will occur at position 4. If there is a 5-methoxy sub-
stitution then coupling will occur at Carbon 2 provided that the
REGULATION OF SERUM MELATONIN AND N-ACETYLSEROTONIN 245
CH 0C~NHCOCH3
3 ~I
~
_.R,.u_
N CH2
I I
H Protein
M elatonin-M-Protein
Protein
I
CH2
HO~NHCOCH3
~ I N
I
I
H
N-acetylserotonin-M-Protein
Fi gure 1
indole nitrogen is unsubstituted. The above holds true for N-

acetylated compounds. It should be pointed out that the formalde-
hyde reaction occurs readily with free amino groups. Therefore,
a free amino on the side chain of an indole will also react with
formaldehyde to produce either polymerization of the indoles and/
or coupling to the phenolic hydroxy groups of tyrosine in the
protein. Furthermore, under certain conditions B-carboline may be
formed if the amino group is free.
Based on the foregoing it is possible to devise a generalized
strategy using the formaldehyde condensation reaction to produce
antisera that will bind individual indolealkylamines: reversably
block the terminal amino group and the 5-hydroxylated substitution
before coupling with formaldehyde. This approach ensures that
coupling occurs at Carbon 2. The blocks are removed after con-
jugation to provide a hapten - protein conjugate with a hapten
of the desired configuration. The major advantage of this
approach is that coupling of indolealkylamine to protein using
this strategy will result in haptens where the site of coupling
of hapten to protein occurs at relatively great distances from
those groups which differ among the various indoleamines and

which are the active metabolic sites in vivo. One should there-
fore be able to produce antisera that specifically bind individual
biologically significant, indolealkylamines.
RADIOIMMUNOASSAY OF N-ACETYLSEROTONIN AND MELATONIN USING A SUB-

TRACTION METHOD
1. Validation
Using the two antisera described above, radioimmunoassays for
MEL and for N-acety1serotonin plus melatonin were developed. For
melatonin detailed cross-reactivity studies were completed, satis-
factory parallelism was demonstrated, and satisfactory data on
assay variation were presented (Pang et al., 1977). In addition,
melatonin estimates in pineal were crossvalidated by GCMS (Pang
et al., 1977). Crossvalidation of these radioimmunoassays in
serum however, has been difficult because of lower serum levels of
MEL. For human serum, crossva1idation with a number of other MEL
radioimmunoassays has been accomplished (Wetterberg 1977, Wetter-
berg and Eriksson 1981); however, the comparison of results ob-
tained using such similar techniques may not permit the identifi-
cation of problems unique to radioimmunoassay. The recent develop-
ment of a more sensitive negative chemical ionization gas chroma-
tographic-mass spectrometry assay for MEL (Lewy and Markey 1978)
permits the crossvalidation of the radioimmunoassay using a tech-
nique based on totally different physical-chemical principles.
We have assayed 6 rat serum samples for MEL both by radioimmuno-
assay and gas chromatography-mass spectrometry and found a corre-
lation of 0.983 (Grota et al 1981).
N-acetylserotonin in serum and pineal tissue has been esti-
mated by a subtraction method in which assayed melatonin values
are subtracted from the estimates of N-acetylserotonin plus mel-
atonin (Pang et al., 1977). Validation of NAS assays are dis-
cussed below.
In an investigation by Yu et al., (1981), circulating mel-
atonin following pinealectomy was found to be reduced but not
abolished. This study, however, did not settle the issue of the
source of circulating melatonin. Lewy et al., (1980) using GCMS
assay for MEL, reported that pinea1ectomy eliminated circulating
melatonin. In a separate study we have assessed the effect of
pinealectomy on circulating melatonin using the melatonin assay
which had been crossvalidated with GCMS (Grota et al., 1981). In
those studies melatonin levels were reduced to below the limit of
sensitivity in pinealectomized rats (Harvey et al., 1981) indicat-
ing that the pineal is the major (or only) source of circulating
melatonin.
Studies on Regulation of Serum Melatonin

Our initial studies showed that MEL levels in albino rat
pineals displayed a diurnal rhythm with high levels during dark
and low levels during light (Pang et al., 1977). Rats were housed
under a 12:12 light: dark cycle and sacrificed at 4 hour intervals.
No rise was seen 2 h after dark onset and the peak occurred 6 h
after dark onset. In another study the 24 h pattern of pineal and
serum melatonin levels were compared (Brown and Grota, 1980). A
similar night time rise was seen, but peak serum melatonin occurred
later than peak pineal melatonin (10 h after dark onset vs 6 h).
We next examined the influence of feeding time on regulation
of serum MEL and corticosterone (Holloway et al., 1979). Restrict-
ion of food availability to a two hour period during either light
or dark had previously been shown by Krieger (1974) to have a
significant influence on the adrenal rhythm with maximal elevation
occurring just prior to food presentation. We have confirmed this
finding. However, in contrast to corticosterone, serum MEL
rhythms were not modified by the timing of food availability. Our
findings are consistent with those of Moore and Traynor (1976) who
reported no alteration in the pineal N-acetyltransferase rhythm
in rats given 1 h access to food in the morning. These findings
indicate that the MEL rhythm is more closely linked with the light
jdark cycle than is the adrenal rhythm.
In several studies we examined the influence of shortening
the photoperiod on the serum melatonin rhythm. In rats maintained
in light:dark 12:12 and 2:22 or 1.5:22.5 cycles, a 24 hour rhythm
was observed with the peak consistently occuring 18 h after
light onset. (Brown et al., 1980a, b, Grota et al., 1982). If
light onset were considered to be the synchronizer, the overall
24 hour patterns did not differ. Furthermore, in the hamster
maintained under identical conditions, 24-hour serum melatonin
patterns also did not differ (Brown et al., 1980 b, Brown et al.,
1982), although in the short photoperiod overall melatonin levels
were reduced. Thus, in both rat and hamster there is evidence
that the 24 hour serum melatonin rhythm is cued by light onset.
These findings are of additional interest because of the
pronounced endocrine effects produced in the hamster and the rat
by exposure to short photoperiod (Brown et al., 1982). These
changes have been attributed to an altered 24 hour pattern of
melatonin and notably to an early rise in melatonin (Reiter 1980).
We have found no evidence of such a change in the 24 hr. pattern of
melatonin. Since our studies have been conducted at only certain
intervals after exposure to short photoperiod, (3, 4 and 7 weeks
in the rat, and 7 and 12 weeks in the hamster) it remains possible
that 24 hour serum melatonin patterns may show alterations at
other intervals.
248 G. M. BROWN ET At.
Nonetheless, in the hamster when there is testicular regress-

ion and a drop in circulating testosterone induced by either 7 or
12 weeks of short photoperiod there is no evidence of an early
rise in serum melatonin and, in fact, melatonin levels are reduced.
In the rat, olfactory bulbectomy increases the inherently
low sensitivity of the reproductive axis to a short photoperiod.
In bulbectomized rats, reduction of seminal vesicle and prostate
but not testis weights relative to body weight can be detected
after 3 and 7 weeks on a L:D 2:22 light cycle (Harvey et al.,
1981). Absolute testis weights were reduced at 7 weeks but still
showed histological evidence of sperm production. These effects
were partially or totally reversed by pinealectomy. However,
while pinealectomy reduced serum melatonin to undetectable levels,
there were no significant differences in the height or duration of
the melatonin peak between anosmic animals showing gonadal re-
gression and intact controls.
Studies On Regulation of N-acetylserotonin
Diurnal rhythms of immunoreactive NAS and MEL have been ex-
amined in the serum of rats on a 12:12 light:dark cycle (Pang et
al., 1980). In two separate experiments in which lights were on
from 0600 to 1800 h, maximum levels of NAS were found at 800 h; with
a minimum at 1200 and 1600 h. The patterning of NAS and of MEL
differed somewhat as the MEL peak preceeded the NAS peak by 4 hours.
In order to determine whether the circulating MEL and/or
NAS originated at least in part from extra pineal sources, the 24
hour pattern was examined in pinealectomized animals on the same
light/dark cycle. Concentrations of circulating NAS were signi-
ficantly reduced after pinealectomy (Yu et al., 1981), suggesting
that the pineal contributes a significant amount of this sub-
stance to the blood. However, NAS was not abolished, suggesting
the existence of extra pineal sources capable of secreting this
substance.
DEVELOPMENT OF SPECIFIC NAS ANTISERA

Because of the finding that N-acetylserotonin is present both
in the pineal and circulation and because of limitations inherent
in estimating NAS by a subtraction method we next undertook to
develop a specific NAS radioimmunoassay.
Using fvlEL as a model substance, a variety of approaches for
the development of specific antisera were explored. In addition
to the approaches using formaldehyde condensation as described
above, we have produced antisera using melatonin conjugated to
albumin by means of diazotized p-aminobenzoic acid, Na-p-carboxy-
benzyl, and Na-propionic acid bridges (Wurzberger et al., 1976,
3 7' I
CH O~NHCOCH3 CH30~NHCOCH3
~ I ~I I
o
N N
I I
CH2
I
CH2
I
C=O
I
NH
I C=O
Protein I
NH
I
Melatonin-PA-Protein Protein
Melatonin-PCB-Protein
Figure 2
DeSilva and Snieckus 1978, Blair and Seaborn 1979, Brown and
Grota 1980, Grota et al., 1981) (Fig. 2). Crossreactivity of com-
pounds with structural similarity to melatonin was assessed using
tritiated melatonin as the ligand. For these three classes of
antisera, N-acetylserotonin and 6-hydroxymelatonin had the great-
est crossreactivity which was 3% or less for each of the antisera.
Thus, including the Mannich approach, we have assessed four diff-
erent approaches, each of which is capable of producing anti-
bodies to indole haptens which have sufficient specificity to be
useful in radioimmunoassay. However, there are differences be-
tween these techniques. The Mannich reaction is carried out
readily in aqueous solutions but the rate of reaction is low and
it is difficult to control. Both the Na-p-carboxybenzyl and the
Na-propionic acid derivatives can be prepared in crystalline form
and conjugated to antigenic protein using water soluble carbodii-
mide. One disadvantage of this approach is the relatively poor
solubility of these derivatives. Coupling via diazodized p-amino-
benzoic acid is quick and done readily. However, we have found
that this reaction yields numerous side products in model reac-
tions and hence the antigen may be a heterogeneous one.
HO~NHCOCH3
~I N
I
Q
I
c=o
I
NH
I
Protein
N-acetylserotonin-PCB-Protein
Figure 3
We have chosen to use the Na-p-carboxybenzyl (Pcb) approach

for the preparation of specific antiserum to N-acetylserotonin
(DeSilva and Snieckus 1978, Pang et al., 1982). Serum harvested
from animals immunized with N-acetylserotonin - pcb - BSA (Fig.
3) was incubated with tritiated N-acetylserotonin (35Ci/mmol)
obtained from Amersham. Indoleamines related to N-acetylsero-
tonin were evaluated for their ability to inhibit binding of
tritiated N-acetylserotonin. Of the naturally occuring sub-
stances tested 6-hydroxymelatonin had .5% crossreactivity while
other indoleamines had less. Parallelism with the standard N-
acetyl serotonin was shown for rat, hamster, and rabQit serum, for
rat and hamster serum extract and for rat brain, retina and
pineal extract. Sensitivity of the assay was 10 - 25 pg/tube
based on the dose of NAS producing 12% inhibition of binding
(twice the standard deviation of the binding at zero concentra-
tion). Within and between assay co-efficient of variation for
a rat serum control containing 142 picograms per tube were 8.4% and
7.7% respectively in 15 assays. An extracted sample of rat
serum was chromatographed on high pressure liquid chromatography
(Anderson et al., 1981). Radioimmunoassay of the aliquot gave
only one peak which corresponded to authentic N-acetylserotonin
and the radioimmunoassay value before and following chromato-
graphy agreed within 5%. To date, it has not been possible to
cross validate this assay using GCMS as the appropriate GCMS
methodology has yet to be developed.
STUDIES ON REGULATION OF SERUM N-ACETYLSEROTONIN

Because data from the scheduled feeding study indicated that
the melatonin 24-hour rhythm was tightly coupled to the 24-hour
light-dark rhythm we have used different light-dark rhythms to
determine whether NAS and MEL in serum could be dissociated. In
one study rats were maintained for 8 weeks under either a 12:12
light:dark cycle {LD 12:12} or under LD 2:22. At various times
during a 24-hour period animals were killed and the serum examined
for NAS and MEL. As observed earlier using the subtraction method
{Pang et al., 1977}, when animals were kept under a LD 12:12
cycle, both NAS and MEL have a crest late in the dark period.
However, parenthetically, it should be noted that the crest for
NAS is at a level of 2 to 2.5 ng/ml while for MEL a maximum level
is reached at approximately 40 to 50 pg/ml. In contrast to these
data we now found dissociation of the 24 hour patterns of NAS and
MEL in serum among animals maintained under LD 2:22. For MEL there
was no change in the rhythm relative to the LD 12:12 animals. How-
ever, the serum rhythm of NAS showed a shift to an earlier time
period such that in both LD 12:12 and LD 2:22 the crest occurs
approximately 6 to 8 hours after the onset of darkness. In
addition, NAS levels "Jere elevated with a maximum of approximately
3.6 ng/ml. These data are shown graphically in Fig. 4.
4.0
3.6
\
1\
I \
3.2 I \
I \
I \
! I \
}-+
I
2.8
E'"
I
I
I
Z I
Z 2.4
I
I
0 I
l- \
0 I \
a:: 2.0 I \
w I
en
...J
~
W
U
«
2:
.8
.4 ------.. to 12:12
0-----0 lO 2:22
0 1200 2400
TIME OF DAY
Figure 4 The 24 hour serum N-acetylserotonin rhythm in

the rat differs in short and long photoperiods.
A second experiment examined the rate at which MEL and NAS

in serum synchronized to 12 hour light dark cycles after a 30 day
period in constant light. Constant light was used to eliminate
the lighting cue and to reduce the production of NAS and MEL. As
can be seen in Fig. 5, at the end of 30 days of constant light
MEL levels were approximately 10 pg/ml. Within two hours of the
first exposure to darkness serum MEL levels were elevated and
there is evidence of synchronization of MEL levels to the
dark periods, in that during darkness MEL levels are elevated
above levels in the light. One data point differs for no known
reason. In contrast to MEL, NAS shows no synchronization to
periods of light and dark even after 6 days in the 12 hour light:
dark cycle (Fig. 5). These two studies clearly show that the
daily pattern of serum MEL can be dissociated from the pattern
of serum NAS. Such data imply independent regulation of these
two substances. The dissociation of these two substances in
serum, even though they are related biosynthetically, may occur
because the precursor NAS is 50 times more concentrated in serum
than MEL. This fact means that the total serum variation for day
and night MEL remains within the range of biological variability
for NAS even at its lowest levels. Because these studies were
done in only one species, the albino rat, we are pursuing essent-
ially the same questions in the hamster to increase the general-
izeability of these data. Only pilot data is available at this
time but as for the rat, the patterning of MEL and NAS in short
photoperiods appears different for the two hormones. Thus, in
the hamster as well as the rat, serum levels of HAS and MEL can
be dissociated.
At the present time the significance of the NAS rhythm is
unknown. In fact, at this point we do not know with any certainty
whether the NAS rhythm originates from the pineal or from another
site of production as the enzyme N-acetyltransferase is found in a
variety of tissues.
Nonetheless, the pronounced 24 hr rhythm of NAS in serum
together with its dissociation from the MEL rhythm under short
photoperiodic conditions in the rat suggests that synchrony and
desynchrony of these rhythms may have a functional significance.
CONCLUSION
Using antigens in which melatonin and N-acetylserotonin are
coupled to protein at or adjacent to the ring nitrogen we have
produced antisera to both substances which are sufficiently
specific to be useful in radioimmunoassays.
Radioimmunoassays for serum MEL and NAS have been developed
and validated. Studies of serum melatonin have shown that this
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CONSECUTIVE 12 HR LIGHT/DARK CYCLES
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CONSECUTIVE 12 HOURS LlGHT·DARK PERIODS
Figure 5 The 24 hour rhythm of serum melatonin but not N-acetyl-

serotonin resynchronizes to the light cycle promptly at
the end of 30 days of constant light.
substance is tightly regulated by the light/dark cycle. The

peak in serum MEL was consistently found 18 h after light onset
in both rat and hamster in both 12:12 light:dark cycles and in
2:22 cycles. These findings suggest that light onset serves as
the synchronizer for the serum MEL rhythm. In contrast to MEL,
the serum NAS rhythm in the rat shifts when the photoperiod is
shortened, so that it peaks 6 to 8 hours after the onset of
darkness. It is speculated that association and dissociation of
serum NAS and MEL rhythms may have a functional significance.
ACKNOWLEDGEMENTS
Investigators wish to acknowledge technical assistance of
L. Campbell, K. Caskey and M. Joshi together with the secretarial
assistance of L. Koutalos.
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32: 262.
DIFFERENTIAL LOCALIZATION OF MELATONIN
AND N-ACETYLSEROTONIN IN BRAIN
G.M. Brown, O. Pulido, L.P. Niles, S. Psarakis,

A. Porietis, G.A. Bubenik*, and L.J. Grota**
Department of Neurosciences
McMaster University, Hamilton, Onto
*Department of Zoology, University of Guelph, Guelph, Onto
**Department of Psychiatry, University of Rochester
Rochester, N.Y. 14624
INTRODUCTION
Until 1974 quantitative and qualitative localization of mel-

atonin (M) had been performed mainly by bioassay (Ralph and Lynch,
1970). As this technique is relatively insensitive, the detection
of M outside of the pineal gland was infrequent (Pang et al. 1974).
This led to the conclusion that the epiphysis cerebri was the uni-
que source of M and its derivatives (Reiter, 1973). This firm opi-
nion persisted for more than 15 years after the discovery of M in
1958 (Lerner et al. 1958) despite numerous reports of extrapineal
detection of hydroxyindole-O-methyl transferase (HIOMT) the enzyme
essential for M synthesis (Quay, 1965; Cardinali and Rosner, 1971a;
Cardinali and Wurtman, 1972) as well as the demonstration of syn-
thesis of melatonin in the retina (Gern et al. 1978, Pevet et al.
1980).
IMMUNOHISTOLOGIC STUDY USING BISPECIFIC ANTISERUM
With the development of the first bispecific antiserum (bind-

ing M and its precursor N-acetylserotonin equally well) by Grota
and Brown (1974), the way was opened for development of not only
radioimmunoassay (RIA), but also for immunohistology of N-acetyl-.
ated indolealkylamines (INAI). In 1974 we tested this antiserum
on frozen sections of rat brain and pineal tissues. Using the
modified double antibody technique developed by Coons and co-work-
ers (1955) positive staining was first detected in the parenchymal
cells of the pineal gland. In addition to this expected locali-
257
zation of immunoreactive N-acetylated indolealkylamines (INAI),

however, positive staining was also detected in the granular layer
of cerebellar cortex which was present in the same cross section
of the rat brain as was the pineal gland. The staining was consis-
tent using both fluorescein isothiocianate and UV light as well as
peroxidase and diaminobenzidine reaction systems. Saturation of
antiserum by an excess of M as well as replacement of first anti-
serum by a nonspecific serum abolished the staining (Bubenik et al.
1974). Consistent staining was also observed in the habenula (an
area adjacent to the pineal gland) as well as in the fimbria
hippocampi. However, in these tissues the saturation of antiserum
by M did not completely abolish the staining and these areas were
not further investigated.
The retina had been found to contain HIOMT (Cardinali and

Rosner, 1971a) as well as to have the ability to synthesize M from
serotonin in vitro (Cardinali and Rosner, 1971b). In addition,
the retina has a close developmental relationship to the epiphysis
cerebri (Kappers, 1976). Our successful localization of INAI in
the outer nuclear layer of the retina (Bubenik et al. 1974) is
therefore not surprising.
STUDIES USING SPECIFIC MELATONIN ANTISERUM
Two years later, specific anti-M antiserum became available,

All tissues identified as INAI positive were re-tested. Using both
the bispecific and the anti-M antisera, the proportion of immuno-
reactive melatonin (1M) and its precursor, N-acetylserotonin (NAS),
in tissues were estimated. Thus the intensity of staining obtained
with bispecific antiserum, minus the staining with monospecific
anti-M antiserum indicated the amount of immunoreactive NAS (INAS).
Predominantly M staining was detected in the optic nerves, chiasm
and tract and the suprachiasmatic nucleus. Almost equal amounts
of 1M and INAS was estimated in the retina and the pinealocytes.
In addition, to these CNS tissues, 1M was also identified in the
Harderian gland (Bubenik et al. 1976b) which is another tissue
where HIOMT had previously been reported (Cardinali and Wurtman,
1972). The localization of melatonin in the retina was later con-
firmed by other colleagues using immunohistology (Vivien-Roels et
al. 1981) TLC (Pevet et al. 1980) or RIA (Pang et al. 1977; Pang
et al. 1980) employing a variety of different antisera. A similar
confirmation of our findings carne later for the localization in
the pineal (Pang et al. 1977; Freund et al. 1977; Pevet et al.
1980; Vivien-Roels et al. 1981). In contrast, only INAS was
detected in the granule and Golgi II cells of cerebellum, the axons
of pontal reticular formation and the axons of the spinal tract
of the trigeminal roots in the caudal pons and the rostral medulla
oblongata (Bubenik et al. 1976a).
Because other detection techniques were not sufficiently

sensitive at that time, immunohistology was also used in order to
LOCALIZATION OF MELATONIN AND N-ACETYLSEROTONIN 259
estimate possible circadian variation in these tissues as well

as to study the ontogeny and the sites of possible M synthesis.
Our conclusion, that extrapineal synthesis of melatonin may exist
and that this might exhibit diurnal variation independent of the
pineal gland was subsequently confirmed both by the presence of
melatonin in tissues following pinealectomy and by documentation of
synthesis in the retina in several studies (Gern et al. 1978; Gern
and Ralph, 1979; Pang et al. 1980; Pevet et al. 1981; Holloway et
al. 1980; Yu et al. 1981; Grota, Holloway and Brown, 1982).
Finally, the results of immunohistological investigations of

retinas and Harderian glands of rats injected or implanted with M
and killed during the daytime revealed a several fold increase in
intensity of immunofluorescence as compared to control animals.
The increase in intensity of staining vastly exceeded the highest
levels observed during the night time. It has been hypothesized
that the retina and the Harderian gland possess specific receptors
which have spare capacity to pick up amounts of M which are higher
than the normal physiologic levels produced locally (Bubenik et al.
1978). Receptors for M have been reported by several groups
(Cohen et al. 1978; Niles et al. 1979; Cardinali et al. 1979;
Holloway et al. 1979; Niles et al. 1980), however, so far none
of these investigators have reported M receptors in the retina or
the Harderian gland.
Why is there production of M in multiple sites? It has been

speculated that M was originally produced in a number of organs
for local neuromodulator (Bubenik and Purtill, 1980). However,
with the increase in size of the pineal during phylogenetic
development, the production of M there augmented to the point where
it began to escape into the peripheral circulation and to assume
hormonal properties (Bubenik et al. 1980; Quay, 1980). Thus the
pineal gland became the main source of NAI in most mammalian
species. However, local production may still continue in the
retina, brain tissue and the intestines (Bubenik et al. 1977; Gern
et al. 1978; Gern and Ralph, 1979; Pevet et al. 1981). These other
organs are potential sources of circulating M if the primary source
is lost (Kennaway et al. 1977; Yu et al. 1981). However, no cir-
culating melatonin is detectable by GCMS following pinealectomy in
the rat (Lewy et aI, 1980).
STUDIES USING NAS SPECIFIC ANTISERA
With the recent availability of specific antisera binding

N-acetylserotonin (NAS) (Brown and Grota, 1980; Pang et al., 1981),
specific immunohistological localization of NAS has become possible.
(Pulido et al. 1981). NAS-immunoreactivity has been demonstrated
only in unfixed frozen sections. At the present time, no fluo-
rescence has been observed in cryostat sections of tissues fixed
by perfusion with: a) 4% Paraformaldehyde in PBS buffer pH 7.3
(Steinbusch, 1981): b) periodate-lysine paraformaldehyde (Mclean
and Nakane, 1974) or c) 0.35% glutaraldehyde in Tris-HCL buffer

(Willingham and Yamadi, 1979). We are aware that fixation would
be advantageous for increased morphological detail and tissue
preservation. Further experimental testing of several fixation
combinations and concentrations are in progress. Despite this
limitation, the application of NAS antisera to immunohistochemistry
using unfixed frozen sections has proven to be a reliable and
specific method for the localization of INAS in brain tissue.
Specificity of INAS Immunohistology
Specificity of the NAS antisera has been evaluated using the

following parameters: 1) radioimmunoassay, 2) immunohistochemical
controls using PBS and non-immune serum; 3) absorption tests using
different concentrations of NAS; 4) inhibition experiments using
different concentrations of different substances that might
crossreact; 5) pharmacological treatments that might alter NAS
dynamics in the brain; 6) comparison of NAS distribution with that
of 5-HT and melatonin; 7) immunohistological evaluation of the same
brain regions using melatonin antisera.
Each anatomically distinct area where positive INAS was iden-

tified, was evaluated for non-specific binding, cross-reactivity
and melatonin immunoreactivity. Non-specific binding after exposure
of slides to NRS was observed in the wall of the blood vessels,
the ependymal lining, and the Pia mater. Areas with positive INAS
showed strong inhibition of the fluorescence intensity after pre-
incubation of the antiserum with concentrations of NAS over l~g/
lOO~l whereas no inhibition was seen with concentrations of
400~g/lOO~1 of melatonin, serotonin, 5-hydroxytryptophol,
3-methoxytyramine, N-acetyltryptophan, dopamine, norepinephrine.
These data indicate that this immunohistologic method is highly
specific for immunoreactive N-acetylserotonin.
Mapping of INAS
Identification of INAS was confirmed in the granular layer

of the cerebellum, tractus spinalis nervi trigemini and pontine
reticular formation. INAS was also observed in the Purkinje cells
and the neuronal cell bodies of the following area: colliculus
superior and inferior, nucl. originis nervi trigemini, nucl.
originis nervi facialis, nucl. cochlearis, nucl. raphae pontis,
nucl. corporis trapezoidei, nucl. interpeduncular is , locus coeru-
leous, nucl. tractus mesencephali, superior olivary complex, nuclei
pontis, nucl. ruber, nucl. reticularis gigantocellularis, nucl.
reticularis pontis caudatis and oralis.
With the technique employed, INAS has been mostly visualized

in neuronal cell bodies in the cerebellar cortex and brain stem
(Fig 1, 2). Dendritic projections are distinguishable within some
of the Purkinje cells. (Fig. 1) Stem processes are identified in
some neurons (Fig. 2). Poorly defined filamentous structures are

seen intermingled with neuronal cell bodies in areas such as nucl.
pontis and nucl. interpenduncularis. Whether those fibers represent
nerve processes, glial filaments or deposits of INAS within the
extra cellular matrix remains to be determined.
Figure 1: Photographs of frozen sections from rat cerebellar

cortex after incubation with: a) NAS-antiserum
~ NAS-antiserum with NAS, c) High magnification of a
Purkinje cell showing specific NAS-immunofluorescence.
A dendritic projection is indicated by an arrow.
Bar = 50 urn.
Figure 2: Photographs of frozen sections from rat brain stem

showing specific NAS-immunofluorescence. a) Pontine
Reticular Formation, b) Nucl. vestibularis, c) Nucl.
Locus Coeruleus. Arrows indicate stem processes
from neuronal cell bodies.
Bar - 75 um.
Relationship of INAS to Serotonin and Melatonin
Although serotonin appears to be present in many areas of the

central nervous system (Saavedra, 1977, Steinbusch, 1981), major
differences have been found between the distribution of INAS con-
taining structures as compared to 5-HT.
INAS is present in significant amounts in the cerebellum where

it is localized in the granular layer of the cortex, Purkinje cells
and cerebellar nuclei. No melatonin containing structures have been
visualized. Serotonin in cerebellum has been reported as present
in very low amounts, being seen only in a few nerve terminals in
the molecular layer of the cortex and in the cerebellar nuclei.
Although, there are regions ill the brain stem where both INAS
and 5-HT are visualized, in many of these regions, 5-HT is locali-
zed in nerve terminals whereas INAS is mostly identified in cell
bodies i.e. nucl. originis nervi occulomotor, nucl. originis nervi
trigemini, nucl. tractus mesencephali, nucl. originis nervi
facialis, nucl. olivaris superior, nuclei pontis, nucl. reticularis

giganto-cellularis, nucl. reticularis parvocellularis, nucl. reti-
cularis pontis caudalis and oralis, nucl. vestibularis lateralis.
Other areas such as the nucl. cochlearis dorsalis and ventralis do
not have any or have very poor 5-HT innervations, whereas numerous
INAS structures are visualized. Both INAS and 5-HT are visualized
in cell bodies of the following regions: nucl. interpeduncularis,
nucl. ruber, nucl. locus coeruleous, nucl. vestobularis medialis
and nucl. raphae pontis. Whether in these regions both substances
are present within the same cell or each substance has a different
cell population, has yet to be investigated. No melatonin contain-
ing structures have been visualized in these regions.
These data, together with a lack of melatonin immunoreactivity

in slides from the hindbrain indicate that major differences exist
in the localization and distribution of INAS as compared to 5-HT
and melatonin.
Localization of INAS in Hippocampus
Following preliminary observations of NAI localization in the

hippocampus (G.A. Bubenik, unpubl.) the two-antisera technique was
applied to this area (Porietis et al., 1978). Our data confirmed
the presence of NAI's in hippocampus and identified the acetylin-
dole there to be largely, if not exclusively, INAS. Presence of
hippocampal NAS was subsequently confirmed by gas chromatography-
mass-spectrometry (GCMS) (Brown et al., 1981). Serotonin locali-
zations have also been widely reported in the hippocampus (Lidov
et al., 1980; Steinbusch, 1981).
The recent advent of NAS-specific antisera (Brown and Grota,

1980; Pang et al., 1981) resulted in a continuation of these local-
izations in detail. In hippocampus, using both fluorescent and
enzyme (Protein-A peroxidase) markers (Nakane and Kawaoi, 1974),
INAS, but not 1M, was localized within the pyramidal cell layers
of CAl and CA3, as well as in the polymorph and granular cell
layers of the CA4/dentate region. In CAl and CA3 the localization
was in small units, submicron sized, external to but in close
apposition to the large pyramidal cells. There is no evidence at
this time to support localization within these cells. Occasional
fibrous structures were observed coursing between the pyramidal
cells in CAI/CA3. In the CA4/dentate region, NAS was localized in
two distinct populations of structures, cell bodies and apparent
processes/terminations. Cell bodies were identified in the poly-
morph cell layer, especially adjacent to the granule cell layer.
The frequency of cell body staining was low. The occurence of
small deposits of markers occurred more frequently throughout the
polymorph cell layer, extending into the granule cell layer.
Fibrous structures were seen in both the granule and polymorph cell
layers, in the latter sometimes associated with stained cell bodies.
Earlier observations with the anti-melatonin sera failed to

demonstrate any immunoreactive melatonin in the hippocampus.
Likewise, the localization of serotonin as reported by others
(Lidov et a1., 1980; Steinbusch, 1981) describe a diffuse term-
ination of 5HT neurons of brainstem origin in the basal dendritic
fields of stratum oriens of CAl and adjacent to the dentate
granule cell layer. These authors do not describe 5HT-containing
cell bodies within the hippocampus.
The serotonergic input to the hippocampus is largely from

brainstem via the fimbria-fornix pathway. Lesions of these nuclei
or of the fimbria-fornix typically result in degeneration of
presumed 5HT neuron terminals in the hippocampus (Pasquier and
Reinoso-Suarez, 1978). The pattern of neuronal degeneration
within hippocampus reported by these authors does not resemble
the distribution of INAS-containing terminals reported here.
Furthermore, lesions of the fimbria-fornix, either unilateral
or bilateral, do not affect the content or distribution of INAS-
immunoreactivity within the hippocampus (Porietis et a1., in
preparation).
Development of hippocampal INAS
In pilot studies we identified INAS in the granular cell layer

of the dentate gyrus as early as 20 days postconception. In an
effort to examine the appearance of INAS in the CNS, the hippo-
campus was chosen as the logical place for its study as a function
of cortical development.
The hippocampus is that horn-shaped part of the cerebral

cortex that lies along the curvature of the lateral ventricle.
This formation, which is continuous with the entorhina1 cortex, is
further divided into the hippocampus proper (Ammon's horn), the
fascia dentata (dentate gyrus) and the subicular complex (Storm
Mathisen, 1977).
The dentate gyrus was chosen for study because it is an

easily distinguishable component of the hippocampus under the
light microscope and it is feasible to follow its development as
its formation is completed postnatally. Altman and his colleagues
(O'Keefe and Nadel, 1978) have shown that its development lags well
behind the rest of the hippocampus and the rest of the brain in
general.
A specific NAS-antiserum and fluorescein-labelled second

antibody were employed to investigate the presence of INAS in the
dentate gyrus as a function of age. The hippocampus of rats at
18, 20, 22, 30 and 38 days postconception as well as that from
adult animals were studied. Specificity of the NAS-antiserum had
previously been evaluated, both by RIA and immunohistology, the
results of which indicated that the NAS-antiserum was saturable
using the NAS ligand, but not with melatonin, 6-hydroxymelatonin,

N-acetyltryptamine, serotonin or other ligands (Pulido et al.
1981). The specificity of the NAS-antiserum was re-confirmed in
the hippocampus by immunohistologic inhibition experiments using
sections from adult, 22 and 38 day postconception rats treated
with an NAS-antiserum that had previously been incubated with NAS,
melatonin or serotonin. NAS-antiserum pre incubated with NAS dem-
onstrated immunofluorescence levels characteristic of non-specific
binding as observed in tissues treated with non-immune serum.
Both melatonin and serotonin in comparable concentrations failed
to decrease the observed levels of fluorescence.
Regardless of age, INAS was observed primarily in the cell

bodies of the granular cell layer. This distribution was diff-
erent from that of serotonin which is present in the nerve term-
inals of the molecular layer and the hilar zone beneath the
granular layer (Moore and Halaris, 1975). Furthermore, INAS
appeared to be age-dependent reaching adult levels by 38 days
postconception (Table 1). Our data showed characteristic changes
during development in the rat which tend to parallel among other
things concurrent changes in granular cell number. The low levels
of INAS observed in the neonatal dentate are consistent with the
small number of functional neurons present in the granular jell
layer at birth. Bayer and Altman (1975) have shown using ( H)-
thymidine autoradiography and low-level X-irradiation that 85
percent of dentate granular cells originate after birth as compared
to the majority of cells in the molecular and hilar layers of the
dentate area that originate during embryonic life.
Crain et al., (1973) have likewise shown that synapse forma-

tion between the perforant path axons from the entorhinal cortex
and the dendrites of the granular cells takes place concurrently
with cell formation with the most active synaptogenesis occurring
between 25 and 32 days postconception. Our findings also correlate
with the appearance and subsequent increases of N-acetyltransferase
in the hippocampus (Hsu and Mandell, 1981) suggesting, perhaps, a
role for NAS in the normal functioning of these cells.
A more rigorous examination of the dentate gyrus is required

in ordered to further elucidate this correlation. It may be more
meaningful, perhaps, to examine NAS content per neuron rather than
per tissue as the content per neuron may not be influenced by
developmental changes in neuron numbers. Such information there-
fore may be more likely to reflect change or lack of change in
NAS biosynthesis as a function of age. In order to accomplish this
end it may be necessary to obtain a more accurate estimate of the
total number of granular cells as a function of age. Bayer et al.
(1982) have recently shown, using volumetric estimates, that the
total number of granular cells increases linearly in rats between
1 month and 1 year of age thereby adding substantially to the
existing population of neurons.
TABLE I
NAS-IMMUNOFLUORESCENCE AS A FUNCTION OF AGE IN THE DENTATE GYRUS

OF THE HIPPOCAMPUS OF THE RAT
AGE (POSTCONCEPTION) INTENSITY OF FLUORESCENCE

(PHOTOMETER READING)
18 days
20 days 1l.24 ± 2.0 *
22 days 23.78 ± 2.0
30 days 23.32 ± 2.6
38 days 54.0 ± J.8
Adult 56.37 ± 5.8
*standard Error of the Mean
To date, the function of the hippocampus is still unknown.

Selective ablation of dentate granular cells in the hippocampus
of infant rats produced by surgical destruction of the area
results in increased locomotor activity (Bayer et al., 1973).
Furthermore some functions of the hippocampus are reported to
develop in synchrony with the maturation of the dentate gyrus
(Douglas, 1975). Perhaps there is some function for NAS in
hippocampal activity.
Pharmacologic studies on cerebellar INAS
The formation and metabolism of NAS in the CNS has not been
determined. Synthesis of NAS in the brain may follow a pathway
similar to that described for pineal tissue (Brownstein, 1975;
Wurtman and Moskowitz, 1977), i.e., tryptophan+5-hydroxytryptophan+
serotonin+NAS. To test this possibility, rats were treated with
either parachlorophenylalanine (PCPA) or 6-fluorotryptophan, both
of which are potent inhibitors of tryptophan hydroxylase, the
enzyme necessary for the synthesis of serotonin from tryptophan
(Deguchi and Barchas, 1973; Miller et al. 1970, McGeer, 1973).
Tryptophan hydroxylase has wide distribution in the CNS and

the activity of this enzyme is highest in nuclei containing large
amounts of serotonin (Brownstein et al., 1973; Saavedra, 1977).
Tryptophan hydroxylase has also been found, to some extent, in all
areas where NAS has been visualized. To inhibit the activity of

this enzyme we used PCPA (320 mg/kg b.w.) and 6-fluorotryptophan
(115 mg/kg b.w.). Semiquantitative evaluation of the NAS-
fluorescence intensity in cerebellar cortex, using a photo-
multiplier-photometer (Photovolt 520M, Wild-Leitz, Canada) showed
a significant (p < 0.001) inhibition of the NAS-fluorescence in-
tensity in animals treated with PCPA (INAS-fluorescence: 52.4 ± 11)
or 6-fluorotryptophan (INAS-fluorescence: 36.6 ± 5.2) as compared
to the saline controls (INAS-fluorescence: 108.3 ± 11.5). Results
are expressed as mean ± standard error of the mean. These data
show that both tryptophan hydroxylase inhibitors significantly
reduce cerebellar NAS-immuno-fluorescence intensity relative to
saline treated controls.
In the pineal gland the rate limiting and final step in N-

acetylserotonin (NAS) synthesis is the acetylation of serotonin
by N-acetyltransferase (NATase, E. C. 2.3.1.5) (Brownstein, 1975).
NATase has been demonstrated in a wide variety of brain areas,
with the highest concentration in the cerebellum (Hsu and Mandel,
1981; Paul et al., 1975; Yu and Boulton, 1979). Brain NATase
activity significantly increases following the administration of
S-adrenergic agonists suggesting that extra-pineal brain NAS
synthesis may be under noradrenergic control, as in the pineal
gland (Friedhoff and Miller). This hypothesis is supported by our
observations that NAS-containing structures in areas such as
cerebellar cortex and cochlear nuclei are adjacent to a rich nor-
adrenergic (NE) innervations (Pulido, 1981). There is also evi-
dence that NE exerts a significant action on Purkinje cells
(Freedman et al., 1976; Hoffer et al., 1973) and we have shown that
Purkinje cells have high NAS-immunofluorescence. To test the
possibility of a functional relationship between NAS and NE in the
rat CNS, animals were treated with isoproterenol (10 mg/kg b.w.),
a S-adrenergic agonist, and/or with propranolol (20 mg/kg b.w.), a
S-adrenergic blocking agent, and NAS-immunofluorescence in the
cerebellum assessed semiquantitatively.
We found significant (p < 0.002) increases in cerebellar

NAS-immunofluorescence (155.8 ± 10.2) after I-isoproterenol treat-
ment as compared to control fluorescence of 85.0 ± 9.1. The effect
was completely blocked by ,the S-adrenergic antagonist, propranolol
which had fluorescence of 108.5 ± 14.7. These data suggest that
the synthesis of NAS in the cerebellum is mediated by NATase acti-
vity and probably regulated by a S-adrenergic mechanism which may
be similar to that in the pineal gland. This interpretation is
supported by the wide distribution of both typrophanhydroxylase and
N-acetyltransferase in brain tissue and by the observation that the
activity of N-acetyltransferase in the rat brain is increased by
pretreatment with isoproterenol. (Friedhoff and Miller 1977).
268 G. M. BROWN ET Al.
3H N-ACETYLSEROTONIN BINDING IN 1~1ALIAN BRAIN
The specific binding characteristics of tritiated N-acetyl-

serotonin (29.5 Ci/mmol, Amersham) were examined in synaptosomal
fractions prepared from human, calf and rat brain tissues as
previously described (Niles et al., 1982). Fresh or frozen (-20 o C)
tissues were homogenized in 10 volumes of ice cold 0.32M sucrose
and following centrifugation (1000 x g for 10 minutes at 4°C) the
nuclear pellet was discarded. The supernatant was spun at
20,000 x g for 20 min and the crude synaptosomal fraction (P2) was
suspended in 20 volumes of distilled water to allow lysis to occur
and subsequently washed three times in distilled water with centri-
fugation at 48,000 g x 10 min. The final pellet was suspended in
80 - 100 volumes of 0.05M Tris-HCL buffer (pH 7.4) and used in
receptor assays.
Binding assays were performed by incubating 1 ml aliquots of

tissue in triplicate with 3H NAS in the presence or absence of a
large excess of unlabelled NAS (10-~). For inhibition experiments,
various drugs in concentrations ranging from 10- 10 - 10-5M were
also added to incubation tubes. After incubation at 37 0 C for 30
min., samples were rapidly filtered on GF/B filters by vacuum and
then rinsed with 4 x 3ml aliquots of ice cold Tris buffer. Filters
were equilibrated overnight in PCS cocktail and then counted by
liquid scintillation overnight in PCS scintillation cocktail and
then counted by liquid scintillation spectrometry. Tissue protein
concentrations were determined as described by Lowry et al. (1951)
using bovine serum albumin as standard. Less than 0.2% of 3H NAS
was non-specifically bound to filters in the absence of tissue.
Specific 3H NAS binding, defined as that displaced by excess

unlabelled NAS, was about 70% of total binding and was found in
synaptosomal fractions from rat, calf and human brain as shown in
Table 1. Scatchard analysis of binding data for fresh membranes
from male or female rats indicated the presence of one population
of high affinity binding sites with a dissociation constant (Kd)
of 3-7 nM and a maximal binding site concentrations of 250-400
fmol/mg protein. In contrast to fresh tissues, frozen membranes
produced higher levels of specific binding which yielded biphasic
Scatchard plots. Graphic analysis (Rosenthal, 1967) indicated
the presence of two receptor classes.
The high affinity (Kdl) and low affinity (Kd2) sites with
their respective Bmax values are indicated in Table!! for frozen
membranes from rat, calf and human cerebellum. The similarity in
the high affinity Kd and Bmax values for fresh and frozen membranes
from rat brain suggests that the increased binding observed in
frozen membranes is due to exposure of a low affinity, high
capacity binding component.
TABLE II
REGIONAL DISTRUBUTION OF SPECIFIC [3HJ NAS AND [3HJ 5-HT BINDING IN

THE RAT CNS
SPECIFIC BINDING
CNS REGION [3 HJ NAS [3 HJ 5-HT
Fmoles/mg Erotein
STRIATUM 34.12 ± 0.86 197.91 ± 2.34
CORTEX 32.83 ± 4.94 185.13 ± 8.10
HYPOTHALAMUS 22.69 ± 2.30 167.92 ± 8.89
CEREBELLUM 22.43 ± 1. 73 91.39 ± 27.07
PONS-MEDULLA 15.69 ± 2.00 48.54 ± 5.78
HIPPOCAMPUS 13.93 ± 0.34 141.49 ± 12.05
MIDBRAIN 12.55 ± 4.36 50.01 ± 4.38
S~aptosomal ~embranes were incubated in triplicate with 5nM of

[ HJ NAS or [ HJ 5-HT at 37 0 C for 30 minutes. Nonspecific binding
was measured in the presence of 19 ~M nonradioactive NAS or 5-HT.
The correlation coefficient (r) [ HJ 5-HT binding is 0.63. Means
± SEM are presented.
Regional Distribution of Binding
Regional binding studies carried out with fresh P2 fractions

from rat brain indicated that 3H 5-HT binding was about 3-10
fold greater than 3H- NAS binding in all the CNS areas examined
However, similar relative levels of binding (94 - 100%) were found
for both radioligands in the striatum and frontal cortex. With
striatal binding taken as representative of 100% binding for both
radioligands, there were marked differences in the relative bind-
ing observed in other brain areas. The relative binding levels
of 3H-NAS in the cerebellum pons-medulla and midbrain were 66%,
46% and 37% while corresponding values for 3H-5-HT were 46%3 25%
and 25%. Conversely, in the hypothalamus and hippocampus, H-5-HT
binding was higher at 85% and 71% respectively, as compared with
67% and 41% for 3H- NAS . Thus, relatively more 3H- 5- HT binding
was present in the limbic areas examined while more 3H- NAS binding
occurred in the cerebellum and brainstem.
Structural Characteristics of Binding
Inhibition experiments were carried out with fresh rat cere-

bellar synaptic membranes using a variety of 5-HT related compounds
and other drugs (see Table III. Among the drugs tested, NAS and
5-HT had the highest affinity and were equipotent inhibitors of
binding. The Hill number for NAS did not differ from unity but
lower nH value for 5-HT suggests interaction at multiple binding
sites. This is also suggested by the low Hill numbers for the
5-HT agonist, quipazine, which showed low affinity and the 5-HT
antagonist, methysergide, which showed mbderate binding affinity.
TABLE III
INHIBITION OF [3H]NAS BINDING IN RAT CEREBELLUM
DRUG nH
NAS 10.19 ± 2.51 0.95 ± 0.17

5-HT 16.61 ± 7.82 0.78 ± 0.11
QUIPAZINE 2015 0.67
5-METHOXYTRYPTAMINE 268 0.28
MELATONIN >10000 0.29
N-ACETYLTRYPTAMINE ND
METHYSERGIDE 173 0.57
Fresh cerebellar membranes were incubated with 2.5 nM [3H]NAS and

at least five concentrations (10- 10 - 10- 5M) of each drug. The
concentration (IC SO ) of each drug which displaced 50% of bound
radio1igand was determined from logit-1og inhibition plots. Hill
coefficients (nH) represent the slopes of these plots. Means of
2-5 experiments are presented. ND = no displacement.
Replacement of the 5-hydroxy1 group possessed by both NAS and

5-HT with 5-methody resulted in a 15-25 fold decrease in affinity
as found for 5 methoxytryptamine. Melatonin, which has an N-acetyl
group in addition to the 5-methoxy group showed even less binding
affinity while N-acety1tryptamine, with only the N-acetyl group,
failed to inhibit binding.
In summary, saturable, high affinity binding of 3H- NAS is

present in rat, calf and human brain. The regional distribution of
this binding in the rat brain indicates that relatively more 3H- NAS
than 3H-5-HT is bound in the cerebellum and brain stem, areas which
have also been reported to contain INAS (Pulido et al., 1981). The
pharmacology of 3H- NAS binding indicates that the 5-hydroxyl group
is necessary for high affinity binding which appears to occur pri-
marily at central serotunergic binding sites (Niles et al., 1982).
However, the complexity of binding suggests that 3H- NAS also labels
other non-serotonergic binding sites in the cerebellum.
CONCLUSION
A. Melatonin is distributed in pineal, harderian gland,

hypothalamus, retina, and gut tisses. The function of melatonin in
these tissues is unknown. Apart from the hypothalamus, melatonin
is not found in the CNS. The distribution of IM follows previous
localization of HIOMT (Axelrod et al. 1961), the enzyme converting
NAS to melatonin.
B. A unique distribution of INAS has been defined in the

central nervous system by two different immunohistological app-
roaches. This distribution differs significantly from that of both
melatonin and serotonin. The presence of NAS in brain tissue has
been confirmed by GC-MS (Brown et al., 1981) and by radioimmuno-
assay (Pang et al. 1981). Studies on INAS in brain have demons-
trated that tryptophan hydroxylase may be essential for its syn-
thesis and that a-adrenergic stimulation increases INAS. Within
the hippocampus the quantity of INAS correlates with development
of the dentate gyrus Saturable high affinity binding of tri-
tiated NAS in brain has been demonstrated; a binding which has a
serotonergic component but which is relatively greater than [JHJ
5-HT binding in brain areas that are richest in INAS. Taken
together, these findings suggest that N-acetylserotonin may have
a function in brain separate from that of either melatonin or
serotonin.
ACKNOWLEDGEMENTS
The expert technical assistance of S.L. Campbell and

secretarial help of L. Koutalos is acknowledged. This work has
been supported in part by the Medical Research Council of Canada
and The Ontario Mental Health Foundation.
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transferase from rat brain and liver. Can. J. Biochem., 57:
1204-1209.
MELATONIN ACTION: SITES AND
POSSIBLE MECHANISMS IN BRAIN *
**. **
D.P. Cardinali. Marla 1. Vacas, Marla I. Keller
.
Sarmiento and E. Morguenstern ***
Centro de Estudios Farmacologicos y de Principios

Naturales (CEFAPRIN), Serrano 665/669, 1414 Buenos
Aires, Argentina
INTRODUCTION
Knowing what the pineal gland does is a major step to-

wards understanding its physiological function in the body or
what it is for. What a hormone-secreting organ like the pineal
does can be characteriaed by defining the nature and quantita-
tive aspects of its input-output relationships, that is what
the gland secretes and in response to what. Definition of spe-
cific target tissues and intracellular mechanisms of action
for the hormone involved should be also included in the de~
scription.1
The pineal contains a number of biologically active com-
pounds all or several of which could be the factor(s) respon-
sible for the neuroendocrine effects of the gland. Indoles
(often represented by melatonin 2 ) and polypeptides (most of
which are still unidentified) are the compounds usually con-
sidered within this context, although the requirements set by
Bayliss and Starling's definition of a hormone, i.e. "a sub-
stance produced in one organ which after being transported by
the blood (or CSF) acted upon a distant organ to alter its
function" remain to be satisfied for most of them. In the case
of melatonin current experimental evidence indicate that it
* Supported by grants from Consejo Nacional de Investigacio-

nes Cientificas y Tecnicas (CONICET) and Fundacion "Alberto
J.Roemmers", Buenos Aires, Argentina.
** Established Investigator, CONICET.
*** Research Fellow, CONICET
277
is, in fact, a pineal hormone. Melatonin is synthesized and

secreted by the pineal gland of all vertebrates tested so far,
it circulates in blood with a characteristic circadian pattern
and it influences the function of a variety of endocrine or-
gans, mimicking s~veral neuroendocrine effects attributed to
the pineal gland. 2,3
Since pineal's effects tend to be modulatory rather than

primary (that is, to change the timing or amplitude of responses
rather than to generate such responses) considerably more dif-
ficulties are often faced by the investigator to design suc-
cessful experiments exhibiting melatonin activity, than, for
example, experiments showing roles for pituitary hormones. In
hamsters, massive daily doses of melatonin can be devoid of
activity,4 while very small doses, presumably physiological,
given daily at a critical time have marked effects on neuro-
endocrine function. 3 ,5,6 Since the catabolism of melatonin
remains fairly constant under a variety of circumstances, the
logical locus for such dramatic changes in exogenous melatonin
activity is at the target organ level, perhaps on receptor
and/or post-receptor mechanisms.
The present article discusses the current knowledge on

the sites and possible subcellular mechanism subserving the
neuroendocrine activity of melatonin. The interested reader
is referred to recent reviews 2 ,7,8 as well as to an accompany-
ing one 9 for additional discussion of the neural and hormonal
factors controlling melatonin synthesis and release in mammals.
METABOLIC FATE OF MELATONIN
Although the matter remains unsettled it is generally ac-

cepted that the blood rather than the CSF is the primary site
of secretion of pineal melatonin. In rats the concentration of
melatonin in the blood collected from the confluens sinuum is
approximately 8 times greater than that in the trunk blood;
moreover darkness induces increases in plasma melatonin levels
wi thout concomitant irlcreases in CSF. 10 These observations
together with the previous reports on CSF melatonin concentra- 2
tions lower than the plasmatic ones in humans,11 rhesus monkey1
and sheep1j have strengthened the opinion that the normal route
of melatonin comprises the pineal capillaries draining into
the surrounding venous sinuses. Still this remains to be dem-
onstrated in a direct way.
In accordance with recent estimates in rat 14 and sheep,15

melatonin has in blood a half-life of about 20 min. In neonatal
rats melatonin half-life is considerably longer probably due
to a low catabolic activity of the liver. 16 'Rapid hepatic
MELATONIN ACTION: SITES AND POSSIBLE MECHANISMS 279
hydroxylation at position 6 accounts for most of melatonin

inactivation in rat,17 sheep,18 monk ey 19 and man. 17 ,18 Most
(70%) of the 6-hydroxymelatonin is then conjugated to sulfate
while a much smaller amount is conjugated with glucuronic
acidj both conjugates are released from the body in urine and
feces. 17 Other pathways for melatonin degradation include
limited deacetylation by a hepatic aryl acylamidase to yield
5-methoxytryptamine 2D and clsavage of the pyrrole ring by indole-
amine-2,3-dioxygena~i to kynurenamine derivatives (presumably
only in the brain).
About 60-70% of circulating melatonin is bound to plasma
proteins (mainly albumin) and none is bound to protein in
CSF.22 Plasma protein binding does not impede melatonin trans-
port into the brainj 23 the mechanism accounting for the in-
ability of CSF melatonin to attain equilibrium with plasma
melatonin remains unknown. The active accumulation of circulat-
ing melatonin by rat and cat choroid plexus has been regarded
as th~ first step for the secretion of the hormone into the
CSF.24 However, in the frog, the unidirectional flux across
the IV ventricular plexus from the CSF to blood is considerably
higher than that in the opposite direction. 25 The relevance
of these observations in mammals awaits further exploration.
After systemic injections radioactive melatonin enters all
tissues including the brain. 17 One hour after an i.v. injec-
tion to cats melatonin ~s concentrated in the pineal gland,
iris-chroid and ovary.2 Other endocrine organs (like testis,
adrenal and thyroid) and the sympathetic nervous chain also
take up 3H-melatonin. 26 Labeled melatonin injected into the
CSF is taken up unevenly by the brain ~n~ becomes concentrated
within the hypothalamus and midbrain. 2 , 8 Mel~tonin was iden-
tified in the rat hypothalamus by either GCMS2 o~ immunohisto-
chemistry.3D By the latter technique melatonin is also detec-
table in the retina, optic nerve and tract, suprachiasmatic
nucleus and pineal gland. 3D
After a bolus intracisternal injection melatonin disap-
peared very rapidly from the brain, its decay being multi-
phasic. 28 Only 6% of the administered dose was present in the
brain as unchanged melatonin 5 min after injection and only
0.8% remained after 20 min indicating the rapid exchange between
CSF and vascular compartments. The concentration of melatonin
~~ the hypothalamus was 4-5 times that in the rest of the brain.
In the CNS melatonin is readily oxidized to N-acetyl-5-
methoxykynurenamine, which accounts for about 15% of total
urinary melatonin metabolites. 21 The link between brain me-
latonin metabolism and hormone's mechanism of action remains
unknown.
MELATONIN NEUROENDOCRINE ACTIVITY: CENTRAL OR PERIPHERAL?
For many species at temperate latitudes light is the

principal factor which ini~tes and synchronizes the timing
of the reproductive seasonal rhythm. 3 Hamsters, voles, mice
and gerbils, as well as rats under particular circumstances
(e.g. anosmia), exhibit gonadal regression when exposed to
short-day photoperiods, an effect needing the pineal gland
and mimicked by melatonin administration in appropriate amounts
and time schedules,2,3 A number of aspects of the hypothalamic-
hypophysial-gonadal axis are also depressed by melatonin treat-
ment in sheep, steer, dog, rabbit, hare, monkey and weasel
(for references see ref. 2). Additionally, other effects of
short days on endocrine and behavioral function are also in-
fluenced by the pineal gland and melatonin.
The importance of target organ sensitivity in determining
the effects of melatonin mu~t be stressed. While appropriately
timed (late evening) injections of small, physiological amounts
of melatonin can induce gonadal atrophy in male hamsters main-
tained on long days,3,5,6 similar injections can inhibit the
testicular regression induced by short days.31 Melatonin bees-
wax pellets or capsules that deliver a constant amount of me-
latonin prevent the short day-induced gonadal regression as
well as that following evening melatonin injections; they can
also stimulate gonadal growth in hamsters with regressed testes
that are maintained on short days after capsule implantation.
3,32 Massive daily doses of melatonin, even at evening hours
were without effect on the hamster reproductive system. 4 Thus
a wide variety of circumstances (photoperiod length, dosage,
time and way of administration during daily photoperiod)
govern the presence or absence of melatonin antigonadal effects.
The common factor for all of them can be the extent and length
of exposure of the target cells to melatonin. As discussed
below endogenous melatonin via down regulation of receptors or
at post-receptor sites could modulate organ's sensitivity to
subsequently injected exogenous melatonin in hamsters. This
phenomenon can be a critical event in the sensitivity of the
hamster CNS to interpret photoperiodic information in terms of
neuroendocrine and behavioral responses.
Although there is little doubt that melatonin exerts
anti gonadal effects when appropriately injected, two groups
of investigators published ~onflitti~g results with this view
after active immunization of rats or hamsters against melatonin-
protein conjugates. 3o ,33 Melatonin immunization did not prevent
the dark-induced gonadal atrophy; moreover immunization induced
testicular regression in hamsters kept in long photoperiods, a
finding which was interpreted as indicating a progonadotropic
rather than antigonadotropic effect of melatonin. Further
experiments are needed to rule out a possible deleterious ef-

fect of the immunization procedure per se on the sensitivity
of melatonin target organs, as well as to demonstrate that
all the endogenous production of melatonin is effectively
neutralized by that procedure.
The anatomical sites implicated in melatonin antigonadal

activity may include one or a variety of tissues such as the
CNS, the adenohypophysis or the gonads. 2 Since a significant
reduction in gonadotropin output is observed in most experi-
mental paradigms explored, it is reasonable to assume that
melatonin affects neuroendocrine activity by acting centrally
on the hypothalamic-hypophysial axis. Fraschini and co-workers
34 were the first to report that the stereotaxic implant of
melatonin in rat medial basal hypothalamus (MBH) depressed pi-
tuitary and plasma LH levels. Subsequently several investiga-
tors observed that the cerebroventricular injections of mela-
tonin decrease LH and FSH levels. With few exception (e.g.dogs
35), melatonin administered intrapituitary as portal vessel
infusions or systemically preceding an LHRH injection, failed
to modify gonadotropin release. 2These observations strongly
favor a central rather than a pituitary site of action for the
hormone.
In white-footed mice anterior hypothalamic melatonin-bees-
wax implants that released very small amounts (i.e. less than
100 ng/day) of melatonin induced significant reproductive organ
collapse, an effect ~gat was not reproduced by s.c. implanta-
tion of the pellets. In hamsters exogenous melatonin failed
to inhibit gonadal activity if the animals were griorly sub-
jected to anterior hypothalamic deafferentation 37 or to lesion
of the suprachiasmatic nucleus. 38 Thus in mice and hamsters
melatonin appears also to act on central sites to depress gona-
dal growth.
The lack of direct effects of melatonin on the adenohypo-
physial function in rats is an age-related process. In neonatal
but not in adult pituitaries melatonin (10- 8M) inhibited the
stimulation of LH and FSH release brought about by LHRH in
vitro. This effect of melatonin in infant rats was quite spe-
cific inasmuch as the pineal hormone did not alter either the
basal and TRH-stimulated release of prolactin j~d TSH, or the
basal and somatostatin-inhibited GH secretion. In rats pinea-
lectomy or constant light advances the time of puberty,3 proba-
bly by advancing the maturation of gonadotropin control by
the steroids,40 while short photoperiods delay it. Melatonin
injection of prepuberal rats disrupts partially steroid-induc-
ed gonadotropin release and inhibits reproductive organ growth.
Perhaps the impending age-related adenohypophysial insensiti-
vity to melatonin in rats is one of the signals to induce
puberty onset.
Quay41 first suggested that the pineal gland itself can

be a target organ for melatonin, with resultant effects else-
where in the body representing modification of the pineal re-
lease of other hormonally-relevant compounds. Indeed the pres-
ence of an intact pineal gland was needed in order for single
daily afternoon injections of melatonin to cause gonadal atro-
phy in hamsters 3 ,6,42 or to affect prolactin release in under-
fed rats 43 • However repetitive injections of melatonin resem-
bling the diurnal rhythm in hormone secretion to pinealecto-
mized, ganglionectomized or suprachiasmatic-lesioned hamsters,
reproduce the productive collapse seen in intact hamsters in-
jected at the evening. 3 ,6,42 These observations constitute
another evidence on the importance of target organ sensitivity
as regulated by the pineal gland and melatonin itself. Data on
brain melatonin receptors discussed below offer a basis for
explaining these results.
~lthough the foregoing discussion imputes the hypothalamus

(and depending upon age in rats, the adenohypophysis) as the
most likely primary target organs for melatonin endocrine ac-
tivity, it should be noted that the pineal hormone could also
act peripherally. A few studies have shown that the injection
of melatonin into hypophysectomized rats or mice inhibited
HCG-induced gonadal and accessorv organ growth, perhaps by
inhibiting gonadotropin uptake. 44 - 45 Similarly melatonin di-
minished the growth of genital accessory organs in response
to testosterone in rats. 47 Melatonin (10- 6M) depr~ssed the in
vitro biosynthesis of androgens in rat 48 and duck 49 testes as
well adrenal steroid metabolism. 50 In lower concentrations
(10- 8 M or higher) melatonin also decreased the motility of the
spayed rat uterus. 51 Inasmuch as alleged hormone receptors are
detectable in the gonads and uterus 52 ,53 perhaps the melatonin-
mediated effects of the pineal gland on the reproductive system
in rodents may be the consequence of an overall influence on
CNS, pituitary and peripheral structures.
Biosynthetic melatonin capacity as well as immunoassayable
or immunohistochemically detectable melatonin have been shown
in the mammalian retina,Harderian glands and certain parts of
the gut. 30 ,54,55 In the latter the distribution of melatonin
corresponds closely to the localization of serotonin-producing
argentaffin cells; this observation led to the hypothesis that
melatonin-synthesizing cells are part of the APUD system. 55a
Thus melatonin may have not only a classical endocrine role
but also local neuromodulatory functions in certain tissues.
MELATONIN RECEPTORS
Early work supported the existence of a saturable melato-
nin uptake mechanism in brain. The intracisternal injection of

excess amounts of non-labeled melatonin to rats resulted in a
significant depres~ion of the subsequent brain uptake of radio-
active melatonin. 2 The logical corolary of those observations
in vivo was to search for melatonin receptors in brain subcel-
lular fractions. As soon as a radioactive melatonin of very
high specific activity was available this goal could be achiev-
ed.
In 1978 two Laboratories published the first description-
of the occurrence of specific binding sites for melatonin in
its target tissues. Cohen and co-workers 52 observed cytosol
binding sites for this hormone in rat and hamster peripheral
tissues like the ovary, uterus, testes, liver and eyes with
dissociation constant (Kd)= 6 nM for high affinity sites and
550 nM for low affinities sites. No melatonin binding was
detectable by the dextran-charcoal procedure in brain. struc-
tural specificities of the high affinity sites suggested a
requirement for the indole group and decreased affinity for
indole derivatives lacking the N-acetyl group. Cardinali et
a1 56 found melatonin binding activity in crude membrane frac-
tions of bovine M3H with Kd= 12 nM and binding site concentra-
tion of about 15 fmoles/mg prot. Binding affinities of mela-
tonin analogues indicated that the 5-methoxy and N-acetyl
groups were needed for full expression of binding to MBH mem-
branes and that binding was abolished by the presence of a 6-
hydroxy group. In a subsequent and more detailed description
of these binding sites ionic requirement5~ subcellular local-
ization and protein nature were studied. Binding was in-
creased by adding calcium, was inhibited by either sodium or
potassium, and was located mainly in the crude mitochondrial
pellet (P2). Other areas of the bovine brain also exhibited
melatonin binding activity, being 73% and 34% that of MBH in
the occipital and cerebellar cortexes, whereas it was unde-
tectable in amygdala, striatum or pons.
Differing from Cohen et aI's report, Brown and co-workers
30 did find, by using the dextran-charcoal technique, cytoplas-
mic melatonin binding in rat, human and bovine brain. The Kd
observed varied from 9 nM (hypothalamus) to 302 nM (midbrain).
As in bovine striatum 57 no saturable binding of melatonin to
rat striatal membranes was observed 30 •
The possibility that cytosol and membrane melatonin bind-
ing sites co-ex~t in the same tissue is supported by observa-
tions on the rat liver. 53 In this tissue membrane and cytosol
binding sites were found, the former being calcium dependent.
Kd values were 8 nM for membrane binding sites and 20 nM for
cytosol binding sites. Concentration of sites in liver membranes
was about 10% that in the cytosol. Among the various tissues
examined the highest membrane melatonin receptor concentration
was found in the rat hypothalamus and pituitary gland. 53 Obvi-
ously additional studies are needed to establish whether one
or both melatonin binding sites (cytoplasmic or membrane) are
the physiologic receptors. In this sense it is interesting to
note that the correlation of structure-ac~~vity relationships
for melatonin analogues in amphibian skin with that for bins-
ing to bovine MBH membranes 56 ,57 or to rat and hamster periph-
eral tissues 52 is fairly good, suggesting that a common recep-
tor may mediate melatonin effects on brain and periphery. Un-
fortunately no data have yet been published on structure-activ-
ity relationships for melatonin binding in brain cytosol.
A number of reports indicated that both pineal biochemis-

try and morphology are affected by the exogenous injections
of melatonin (for references see ref. 2). If the effect of
melatonin is exerted directly on pineal cells rather than in-
directly via the pineal sympathetic nerves or through a second
hormone acting on the pineal gland, melatonin receptors should
be present in the gland. Such specific melatonin binding sites
were detected by conventional radiochemical techniques in bovine
pineal membranes 59 and by a semi-quantitative immunohistolog-
ical procedure in rat pineal glands. 3o Membrane pineal binding
sites differed in a number of properties from those of MBH,
including their high Kd (700 nM), ionic dependence and struc-
tural requirements; whether or not they represent pinealocyte
autoreceptors remains to be defined.
Another potentially relevant site of action for melatonin

is the retina. The melatonin forming enzyme hydroxyindole-o-
methyl transferase is present in the mammalian retina, and
the rat retina in vitro is capable of converting serotonin
into melatonin. 54 ,55 Since these initial observations several
publications have strengthened the concept that the mammalian
retina is a physiological, significant site of synthesis and
action for melatonin (for references see ref. 60). Melatonin
has been shown to affect the aggregation of melanosomes in
trout and guinea pig pigmented-epithelium; recently presump-
tive melatonin receptors exhibiting a Kd= 1-2 nM and a struc-
ture requirement resembling those of other brain areas have
been identified in retinal tissue preparations. 61
For all the aforementioned melatonin binding sites the

rigorous demonstration of their receptor nature is still
lacking. However circumstantial evidence has been accumulated
in favor of this view for melatonin binding sites in brain
membranes. A first example is given by starvation experiments
in rats. Protein deprivation in these species is accompanied
by significant increases of the number of melatonin receptor
sites in brain membranes without changing their Kd for the

radiolabeled compound. 62 Since several investigators have
shown an increase in the sensitivity of the reproductive sys-
tem to exogenous and presumably endogenous pineal melatonin
in underfed rats,3,43 binding and biological data correlate
under these circumstances.
Another example is given by the daily rhythm of melatonin
sensitivity in hamster and rat brains. As Tamarkin et al. 5
first showed, injection of melatonin is highly effective to
mimic short photoperiods in hamsters if repeated daily at
evening hours, but not during forenoon. Melatonin effect was
blocked after pinealectomy or pineal denervation but in this
case it was found that three injections per day restored the
gonadal response; in contrast to the intact animals, the pine-
alectomized hamsters did not display a diurnal sensitivity to
melatonin. 3 ,6,42 Pinealectomy also blocked the effect of daily
evening injections on prolactin release in underfed rats. 43
These observations suggested that the diurnal rhythm in sensi-
tivity to melatonin in hamsters and rats is influenced somewhat
by the pineal itself. The possibility that the affinity or
number of presumptive melatonin membrane receptors in hamster
and rat brains could change accordingly at times when melatonin
is biologically active or inactive was explored by us. 63 Inter-
estingly enough receptors are maximally concentrated at a time
during the lighting cycle when exogenous melatonin is effective
in inducing gonadal atrophy; conversely the receptor number is
low at a time when melatonin injection is uneffective in inhibit-
ing reproductive physiology. In rat pineal the binding capacity
of melatonin also increased markedly during the light phase of
the day.3D Suppression of melatonin rhythmicity by pinealectomy,
pineal denervation or continuous exposure to light eliminated
both the antigonadal effect of daily evening melatonin injection
3,42,43 and the morning-evening differences in brain membrane
receptor number. 62 The hypothesis explaining these results
implies a rhythm in melatonin receptors maintained in part by
circulating melatonin (Fig. 1). Exposure to high melatonin
levels during the dark phase of daily photoperiod causes de-
sensitization of the neuroendocrine system by down-regulation
of receptor binding sites whereas during daytime the number of
receptors increases because melatonin levels are low, and
restoration of sensitivity occurs at the end of the light phase.
3,63
It is important to note that when the effects of daily

night-time injections of melatonin are assessed in hamsters,
the animals injected at the initiation or 2 h before the end
of the dark phase showed gonadal involution, whereas after
2 or 4 h of dark the hormone failed to affect gonadal function.
64 Although the hypothesis was put forth that injections of
HAMSTER
• DOWN-REGULATION
600 PINEAL PERIOD
MELATONIN
..,c
"
a.
.....
w
~ 100%
~
(J)
w
a:: 50%
~
~
(!)
a
~nl l'~
-
,/ \, "
\\, .. _--_ .. ,.' ~~'''''- ---_ ....,,/ \\, .......
0600 2000 0600 2000

OLD L 0
Fig. 1 A hypothesis to explain the diurnal rhythm in

sensitivity of the hamster neuroendocrine system
to single daily injections of melatonin. Gonadal
response (inhibition) is found only after late
evening or late night injections (central panel).51,64
The lack of effect of early light or middle night
melatonin injections is attributed to down-regula-
tion of receptors elicited by high pineal or extra-
pineal melatonin secretion, respectively. For other
details see text.
melatonin near the middle of the dark phase were ineffective

to inhibit gonadal growth because circulating melatonin levels
were already elevated at this time, subsequent observations
did not support such a view. Differing from other mammals like
sheep or calves, pineal and plasma melatonin in hamsters re-
main low for at least 4-6 h after the outset of darkness~,30
Why then a subsensitivity to the hormone occurs at earlier

times in the scotophase ? A hypothesis is advanced in Fig. 1.
Down regulation of receptors (and therefore subsensitivity to
melatonin) is considered to be the consequence of increased
melatonin secretion by pineal or extrapineal sources. Thus a
hypothetical extrapineal site of origin of melatonin is postu-
lated as responsible for the subsensitivity of hamster repro-
ductive system to exogenous melatonin when pineal melatonin
secretion is low. Plasma levels of melatonin mayor may not
reflect changes in melatonin production by those presumptive
extrapineal sites. In rats an increase in plasma melatonin
(but not in pineal melatonin) preceding the initiation of the
dark period was reported 1o ; additionally the retinal capacity
to synthesize melatonin shows peak values at late evening and
early night. 2,65 Obviously many aspects of the interaction of
melatonin with its brain receptors remains to be examined.
Alternative explanation to those given in Fig. 1 are also pos-
sible, like for example to consider the subsensitivity to mela-
tonin as depending partly on intrinsic properties of the hams-
ter brain rather than exclusively on circulating melatonin
levels. In addition it is not known whether true decreases of
melatonin binding occur in hamster brain at the initiation of
the dark phase. It should be also stressed that hamster and
rat data may not be easily extrapolated to other species, in
which a down-regulation of melatonin receptors is not readily
apparent. For example constant release s.c. implants of mela-
tonin or MBH implants in white-footed mice produce inhibition
of gonadal function. 36
POSSIBLE MECHANISM OF ACTION OF MELATONIN IN BRAIN
A great deal of attention have concentrated on melatonin-

sensitive events at the hypothalamic level; this is not un-
reasonable considering the role that the hypothalamus plays
in the control of pituitary function.
The following mechanisms have been advanced to explain
the action of melatonin on the hypothalamus:action upon sero-
tonin receptors 27 ;bihdingtospecific receptors in MBH;56,57
interaction with benzodiazepinereceptors;66 inhibition of
hypothalamic tubulin synthesis (with concomitant impairment
of fast axonal transport and induction of ultrastructural
changes in nerve endings of M~H);67 reduced number of estradiol
receptors;68 MAO inhibition;6 depressed synaptosomal uptake
and increased release of serotonin;7o depressed release of
dopamine;71 increased GABA content;72 increased release of
2BB D. P. CARDINALI ET AL.
Table 1. Similarities between melatonin and in-

domethacin.
1. Structural
Antibodies to indomethacin-HSA used in mel-
atonin radioimmunoassay.87
2. In vivo effects
Inhibition of post-castration and steroid-
induced LH release by systemic injection in fe-
male rats. 2 ,3,83
Depression of eerum lH levels by cerebro-ven-

tricular injection or MBH implant. 2,3,34,83
No effect on LH response to LHRH injection in

adult rats. 2 ,3,83
Depression of PGE2 release by CNS.78
3. In vitro effects
Depression of PGE2 release by MBH explants 85

and of basal and oxvtocin-induced uterine mo-
tility in rats. 51 ,81
Inhibition of thromboxane synthesis and ADP-

induced aggregation in human platelets. 51 ,82
neuropeptides j 73-75 increased number of immunoreactive LHRH cellsj';lj

inhibition of PGE 2 ,77-78 and cAMP synthesi sj 79 stimulation of
cGMP synthesi sj 79 increased rate of neuronal discharge ~B ros-
tral hypothalamus and reduced catecholamine inhibition. Of
course many of these mechanisms are probably not mutually ex-
clusive.
Very few biological effects of melatonin on brain tissue
have been obtained in vitro at the concentrations of the Kd
of the presumptive receptor sites above described. One of them
is the inhibition of PG (particularly PGE2) synthesis in MBH
of rats. Based on the structural similarities between melato-
nin and the inhibitor of PG synthesis indomethacin (N-p ~hloro
benzo yl J -2-meth yl-5-methox Aindole-3-acetic acid), Gimeno et ale
81 and Leach and Thornburn 2 put forth independently the
hypothesis that melatonin impairs PG biosynthesis in target
tissues. Melatonin (10- 8 M or higher) inhibited the spontaneous
motility of the spayed rat uterus as well as its reactivity
towards added oxytocin (both PG-mediated effects); it also in-

hibited PGE and PGF release to the medium. 81 Thromboxane re-
lease from platelets 82 and platelet aggregation (a thromboxane-
dependent phenomenon)51,82 are also inhibited by melatonin.
Melatonin (10- 8 M or higher) depressed the spontaneous release
of PGE2 from rat MBH, as well as that elicited by adding NE to
the medium. 77 Melatonin treatment of rabbits prevented the
release of E prostaglandins into the cerebrospinal fluid after
cervical stimulation. 78
There is considerable evidence from pharmacological stud-
ies employing cerebroventricular and systemic injections of
PGs and PG synthesis inhibitors to suggest that PGs are involved
in the regulation of adenohypophysial hormone secretion. PGs
(particularly of the E series) were found to stimulate pituitary
hormone release; PGs appear to affect LH, FSH and prolactin
release by acting on neural sites, TSH by acting at the pitu-
itary level, and GH and ACTH by affecting both pituitary and
hypothalamic sites. 83 The best characterized effect of PGE2
on hypothalamic neurohormone release is that on LHRH. PGE2
induces LHRH release to the rat portal blood in vivo,84 and
into the culture medium of rat MBH in vitro.85 PG synthesis
blockage by indomethacin prevents NE-induced LHRH release in
vitro. 85 An increase in PGE 2 synthesis of rat MBH occurs
simultaneously with LH release in estradiol-primed rats. 86
Since the administration of monoaminergic blockers did
not affect PGE2-stimulated LH release in rats, the hypothesis
was put forth that PGE2 acts directl~ on the LHRH secreting
neurons rather than presynaptically. 3 Of course one can vi-
sualize other presumptive sites of action of PGE2 on the LHRH
secreting mechanism. For example the effect of PGE 2 on LHRH
release may be exerted presynaptically on neurons impinging
on neurosecretory cells that use a non- monoaminergic trans-
miter like opioids,GABA or glutamate; alternately PGE2 may af-
fect primarily glilli rather than LHRH containing cells in MBH.
Since melatonin inhibits basal and NE- stimulated PGE2
release by MBH in vitro77 and by brain tissue in vivo 78 the
possibility should be considered that melatonin effect on
pituitary hormone release is mediated by PG synthesis inhibi-
tion. Indeed there are striking similitudes between the neuro-
endocrine effects of melatonin and indomethacin (Table 1).
However as far as LHRH secretion is concerned the data are
conflicting with the view that melatonin depresses neurohormone
secretion, inasmuch as increases, rather than decreases of
neuropeptide release b~ M~~ have been observed after exposure
to melatonin in vitro. 3- On the other hand melatonin treat-
ment resulted in increase of LHRH content of neurosecretory
neurons, as assessed by immunohistochemistry,76 a finding
MElAlONIN---!
reIeaIe
I~
®Ii)
• ••
®
sYI'fI*Is CI> •
~L:.. presynaptic
metabolism __
MELAlONlN
Fig. 2 Possible cellular and subcellular melatonin sites

of action in MBH. Melatonin may affect the neuro-
secretory cell by interacting with membrane (A,F)
or cytoplasmic (B) receptors; it may also change
the activity of neurons impinging on neurosecretory
cells (C), or act on the glial cells (D,E). In a
hypothetical target neuron melatonin could affect
synthesis and transport of neurotransmitter (1),
its storage (2), release (3), presynaptic metabolism
(4) or reuptake (5)~ or any of the postsynaptic
events (6).
compatible with inhibition rather than stimulation of LHRH re-

lease. Therefore, studies on MBH fragments are beset by the
vagaries of the cellular complexity of the preparation used
and are difficult to interpret in terms of the cellular com-
ponent or subcellular compartment affected (Fig. 2).
Another effect of melatonin exhibiting kinetics properties
and structural-activity relationships compatible with the inter-
action with melatonin receptors is the modifications in cyclic
nucleotide synthesis. Injections of 1 ug of melatonin in the
cisterna magna of rabbits elicited a significant rise in cGMP
• MELAlONIN
o 5 MTOH
A6FMEL
*
200 '" 6 OH MEL
11..
~
l!)
u
I
co 150
~
.!:
III
CI)
C'
c:
0
-
.s::
u
c CI)
u
"-
50
~
o~------------~------------~~
-7 ~5
Concentration in medium, log M
Fig. 3 In vitro effect of melatonin and 5-methoxytryptophol

on cGMP accumulation in rat MBH.79 Explants were in-
cubated for 10 min at 37 C in KRB buffer containing
1 mg/ml glucose and 1 mM theophylline.cGMP concentra-
tion was determined by RIA (basal amounts: 0.23 ± 0.02
pmol/mg protein) (*) p <0.05, ANoVA.
levels of the CSF.88 High concentrations (more than 10-4 M) of

melatonin also increased cGMP levels in the rat testis 89 and
human monocytes. 9o In addition the inhibition by melatonin of
tyrosinase activity in hair follicle cultures in vitro is
mimicked by cGMP.91
In 1981 two reports were published indicating effects of

physiological concentrations of melatonin on ~GMP synthesis in
vitro. Vessely92 found that melatonin (10- 8 M) increased by 2
to 3-fold guanylate cyclase activity in rat ad;~ohypophysis,
testis, ovary, thyroid and liver. Vacas et ale observed that
melatonin (10- 8 M or higher) increased significantly cGMP in
.MELATONIN
100 .SMTCH
~ "6FMEL
a. ~
::!: A60HMEL
<t
\'
o
80 ""
""
"",," ,
.5
en 6 ,, ,,
,, 'I
,
QI
co
-- -- **
C
~ ,, .. *
-
o 40 " . -- -----I *
!!---------1*
20
o~------------~------------~
-7 -5
Concentration in medium, log M
Fig. 4 Effect of melatonin, 5-methoxytryptophol (MTOH),

6-fluoromelatonin (6F-MEL) and 6-hydroxymelatonin
(6-HO-MEL)on cAMP levels in rat MBH. Experimental
details as in Fig. 3. Basal cAMP concentration:
128 ± 13 pmol/mg prot. (*) p (0.05, ANOVA.
rat MBH (Fig. 3). This effect of melatonin was shared to a

less extent by the melatonin analogues 6-fluoromelatonin and
5-methoxytryptophol (Fig. 3) while 6-hydroxymelatonin, the
biologically inactive metabolite devoid of activity in recep-
tor studies,57 did not affect cGMP levels. 79
Melatonin also decreased MBH cAMP accumulationj 79 in this
case melatonin activity was less potent than that of methoxy-
tryptophol or 6-fluoromelatonin, but greater than that of 6-
hydroxymelatonin (Fig. 4). As shown in Fig. 5 this effect of
melatonin on MBH cAMP synthesis was also observed in the cere-
bral cortex.
The relationships of cGMP and cAMP changes induced by
melatonin in MBH to the mechanism of action of the hormone is
not known. Strong evidence exists on the function of cAMP as
a second messenger in transducing specific neurotransmitter

actions onto target cells. 93 Other studies suggest that cGMP
is involved in physiological processes at synapses in both
peripheral and .central nervous tissue. cGMP has been shown to
accumulate in the superior cervical ganglion and different
brain areas in response to electrical stimulation,applied
acetylcholine or other depolarizing agent sj 93 of much interest
for the physiological significance of the melatonin-induced
reduction in MBH cAMP synthesis is the demonstration that PG-
synthesis mhibitors greatly reduced the cAMP accumulation elic-
ited by NE in incubated rat hypothalamic and cerebral cortex
slices,94 The cAMP levels could be restored by addition of very
low concentrations of PGE2i the effect was found for o£-adreno-
ceptors, and suggested a calcium-dependent mediation of PGE2
in the stimulation of cAMP by oZ-adrenergic agonists. It is
tempting to speculate that many of the central actions of me-
latonin could be explained in terms of PGE2 synthesis inhibition,
having a physiological role in the CNS as co-regulator of
monoaminergic pathways.
Because of its nature as a rather atypical microtubule-
active drug 95 melatonin could affect contractile protein-
dependent mechanisms in brain. Indeed treatment with the hor-
HYPOTHALAMUS CEREBRAL CORTEX
K100
0>
E
"-
III
~
o
E
a.
50
* *
DO
VI
-l
UJ
>
W
-l 10
c..
:l:
<1:
u
o
8 6 8 6
MELATONIN CONCENTRATION IN MEDIUM (- LOG M)
Fig. 5 In vitro effect of melatonin on cAMP accumulation

in rat MBH and cerebral cortex. Details of the
experiment as in Fig. 3. (*) p <0.05, ANOVA.
mone 67 or induction of endogenous hormone release by injectioncr

~ -adrenergic agonistg6 depressed tubulin concentration in
MBH. Additionally melatonin locally applied to retinal cells 67
or to sciatic nerve fibers g7 impaired fast axonal transport,
a microtubule-dependent process. Peripherally, some effects
of melatonin, e.g. inhibition of vasopressin-stimulated water
transport g8 , interference with antimitotic drug activity,g5
have been ~lso explained in terms of melatonin interaction
with tubulin. Further data are needed to establish the general
validity of this interaction.
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SEASONAL REPRODUCTIVE EVENTS RELATED TO THE PINEAL GLAND
Russel J. Reiter
INTRODUCTION
Under natural environmental conditions reproduction, and

especially birth of the young in mammals, is carefully regulated
so that these events coincide with the seasons of the year which
will most likely ensure the propagation of the species. The most
important physiological event in the annual reproductive cycle is
obviously the actual delivery of the young. To optimize the
survival of the newborn, animals in their natural habitat
characteristically deliver their young in the spring or early
summer. It is at these times that environmental temperatures and
food availability are such that the young will have the maximal
chance of survival. In order for these species to ensure spring
delivery of the newborns they must synchronize their breeding
habits accordingly. Obviously, if the animals were sexually
competent the entire year they would breed indiscriminately with
regard to season. Thus, evolution has provided them with a
system to restrict their reproductive habits to specific times of
the year. In many mammals it is the environmental photoperiod
which determines their reproductive capability; when this is the
case, the pineal gland serves as the intermediary between the
changing daylengths and the neuroendocrine-reproductive system.
Dependence on the photoperiodic fluctuations allows animals to
not only be in the proper sexual condition at a given time but to
actually anticipate the upcoming season and to make the necessary
physiological adjustments. In the absence of the pineal gland
photosensitive species simply cannot make the necessary
alterations and, consequently, they become continual as opposed
to seasonal breeders (Reiter, 1973/74). This affords them no
advantage under natural conditions and, if it persisted, the
303
304 R. J. REITER
species as a whole could become extinct. The data illustrating

photoperiodic-pineal-reproductive interactions in rodents are
summarized below.
DAYLENGTH MEASUREMENT BY PHOTOSENSITIVE SPECIES
Mammals are generally divided into two broad categories in

terms of their breeding habits; thus, there are long day breeders
and short day breeders. In the present discussion the long day
breeders with short gestation periods primarily will be
considered. Many of the rodents fall into this category. Much
of what has been learned about these interactions has been
derived from studies on the Syrian hamster {Mesocricetus
auratus}. As a result, the following resume will consider this
species in some depth.
The photoperiodic response of the Syrian hamster seems to
depend on a circadian rhythm of sensitivity to light. When light
is present during the insensitive phase of the rhythm the hamster
interprets the day as IIshort". It has been established that when
the daylength is less than 12.5 hours a day, the animal measures
the day as "short" and the pineal gland suppresses the reproduc-
tive system {Elliott, 1976}. Conversely, i f light is present
during the sensitive phase of the circadian rhythm the day length
is interpreted as long and high reproductive activity is main-
tained. Slight experimental perturbations of the circadian
rhythm of sensitivity to light may prevent gonadal regression in
animals kept under what would normally be interpreted as a long
day {Eskes and Zucker, 1978}.
A resonance paradigm has been used to examine the photo-

sensitivity rhythm of the Syrian hamster. The paradigm uses a
fixed duration of light with different lengths of the dark period
to generate a cycle of 24 hours or more. In the experiments in
question, the authors (Elliott et aL 1972; Stetson et aL,
I
1975) used 6 hours of light followed by one of several different

lengths of darkness. Hamsters exposed to light:dark (LD) cycles
of either 6:18 or 6:42 experienced gonadal involution. On the
other hand, when the animals were maintained under either LD 6:30
or LD 6:54 they interpreted these as long photoperiods and active
gametogenic activity was maintained. Besides being capable of
determining the reproductive state of the animal, light also
entrains the circadian locomotor system of this species. When
light is present during the latter half of the hamster's 24 hour
circadian locomotor rhythm, the day is interpreted as long and
the gonads do not regress.
This type of mechanism for maintaining high reproductive

function in the hamster may have adaptive value for the animal in
SEASONAL REPRODUCTIVE EVENTS RELATED TO PINEAL GLAND 305
its natural habitat. Even a brief exposure to light during the

photosensitive phase of the animal is most likely to occur during
the long solar days (spring and summer); this likely assists in
adjusting the animals reproductive state to the appropriate
seasons. Judging from the studies of Elliott et al (1972) and
Stetson and co-workers (1975), light exposure during the photo-
sensitive phase need not be a daily occurrence.
In general, what the data summarized in the above paragraphs

have been interpreted to mean is that hamsters must be exposed to
12.5 or more hours of light daily in order to maintain their
reproductive systems in an active state. This is not actually
the case; what they need is either at least one 12.5 hour light
period or two (or more) shorter intervals of light during a 12.5
hour period. Interestingly, 15 minute periods of light at 6 hour
intervals throughout a 24 hour period not only prevent gonadal
atrophy in hamsters but also curtail the daily rise in N-acetyl-
transferase activity (NAT), the alleged rate limiting enzyme in
melatonin production, in the hamster pineal gland (Rudeen and
Reiter, 1979; 1980). Similar observations in reference to the
state of the reproductive organs were made by Hoffman and Melvin
(1974). The implication is that melatonin is the pineal hormone
responsible for gonadal regression in short day exposed hamsters.
Although there is general acceptance of this conclusion (Reiter,
1980a), there are some who doubt it (Brown et al., 1981).
REPRODUCTIVE CHANGES INDUCED BY PHOTOPERIOD AND PINEAL
In the laboratory setting with controlled photoperiods and

temperature, the maintenance of hamsters in less than 12.5 hours
of light daily causes reproductive collapse in both males and
females of the species. These changes are clearly a manifesta-
tion of pineal hormonal activity since pinealectomy prevents
darkness-induced gonadal involution in both sexes (Hoffman and
Rei ter, 1965; 1966). In males kept under short day conditions
spermatogenesis is arrested (Gaston and Meanker, 1967; Reiter,
1967) while females under similar conditions do not ovulate;
additionally, their ovaries exhibit a lack of antral follicles
and a marked proliferation of the interstitial tissue (Reiter,
1968).
In male hamsters, the gonadally-related hormones seem to

change in a predictable manner during darkness-induced testicular
atrophy (Fig. I). During regression, there is usually a gradual
reduction in circulating levels of luteinizing hormone (LH) ,
follicle stimulating hormone (FSH) , and prolactin (Reiter, 1973;
Berndtson and Desjardins, 1974; Turek et al., 1975; 1976;
Tamarkin et al., 1976). However, these changes are not as
uniform as might be predicted on the basis of the markedly
306 R. J. REITER
4.0 Testes 0.4 Ace. Organs LH

125
3.0 0.3 100
_ 75
co 2.0 co 0.2 E
"
I I
co
c
50
1.0 0.1
25
0 I 0 0
FSH 4 PRL 2.0 TES

500
400 3 1.5
E
300 Q:
E E 1.0
~2 "coc
"coc
I
en
200 ~
- I
0.5
100
0 tNT tNT PlNX SCGX 0 tNT tNT PlNX SCGX o tNT tNT PlNX SCGX
'--' , L........JI '--',
Long Shorl Long Short Long Short
Fig. L Testicular and accessory sex organ weights (combined

weight of seminal vesicles and coagulating glands) and
plasma levels of luteinizing hormone (LH) , follicle
stimulating hormone (FSH), prolactin (PRL) and
testosterone (TES) in male Syrian hamsters exposed to
either long or short days. INT = intact; PINX =
pinealectomYi SCGX = superior cervical ganglionectomy.
atrophic reproductive system (Reiter, 1980b). More consistently,

the concentrations of gonadotrophins within the pituitary
decrease in male hamsters maintained under reduced photoperiodic
conditions (Reiter and Johnson, 1974a)~ The most consistent and
dramatic changes in male hamsters may be those of prolactin.
Both serum and pituitary levels of this constituent decrease
dramatically as a result of pineal-induced testicular involution
(Reiter, 1980b). Treatments which restore circulating prolactin

levels also markedly stimulate spermatogenic and endocrine
activity of the testes in hamsters maintained under short day
conditions (Bex et aL, 1978; Matthews et aL, 1978; Chen and
Reiter, 1980). These observations have led to the tentative
conclusion that prolactin is a key hormone in maintaining the
normal spermatogenic and endocrine activity of the testes.
The hormonal changes which occur in females experiencing

pineal-induced gonadal atorphy are markedly different from those
that occur in males under the same conditions. Rather than being
depressed, pituitary levels of LH and FSH actually increase
(Reiter and Johnson, 1974b) while there are daily afternoon
surges of each of these constituents in the blood (Goldman and
Brown, 1979). Normally, the gonadotrophin surges occur on the
afternoon of every fourth day. The net effect of the change in
hormonal patterns in females in an acutal increase in circulating
titers of LH and FSH. Despite this, the ovaries and uteri of
these animals functionally involute although the size of the
ovaries may actually increase due to the proliferation of the
interstitial elements of the organ (Reiter, 1968). The
significance of the daily afternoon rises in LH and FSH in the
presence of the depressed sexual function remains unknown.
In both males and females of this species, surgical removal

of the pineal gland prevents darkness from inducing both the
morphological involution and the physiological inactivation of
the neuroendocrine-gonadal axis. Indeed, all of the inhibitory
effects of darkness on the reproductive system of photosensitive
species seem to be overcome by either the surgical ablation of
the gland or by its sympathetic denervation (Reiter and
Sorrentino, 1970; Reiter 1980b).
PHOTOPERIOD, PINEAL AND SEASONAL REPRODUCTION
Animals maintained in their natural habitat are exposed to

seasonal fluctuations in daylength. The degree to which the
actual daily photoperiod changes throughout the year depends on
the latitude at which the measurements are made. When Syrian
hamsters are maintained under natural environmental conditions at
about 30 0 N latitude the longest days in the summer approach 14
hours while the shortest days in the winter are of roughly 10
hours duration. Since, as noted above, hamsters require about
12.5 hours of light daily to maintain an active reproductive
system, their exposure to the natural seasonal functions
described above also are associated with appropriate adjustments
in the reproductive system (Reiter, 1973) (Fig. 2). For
descriptive purposes, the annual cycle of reproductive capability
has been divided into the following four phases: the inhibition
308 R. J. REITER
Period of
o.vershoot
,--,
~ 3.0.
8'
CD
25
14
ci. 2.0 13 ] '

.sui 1.5
.£:
c;,
j 12 c:
elntoct ~
~
0 1.0
'5 o PIN X or SCGX >-
II
.!.1
u; 0.5 8
f!!
0. 10.
Aug Sep Oct Nov Dec Jon Feb Mar Apr May Jun .luI Auq
I I I t
Auturmol Winter Vernal Surrvner
Equinox Solstice Equinox Solstice
Period of Atrophy
Fig. 2. Seas.onal changes in day length and testicular weights in

male hamsters kept under natural ph.ot.operi.odic c.ondi-
ti.ons at ab.out 30° N latitude. The seas.onal changes in
repr.oducti.on are negated if the animals are either
pinealect.omized (PINX) .or superi.or cervical
gangli.onect.omized (SCGX).
phase, the sexually quiescent phase, the restoration phase, and

the sexually active phase (Reiter, 1975a) (Fig. 3).
Inhibiti.on phase
In the fall .of the year day lengths eventually fall below the
12.5 hours .of light daily required t.o maintain the g.onads in a
highly active state. Additi.onally, inasmuch as Syrian hamsters
are believed t.o be hibernat.ors they presumably spend pr.ogressively
m.ore time in lightless undergr.ound burr.ows in preparati.on f.or
hibernati.on. At any rate, the increased dark exp.osure stimulates
the pineal gland and repr.oductive inv.oluti.on ensues. The end
.organ reSP.onses as well as the h.orm.onal changes which .occur under
naturally decreasing day lengths are the same as th.ose described
ab.ove which f.oII.oW the exp.osure .of hamsters t.o artifically sh.ort
Sexually Sexually
Inhibition Quiescent Restoration Active
,,
Phase, Phase Phase Phase
>-
, , I
·u
o
a.
___l---...Ii--------
o
U
'"
.2:
-0
::J
-0
o
Q.
a::'" Pineal Intact
....o
Pinealectomized
Q;
>
'"
..J
OJ
o Period of Hibernation
.J
Refractory Period
Month of Year
I Aug I Sep I Oct I Nov I Dec i Jan Feb ' Mar ' Apr ' May Jun ' Jul' Aug' Sep' Oct
Fig. 3. Various stages of the annual cycle of reproduction.

Gonadal atrophy coincides with the period of
hibernation.
days of the laboratory (Reiter, 1974; Stetson and Tate-Ostroff,

1981). The generalized reduction in sexual physiology during the
inhibition phase of the cycle does not occur in the absence of
the pineal gland even though the days may be short and the
tempera tures may be cold (Reiter, 197 Sb; 1980b). The specific
mechanisms involved in pineal-induced atrophy of the reproductive
system under these conditions have not been elucidated; it has,
however, been proposed that the antigonadotrophic hormone of the
pineal gland, possibly melatonin, increases the sensitivity of
the hypothalamo-pituitary unit to the inhibitory feedback effects
ofJ gonadal steroids on LH and FSH (Turek and Campbell, 1979).
The results on which this conclusion was based, however, were
obtained from castrated animals kept under short days in the
laboratory. Hence, whether a change in feedback sensitivity
helps to explain reproductive regression in intact hamsters in
the field remains to be demonstrated. Also, the changing
feedback sensitivity theory does not take into account the role
of prolactin reduction in causing testicular involution in Syrian
hamsters.
The period of time required for the gonads of hamsters to

undergo maximal involution during the fall of the year is
believed to be on the order of 8 weeks. Hence, the inhibition
phase presumably monopolizes about a 2 month period in the case
of the hamster; the duration of this phase undoubtedly varies in
other species. By mid to late autumn the gonads are atrophic and
310 R. J. REITER
the animals presumably are physiologically prepared for hiberna-

tion (Reiter, 1981).
Sexually quiescent phase
Once the gonads are totally involuted the animals enter a

prolonged period during which the testes of the males and the
ovaries of the females are minimally active. This is the
sexually quiescent phase and it coincides with the short, cold
days of the winter. During this phase the animals are obviously
incapable of sexual reproduction; this prevents the birth of
young at a time when they would be exposed to inclement weather
conditions and reduced food availability. It probably allows for
successful hibernation since high levels of gonadal steroids seem
to impede hibernatory processes (Hall and Goldman, 1980).
During the sexually quiescent phase the pineal gland

actively suppresses the growth and function of the neuroendo-
crine-reproductive axis. Thus, if hamsters with infantile
reproductive organs are either pinealectomized or moved to the
laboratory and exposed to long days the gonads initiate
regeneration (Reiter, 1969); indeed, they become sexually
competent within a relatively short time. During the period of
sexual quiesence infantile reproductive organs are ensured by
virtue of the fact that the animals remain in virtually the
lightless environment of their subterranean burrows. Even if the
animals were to emerge above ground during this period, the
daylengths would be less than the mlnlmum 12.5 hours daily
required to maintain the sexual organs in a highly active state.
At some point after the winter solstice the gonads begin

regenerating; by definition, this terminates the sexually
quiescent phase. Apparently the neuroendocrine-gonadal axis, by
mechanisms which are yet to be elucidated, becomes refractory to
the inhibitory influence of the pineal gland and gonadal
restoration ensues (Reiter, 1975b).
Restoration phase
In the case of Syrian hamsters, the restoration phase, i.e.,

the regrowth of the reproductive organs, is accomplished while
the animals are still in the total darkness of their burrows
(Reiter, 1975c). Throughout this phase of the cycle the pineal
gland continues to synthesize and presumably secrete melatonin
(Rollag et al., 1980; Brainard et al., 1983). Assuming melatonin
is the pineal antigonadotrophic agent it is certainly not a
cessation of melatonin production WhlCh results in the initiation
of the restoration phase. Rather the recrudescence is most
likely due to the fact that the reactive site no longer responds
to the inhibitory signal produced by the pineal, i.e., the system
becomes refractory. It was initially proposed that it was the

increasing day lengths in the late winter and spring which caused
full recrudescence of the reproductive organs (Turek et aL,
1975). However, increasing daylengths is insufficient to cause
the regrowth (Reiter, 1980c}i furthermore, the reconstitution of
a functional reproductive system is not impeded even if hamsters
are totally deprived to light by surgical removal of the pineal
gland (Reiter, 1975c). Thus, Reiter (1975c) feels that the
recrudescence is a consequence of an endogenous phenomenon that
is independent of the photoperiod. Since, as noted above,
gonadal steroids impede hibernation, the restoration of
functional reproductive organs, in addition to ensuring the
production of mature gametes, also serves to terminate
hibernation. Once the gonads reach maturity the restoration
phase of the cycle is complete and the animals emerge above
ground, ready to mate (Reiter, 1980b).
Sexually active phase
The restoration of reproductive maturity initiates the

sexually active phase of the annual cycle. Shortly after the
gonads recrudesce the animals mate and, because of the short
gestation period of many of the animals in question, the young
are born soon thereafter. Hence, the young are born in the
spring the time that their chances of survival are the
greatest.
The refractoriness which is initiated late in the sexually

quiescent phase and which ensures endogenous gonadal
recrudescence extends into the sexually active phase (Fig. 3).
Hence, if sexually mature male or female hamsters are deprived of
light shortly after their gonads have regenerated, the sexual
organs do no involute (Reiter, 1975bi 1975c). Because of the
continuance of the refractory condition into the sexually active
phase, long days are really not required at this time of the year
to maintain the high functional state of the reproductive organs.
Long days of the summer, however, are important in that they
interrupt the refractory condition and, thereby, render the
hypothalamo-pituitary-gonadal axis sensitive to the inhibitory of
short days during the subsequent fall (Reiter, 1975bi Stetson et
aL, 1976). Indeed, the animals must be exposed, during the
summer months, to 12 or more weeks of long days before the
refractory condition is interrupted (Fig. 4).
Throughout the sexually active phase the females probably

experience several pregnancies. As fall approaches, however, the
reproductive system is shut down and the annual cycle is
repeated. Obviously, both light and darkness play major roles in
determining the annual fluctuation in reproduction in photo-
periodic species. The changes are mediated by the pineal gland
312 R. J. REITER
~~~~>~::~.L>
o I I I I I I I I I
o 0 ro ~ ~ ~ ro w ~
Weeks
Fig. 4. Refractoriness of the reproductive systems of Syrian

hamsters to short days is interrupted if the animals
are exposed to long day conditions for 12 or more
weeks. Short periods of long day exposure, e.g., 1 or
10 weeks (middle two panels), does not interrupt the
refractory period to darkness.
and, as a result, the removal of this organ completely eliminates

seasonal variations in sexual competence (Reiter, 1980b). In
effect, pinealectomy allows hamsters to be continual as opposed
to seasonal breeders even when they are exposed to natural
environmental conditions.
CONCLUDING REMARKS
For the survival of the species, seasonal fluctuations in
reproductive competence are absolutely essential for animals
subjected to the vissitudes of nature. It is essential that the
young be born at the time of the year which is most compatible
with their survival; this is the spring. To ensure spring
delivery of the young, hamsters as well as many other species have
evolved systems for limiting their reproductive capability to
very restricted portions of the year. To do this the photo-
sensitive species rely on the regularly changing daylengths to
signal their pineal gland which, in turn, has the capability of
inducing regression of hypothalamo-pituitary-gonadal axis. Thus,
the pineal is an absolutely necessary intermediary between the
environment and the reproductive system. Without it, hamsters do
not respond to the prevailing photoperiodic conditions and, as a
result, they are continual, as opposed to seasonal, breeders.
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Berndtson, W. W., and Desjardins, C., 1974, Circulating LH and
FSH levels and testicular function in hamsters during light
deprivation and subsequent photoperiod stimulation,
Bex, F., Bartke, A., Goldman, B. D., and Dalterio, S., 1978,
Prolactin, growth hormone, luteinizing hormone receptors,
and seasonal changes in testicular activity in the golden
hamster, Endocrinology, 103: 2069.
press.
Chen, H. J., and Reiter, R. J., 1980, The combinaton of twice
daily luteinizing homone-releasing factor administration and
renal pituitary homografts restores normal reproductive
organ size in male hamsters with pineal-mediated gonadal
atrophy, Endocrinology, 106: 1382.
Brown, G. M., Tsui, H. W., Niles, L. P., and Grota, L. J., 1981,
Gonadal effects of the pineal gland, in: "Pineal Function",
C. D. Matthews and R. F. Seamark, ed., Elsevier/North
Holland, Amsterdam.
Elliott, J. A., Stetson, M. H., and Menaker, M., 1972, Regulation
of testis function in golden hamsters. A circadian clock
measures photoperiodic time, Science 178: 771.
Elliott, J., 1976, Circadian rhythms and photoperiodic time
measurement in mammals, Fed. Proc. Amer. Soc. Exp. BioI.,
35: 2339.
314 R. J. REITER
Eskes, G. A., and Zucker, I., 1978, Photoperiod regulation of the

hamster testis: Dependence on circadian rhythms. Proc.
Nat. Acad. Sci., 75: 1034.
Gaston, S., and Menaker, M., 1967, Photoperiodic control of
hamster testis, Science, 158: 925.
Goldman, B., and Brown, S., 1979, Sex differences in serum LH and
FSH patterns in hamsters exposed to short photoperiod.
J. Steroid Biochem., 11: 531.
Hall, V., and Goldman, B. D., 1980, Effects of gonadal steroid
hormones on hibernation in the Turkish hamster (Mesocricetus
brandti), J. Compo Physiol., 135: 107.
Hoffman, R. A., and Melvin, H., 1974, Gonadal responses of
hamsters to interrupted dark periods, BioI. Reprod., 10: 19.
Hoffman, R. A., and Reiter, R. J., 1966, Responses of some
endocrine organs of female hamsters to pinealectomy and
light, Life Sci., 5: 1147.
Matthews, M. J., Benson, B., and Richardson, D. L., 1978, Partial
maintenance of testes and accessory organs in blinded
hamsters by homoplastic anterior pituitary grafts or
exogenous prolactin, Life Sci., 23: 1131.
Reiter, R. J., 1967, The effect of pineal grafts, pinealectomy
and denervation of the pineal on the reproductive organs of
the male hamster, Neuroendocrinology, 2: 138.
Reiter, R. J., 1968, Changes in the reproductive organs of
cold-exposed and light-deprived female hamsters
(Mesocricetus auratus), J. Reprod. Fertil., 16: 217.
Reiter, R. J., 1969, Pineal-gonadal relationships in male
rodents, in: "Progress in Endocrinology", C. Gual, ed.,
Excerpta Medica, Amsterdam.
Reiter, R. J., 1973, Pineal control of a seasonal reproductive
rhythm in male golden hamsters exposed to natural daylight
and temperature, Endocrinology, 92: 423.
Reiter, R. J., 1973/74, Influence of pinealectomy on the breeding
capacity of hamsters maintained under natural photoperiodic
and temperature conditions. Neuroendocrinology, 13: 366.
Reiter, R. J., 1974, Circannual reproductive rhythms related to
photoperiod and pineal function: A review, Chronobiologia,
1: 365.
Reiter, R. J., 1975a, The pineal gland and seasonal reproductive
adjustments, Int. J. Biometeorol., 19: 282.
Reiter, R. J., 1975b, Evidence for refractoriness of the
pituitary-gonadal axis to the pineal gland in golden
namsters and its possible implications in annual reproduc-
tive rhythms, Anat. Rec., 173: 365.
Reiter, R. J., 1975c, Exogenous and endogenous control of the
annual reproductive cycle in the male golden hamster:
Participation of the pineal gland, J. Exp. Zool., 191: Ill.
Reiter, R. J., 1980a, The pineal gland: A regulator of

regulators, Progr. Psychobiol. Physiol. Psychol., 9: 323.
Reiter, R. J., 1980b, The pineal and its hormones in the control
Reiter, R. J., 1980c, Reproductive involution in male hamsters
exposed to naturally increasing daylenghts after the winter
solstice, Proc. Soc. Exp. BioI. Med., 163: 264.
Reiter, R.J., 1981, Seasonal aspects of reproduction in a
hibernating rodent: Photoperiodic and pineal effects, in,
"Survival in Cold", X. J. Musacchia and L. Jansky, eds.,
Elsevier/North Holland, Amsterdam.
Reiter, R. J., and Johnson, L. Y., 1974a, Depressant action of
the pineal gland on pituitary luteinizing hormone and
prolactin in male hamsters, Horm. Res., 5: 311.
Reiter, R. J., and Johnson, L. Y., 1974b, Elevated pituitary LH
and depressed pituitary prolactin levels in female hamsters
with pineal-induced gonadal atrophy and the effects of
chronic treatment with synthetic LRF, Neuroendocrinology,
14: 310.
Reiter, R. J., and Sorrentino,S., 1970, Reproductive effects of
the mammalian pineal, Amer. Zool., 10: 247.
Rollag, M. D., Panke, E. 5., and Reiter, R. J., '1980, Pineal
reproductive cycle, Proc. Soc. Exp. BioI. Med., 165: 330.
Rudeen, P. K., and Reiter, R. J., 1979, Pineal N-acetyltrans-
ferase activity in hamsters maintained in shortened light
cycles, J. Endocr. Invest., 2: 19.
Rudeen, P. K., and Reiter, R. J., 1980, Influence of a skeleton
photoperiod on reproductive organ atrophy in the male golden
hamster, J. Reprod. Fertil., 60: 279.
Stetson, M. H., Tate-Ostroff B., 1981, Hormonal regulation of the
annual reproductive cycle of golden hamsters, yen. Comp.
Endocr., 5: 329.
Stetson, M. H., Elliott, J. A., and Menaker, M., 1975, Photo-
periodic regulation of hamster testis: Circadian
sensitivity to the effects of light. BioI. Reprod., 13:
329.
Stetson, M. H., Matt, K. 5., and Watson-Whitmyre, M., 1976,
Photoperiodism and reproduction in golden hamsters:
Circadian organization and the termination of photore-
fractoriness, BioI. Reprod., 14: 531.
Tamarkin, L., Hutchinson, J. 5., and Goldman, B. D., 1976,
Regulation of serum gonadotropins by photoperiod and
testicular hormone in the Syrian hamster, Endocrinology, 99:
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Turek, F. W., and Campbell, C. 5., 1979, Photoeriodic regulation
of neuroendocrine-gonadal activity, BioI. Reprod., 20: 32.
Turek, F. W., Alvis, J. D., Elliott, J. A., and Menaker, M.,
1976, Temporal distribution of serum levels of LH and FSH in
316 R. J. REITER
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periods, Biol. Reprod, 14: 630. .
Turek, F. W., Elliott, J. A., Alvis, J. D., and Menaker, M.,
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MELATONIN AS THE HORMONE WHICH MEDIATES THE EFFECTS OF THE PINEAL
GLAND ON NEUROENDOCRINE-REPRODUCTIVE AXIS OF THE SYRIAN HAMSTER
Russel J. Reiter
INTRODUCTION
Melatonin is the most widely investigated pineal compound.

Since its discovery in 1958 by Lerner and colleagues, it has been
the subject of numerous biochemical and physiological investiga-
tions. Its synthesis within the pineal gland has been
extensively studied and a great deal is known of the factors
which control its production (Smith, 1981). Although its effects
on the endocrine system have been investigated in many different
experimental paradigms (Cardinali, 1981), its sites of action and
mechanisms remain obscure. Despite its prominence in the
scientific literature, its specific role as a hormonal envoy of
the pineal gland continues to be questioned (Brown et al., 1981ai
Pevet et al., 1981a). If not melatonin, the hormonal product of
the pineal could prove to be another indole (Reiter and Vaughan,
1977), a polypeptide (Benson, 1977 i Benson et aL, 1981), or
another compound (Ebels, 1979). The following paragraphs
summarize what is known of synthesis and physilogical actions of
melatonin. The actions of the indole are extremely complex and,
at times, appear to be contradictory. Many of these apparent
contradictions can be explained by what has come to be known as
the down-regulation hypothesis. Since many of the studies have
dealt with the actions of melatonin on reproduction in the Syrian
hamster, these are data that will be considered in greatest depth
herein.
317
318 R. J. REITER
MELATONIN PRODUCTION IN THE SYRIAN HAMSTER PINEAL
Inasmuch as the activity of serotonin N-acetyltransferase

(NAT) activity was considered to rate limit melatonin production
(Klein and Weller, 1970), studies were initially directed at this
enzyme. From these studies it rapidly became apparent that if
there was a relationship between the activity of the acetylation
of serotonin and the actual melatonin produced in the hamster
pineal, then melatonin synthesis was clearly higher at night than
dur ing the day (Rudeen e t aL, 1975; Rudeen and Re i te r, 1977).
& Hamsters
0.5 100()
-NAT
""
....
.<= --- Melatonin
- Period of darJcness
.,
"5
c
0..4 BOO
.....,
.0.
"0
c
c
.~ 0..3 600 ~
% ·cc
.g
~ c
~
u
0..2 400 ~
c co
:Z., 0.
u
~ 0..1 /-1 200
:::; L ___ ~ __ -¥""
c
0. I I I I I I I 0.
1200 1600 2000 2400 0400 0600 1200
Time
Fig. 1. Pineal N-acetyltransferase (NAT) activity and melatonin

content in the pineal of the Syrian hamster throughout
a 24 hour period. Characteristically, there is a
strong correlation between the activity of the
acetylating enzyme and melatonin levels.
Soon thereafter, when a sensitive radioimmunoassay for melatonin

became available, a nocturnal rise in pineal melatonin in the
Syrian hamster was demonstrated (Panke et al., 1978; 1979;
Tamarkin et al., 1979) (Fig. 1). Subsequently, serum levels of
melatonin were also reported to be higher at night than during
the day in the hamster (Brown et al., 1981b; Reiter, 1982). Of
some interest is that photoperiods which are known to suppress
EFFECTS OF THE PINEAL GLAND ON REPRODUCTIVE AXIS 319
the reproductive physiology of hamsters do not change

dramatically the apparent production of the compound within the
pineal gland (Reiter, 1982).
Besides serotonin being metabolized to melatonin in the

pineal, it is also converted to a number of other indoles (Fig.
2). Some of these may have antigonadotrophic actions (Reiter and
Vaughan, 1977). Recently, 5-methoxytryptamine has received some
interest in this regard (Pevet et al., 1981b).
H H
I I
~-C.C-NHI
t.J-,.) ~ /-'0
~ C)H
II
Tryptophan
~(D H H H H H II
IKI-f""ir--rH-NHI HO-(O-H-OH ~CHP-(O+f-OH
~) HC=O H H I H H
~ OH N
I
N
I
II H H
S-Hydroxytryptophan S-Hydroxytryplophol S-Methox.ytryplophol
~w f(61 H 0
OH·Cb+{-NHI -
H H
(51
OH
1""'ir--n
'vl)-~-C-H I "
H H
N
I. I
II
s- Hydroxytryptamine
1
S-Hydroxyindolc acetaldehyde
(b llill -COlli CtJI'

(Serotonin)
I(l) H II II 0 (6) H 0 4 H 0
HO- "" -~ -~ -N-C-CH, OH "" I (I
-f-C-OH _CHP- "i'-C-OH
N HH H ... N H
I I I
II h II
""-Act'lyl-S-hydroxylryplamine S-Hydroxyindole .cetic I<:id S,....Methoxyindolc acetic acid
(N-Acelylserotoninl
~(4) ~ ~ ~ II
CIIP-Cb-~-~-N-C-CH,
I
II
S--Methoxy-N-.«tyltryptamine
(Melaloninl
Fig. 2. A summary of tryptophan metabolism in the pineal gland.

The enzymes involved include (I) tryptophan
hydroxylase, (2), aromatic L-amino acid decarboxylase,
(3), N-acetyltransferase, (4), hydroxyindole-O-methyl-
transferase, (5), monoamine oxidase. 5-hydroxyindole
acetaldehyde (6) is an unstable intermediate and is
either oxidized to 5-HlAA or reduced to 5-hydroxy-
tryptophol.
ANTIGONADOTROPHIC ACTIONS OF .MELATONIN
Although the inhibitory effect of the pineal gland on the

reproductive system of the hamster was demonstrated in the mid
1960s (Czyba et al., 2964; Hoffman and Reiter, 1965; 1966), early
attempts to illustrate the antigonadotrophic actions of melatonin
320 R. J. REITER
in this species failed (Reiter, 1974). In these early studies

melatonin was administered in a variety of ways (injected daily
or subcutaneously implanted); however, the proper experimental
protocol for uncovering its gonad-inhibiting action escaped
detection. Finally, in 1976 Tamarkin and colleagues finally
identified a treatment schedule which inhibited the reproductive
system of hamsters to the same degree as did exposure to short
days. A chief requirement for the antigaondotrophic action of
melatonin in hamsters is its time of administration. In line
with this, Tamarkin et al (1976) kept hamsters under light:dark
(LD) cycles of 14:10 (lights on daily at 0600 hours) and injected
them with 25 I-lg melatonin at either 1000 hours or 1600 hours
daily. Whereas the morning injections, as had been reported
previously (Reiter, 1974), completely failed to alter the sexual
status of the animals, the same dosage of melatonin given in the
afternoon totally suppressed the activity of the neuroendocrine-
reproductive axis. Thus, hamsters obviously have a period when
they are insensitive to melatonin and a period when they respond
to the indole. The changing sensitivity of the system to
melatonin may be regulated by melatonin itself (Reiter, et al.,
1978; Reiter, 1980a). This aspect of melatonin's action will be
discussed later in the present report.
The observations of Tamarkin and co-workers (1976) were

readily confirmed in an independent laboratory in which single
daily injections of melatonin were given to intact,
pinealectomized or pineal denervated hamsters at 1900 hours daily
(Reiter et al., 1976). The purpose of this study was to
determine whether the ability of afternoon melatonin injections
to inhibt reproduction depended upon an intact pineal gland.
Indeed, melatonin could inhibit the functioning of the neuro-
endocrine-reproductive system only in those hamsters in which the
pineal was intact and sympathetically innervated. This implied
that the exogenously administered melatonin was either acting
directly on the pineal gland to release some other gonad-
inhibiting factor or that melatonin administered in the afternoon
was synergizing with endogenously produced melatonin (or some
other pineal compound) released at night. Judging from more
recent investigations, it appears that any procedure which
interferes with the nighttime rise in melatonin also curtails the
ability of nighttime melatonin injections to suppress the
reproductive system (Reiter et aL, 1982). The implication of
these results is that exogenously administered melatonin probably
acts in concert with endogenously produced melatonin to induce
regression of the reproductive system. Apparently in this
scheme, the two pulses of melatonin must be separated by at least
several hours. The need for spacing the two injections is also
supported by the findings that melatonin can induce reproductive
involution in pinealectomized hamsters provided it is given as
widely-spaced daily injections (Tamarkin et al., 1977).
Melatonin, administered by the means described above, is

equally as effective as darkness in inhibiting reproduction in
the Syrian hamster (Reiter, 1980b) (Fig. 3); likewise, where they
have been measured the hormonal changes induced by repeated
melatonin injections are similar in timing and magnitude to those
caused by reduced photoperiods (Reiter, 1981). This is strong
evidence for the conclusion that melatonin is the factor wich
mediates the inhibitory effect of the pineal gland on repro-
duction in this species. Finally, the quantity of melatonin
required to inhibit reproduction has been established (Rollag et
al., 1980b). It appears that hamsters must receive on the order
of 1.6 jJg melatonin daily as a subcutaneous injection in order
for reproductive collapse to ensue. This is somewhat more than
is produced in the hamster pineal gland on a daily basis (Rollag
et aL, 1980b).
COUNTER ANTIGONADOTROPHIC ACTIONS OF MELATONIN
Not in all cases is melatonin inhibitory to the

neuroendocrine-reproductive axis of Syrian hamsters; indeed, in
some cases the indole actually prevents the suppressive influence
of darkness on the reproductive system. When these observations
were made they raise serious doubts about the normal role of
melatonin in mediating pineal induced gonadal involution. The
observations were made in 2 species, the Syrian hamster and the
Djungarian hamster (Phodopus sungorus) , within the same year. In
both species melatonin reservoirs, placed subcutaneously, negated
the inhibitory effect of short day exposure on reproduction
(Hoffmann, 1974; Reiter et aL, 1974). Although the .minimal
effective daily dose of melatonin required to prevent the
inhibitory action of short days was never definitively
established, it appears that less than 3.6 jJg released from a
subcutaneous deposit easily negates the suppressive action of
darkness and the pineal gland on the neuroendocrine-reproductive
axis of hamsters (Reiter et al., 1975).
To this point we had assumed that darkness mediated

reproductive collapse in the Syrian hamster was a consequence of
melatonin synthesis and release from the pineal gland. However,
the results summarized above revealed just the opposite, namely,
that melatonin, continuously available, prevented the pineal
gland from inducing regression of the testes or ovaries of
hamsters exposed to short days (Fig. 3). These results seem to
mean that either melatonin was not the pineal antigonadotrophic
factor or that a continual uniform level of melatonin, as opposed
to a daily nocturnal rise, somehow interfered with the normal
mechanisms governing darkness induced reproductive collapse.
Also, the interesting possibility was raised that subcutaneous
melatonin deposits may even prevent daily afternoon injections of
322 R. J. REITER
Reproductive Organs·d Syrian Hamsters
0 ~
TES ASO
3.2 04
24 0.3
0 ~
'" 1.6
E 0.2 E '"
~ ~
ri
'" '"
08 0.1
0.0 00
Long Days Short Days Short Days Short Days
INT INT PINX MEL PEL
3.2 04
24 0.3
0 ~
'" 1.6
E '"
02 E
0 0
0- 0-
ci
0.8 0.1
00 0.0
AM mel PM mel PM mel PM mel
INT INT PINX MEL PEL
Fig. 3. Testes (TES) and accessory sex organ (ASO) weights in

male hamsters kept in either short or long days or
given morning (AM mel) or afternoon (PM mel) melatonin
injections. Both pinealectomy (PINX) or subcutaneous
melatonin pellets (MEL PEL) prevent the action of short
days or afternoon melatonin inejctions. INT = intact
animals.
mela tonin from causing gonadal regression, i. e. , perhaps

melatonin could inhibit its own action. When tested this was,
indeed, found to be the case (Reiter et aL, 1977). Thus, if
long day exposed male hamsters received a subcutaneous reserve of
melatonin (in beeswax) and, additionally, were injected with 25
~g of melatonin each afternoon the reproductive system remained
highly functional.
Contrary to the initial impression, these collective results

are strongly supportive of the idea that melatonin is the pineal
antigonadotrophic factor in the Syrian hamster for the following
reasons. Obviously, properly administered melatonin causes
gonadal regression in this species (Tamarkin et al., 1976; Reiter
et aL, 1976); on the hand, continually available melatonin
prevents darkness induced gonadal regression (Hoffmann, 1974;

Reiter et aL, 1974). Most interestingly, melatonin deposits
under the skin also negate the action of afternoon melatonin
injections (Reiter et aL, 1977). This implies that gonadal
regression due to darkness and to afternoon melatonin injections
are caused by the same compound, i.e., melatonin. Continually
available melatonin apparently interferes with the ability of
cyclic melatonin (either endogenous from the pineal or exogenous
following an injection) to modify the reproductive system.
Presumably, melatonin may have these apparently paradoxical
effects by virtue of the fact that it alters the sensitivity of
its own receptors. This has come to be known as the down
regulation hypothesis and is discussed below.
DOWN REGULATION HYPOTHESIS OF MELATONIN) S ACTION IN THE SYRIAN

HAMSTER
The concept that melatonin regulates the sensitivity of its

own recepetors was first proposed in 1978 by Reiter and colleagues
and has been further elaborated in recent years (Reiter, 1980a;
Cardinali, 1981). The theory relies on the idea that when
melatonin acts on its receptors it temporarily desensitizes (or
down regulates) them to further melatonin for a period of time.
The theory explains most of the known actions of the indole in
terms of the reproductive system of the Syrian hamster. These
interactions are summarized in figure 4. When hamsters are kept
under long day conditions the endogenous melatonin peak occurs
late in the dark phase; when the indole is released it
theoretically acts on and down regulates its receptors. As a
result, early in the light phase the melatonin receptors are in a
desensitized state (Fig. 4, panel A). This explains why daily
melatonin injections in the morning are incapable of inducing
reproductive collapse. By late in the light period the melatonin
receptors have regenerated their sensitivity, i.e., they are no
longer down regulated, and daily injections of the indole at this
time leads to reproductive inhibition (Fig. 4, panel B). This
theory also explains why continually available melatonin prevents
both dark-induced and afternoon melatonin injection-induced
gonadal regression. When melatonin is persistently available
from a subcutaneous reservoir, its receptors are always in a down
regulated state (Fig. 4, panel C). Down regulation of the
melatonin receptors could, in fact, be a consequence of a change
in sensitivity (Reiter, 1980a) or an alteration in their number
(Vacas and Cardinali, 1970; Cardinali, 1981). Regardless, the
net effect is the same.
There have been a number of physiological tests of the down

regulation theory. We predicted that whereas large doses of
324 R. J. REITER
2S,..q
c::::::::J Ught A me'
8
c _Dark , inj
. E. ,------------..
~ i ,r-~, ,,
Ql : ,,
i \
::;; I
~ i
! j------j
I : I
I gonadal \ :
'..._--
_____i atrophy '-.j
i I I i i I ; i j , I j : i ,
0800 1200 1600 2000 2400 04p0 0800 0800 1200 1600 2000 2400 0400 0800
25f1-Q 'm. 25f1-g o

c
me'
deposit
me'
ini
me'
inj
me'
'-4I
"C' ioj
,.
12
o
i I I I , I ,,
W
::;;
I
,,
i i
.,~
C
"
nono
" none' I
.g'"
gonadal / gonadal / i' '\
_--------------,
response response , I
C ... '.._--
W
I I i i I I I I I , Iii I
0800 1200 1600 2(X){) 2400 0400 oeoo 0800 1200 1600 2000 2400 0400 0800
Time Time
Fig. 4. Summary of the down regulation theory of melatonin

action. See text for details.
melatonin, given in the morning, would not induce gonadal regres-

sion (since receptors were down regulated), they would prevent
the afternoon injection of the indole from causing reproductive
collapse since they would prolong the desensitization of the
melatonin receptors (Fig. 4, panel D). This is indeed the case.
When 1 mg or less of melatonin was injected at 1100 hours (lights
on at 0600 hours), it prevented the inhibitory action of smaller
doses of melatonin at 1700 hours on reproduction (Chen et al.,
1980; Richardson et al., 1981). Presumably, melatonin in the
morning prolonged the densensitized state of the melatonin
receptors and prevented the later injections of the indole from
causing an effect.
Finally, in further tests of the theory it has been found

that very large doses of melatonin even when they are given in
the afternoon prevent darkness-induced gonadal atrophy {Turek and
Pappas, 1980; Richardson et al., 1982}. Likewise, very large
doses of melatonin given in the afternoon to hamsters maintained
under long days does not cause gonadal atrophy (Chen, 1981).
Presumably, the large bolus of the indole desensitizes the
receptors much like does melatonin from a subcutaneous reserve.
This further indicates that darkness mediated shut down of the
reproductive system in the hamster is due to endogenously
produced melatonin. It is important to emphasize that the down
regulation theory of melatonin's action applies, at this point,
to the Syrian hamster. In other species melatonin may be

completely incapable of altering the sensitivity of its own
receptors.
ROLE OF MELATONIN IN SEASONAL REPRODUCTION
In photosensitive rodent species, the light:dark cycle

acting by way of the pineal gland is clearly involved in
determining seasonal variations in reproductive competence
(Reiter, 1980a; 1982; Stetson and Tate-Ostroff, 1981). The
specific hormone responsible for regulating the seasonal
reproductive cycle may be melatonin. Certainly, it causes many
of the same changes as does dark exposure (Tamarkin et al., 1976;
Trakulrugnsi, et al., 1979). Also, when administered by means of
the proper paradigm it can actually duplicate the annual cycle of
reproduction in the laboratory. As noted above, afternoon
injections of melatonin eventually cause a total collapse of the
gonads. After the reproductive organs are in an atrophic state,
either short day exposure (Reiter, 1973) or continued melatonin
injections (Tamarkin et aL, 1976) maintain the reproductive
system in a quiescent state for a period of time. Furthermore,
if either short day exposure (Reiter, 1972; Stetson et al., 1976)
or daily afternoon melatonin administration (Tamarkin et aL,
1976) are continued, the neuroendocrine-gonadal axis endogenously
regenerates to a functionally mature state. Thus, the
reproductive system is capable of becoming refractory to both
short days and to melatonin. Once the gonads have recrudesced
they remain refractory to both darkness (Reiter, 1975) and
melatonin (Bittman, 1978; Reiter et al., 1979) for a period of
time. Finally, the refractoriness to both short daylengths and
melatonin administration is interrupted by the maintenance of
hamsters under long day conditions for a prolonged period
(Reiter, 1972; Reiter et al., 1979). The interactions of the
photoperiodic changes and melatonin administration on the
reproductive system of the Syrian hamster are compared in figure
5.
The similarities between darkness induced and melatonin-

mediated gonadal regression as well as the development of
refractoriness to both conditions makes it seem likely that they
are both caused by the same hormone, Le., melatonin.
Considering that the animals become refractory to melatonin, the
amount of melatonin produced by the pineal gland does not have
to change throughout the year; rather, the responsiveness of the
animal to melatonin changes. When examined, it was found that
the pineal gland produces ample amounts of melatonin during all
stages of the annual reproductive cycle (Rollag et aL, 1980a;
Brainard et aL, 1983) although under natural photoperiodic
326 R. J. REITER
REACTIONS TO PHOTOPERIOD
I
I I
~~~ i
" q,.1
I
I lNHIaTlON
I PHASE
1
,I
REACTIONS 10 MELATONIN
;;j
...>
..J
FALL WINTER SUMMER
Fig. 5. Summary of the actions of photoperiod or melatonin

treatment on seasonal reproduction in hamsters. When
properly administered, melatonin duplicates the effects
of seasonally changing photoperiods.
conditions there may be slightly less melatonin produced during a

given day in the summer than in the winter (Brainard et al.,
1983).
CONCLUDING REMARKS
Although compounds other than melatonin have been recovered

from pineal tissue, none have been shown to produce the dramatic
reproductive effects that melatonin does. The changes induced by
the daily afternoon injections of melatonin into Syrian hamsters
are similar to those produced by short day exposure. On the
other hand, the continual availability of melatonin from a
subcutaneous reserve actually counteracts the inhibitory effect
of dark exposure on reproduction as well as negating the
inhibitory influence of daily afternoon melatonin injections on
gonadal physiology. Apparently, melatonin has the capability of

desensitizing its own recepto~s in the Syrian hamster. There is
now strong evidence that melatonin may mediate the seasonal
changes in reproduction which hamsters experience when they are
maintained under natural photoperiods throughout the year.
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tives", N. Birau and W. Schloot, ed., Pergamon, New York.
Stetson, M. H., and Tate-Ostroff, B., 1981, Hormonal regulation
of the annual reproductive cycle of golden hamsters,
Gen. Compo Endocr., 45, 329.
Stetson, M. H., Matt, K. S., and Watson-Whitmyre, M., 1976,
Photoperiodism and reproduction in golden hamsters:
Circadian organization and termination of photorefractor-
iness, BioI. Reprod., 14: 531.
Tamarkin, L., Westrom, W. K., Hamill, A. I., and Goldman, B. D.,
1976, Effect of melatonin on the reproductive systems of
male and female Syrian hamsters: A diurnal rhythm in
sensitivity to melatonin, Endocrinology, 99: 1534.
Tamarkin, L., Hollister, C. W., Lefebvre, N. G., and Goldman, B.
D., 1977, Melatonin induction of gonadal quiescence in
pinealectomized Syrian hamsters, Science, 198: 953.
Tamarkin, L., Reppert, S. M., and Klein, D. C., 1979, Regulation
of pineal melatonin in the Syrian hamster, Endocrinology,
104: 385.
Trakulrungsi, C., Reiter, R. J., Trakulrungsi, W. K., Vaughan, M.
K., and Waring-Ellis, P. J., 1979, Interaction of daily
injections and subcutaneous reservoirs pf melatonin on the
reproductive physiology of female Syrian hamsters, Acta
Endocr., 91: 59.
Tur~k, F. W., and Pappas, P., 1980, Daily melatonin injections
inhibit short day-induced testicular regression in hamsters,
Experientia, 36: 1346.
Vacas, M. I., and Cardinali, D. P., 1979, Diurnal changes in
melatonin binding sites of hamster and rat brains.
Correlations with neuroendocrine responsiveness to
melatonin, Neurosci. Letters, 15: 259.
THE 5-METHOXYINDOLES DIFFERENT FROM MELATONIN:
THEIR EFFECTS ON THE SEXUAL AXIS
1 2
Paul Pevet '
1) The Netherlands Institute for Brain Research

Amsterdam, and 2) Dept. of Anatomy and Embryol-
ogy, University of Amsterdam, The Netherlands
From the time of Lerner's discovery of melatonin

(Lerner et al., 1958) to the present, much pineal re-
search has been centered on the study of the effects of
melatonin, and to date this 5-methoxyindole is considered
by many authors as the pineal hormone responsible for in-
hibiting the reproductive system (details and references
in Reiter, 1980) or the pineal factor conveying the
photoperiodic message (see details and references inHoff-
mann, 1981.
Melatonin, however, is only one of the numerous in-

doleamines synthesized in the pineal gland. In the
gland, indeed the amino-acid tryptophan is converted to
5-hydroxytryptophan by hydroxylation (Lovenberg et al.,
1967; Jequier et al., 1969). Decarboxylation of this in-
dole derivative (Lovenberg et a1., 1962; Snijder and
Axelrod, 1964) leads to the formation of 5-hydroxytryp-
tamine (serotonin). Serotonin, then, can be acetylated
to N-acetylserotonin (Weissbach et al., 1960), oxidized
to 5-hydroxyindole-3-acetic acid (Lerner and Case, 1960),
or metabolized to 5-hydroxytryptophol (McIsaac and Page,
1959). The 5-hydroxyindoles, 5-hydroxytryptophan, 5-
hydroxytryptamine (serotonin), 5-hydroxy-indo1e-3-acetic
acid, 5-hydroxytryptophol and N-acetyl-5-hydroxytryp-
tamine (N-acetylserotonin) can be methylated by hydroxy-
indole-O-methyl transferase (HIOMT) (Axelrod and Weiss-
bach, 1960, 1961; Axelrod and Lauber, 1968; Cardinali
and Wurtman, 1972). This means that at least 5 different
methylated products, 5-methoxytryptophan, 5-methoxytryp-
tamine, 5-methoxyindole-3-acet1c acid, 5-methoxytrypto-
331
332 P.PEVET
phol and melatonin, can be formed in the pineal (for more

details, see Balemans, 1979, 1981), the list probably be-
ing not complete since O-acetyl-5-methoxytryptophol might
also be present in the pineal (Smith et al., 1979). All
these 5-methoxyindoles and 5-hydroxyindoles are potential,
active factors.
In the present review we will examine the physiolog-

ical properties of the 5-methoxyindoles only, and we will
concentrate our analysis on their effect on the sexual
axis.
I 5-METHOXYTRYPTOPHOL
5-Methoxytryptophol (5-MTL) has been first identified

in bovine pineal by McIsaac et al. (1965) and later demon-
strated to be present in numerous other species (Table 1).
Contrary to melatonin (MEL), however, its synthesis occurs
during the light phase of the light:dark cycle, at least
in the hamster (Pevet et al., 1980). As Carter et al.
(1979) observed in the rat that during the light period
the plasmatic concentration is significantly higher than
during the dark period, it would be tempting to correlate
these two observations and to conclude that the circula-
ting 5-MTL originated from the pineal. However, in our
opinion, to date we cannot raise this conclusion, not on-
ly because it is difficult to compare results obtained in
rat and hamster, but especially because it is well-known
that 5-MTL, like MEL, is also synthesized in different
other organs such as, for example, the retina and the
Harderian gland (Balemans et al., 1981, 1982; Pevet et
al., 1980, 1981a).
Looking at the literature, it appears that 5-MTL,

like MEL, is capable of modifying sexual development and
reproduction, especially in birds and mammals. In juven-
ile chicken, for example, the gonadal growth is stimula-
ted by 5-MTL and MEL, whereas in maturing and adult ani-
mals both indolic compounds show an inhibitory effect,
that of 5-MTL however being more specific than that of
MEL (Balemans, 1972; Balemans et al., 1977a,b). A similar
stimulatory or progonadotropic effect of 5-MTL during
early post-hatching stages of development has been de-
scribed in the quail and, interestingly, in this bird
the effect appears to be sex-, age- and photoperiod-
dependent. 5-MTL, indeed, was found to stimulate ovarian
growth in young females kept in l6L/8D or l2L/12D but not
in 8L/16D, and did not have any effect in males (see
details in Alexander, 1969).
5-METHOXYINDOLES: THEIR EFFECTS ON THE SEXUAL AXIS 333
Table 1. Pineal 5-methoxytryptophol (5-MTL) content in

different mammals
F: fluorometry, GCMS, gas chromatography-mass spectrometry,

WT: wet tissue
Technique
Species Concentration References
used
Cow 1260 ~ 150 pmol/g WT GCMS Beck et al., 1981
Dog 170 ~ 10 ng/g WT F Miller and Maickel, 1970
Pig 15.1 ~ 2.3 pmol/g WT GCMS Beck et al., 1981
Rat 480 ~ 90 ng/g WT F Miller and Maickel, 1970

154 pg / pineal gland GCMS Carter et al. , 1979
Sheep 370 + 90 pmol/g WT GCMS Beck et al., 1981
Considering mammals, it appears also that very often

5-HTL presents the same effects as MEL. Both compounds,
for example, inhibit in mouse ovarian and uterus hyper-
trophy induced by an injection of HCG or PMSG (Vaughan et
al., 1967), inhibit the compensatory ovarian hypertrophy
occurring after unilateral gonadectomy (Vaughan et al.,
1972), decrease ovarian weight, and inhibit estrus in the
rat. In these last two experiments 5-MTL was found to be
even more effective than MEL (McIsaac et al., 1964). This
is also true when the effects of both compounds implanted
in the median eminence or in the reticular formation on
the pituitary LH content of castrated male rats are con-
sidered. 5-MTL induced indeed a decrease larger than
that obtained with MEL (Fraschini et al., 1970). Admin-
istration of melatonin (Debeljuk et al., 1970) or 5-MTL
(Talbot and Reiter, 1973/1974) induces a similar decrease
in the rat pituitaryFSH level., but when implanted in the
median eminence only 5-MTL reduces FSH secretion in rats
(Fraschini, 1969; Fraschini and Martini, 1970). When in-
jected intracisternally into young female rats, both com-
pounds delay similarly the time of vaginal opening (Fra-
schini et al., 1971), They also delay light-induced
estrus in the ferret, but here 5-MTL is less effective
than MEL (Herbert, 1971). 5-MTL is also known to block
copulation-induced release of gonadotropin in adult rats
334 P.PEVET
Table 2. Pineal 5-methoxytryptamine (5-MT) content in different

mammals
F: fluorometry, GCMS: gas chromatography-mass spectrometry,

WT: wet tissue
Technique
used
Cow 2.63 pmol/g WT GCMS Beck et al., 1981

117 ~ 48 pmol/g WT GCMS Bosin et a1., 1979
Baboon 2400 ng/g WT F Prozialeck et al. , 1978
Dog 740 ng/g WT F Prozialeck et al., 1978

740 ~ 40 ng/g WT F Miller and Maickel, 1970
Man 0-152 pmol/g WT GCMS Bosin and Beck, 1979

12.8 + 4.9 ng/g WT F Prozialeck et al., 1978
Pig 26.4 + 3.9 pmol/g WT GCMS Beck et al., 1981

228 + 119 pmol/g WT GCMS Bosin et al., 1979
Rat
(Wistar)
0.035 pmol/g GCMS Beck et al., 1981
3010 + 1700 ng/g WT F Prozialeck et al., 1978
3700 ~ 300 ng/g WT F Miller and Maickel, 1970
4200 ~ 900 ng/g WT GCMS Cattabeni et al., 1972
2000 ng/g WT F Maickel and Miller, 1968
(Sprague-Daw ley)
0.028 + 0.005 pmol/g WT GCMS Beck et al., 1981
Sheep 1.78 ~ 0.53 pmol/g WT GCMS Beck et al., 1981

545 + 180 pmol/g WT GCMS Bosin et al., 1979
(Farrel et al., 1968) and, when regularly injected, to in-

hibit spermatogenesis (Mas andOakin, 1977).
5-MTL appears thus to be a very active principle
with clear antigonadotropic properties. Surprisingly, how-
ever, and although some of these findings (e.g. McIsaac
et al., 1964) are contemporary with those made with mel-
atonin, the study of the endocrine properties of 5-MTL
has been neglected by most pinealogists. The reason for
this disinterest, which is now considered to be due to

the fact that 5-MTL has not been shown to be capable of
inhibiting the hypothalamo-hypophyseo-gonadal axis of the
hamster (Sackman et al., 1977), is not clear and especial-
ly illogical when we remember that it was only in 1976
that Tamarkin et al. clearly demonstrated that MEL is
able to induce gonadal atrophy in the hamster. Moreover,
5-MTL is known to have a strong effect on the sexual ac-
tivity of the hamster. Indeed, Reiter et al. (1975) have
reported that weekly implantation of 5-MTL .in male ham-
sters exposed to a light/dark cycle of 1:23 prevented the
short photoperiodically induced depressiqn of pituitary LH
and prolactin, and testicular and accessory sex organ re-
gression. This effect which, depending of the author, is
called a progonadal or a counterantigonadal effect, is
- in our opinion - very important. Indeed, if we consider
with Hoffmann (1981) that the pineal is not an antigonado-
tropic organ but a gland involved in conveying the effect
of photoperiodic stimuli upon the neuroendocrine axis,
or with Pevet et al. (1981c) and Pevet and Haldar-Misra
(1982a) that all 5-methoxyindoles synthesized by the pin-
eal as well as by other organs such as the retina and the
Harderian gland are implicated in a system by which the
pineal and some other organs such as the brain are able
to perceive, to differentiate and to integrate environ-
mental information (photoperiod, temperature, etc.), the
results obtained by Reiter et al. (1975) could be inter-
preted by the fact that, when constantly released, 5-MTL
prevents the detection by the animal of the short photo-
period. This would mean that 5-MTL, like MEL or 5-methoxy-
tryptamine (see next subheading), is deeply implicated
in the control of seasonal reproductive activity, at least
in the hamster.
II 5-METHOXYTRYPTAMINE
5-Methoxytryptamine (5-MT) is known to be ?resent

and synthesized in the pineal gland (Table 2) (Balemans
et al., 1980a,b, 1982; Pevet et al., 1981a) and also,
like MEL, in the, Harderian gland, intestine and retina
(Prozialeck et al., 1978; Pevet et al., 1981a; Balemans
et al., 1982). Comparing the results obtained, for exam-
ple, by the same team and with the same technique, it
appears that the concentration of 5-MT in the pineal is
higher than that of MEL, at least in the rat (Cattabeni
et al., 1972; Maickel and Miller, 1968), a result which
is probably due to the higher 5-MT synthesis, as was
demonstrated in mole, hamster and Spalax pineals (Bale-
mans et al., 1980b, 1982; Pevet et al., 1981a). As 5-MT
336 P.PEVET
has been detected in human plasma (Franzen and Gross,

1965; Prozialeck et al., 1978) - and with a higher value
during the night (Hooper et al., 1981) - in cerebrospinal
fluid (Prozialeck et al., 1978) and urine (Prozialeck et
al., 1978; Haddox and Saslaw, 1963), and as the transforma-
tion of a small fraction of circula~ing melatonin into 5-
MT (see below) cannot explain the large amount of 5-MT
found in the blood (Hooper et al., 1981), it is very prob-
able that 5-MT, like MEL, is released from the pineal
and/or other organs into the general circulation.
In the past few years 5-MT has attracted a great deal
of interest, mainly because (1) it is a potential precur-
sor in the biosynthesis of the psychomimetic agent 5-meth-
oxy-N,N-dimethyltryptamine (Holmstedt and Lindgren, 1967)
- a pathway shown to be present in the human pineal (Guch-
hait, 1976) - (2) it possesses psychomimetic properties of
its own (De Montigny and Aghajanian (1977), and (3) it
mimics the action of serotonin on brainstem neurons (Brad-
ley and Briggs, 1974). At the level of the sexual axis
the relatively small amount of investigations has permit-
ted to clearly demonstrate that 5-MT has a profound in-
fluence on sexual development and reproduction. In the
quail, for example, 5-MT influences gonadal maturation and
activity. This effect, however, is highly age-, sex- and
photoperiod-dependent (Alexander, 1969). Low doses of 5-MT
(10 ~g/day) stimulate the rate of sexual maturation in
juvenile females kept in 16L:8D but not in 12L/12D or 8L:
16D. Large doses (100 ~g and 500 ~g) are less effective
(Alexander, 1968). In adult females 5-MT inhibits ovarian
growth when they are exposed to a photoperiod of 12L:12D
but not when exposed to 8L:16D (Alexander, 1969). 5-MT was
also found to prevent delayed maturation induced by pro-
lactin treatment, indicating so that prolactin and 5-MT
exert antagonistic effects on a common mechanism control-
ling gonadal secretion, at least in the female quail
(Alexander, 1969). In male quails 5-MT does not seem to
stimulate sexual maturation but was found to lower testic-
ular weight at 45 but not at 35 days of age (Alexander,
1969). Concerning mammals, 5-MT appears to be also very
potent. In young mice, for example, it inhibits ovarian
growth induced by injection of gonadotropin (Vaughan et
al., 1976). 5-MT, like MEL, is also known to suppress
the LHRH-induced release of LH from neonatal rat anterior
pituitary glands in organ culture. In this test, however,
MEL appeared to be more effective (Martin et a1., 1977).
When injected daily in the late afternoon in hamsters
kept under long photoperiod (14L:10D), 5-MT, like MEL
(Tamarkin et al., 1976), induces a complete gonadal
atrophy similar to that observed in hamsters maintained in
short light cycles or in darkness (Pevet et al., 1981b).
In contrast to MEL which, under the same experimental con-

ditions, needs the presence of the pineal to be effective
(Reiter et al., 1976), 5-MT acts even in pinealectomized
animals. However, in such operated animals the regression
of the testes is less when compared with that observed in
intact hamsters (Pevet et al., 1981c). It thus appears
that 5-MT - althogh in itself causing a slight antigon-
adotropic effect - is, like MEL, more effective if the
pineal is present. Moreover, like for MEL (Tamarkin et al.,
1976), the reproductive system of the male hamster shows
a diurnal rhythm in sensitivity to 5-MT. Indeed, under
the same experimental condition, when injected in the
morning, 5-MT does not have any effect on testicular ac-
tivity (Pevet et al., 1981c). Subcutaneous deposits of
5-MT in male hamsters prevent the reproductive quiescence
which is the consequence of natural or of experimentally
shortened daily light conditions (Pevet and Haldar-Misra,
1982a). Yet, this result is similar to that obtained with
MEL (Hoffman, 1974; Reiter et al., 1974). This is also the
case when 5-MT is injected daily in the morning in animals
kept under short photoperiod (10L:,14D). Under these con-
ditions 5-MTL, like MEL (Turek and Pappas, 1980), prevents
at least partly the short-photoperiodic~lly induced gon-
adal atrophy (Pevet and Haldar-Misra, 1982b).
Taken together, these results clearly demonstrate
that 5-MT is a physiologically active compound which, if
administered properly, can duplicate most of the effects
of the photoperiod on the reproductive axis of the male
hamster. In the hamster as well as in the rat 5-MT gives
apparently the same physiological response as does MEL.
This raises thus the question of whether these effects of
5-MT and MEL are independent ones and are due to (1) a
direct or indirect transformation of 5-MT into MEL, or,
at the contrary, (2) to a transformation of MEL into 5-MT.
As explained by Balemans et al. (1980a), and al-
though such metabolic pathways have never been demon-
strated, the possibility that exogenously administered 5-MT
is acetylated by N-acetyltransferase and, thus, trans-
formed into MEL in the pineal or in other organs such as
the retina or the Harderian gland, cannot be excluded. The
physiological properties of 5-MT would thus be indirectly
due to MEL. Such a transformation of 5-MT into MEL, for
example, would explain why the antigonadotropic effect of
5-MT injected late in the afternoon can be prevented by
morning injections of large doses of MEL but not of 5-MT
(Pevet and Haldar-Misra, 1982c). Indeed, as the N-acetyl-
transferase activity is activated only during the dark
period of the light/dark cycle (Klein, 1979), the 5-MT
injected in the morning could not be transformed into
MEL. However, such a transformation would not permit to
338 P.PEVET
explain why morning injections of 5-MT prevent short-

photoperiodically induced gonadal atrophy (Pevet and Hal-
dar-Misra, 1982b). For thi~ last-mentioned experiment a
possible 5-MT-induced increase of MEL synthesis, demon-
strated to exist in rat (Trentini et al., 1982), could
perhaps be considered.
5-MT is produced by a direct methylation of serotonin.
In the rat, Fiske and Huppert (1968) have demonstrated
that injection of MEL provokes an increase in pineal
serotonin content. The effects of MEL could therefore be
due to the increase of 5-MT indirectly provoked by the
MEL-induced increase of serotonin production. This could
explain for example why, when injected late in the after-
noon into hamsters kept under long photoperiod, 5-MT but
not MEL is effective in pinealectomized hamsters. It has
been demonstrated in vitro (Rogawsky et al., 1979) as
well as in vivo (Beck and Jonsson, 1981) that a small a-
mount of circulating MEL (0.3 - 0.8% in vitro) is deacetyl-
ated and thus transformed into 5-MT. Considering now that
other organs containing aryl acy1amidase, the enzyme most
probably responsibe of~the deacetylation (Beck and Jons-
son, 1981), such as the kidney (Krebs et a1., i947; Nim-
mo-Smith, 1970; Hoagland and Graf, 1971) might also be
implicated in such a ph~nomenon, the possibility that the
effect of exogenously administered MEL is in fact due
to 5-MT has to be seriously considered. For example, the
observation that three daily injections of MEL are ef-
fective in pinealectomized hamsters could be due to the
fact that so the concentration of 5-MT formed by deacetyl-
ation of MEL would be enough to induce gonadal atrophy.
However, thedeacetylation of the circulating MEL does
not explain why MEL injected in the morning in the male
hamster (Pevet and Haldar-Misra, 1982c) but not 5-MT
prevents gonadal atrophy induced by 5-MT injected in the
late afternoon.
It is very probable - at least, this is our present
opinion - that both compounds have their own effects, but
that these effects are physiologically dependent of each
other. Moreover, it is not possible to exclude that, de-
pending of the time of injection and the experimental
conditions, one of the 5-methoxyindoles, or part of it,
in transformed into the other, and vice Versa, such a
phenomenon being perhaps an integral part of the physio-
logical response.
III 5-METHOXYINDOLE-3-ACETIC ACID
Like other 5-methoxyindoles, 5-methoxyindole-3-

acetic acid (5-MIAA) is present (table 3) and synthesized
in the pineal g~and (Axelrod and Weissbach, 1961; P6vet

et al., 1981a; Balemans et al., 1982) and in several
other organs such as the retina (Pevet et al., 1981a;
Balemans et al., 1982) and the Harderian gland (Balemens
et al., 1982). Such a synthesis, when compared to that of
MEL, appears not neglectable, and for example Lerner et
al. (1960) found that the amount of 5-MIAA in the pineal
is 10 times greater than that of MEL. Beck et al. (1981)
also found a higher concentration of 5-MIAA than of MEL
in the cow and sheep, but not in the pig.
Table 3. Pineal 5-methoxyindole-3-acetic acid (5-MIAA) content in

different mammals.
F: fluorometry; GeMS: gas chromatography-mass
spectrometry, WT: wet tissue
Technique
used
Cow 17600 + 1500 pmol/g WT GCMS Beck et al., 1981

2000 ng/g WT Lerner et al., 1960
Dog 80 ng/g WT * F Miller and Maickel, 1970
Pig 406 + 117 pmol/g WT GCMS Beck et al., 1981
Rat 1000 ng/g WT F Quay, 1964

170 ng/g WT* F Miller and Maickel, 1970
Sheep 22800 + 5400 pmol/g WT GCMS Beck et al., 1981
* concentration of 5-HIAA/5-MIAA
It is very probable that, like the other 5-methoxy-

indoles, 5-MIAA is released from the pineal and/or other
organs into the general circulation. However, this is dif-
ficult to determine since the 5-MIAA in the animal is also
formed by other pathways. 5-MIAA, indeed, is a metabolite
of practically all 5-methoxyindoles (Delvigs et al., 1965),
and almost all 5-methoxyindoles, when administered exo-
genously, are more or less converted to 5-MIAA. For ex-
ample, when administered into rat or human, 5-MTL is met-
abolized in approximately 2 hours. The major metabolite
is 5-MIAA and has been found by isotope dilution tech-
nique to represent 93.3% in rat (Delvigs et al., 1965) and
53-73% in human (Hoskins and Pollit, 1978) of the admin-
340 P.PEVET
istered 5-MTL. Similarly it has been demonstrated that

most of the 5-MT injected or formed by deacetylation of
MEL is rapidly deaminated by MAO and converted to 5-MIAA
(Rogawski et al., 1979; Kopin et al., 1961; Kveder and
McIsaac, 1961). Using radioactive 5-MT, Kveder and Mc-
Isaac (1961) have demonstrated that 96,8% of it was trans-
formed into 5-MIAA. Concerning MEL, the major pathway
for metabolism is hydroxylation in position 6, followed
by sulphate or glucuronide conjugation. However, 0.5 - 2%
of the exogenously administered MEL is also transformed
into 5-MIAA (Kveder and McIsaac, 1961; Kopin et al.,
1961.
5-MIAA appears thus to be a metabolite of the three
5-methoxyindoles known to have a clear effect on the
sexual axis (see before). This raises immediately the
question of whether the observed effects of MEL and
especially of 5-MTL and 5-MT could not be indirectly due
to 5-MIAA. Apparently, this is not the case. Indeed,
5-MIAA injected late in the afternoon into hamsters kept
under long photoperiod is not able, contrary .to MEL and
5-MT, to induce testicular atrophy (Pevet, unpublished
result). It would also be possible to ask whether a com-
pound which is an end-product of 5-MTL, 5-MT and partly
of MEL metabolism, excreted into urine (Delvigs et al.,
1965; Kopin et al., 1961; Kverder and McIsaac, 1961) could
not be simply considered as a product of degradation
without physiological effects. This will explain why 5-
MIAA was found to have no effect in the in vitro rat
pituitary response to LHRH (Martin et al., 1977) and on
the ovarian hypertrophy occurring after unilateral gon-
adectomy in mice (Vaughan et al., 1972). However, as 5-
MIAA is synthesized, like other 5-methoxyindoles, in many
organs (see before) it cannot, in our opinion, simply be
considered as a product of the degradation of the other
5-methoxyindoles. This opinion is supported by the fact
that in young mice 5-MIAA inhibits the growth of ovaries,
induced by injection of gonadotropin (Vaughan et al.,
1976).
IV 5-METHOXYTRYPTOPHAN
To our knowledge, Balemans et al. (1978) were the

first to mention 5-methoxytryptophan (5-MTP) as a 5-meth-
oxyindole possibly formed in the pineal gland. Later it
has been demonstrated that this compound was indeed syn-
thesized in the pineal (hamster: Balemans et al., 1982:
mole: Pevet et al., 1981a; parakeet: Balemans et al.,
1981, and rat: Balemans et al., 1980a) as well as in the
retina (mole, hamster) and the Harderian gland (hamster)
(Pevet et al., 1981a; Balemans et al., 1982) From all

5-methoxyindoles, 5-MTP appears also to be the one of
which the synthesis is the most important. In the mole,
for example, the pineal synthesizes 50 to 100 times more
5-MTP than MEL (Pevet et al., 1981a) while in the hamster
4 to 10 times more 5-MTP than MEL is produced by the
Harderian gland (Balemans et al., 1982). Moreover, looking
at the annual variations of the capacity of the pineal,
retina and Harderian gland to synthesize 5-methoxyindoles
in hamsters kept under natural climate conditions, 5-MTP
appears to be the compound which presents the highest
annual variations. It is thus very probable, at least in
our opinion, that 5-MTP is a 5-methoxyindole of great
physiological importance. Unfortunately, as the proper-
ties of 5-MTP have practically never been studied, no ex-
perimental data support this opinion. For example, 5-MTP
injected in the late afternoon or morning (Pevet et al.,
1981b) or continuously administered (Pevet, unpublished
data) into hamsters kept under long photoperiod does not
have any effect on testicular activity. This, however,
does not mean that 5-MTP has no biological effect. It
simply means that in the experimental model used 5-MTP
has no effect on the parameter studied, the testes of the
hamster.
CONCLUSION
From the experiments described in this review it is

evident that some 5-methoxyindoles different from MEL
greatly influence sexual development and reproduction, at
least in birds and mammals. Therefore, MEL is clearly not
the only indolic compound which has to be considered when
pineal function is studied.
It has been suggested by Pevet and Haldar-Misra (1982
a) that the 5-methoxyindoles would be implicated in a
system enabling the pineal and some other organs such as
the brain to perceive, to differentiate and to integrate
environmental information such as photoperiod, temperature,
food, etc. In response the pineal, then, would synthe-
size and release proteic/peptidic hormone(s) which would
have an effect on the function of the reproductive system.
The question whether all 5-methoxyindoles are im-
plicated in the transfer of all information or whether
each of the 5-methoxyindoles is specialized for one or
more of these parameters, has now to be answered. To date
the experimental data are so rare that it is difficult to
do so. However, it has been demonstrated that both MEL and
5-MT are deeply implicated in the conveyance of photo-
periodic information. Moreover, as 5-MTP is synthesized in
342 P. PEVET
the retina, Harderian gland and pineal of hamsters kept

under natural environmental conditions only during winter,
it would be possible that 5-MTP is implicated in convey-
ing information on temperature. This, however, remains to
be demonstrated.
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Acknowledgement: The author wishes to thank Miss J.Se1s

for skillful secretarial aid.
ACTIONS OF THE PINEAL GLAND AND MELATONIN
ON THE SECRETION OF CEREBROSPINAL FLUID
w. B. Quay
Neuroendocrine Laboratory, Department of Anatomy

University of Texas Medical Branch
Galveston, Texas, U.S.A.
INTRODUCTION
Association of pineal function with flow or content of the

cerebrospinal fluid (CSF) is a very old concept. In western civili-
zation it came into prominent view with the writings of Herophilos
of Alexandria (325-280 BC). However, then and through many subse-
quent centuries, this functional association was based essentially
on philosophical speculations, which were contested and eventually
discredited, bringing down with them many of the notions derived
from, and dependent upon, them. This long and faltering early phase
of "pinealology" corresponds to the first of the three periods in
pineal research as summarized by Kappers (1979).
The present, and third, period in the historical evolution of

pineal research has given us new vistas of the true functional
relations of the pineal. One of these research vistas that I believe
to hold especial importance concerns major functional interrelations
between the pineal and the central nervous system (CNS). I believe
that present evidence suggests that on both the pineal and the nervous
system sides of these interactions, both neural and humoral mechanisms
are involved. By neural mechanisms we mean innervations. By humoral
mechanisms we mean chemical mediations at the tissue humor or parahor-
mone level as well as at the truly hormonal level. The hormonal level
of chemical mediation has the more prominent status in current con-
cepts of mammalian pineal physiology and in the design and interpreta-
tion of experiments. In comparison, the range of probable mechanisms
of mediation involving innervations and local humoral mechanisms is,
I believe, understated. Having said this, we must admit, however,
that the physiological and pharmacological actions we will be discuss-
349
350 W.B.QUAY
ing are known chiefly in terms of a hormonal paradigm of pineal

action. Furthermore, on the pinealopetal side, aside from sympath-
etic innervation, relatively little is known about how information
concerning brain and/or CSF characteristics and composition may be
transmitted to and received by pinealocytes.
It is now clear that the pineal and its hormones have multiple
sites of action and kinds of effects within the CNS. References
are available in the review of Trentini et al. (1979), and in the
present volume those by Cardinali and Mess. My report here is
limited to considerations of evidence of pineal actions on mechan-
isms governing the secretion and composition of CSF, a hitherto
relatively neglected subject. The possible physiological impact,
however, of pineal actions on secretion and composition of CSF may
extend far beyond the particular primary target cells involved in
these actions. This is because controls of neuronal excitability
and transport mechanisms are affected significantly by small changes
in electrolyte concentrations and in intracranial pressure, through
the media of CSF and related compartments within the brain and its
tissue coverings. CSF represents the extra- and intercellular fluid
of the CNS, and is known and studied chiefly as the fluid within the
internal ventricular system and the. external arachnoidal covering
and cisternal systems of the CNS and its meninges.
My concern with a possible pineal role in regulation of the

electrolyte concentrations of the brain started with the obtaining
of unexpected experimental results (Quay, 1965). These showed that
in pinealectomized rats, as compared with sham-operated and non-oper-
ated sibling control animals, a sodium-deficient diet led to a signi-
ficantly lower potassium content in some brain regions (Quay, 1965).
Furthermore, continuous light produced the same effect as pinealect-
omy on brain potassium concentrations in animals on a sodium-defic-
ient diet. But brain potassium concentrations were not lower in
pinealectomized rats following a potassium-deficient diet. These
and other results from experiments and available published informa-
tion led to the hypothesis that in the evolution of the pineal and
the astrocyte system of the brain in tetrapods, the pineal acquired
a role in regulation of electrolyte concentration possibly by acting
on the selective transport and metabolic activities of astrocytes
(Quay, 1965, 1969, 1970b). In studies on temporal relations of
mitotic activity of astrocytes following a standardized forebrain
lesion, there was no generalized effect of pinealectomy. But a
difference was seen in the phase relations of astrocyte mitoses in
the amygdaloid nuclei in pinealectomized animals (Quay, 1974b).
Pinealectomy also failed to modify significantly brain water content
after two kinds of phase-shifts in photoperiod, and at two times in
relation to the timing of the daily photoperiod (Quay, 1970a). It
has been difficult to study quantitatively and with sensitivity the
responses of astrocytes 'since they are usually dispersed among other
cell types within CNS tissue. However, with technical advances in
PINEAL GLAND AND SECRETION OF CEREBROSPINAL FLUID 351
the use of potassium-sensitive microelectrodes, autoradiography and

immunocytochemistry, important research advances now appear more
likely in this area.
The hypothesis that the mammalian and human pinealocyte responds

to changes in intracranial pressure is currently supported by only
circumstantial and indirect evidence. This hypothesis is, however,
worthy of note because it points out one category of relationship on
the afferent side of a pineal role in the control of secretion and
composition of CSF. In early and detailed work on human pineal
pathology Walter (1923) observed that hypertrophy of pinealocytes was
a concomitant of a diverse array of chronic pathological or disease
conditions which were either causes of, or associated with, increased
intracranial pressure. A reevaluation (Quay, 1974a) of a set of
necropsy data on pineal enzyme activities in man (Otani et al., 1968)
also can be interpreted as relevant to this hypothesis.
Our present report and review focuses primarily on recent exper-

imental evidence suggesting that the mammalian pineal through the
agency of at least one of its hormones, melatonin, has a regulatory
role in relation to secretory or transport, and metabolic, activities
of the ependymal epithelial cells of the choroid plexuses of the
lateral ventricles of the brain.
STIMULATORY EFFECTS OF MELATONIN ON CHOROIDAL EPITHELIAL CELLS
Recently we have obtained ultrastructural and morphometric evi-

dence for stimulatory actions of melatonin on choroidal epithelial
cells in male Golden Hamsters (Mesocricetus auratus) (Decker and Quay,
in press). The Golden Hamster is a species in which a greater role
for humoral control of choroid plexuses might be expected on the
basis of the observation by Lindvall et al. (1977) that of ten stud-
ied species, the Golden Hamster had the least evidence of a choliner-
gic nerve supply to its choroid plexuses. It is to be appreciated,
however, that other kinds of innervating fibers are not ruled out,
and that there are differences between the choroid plexuses of the
different ventricles in terms of their characteristics of growth
(Quay, 1972), metabolism, composition (Quay, 1966) and innervation
(Lindvall et al., 1977).
Animals and Procedures
Male Golden Hamsters were injected daily for 28 consecutive

days starting at 4 to 5 weeks of age, with vehicle alone (0.1 ml
0.9% NaCl 30% ethanol), or 25 ~g melatonin (Sigma), or 2500 ~g mela-
tonin in 0.1 ml of vehicle. The animals were kept in a LD 14:10
photoperiod and received their injections in dorsal skin folds on
the body between 13:00 and 13:45 (= Lll.OO to Lll . 75 ). They were
352 W.B.QUAY
killed by cervical dislocation on the afternoon of the day following

the last injection day. Choroid plexuses (CPs) were removed and
fixed within 5 minutes of the time of death. Each of the 16 animals
provided 3 CP samples: (1) CPs of ventricles I and II, the lateral
ventricles, pooled; (2) CP of ventricle III, including its posterior
portion which extends into the suprapineal recess (= dorsal sac);
and (3) CP of ventricle IV. Primary fixation was in 2% glutaralde-
hyde 2% paraformaldehyde in 0.1 M pH 7.3 sodium cacodylate buffer,
first at room temperature and then overnight at 50 C. The CPs sub-
sequently were rinsed in cacodylate buffer and post-fixed in 1% os-
mium tetroxide 1.5% potassium ferricyanide in 0.1 M sodium cacodyl-
ate (Langford and Coggeshall, 1980). Staining en bloc with uranyl
acetate, dehydration through ethanol solutions and passage through
propylene oxide preceded embedding in an Epon/Araldite mixture
(Mollenhauer, 1964). Thick (l~) sections stained with toluidine
blue were inspected and sui table areas were thin-·sectioned and
stained with uranyl acetate and lead citrate (Venable and Cogges-
hall, 1965).
Thin sections were examined with a Philips EM 300 and the first
ten "whole" cells per each animal's regional (ventricular) sample
were photographed. For the purposes of this study, "whole" cells
were those choroidal eperidymal cells that showed within the parti-
cular section field their nuclei and both the apical (ventricular)
and basal plasma membranes. For morphometry, electron photomicro-
graphs were taken at tap 1 (x 3,160) and later enlarged (x 2.8)
giving a final magnification of x 8,850.
Ten choroidal ependymal cells from each of the three ventricu-

lar CP regions (1+11, III and IV) were studied from each of the
animals. Thirteen different kinds of counts or measurements were
made. These were not converted to volumes or volume densities
since sampling was biased in favor of cells sectioned in the verti-
cal system of planes with respect to the plane of the ependymal epi-
thelial layer. Therefore, the significance of the resulting measure-
ments resides chie~ly in their comparative value ~or the purpose o~
testing for kind(s) and magnitude(s) of effect(s) of melatonin.
They may not accurately represent true absolute values of component
volumes within the cells.
Observations
The ultrastructural characteristics of the choroidal ependymal

cells in our Golden Hamsters conformed to what has been described
for these cells in other species (Westergaard, 1970; Davis and Mil-
horat, 1975; Agnew et al., 1980; Koshiba et al., 1980; Allen et al.,
1981). However, our chief interest was in those features that re-
late to functional activities and that demonstrated significant
differences following daily injections of melatonin.
Significant differences were found in four features of the cells

suggesting stimulatory actions by melatonin: (1) increase in cyto-
plasmic volume (Table 1), (2) increase in nuclear volume (Table 1),
Table 1. Effects of Melatonin and Ventricular Origin on Mean

Areal Measurements of Choroidal Ependymal Epithelial
Cells in Male Golden Hamsters (data of Decker and
Quay, in press)
Measurements (~2) Ventricles

Melatonin I + II III IV
llg /day x ± SE x ± SE x± SE
Total Cell Area

0 74.6±3.6 <.001 75.1±4.5 71.8±3.8
25 78.6±1.8 <.001 78.0±4.6 86.2±5.9
2500 115.6±8.4 * 82.9±5.0 78.0±4.0
Cell Area Minus
Nucleus & Blebs
0 58.7±3.8 <.001 60.5±4.0 55.5±3.1
25 60.0±2.6 <.001 63.3±4.5 66.4±7.5
2500 88.4±7.8 * 65.6±3.8 61.3±3.3
Nuclear Area
0 15. 5±1. 5 14.8±2.2 13.7±1.1
25 15.4±0.9 <.05 13.5±1.1 15.9±0.9
2500 20.4±1.8 * 16.3±1.9 15.3±0.9
ap = probability based on Scheffe tests following ANOVA,

in comparisons with starred (*) value in same column and
kind of measurement.
(3) increase in mitochondrial area per cell (Table 2), and (4) in-
crease in length of apical (ventricular) microvilli (Table 3). All
of these effects observed at the ultrastructural level were either
more notable in, or were restricted to, the choroidal ependyma of
the lateral ventricles as compared with those of ventricles III and
IV (Tables 1 through 3).
354 W.B.QUAY

Mitochondrial Area Per Cell, Mitochondrial Fraction
of the Cytoplasm and Area Per Mitochondrion, in
Choroidal Ependymal Epithelial Cells in Male Golden
Hamsters (data of Decker and Quay, in press)
Measurements Ventricles
Melatonin I + II III IV
11g /day x- ± SE pa x± SE x± SE
Mitochondrial Area Per Cell

o 7.34±0.42 * 7.57±0.57 8.65±0.76
25 9.23±0.43 <.05 8.76±0.59 9.28±0.42
2500 9.97±0.50 <.005 9.07±0.57 9.97±0.43
Mitochondrial Fraction of Cytoplasm (1 = 100%)

o .1251±.0068 <.025 .1250±.0073 .1559±.0072
25 .1539±.0066 * .1383±.0083 .1398±.0082
2500 .1128±.0049 <.001 .1382±.0121 .1626±.0090
Area Per Mitochondrion (11m2)

o .1308±.0134 .1099±.0190 .1317±.0058
25 .1417±.0086 .1312±.0173 .1469±.0153
2500 .1346±.0076 .1413±.0095 .1520±.0173
a p = probability based on Scheffe tests following ANOVA, in

comparisons with starred (*) value in same column and kind
of measurement.

Length of Microvilli on the Ventricular Apices of
Choroidal Ependymal Epithelial Cells in Male Golden
Hamsters (data of Decker and Quay, in press)
Melatonin Ventricles
11g/day I +II III IV
x ± SE pa x± SE x ± SE
0 827.2±40.6 <.OOlb,c 1052. 4±lf1. 7 10l2.8±34.1
25 1026.0±30.8 <.OOlc 1053.5±50.5 1027.l±31.9
2500 1245.7±30.8 1146. 8±91. 2 1113.9±46.1
ap = as in Table 2, with comparisons with 25 (b) and 2500 11g (c).

DISCUSSION
The cellular changes produced by melatonin injections may repre-

sent two general categories of cellular response. The increase in
cytoplasmic and nuclear areas or volumes (Table 1) appears to indi-
cate a kind of cellular hydrops. This interpretation is based on the
fact that this swelling of the choroidal ependymal cells occurred
only in animals receiving the very high (2500 ~g/day) dose of mela-
tonin, while the other cellular responses to melatonin had a graded
increase from 0 to 25 to 2500 ~g dose levels. Nevertheless, the
swelling of the choroidal ependymal cells with the high dose serves
to demonstrate regional differences in the sensitivity of the chor-
oidal ependymal cells to melatonin. The results in the top and mid-
dle thirds of Table 1 show that the choroidal ependymal cells of the
lateral ventricles increased about 50% both in total cell area and
in cell area minus nucleus and blebs, while there was no such in-
crease in the comparable cells of ventricles III and IV after the
high dose of melatonin.
The second category of cellular response to melatonin here, is

of a graded nature and may be more confidently interpreted as repre-
senting cellular stimulation by melatonin. The increase in mitochon-
drial area per cell (top third of Table 2) appears to represent an
increased number of mitochondria per cell and not an increase or
change in average size of the mitochondria (bottom third of Table
2). The low value for mitochondrial fraction in choroidal ependymal
cells of the lateral ventricles after high melatonin dosage (middle
third of Table 2) can be attributed at least largely to the afore-
mentioned swelling of these cells. Visual comparisons of the cells
suggest that it is chiefly in the apical cytoplasm, that toward the
ventricle, that the increase in number of mitochondria occurred.
Mean length of apical microvilli also showed a graded response

to melatonin. This too was notable only in the choroidal ependymal
cells of the lateral ventricles. On the basis of circumstantial
evidence this response appears to be interpretable as representing
a stimulatory action of melatonin. Although the morphological evi-
dence is incapable of providing directional determinations of changes
in fluid movement across the choroidal epithelium, the 24 (25 ~g
melatonin) to 50% (2500 ~ melatonin) increase in mean length of the
microvilli suggests increased capacity for such flow or transport.
The recent demonstration of ouabain-sensitive Na+,K+-ATPase in these
apical microvilli (Masuzawa et al., 1981) indicates the involvement
of these structures at this particular location in the secretion of
CSF and in ion transport. The driving force for CSF secretion has
been believed for many years to be the active transport of Na+ across
the choroid plexus cells. According to a model (Wright, 1972) of how
this system works, Na+ enters the choroidal epithelial cell from the
serosal solution in relation to an electrochemical gradient, and is
actively secreted into the CSF across the brush border (= apical
356 W. B. QUAY
microvilli) by a ouabain-sensitive Na+,K+ exchange pump. The loca-

tion of this pump has been demonstrated also by means of autoradio-
graphy of the binding of ouabain from solutions of very low concen-
tration (Quinton et al., 1973). Much remains to be done of course,
in order to determine: (1) the extent to which this pump and its
actions and linkages are influenced by pinealectomy and/or melatonin,
(2) the generality among different species and choroid plexuses of
such pineal or melatonin effects, and (3) the contribution of such
effects to physiological and pathological regulation of CSF volume
and pressure.
Since melatonin in this study was administered systemically, it

appears likely that the accessibility of melatonin to the choroid
plexuses of the different ventricles was equivalent. It may be
functionally significant that the choroidal ependyma at the start of
CSF's pathway of flow (ventricles I + II) was affected by melatonin
while those situated close to the pineal (ventricle III, including
the suprapineal recess) and "downstream" (ventricle IV) were not.
This suggests that at least in the Golden Hamster, if endogenous
melatonin has a role in influencing the CPs of the lateral ventricles
in their secretion of CSF, the effective route of access can be vas-
cular and systemic rather than necessarily either intraventricular
or by a special local vascular route or portal system starting in the
pineal. Melatonin has been found in CSF and blood of man (I. Smith
et al., 1976; Arendt et al., 1977; Vaughan et al., 1978; Brown et
al., 1979; J. A. Smith et al., 1979; Tan and Khoo, 1981) and other
mammals (Withyachumnarnkul and Knigge, 1980; Perlow et al., 1981).
CSF levels of melatonin are generally lower than .blood levels, and
several other lines of evidence have been raised for the conclusion
that at least in man and rat the major route of pineal release of
melatonin is into the bloodstream rather than the CSF (Vaughan et
al., 1978; Brown et al., 1979; Withyachumnarnkul and Knigge, 1980;
Tan and Khoo, 1981). After intravenous injection of ~-melatonin in
rats, melatonin is detectable in CSF within 1 minute and reaches
peak levels in CSF and choroid plexuses at 5 minutes (Mess and Tren-
tini, 1974). However, in the bullfrog melatonin can be taken up
from CSF by the choroidal epithelium by an active transport process
located at the apical brush border (Smulders and Wright, 1980).
Increased intracranial pressure (ICP) either from increased CSF

pressure or increased vascular perfusion pressure is generally
thought to lead to a lowering of the rate of secretion of CSF by the
choroid plexuses (Sahar, 1972b). However, elevation of ICP does not
appear to influence the rate of production of CSF if the cerebral
vascular perfusion pressure is maintained above a particular level,
such as 70 mm of Hg in cats (Weiss and Wertman, 1978). These and
other findings serve as caveats for anyone tempted to take a simpl-
istic view of the control of either ICP or rate of formation of CSF.
In relation to the interpretation of our results with melatonin,
some partial satisfaction may accrue from the general belief that
no bulk absorption of CSF occurs via the choroid plexuses (Sahar,

1972a), and the finding that the potassium concentration of CSF
secreted by the choroid plexuses is actively regulated by them
whether the primary alteration in potassium levels occurs in plasma
or CSF (Husted and Reed, 1976).
Among important questions yet to be addressed are the following:

(1) Are the choroidal ependymal effects of melatonin general in rela-
tion to different species? (2) Are they, like many of melatonin's
effects, dependent upon critical timing within circadian and related
biorhythms? (3) What, if any, kinds of stimuli relating to ICP and/or
CP function serve in the elicitation of melatonin release for regula-
tion of CP cells?
CONCLUSIONS
In one species, the Golden Hamster, melatonin has been shown

here to affect quantitative characteristics of the choroidal ependy-
mal cells of the lateral ventricles, but not those of ventricles III
and IV. Those affects that are increased with dose level appear to
be stimulatory in nature, and to be consistent with the hypothesis
that among other roles for melatonin as a circulating hormone is one
relating it to stimulation of secretion of CSF and possibly of rela-
ted activities linked to regulation of transport of particular ions,
such as K+, through the choroidal ependymal epithelium.
These findings possibly have relevance to melatonin's actions

in humans, and provide a basis for caution in experimental adminis-
tration of large doses of melatonin to human subjects. They may
also provide the basis tor testing humoral and additional pharmaco-
logical approaches to control of CSF secretion and of some types of
dangerous increase in intracranial pressure. Melatonin, other pineal
5-methoxyindoles, and their analogues merit study with regard to
these possible relationships and therapeutic problem areas.
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PINEAL-HYPOTHALAMIC INTERACTIONS: POSSIBLE ROLE OF THE MONO-
AMINERGIC NEURON SYSTEM
B. Messl, G.P. Trentini 2 , C. Ruzsas l ,

and C.F. De Gaetani 2
1. Department of Anatomy, Univ. Med. School P~cs
Hungary
2. Department of Pathological Anatomy, Uni v. of
Modena, Italy
I. INTRODUCTION
Reviewing the program for this training course, it is easy

to realize that many of the lectures are somehow related to the
topics of the present paper. (See lectures of Cardinali, Dema-
ine, Moore, Quay). Therefore, it is difficult to find the
proper boundary between the different aspect of similar re-
search problems. In this view, the general morphological, phy-
siological and biochemical changes in the central nervous sys-
tem, due to the action of the pineal hormones, will be touched
upon only very briefly. Therefore, I would like to concentrate
rather on the problem of the site and mechanism of action of
pineal hormones Qy which this gland influences the function of
the neuroendocrine system. I will attempt to summarize the
data indicating a central nervous point of attack of pineal
indoles that influence the secretion of the hypothalamic re-
leasing hormones.
361
362 B. MESS ET AL.
II. THE ROUTE OF PI~~AL SECRETION.
In the introductory lecture of the Ciba Foundation's Sym-

posium on the pineal gland, held in London, Ariens-Kappers (1971)
devoted a special chapter entitled "Mode of secretionaf pineal
products". This chapter thoroughly discussed the possibility,
whether the pineal gland secretes its products into the capillary
network of the organ, like a classical endocrine gland, or if
these bioactive principles (hormones) are secreted directly into
the cerebrospinal fluid (CSF). In this lecture, and later in the
general discussion of the entire symposium, Ariens-Kappers
strongly stressed that " ••• all light and electron microscopical
data point to the extrusion of the pineal products into the gen-
eral blood circulation •••• pharmacological findings are, in
itself, no reason for accepting that, under physiological cir-
cumstances, the pineal secretory products are extruded into the
cerebrospinal fluid". This view was supported by the reeent
findings of Wi thyachumnarnkul and Knigge (1980). They found that
the melatonin concentration was significantly higher in the
blood of the confluens sinuum than in the peripheral blood. CSF
taken from the cysterna magna had an even lower melatonin con-
centration. The melatonin level was increased in the blood but
not in the CSP in the dark-night per:l.od. Besides these morpho-
logically and physiologically well-founded statements, some
physiological CPraschini et al., 1971), pharmacological (Martini
and Fioretti, 1971), anatomical (Sheridan et al., 1969) or ul tra-
structural (Hewing, 1980) data suggest the possibility of a di-
rect CSF route for pineal secretions.
Rather than belabor the pros and cons concerning this mat-
ter, I would like to briefly 91ll1DBarize our investigations per-
formed in the early seventies to approach this puzzling problem.
PINEAL-HYPOTHALAMIC INTERACTIONS 363
The most direct evidence would be to compare the indoleamine

(e.g. melatonin) level of the general circulation, of the
effluent blood of the cannula ted pineal veins and of the cerebro-
spinal fluid, as had been suggested by the speaker at the above
mentioned pineal symposium in London (Mess 1971). Because of
technical reasons we had to omit the cannulation of the pineal
veins and an alternate method was applied. 3H-Melatonin was
injected intravenously into adult male rats, and the presence
of labelled melatonin was parallelly dete~ed in the peripheral
blood, CSF, choroid plexus, hypothalamus, midbrain and cerebral
cortex at different intervals after 3H-melatonin injection. In
another set of experiments, the tadpole skin-lightening effect
was used for the comparative bioassay of melatonin concentra-
tion of the blood, CSF and choroid plexus of cats.
Table 1. summarizes the results. Only 2 minutes after its
i.v. injection, labeled melatonin appeared in the CSF in high
concentrations. A significant isotope accumulation was detected
in the CSF even after 2 hours. On the other hand, the choroid
plexus showed 2-6 fold higher melatonin content than either
hypothalamic or mesencephalic tissue. To verify that melatonin
passes from the blood stream to the eSF in a biologically active
form, a bioassay CBagnara, 196)) was performed. As demonstrated
in Table 2 •• biologically active melatonin appears in the CSF
and in the choroid plexus in high concentrations 1 and 2 hours
after i.v. injection of purified melatonin. - These data lead
to the conclusion that melatonin is secreted into the capillaries
of' the pineal gland, and then concentrated by the choroid plexus
and secreted into the CSF. It might explain the controversy de-
tailed in the introduction. The pineal secretes its hormones,
like all true endocrine glands, into the capillary network of
the gland, but being concentrated in the esp, they might act
on the central nervous system through the CSP.
364 B. MESS ET AL.
Table 1
Levels of 3H-melatonin in blood, CSF and different areas of the
brain in the albino rat (Dose of me1atonin=40f,lCi/rat)
Minutes CPM/ml of blood and CSF, and CPM/mg

after brain and ghoroid l!lexus
melatonin Blood CSF Choroid Hypotha- Mesence-
injection serum l!lexus lamus l!halon
1 43230 9950 63,0 21,5 18,7
2 29838 10698 54,2 17,3 19,0
5 37 frl 8 20969 66,0 11,4 9,4
10 31093 10119 52,0 11,2 11,4
20 22123 8800 33,7 5,0 4,5
30 16252 5388 34,1 4,7 4,8
60 8046 3801 18,4 4,2 3,4
120 2273 1000 19,8 3,2 1,6
180 2497 1636 9,9 2,1 2,4
360 1624 890 21,4 1,4 1,8
720 873 793 17,8 2,0 2,0
°The statistical difference between the uptake of the choroid

plexus and of the hypothalamus and mesencephalon was equally
highly significant p < 0,001.
III. ACTIONS OF PINEAL BOBIOHES ON THE CENTRAL NERVOUS SYSTEJII
Our results, summarized above, as well as the data of

Cardinali et al., (1973, 1978), clearly indicate that ai ther
i.v. or intracysternally injected melatonin will be stored by
the hypothalamus and mesencephalon in considerably higher con-
centrations than by the cerebral or cerebellar cortex. The
Table 2
Melatonin level in eSF, blood serum and choroid plexus ot mela-
tonin injected cats as assayed by Xenopus laevis skin melanophore
test.
Experimental Melanophore index

group
eSF Serum Choroid Melatonin Untreated
plexus lUg/ml tadpoles
Untreated +3,8
control cats 1,79 4,73 -°,30 2,17 4,73
1 hour atter
500 J,l.g melato-
nin injeetion 1.80 2.2cr 2.0OXX 2.30 4.80
Untreated !,74 ~,96 ~,59
control cats -0,23 -0,34 -0,30 2,35 4,31
1 hour after l,22x 1,76 4,53
1000 ug -0,12
melatonin
injection
Untreated ~,31 t,81 1,68
control cats -0,27 -0,08
x
2 hours after l,83 1,94 4,67
1000 J,l.g -0,12
melatonin
injection
x p < 0,01 compared to the corresponding untreated control

cats
xx p < 0,05 compared to the corresponding untreated control
cats
366 B. MESS ET AL.
melatonin content of different regions of the human brain, de-

tennined by radioimmunoassay, was always many-fold Cabout 50
times) lower than that found in the pineal • No measurable HIOMT
activity has been shown in brain tissue indicating that melato-
nin, which is present in the brain, is not synthetized in situ
CKopp et al., 1980). Since Niles et al. (1919) detected melato-
nin receptors in the brain, it seems to be very probable that
melatonin is taken up and stored by specific melatonin receptors
within the brain tissue. The trapped pineal hormones signifi-
cantly influence the function and metabolism of the brain tisBue.
As mentioned in the introduction, only the major points of these
latter aspects of pineal-hypothalamic interactions will be men-
tioned briefly.
Pinealectomy significantly increases the MAO activity of
the pituitary and less dramatically of the hypothalamus, where-
as melatonin decreases MAO activity in the same organs. It was
suggested that MAO is a terget enzyme for melatonin CUrry and
Ellis, 1915). Data, indicating the role of species differences
in the reaction of the hypothalamus to melatonin under in vitro
conditions, were reported by Frehn et al. Cl914). !J. 4-reductase
activity of the hypothalamus was decreased in the hamster and
increased in the rat when incubated with melatonin CIO- 6mol/l).
Other enzymes of the brain also are influenced by the pineal
gland: Barbanti and Trantini (1965) reported that the ablation
of the superior cervical sympathetic ganglia induced a marked
increase in the acetylcholinesterase activity of the magnocellu-
lar hypothalamic neurosecretory nuclei. Pinealectomy, on the
other hand, depressed the thiamine diphosphat-phosphohydrolase
activity of the same hypothalamic nuclei. This latter affect
Was reversed by melatonin or by 5-methoxytryptophol CDe Vries,
1912).
Not only were different enzyma. tic activities of the brain
influenced by pineal hormones, but the protein synthesis of the
hypothalamus and hypophysis, demonstrated by incorporation of

3H leucine into protein, also was dramatically depressed by
melatonin pretreatment (Orsi et al., 1973). However, the protein
synthetio rate of the pineal, or that of mONIT activity, were
not influenced by melatonin. ~e investigators oonsidered these
results to provide further experimental evidence for the role
of the hypothalamo-hypophyseal system as a target organ for
pineal melatonin.
The functional activity of neurons from different brain
areas also is generally influenced by pineal indoles. Aside from
detailing the well-known data showing that melatonin has a se-
dating effect of the eNS (Anton Tay, 19'14; Marezynski et al.,
1964 etc.), the more recent electrophysiologic results of
Demaine and Kann (1979 a and b) should be mentioned. They studied
the effects of the pineal indoles, melatonin, 5-methoxytryptophol
and 5-hydroxytryptophol, on the electrical activity of neurones
in the preoptic and anterior hypothalamic areas of rats and
hamsters. Upon electrical unit recording of single neurones
wi thin these brain areas, melatonin increased, while 5-hydroxy-
tryptophol decreased the rate of neuronal discharge in the
majority of the cases. Pineal indoles also modified the inhibi-
tory responses to dopamine and noradrenaline. In evaluating
these results, one should take into consideration that only
these areas playa major role in the regulation of the hypotha-
lamo-hypophyseal-gonadal axis.
There are still several important effects indicating the
action of' p:i.neaJ. honnones exerted on the ONS. The main aim of'
this paper, however, is the discussion of a special aspect of
pineal-hypothalamic interplay. In the subsequent part of this
talk, I shall try to s'Wllll18.rize the findings dealing with the
mechanism of pineal-hypothalamic interactions and its integra-
ti ve role in the regulation of the hypothalamO-hypophyseal
neuroendocrine circuit.
368 B. MESS ET AL.
IV. EFFECTS OF PINEAL HORMONES AT THE LEVEL OF THE ADENOHYl'O-

PHYSIS
As discussed above, the pineal gland and its honnones exert

oonsiderable influence on the central nervous system, mainly at
the level of the hypothalamus and mesencephalon. However, the
action of the pineal hor.mones, at the pituitary level also can
not be excluded. The in vivo experiments, performed in immature
(Mieno et al., 1978) and in adult dogs (Yamashita at al., 1978),
seem to verifY this possibility. The rate of secretion of testi-
cular 17-oxosteroids was significantly enhanced after intraca-
rotid injection of synthetic LBRH both in mature and immature
dogs. Melatonin itself did not influence oxosteroid output.
However, the effect of exogenous LHRH was markedly diminjshed
by melatonin pretreatment (10 or 100 P.g/kg intracarotidally 3
hours before LHRH administration). It was concluded by the
authors that melatonin may act directly on the anterior pituitary
gland in dogs to inhibit the IJIRH-indueed release of LH (Fig.
h)·
In addition to melatonin, two other bioactive pineal prin-
ciples, were reported to have similar effects: 5-methoxytryp-
tophol was less effective (Mieno et al., 1981), while arginine
vasotocin CAVT) proved to be as effective as melatonin in the
inhibition of the LHRH-induced rise in testicular secretion of
17-oxosteroids (Miano et al., 1980). It was suggested that 5-
-methoxytryptophol might act on the same anterior pi tui tary
si te as melatonin, whereas AVT might have a different site of
action.
This aspect was systematically investigated in rats both
under in vitro and in vivo conditions by Dr. Martin's group.
LHRH-induced LH release from pituitary cultures donated by rats
2, 5 and 10 days of age, was significantly diminished by adding
-- 100
I
C
·E
~
I
eo LH-RH
!
..:.:
eo 80 T
c
'-'
c
.2
60
~u
0
III
'"0
·0
... 40
2
'"
0
)(
0 20
r!.
.....0
0 0
~ -20-10 0 15 30 60 90 120
IX
Time (min) before and after LH-RH injection
Fig.l. Rate of secretion of 17-oxosteroids by one testis in re-

sponses to an intracarotid injection of luteinizing hormone re-
leasing hormone CLH-RH, 5 p,g/kg body wt) in dogs given melatonin
into the carotid artery 3 h before, expressed as ng produced
per kg body wt per min •• , 100 ug melatonin/kg body wt (seven
dogs); ., 10 t1g melatonin/kg (five dogs); 0, melatonin vehicle
Csix dogs). Each point represents the mean :!:S.E.M. 'p( 0,05;
<
"p 0,01 compared with the corresponding value when LH-RH was
given without pretreatment with melatonin (Yamashita et al.,
1978J.
melatonin to the medium at 10-7_10-10M concentrations. Serotonin

and 5-methoxytryptam1ne were similarly effective, but other
pineal indoles failed to inhibit La release. However, melatonin,
serotonin and 5-methoxytryptamine were ineffective in the inhib-
i ting LH release when pituitary glands from animals 21 and 30
days were incubated (Martin et &1., 1977 J. Comparable results
were obtained under in vivo experimental conditions. Serum LH
levels were significantly lower in the melatonin-pretreated
370 B. MESS ET AL.
LBRH injected male and female neonatal rats than in the solely
LHRH-in3ected anjmaJ s. Melatonin pretreatment, however, failed
to il1hibi t the LHRH-induced IB release in the older (35-44 day-
-old) animals (Martin et a1., 1980 a). It was concluded that
melatonin and serotonin can act directly on the neonatal
pi tui tary gland to suppress LHRH-induced release of LB.
The melatonin metabolite (6-hydrQx;yme1atonin) and a mela-
tonin analog (6 £luoro- and 4-6 d1fluoromelatonin) also were
tested under in vitro condi tiona. The hydroxy-metabolite of
melatonin lost, whereas the £luoro-analog preserved the capa-
bility to decrease LH-release at the pituitary level of neonatal
rats Ckrtin et al., 1980 'b).
!he release of FSH in response to LHRH alse was inhibited
by adding melatonin to the incubation medi,. (Martin and Sattler
1979). However, this effect was inversely related to the age of
young rats used as pituitary donors (Fig.2.). After 20 days of
age, the melatonin sensitivity of the neonatal rat pituitary
rapidly disappeared in this model. In contrast, Orts et ale
(1980) found that a pineal tripeptide, threonylseryllysine, si-
multaneously injected intravenously with s,ynthetic LHRH, caused
a significant delay of the ~SH surge for least 30 minutes after
injection in the adult male rat.
The discrepancy experienced between the findings of Yama-
shita et al. Cl978) and of Mart:i.n et al. (1977) conce:m.ing the
age of the ani mals might easlly be due to species differences
(dog and rat). The importance of species differences in these
types of studies also has been suggested by other investigaiors.
Both in the immature (8 and 12 day of age) and in the adult
Syrian hamster, melatonin had no effect at all on the LKRH-in-
duced LH release from the pituitary, equally under in vivo or
in :vitro experimental conditions (Bacon et al., 1981). Further-
more, Weinberg et ale (1980) also obtained no decrease in IBRH
response following melatonin infusion in young human volunteers.
:z
Z
0
!;i 50
•
~
w
:=E
•
1£ 40
:z
~ 30
I-
CD
~ 20
::r:
c.n 10
.....
I-
:z
w
u 0
a::
w
0-
5 10 15 20 25 30
AGE (days)
Fig.2. Relationship between animal age and percent maximal inhib-

ition by melatonin of pituitary FSH response to LHRH. The dif-
ference in FSH values between groups treated with LHRH alone and
groups treated with LHRH and lO-~ melatonin is expressed for
each age as a percent of stimulation over controls by LHRH al'one
(Martin and Sattler, 1979).
Thus, four different mammalian species with completely different

pituitary reactions to melatonin treatment is a point to be
underscored! One should notice that melatonin does not act at
all at the level of the anterior pituitary in two species
(hamster and man), or aets at this level only in a very limited
perinatal period in the other model (rat), and is fully active
only in the dog. It might be assumed therefore that the direct
372 B. MESS ET AL.
action of melatonin on the adenohypophysial tissue, inhibiting

LH release in response to the LHRH might not play an essential
no less a general role in the regulatory mechanism exerted by
the pineal gland on the neuroendocrine system.
V. PIJIEAL-CNI-GOB'ADO!ROPHIB' CmCUI~
The gonadotrophic bozmone system will be used as typical

example to try to summarize the mechanism of pineal hypotha-
lamic interactions to provide a framework to understand this
pineal-pituitary trophic Ce.g. LH) relationships. This will be
done for two reasons: firstly, the role of the pineal gland
has been most evidently and clearly demonstrated for reproduc-
tive functions and secondly, this phenomenon has been of major
interest to me and my collegues for more than a decade.
Pinealectomy was found to reastablish ovulation and lute-
inization in rats subjected to the constant estrous-anovulatory
(CEA) syndrome (Fig, 3.). This syndrome can be provoked by either
electrolytic lesion of the suprachiasmatic area, or by fro~tal
deafferentation of the hypothalamus (Mess et al •• 1971), It

subsequently was shown that pinealectomy also provokes ovula-
tion and luteinization in the neonatally-androgenize4 CRA
animals CRuzsas et al., 1977). Since the anovulatory animals
ovulated after pinealectomy, though at irregular intervals
(Kess et al., 1973), a preovulatory LR peak was noted (Trentini
et al., 1976), and the majority of these rats became pregnant
following pinea!ectomy (Trentini et al., 1973); thus it ap-
pears that pinealectomy can provoke an increased LH release in
CEA animals.
It is beyond the scope of this paper to discuss all the
considerations and evidence which have led us to the assumption
that this effect of pinealectomy might be mediated by the
Fig.3. Effect of pinealectomy on the hypothalamic lesion induced

polyfollicular ovaries of the rat. a, intact control; b, polyfol-
licular ovary; c, reappearance of corpora lutea in the polyfol-
licular ovary following pinealectomy; d, marked thecal luteiniz-
ation in the polyfollicular ovary following pinealectomy.
374 B. MESS ET AL.
serotoninergic neuron system of the brain stem. ~is assumption

was, however, verified by experiments demonstrating that the
ovulation-inducing effect of pinealectomy can be counteracted
by daily injections of low doses (1 mg/kg day) of serotonin.
Furthermore, the blockade of serotonin biosynthesis by para-
chlaro-phenylalanine (pCPA) treatment or by maintaining CEA rats
on a tryptophan-poor diet (TPD) also provoked ovulation and
luteinization in non-pinealectomized CEA animaJs (Table 3.)
(Tima et al., 1973).
Table 3
Effect of pineal ectomy, serotonin C5-HT) or parachlorophenyl-
alanine CpCPA) treatment, or keeping the animals on tryptophan-
-poor diet CTPD, for 30 days) on the occurrence of luteinization
in the hypothalamic deafferentation-induced constant estrous
anovula tory CCEA) syndrome.
Exp. group Occurrence of corpora lutea

at the end of 4 weeks after stopping
treatment % treatment %
1. CEA+saline 0/47 0,0 0/47 0,0
2. CEA+pinealectomy 11/17 64,7% 11/17 64,7x
3. CEA+pinealectomy+5-HT 2/25 8,0XX: 3/25 12,0XX:
4. CEA+pCPA 11/40 27,~ 21/40 52,~
5. CEA+TPD 11/30
% p < 0,01 va. group 1.

xx: p < 0,01 vs. group 2.
All these data bave been summarized in detail in a mono-

graph (Mess et al., 1978). Only the basic data were reviewed
here, serving as a starting point for our fUrther studies.
It appears curious that pinealectomy was completely inef-
fective in the third type of the CEA syndrome, i.e. in the
light-induced constant-estrous (LeE) animals. In our subse-
quent and presently ongoing experiments, this latter syndrome
proved to be an appropriate model for the investigation of the
mechanism of action whereby seroton1nergic pathways mediate the
effects of the pineal gland on the reproductive system.
~e explanation of the above finding that pinealectomy
failed to induce ovulation and luteinization in the LCE animals,

in contrast to the two other types of CEA syndrome, seemed to
be principle. Wurtman et ale (1964) stated that constant light
exposure reduces considerably 'the enzyme activity CHIOJfT) of
the pineal gland which is necessary for indoleamine production.
Therefore, continuous light exposure was called by the Axelrod-
-Wurtman group as "functional pinealectomy". The surgical extir-
pation of a functionally eliminated gland cannot provoke consid-
erable effects. The problem, however, still remained unresolved
why the removal, or functional inactivation of the pineal gland,
elicites La-release and consequently ovulation in the hypothala-
mically lesioned, or neonatal androgenization-induced CRA
syndrome, and not in the LCE animals? !file seemingly contradic-
tory finding of Reiter et ale (1974) provided one probable way
for an approach to this question. !filey reported that under
special light-mediated experimental conditions, melatonin inhi-
bi ts the antigonadotrophic activity of the pineal gland. In
other words. under some carefully controlled conditions, melato-
nin has, instead of the commonly known antigonadotrophic action,
a "progonadotrophic" function.
376 B. MESS Et Al.
In addition to our own results mentioned above, much data

in the literature (for survey see Kamberi, 1974) have stressed
the role of the seroton1nergic neuron system played in the reg-
ulation of gonadotrophin secretion. It should also be taken into
account that melatonin and serotonin concentrations of the brain
were shown closely correlated (Anton Tay et a1., 1968; Cardinali
1975). On the basis of these latter considerations, we tried to
approach the problem by utilizing melatonin or serotonin sub-
stitution in LeE animals.
Daily subcutaneous injections of 0,1 mg serotonin/rat
elicited luteinization in the ovaries of LeE animals. Neither a
higher (1,0 mg) nor a lower (0,01 mg) dose of this monoamine had
any effect (Hagino et al., 1974) suggesting that just the sub-
su tution of the physiological amount of serotonin JIlight play a
role in this mechanism.
A single intracisternal, but not subcutaneous injection of
50 ~g of melatonin also provoked ovulation in 37,5% of the LCE
animals. Even more pronounced was the effect of chronic melato-
nin administration: daily subcutaneous injection of 2xl00 pg of
this indoleamine, or its subcutaneous implantation in beeswax
pellets, restored the formation of corpora lutea in over 70% of
the LeE rats (Table 4.). The same melatonin doses were, however,
significantly less effective, when serotoninergic receptors were
parallelly blocked by Methiothepin injection, or if the animals
were maintained on a tryptophan poor diet CTrentini et al.,
1918). These results clearly indicate that the increase in the
melatonin level JIlight elicit LH release in the LeE animals on
the one hand, and on the other that this effect of melatonin
requires functionally intact serotoninergic receptors.
Rats with the fully developed CEA syndrome returned to a
normal light-dark rhythm, leads to the reappearance of vaginal
cyclicity and ovulation. However, the daily injection of
Table 4
Luteinization provoked by chronic melatonin treatment (for 20

days) in the light-induced constant estrous anovul.atory (LCE)
animals with or without pinealectomy, and counteraction of this
effect by methiothepin treatment or by feeding the animals with
tryptophanl""Poor diet C!PD).
Experimental group Hats revealil!!i ~uteiniz. Luteiniz-

No. of treated animals atioll
f
LCE+solvent in3ection 0/30 0,0
LCE+Melatonin s.o. 18/25 72,aa
(2%100 I1g)
LCE+Melatonin implanted 11/15 73,3xx
;i,n peeswax (3xl. DIg)
LCE+~elatonin s.c. 11/15 73,3xx
(2%100 I1g J+
+pinealectomy
LCE+Melatonin s.c. 6/20 30,~
(2xlOO I1g) +
+Methiothepin i.p.
(2xl0 mg/kg)
LCE+Melatonin s.c. 7/25 28,OX
C2xlOO I1gJ+TPD
:x: p < 0,01 vs. melatonin treated groups
xx p < 0,01 vs. controls
methiothepin (serotonin receptor blocker), or maintaining rats

on a tryptop~poor diet after replacement to a normal light-
-dark environment,significantly inhibits the restoration of
ovul.a tion. !rhese resuJ. ts again suggest a role of the serotonin-
ergic system in this mechanism. The direct proof for this as-
378 B. MESS ET AL.
sumption was provided by the experimental group, where LeE

animals were kept on TPD after replacement to a light-dark
cyclic environment, but were simultaneously daily injected with
5Omg/kg serotonin. All of the animals of this latter group
ovulated in contrast to the 48% occurrence of oVUlation in the
light-dark replaced TPD fed animals (Mess et al., 1979) (Table
.2.a.J
Table 5
Ovulation provoked by replacement of light-induced constant
estrous anovulatory (LeE) rats into a cyclic light-dark schedule
(LDJ, and the counteraction of this effect by methiothepin
treatment or by a tryptophan-poor diet CTPD) (20 days of treat-
ments).
Experimental group Occurrence of ovulation Statistical

Number Percent significance
1. LeE rats 0/17 0,0

2. LCE+LD 30/45 66,6 p < 0,01 vs.Gr.l.
3. LCE+LD+Methiothepin 2/19 10,5 p < 0,01 vs.Gr.2.
C2%10 mg/kg)
4. LCE+LD+TPD 12/25 48,0 p < 0,05 vs.Gr.2.
5. LCE+LD+TPD+5-HTP 10/10 100,0 P < 0,01 vs.Gr.4-.
(1 mg/kg)
On the basis of this last series of experiments, we pro-

posed a working hypothesis regarding the role of a "threshold
level" of serotonin in the elicitation of ovulation. According
to this hypothesis, a critical serotonin level is required to
induce or permit the activation of the complicated neuroendocri-
ne mechanism leading to the preovulatory LH-peak. Continuous

light exposure suppresses serotonin levels below this threshold
level by reducing melatonin biosynthesis in the pineal gland.
The replacement of the animals into the physiological LD envi-
ronment gradually restores serotonin biosynthesis, the brain
serotonin concentratiQn reaches the "threshold level" and
ovulation can then occur. This working hypothesis is compatible
with the findings of Hagino et al,. (1974) and Reiter et ale
(1974).
Melatonin treatment starting on the first day of continuous
light exposure, and injected at the beginning of the dark phase
(8 PIO, prevents the development of the CEA syndrome CTrentini
et al., 1981). !his latter finding led us to conclude that
melatonin might mimic the dark phase of the nyctohemeral rhytlua
in the CEA rat, therefore counteracting the effects of continuous
light exposure through the serotoninergic neuron system.
Finally, we sought for some morphological proof supporting
our view that serotoninergic neuron system might be the most
important mediator between the pineal gland and gonadotrophin
regulation. Furthermore, we tried to localize the most important
structures in the CNS participating in this serotoninergic
mechanism.
It is generally accepted that the origin of the ascending
serotoninergic pathway terminating in the hypothalamus are the
raphe nuclei of the mesencephalon CUngerstedt, 1971). ~e
serotonin concentration of these nuclei was quantitatively and
comparatively determdned by Palkov1ts et ale (1974) and their
da ta strongly suggested the importance of the midbrain raphe
nuclei in the serotoninergic mechanism outlined above. The
suprachiasmatic nuclear area of the hypothalamus was shown to
possess high concentrations and intensive turnover of serotonin,
which are closely related to neuroendocrine-regulating prope~ties
380 B. MESS ET AL.
of these areas CRa:i,sman and Brown Grant, 1977; Hery et al.,

1981).
On the basis of these findings, we investigated the effect
of electrolytic or of pha.:macological (neurotoxin) lesions of
these two regions of the CBS that are preSlDlled to play an integ-
rative role in the mediation of pineal effects in the eEA
syndrome.
Electrolytic destruction of the median or dorsal raphe
DUcleus or of the suprachiasmatic area failed to counteract
the effect of continuous light CLL) exposure. The occurrence
of corpora lutea was equally very low in each lesion-bearing
group following LL exposure. Under normal light-dark CLDJ
environmental circumstances, only suprachiasmatic lesions re-
duced markedly the occurrence of corpora lutea, as is well known
CHillarp, 1949; Flerk6, 1954). Destruction of the raphe nuclei
failed to diminish luteinization in LD anjmals. However, LL-
-exposed s~operated rats in~ected daily with melatonin re-
vealed luteinization in 75% of the animals. Electrolytic lesion
of the supracbiasmatic area and of the median raphe nucleus
significantly counteracted the luteinization-inducing effect of
melatonin administration in the LL anjmals. A similar, but less
significant effect was e~icited by the dorsal raphe lesions
CRuzsas et al., 1981) C!able 6, J. These results lend support to
the assumption that melatonin influences the reproductive func-
tion of the rat by affecting the activity of the median and
tless pronouncedly) the dorsal raphe nuclei and of the supra-
chiasmatic area.
Electrolytic lesion of these regions also might destroy
some non-serotoninergic structures belonging to the catechola-
minergic or liHRH systems, which could interfere with the effect
of lesions of the raphe system.
Pha:macologic destructions of the serotoninergic neurons
Table 6
Effect of electrolytic lesion of the median raphe CMR), dorsal

raphe (DR) or of the suprachiasmatic nuclei (SeN) on the occur-
rence of corpora lutea in ~exposed rats treated daily with
10 p.g melatonin (Mel).
Experimental Occurrence of corpora lutea Sta tistical

groups No of rats % significance
LD
1. Sham 14/15 93,3
2. MR 10/10 100,0
3. DR 16/16 100,0
4. seN 1/11 9,1 p< 0,01 vs.gr.l.
LL
5. Sham 1/20 5,0 p< 0,01 vs.gr.l.
6. MB. 3/12 25,0 p< 0,01 vs.gr.2.
7. DR 2/21 9,5 p < 0,01 va.gr.3.
8. seN 1/12 8,3
LL+Mel
9. Sham 15/20 75,0 p < 0,01 vs.gr.l
and 5.
10. MR 1/9 11,1 p < 0,01 vs.gr.9.
ll. DR 10/23 43,5 p < 0,05 vs.gr.9.
and 7.
12.sCN 2/21 9,5 p< 0,01 vs.gr.9.
by local microinjections of 5-7 dihydroxytryptamine seemed to

be more specific than electrolytic lesions. This drug was micro-
injected with the aid of stereotaxic instrument into the same
areas of the eNS as were destroyed by electrolytic lesions in
382 B. MESS ET Al.
Table 7
Effect of 5.7-dthydroxytrypt&m1ne lesions of the median raphe
OIR), dorsal. raphe (DR) or of the suprachiasmatie nuclei (SCN)
on the occurrence of corpora lutea in LL-exposed rats . treated
with melatonin (Mel. 10 !-Lg/day).
E%perimsntal Occurrence of corpora lutea Statistical

groups No of rats ~ significance
LD
1. Sham 14/15 93 .3
2. IIR 10/12 83.3
3. DR 9/10 90,0
4. seN 4/7 57,1 p< 0,05 va.gr.l.
LL
5. Sham 1/20 5.0 p < 0,05 va.gr.l.
6 . IIR 1/8 1 2.5 p < 0,01 vs. gr. 2 .
7. DR 0/9 0.0 p < 0,01 VB.gr.).
8. seN 0/8 D,D p < 0,05 va.gr.4.
LL+Mel
9 . Sham 1 5/20 75.0 p < 0,01 vs.gr.1.
and 5.
10. MR 10/18 55.0 p < 0,05 vs.gr.6.
11. DR 11/22 50,0 p < 0 , 05 vs.gr.7.
12. SCN 5/19 26.3 p < 0,01 vs.gr.9.
the previous experiment. Neurotoxic lesions of the raphe nuclei

or of the suprachiasmatlc area did not ~uence the development
of the ORA syndrome (abol ishment of fODlation of corpora lutea)
under LL conditions, similar to the effect of electrolytic
lesi ons. Under LD condi tiona, only suprachiasmatic lesion
influenced luteinization. However. the decreased percentage

occurrence of corpora lutea (57,1%) was less pronounced compar-
ed to that of electrolytic lesions of the same area (9,1 %).
Pharmacologic lesions of all the three investigated brain areas
significantly counteracted the luteinization-inducing effect of
melatonin treatment in the LL exposed animals. There was no
difference between the effect of the dorsal or of the median
raphe lesions (occurrence of corpora lutea 50 and 55% re-
spectively). however. these were significantly less effective
in counteracting the luteinization inducing effect of melatonin
than suprachiasmatic lesion (26,3% occurrence of corpora lutea;
Table 7.) , (Ruzsas et al •• 1982).
Comparing the results obtained by electrolytic and by
neurotoxin intervention. two main differences should be' stress-
ed. The neurotoxin lesion of the suprachiasmatic area was less
effective in provoking the CEA syndrome under LD conditions
than was electrolytic destruction. This might be explained by
presuming that the electrolytic lesion destroyed not only
serotoninergic terminals but some other structures, probably
belonging to the LHRH system. This interpretation is supported
by the findings of Mess et al. (1966), Palkovits et al. (1974)
and Samson et al. (1980) showing that the suprachiasmatic area
possess high LHRH content. Pharmacological lesions of the median
raphe nucleus was significantly less effective in counteracting
the luteinization-inducing effect of melatonin in LL animals
than was the electrolytic damage of the same nucleus. It is
conceivab~e that the electrolytic lesion also destroyed some
noradrenergic fibres and terminals in the vicinity of the
median raphe nuclei (Roizen and Jacobowitz 1976) CFig.4.).
The function of the pineal-serotoninergic system-hypot~
lamic-gonadotrophincircuit could be schematically summarized
in the following way: Environmental impulses Ce.g. light sti-
384 B. MESS ET AL.
% lD II Ll + Mel
20
"
100 16
Electrolytic 21 12
lesion
12
x
i
23
50
x
20
15 10 16
Sham MR DR seN Sham MR DR SeN MR DR seN
% 9 8
100 20
5,7- DHT
lesion
19
x
50 22
12
15
Sham SeN Sham MR
Fig.4. Diagrammatic representation of the occurrence of the

constant estrous-anovulatory (CEA) state in rats maintained
in normal light-dark cycles (LD) or in continuous light (LL),
treated daily with 10 .... g melatonin (Mel) and bearing either

electrolytic destruction or 5,7-dihydroxytryptamine (5,7-DHT)
lesion in the median raphe (MR), dorsal raphe (DR) or in the
suprachiasmatic nuclei (SCN).
p < 0, 01 vs. sham LL+Mel

p < 0,01 vs. the corresponding LL and LD
p < 0,05 vs. the corresponding electrolytic lesioned group
mu1.i) regulate the secretory activity of the pineal gland;

Melatonin acts upon the serotoninergic neurons of the midbrain;
Ascending serotoninergic fibres terminate in the hypothalamus,
mainly in the suprachiasmatic area; The serotoninergic inputs
might regulate LHRH release (Fig.5.).
/
/
I
I
I FX / "
I I ..... - - - ...
I I " .....
I I /
, / I
,,, I
THALAMUS
I
I
LIGHT
-- --- -
\ /
......
.....
"
Fig.5. Diagrammatic representation of the postulated mechanism

by which light impulses might influence gonadal activity
through melatonin secretion.
SUMMARY
It is proposed that the pineal might secrete its hormones

towards the capillary system of the gland. Pineal indoles
(me1atonin) are then concentrated by the choroid p1exus into
the cerebrospinal fluid. Since the me1atonin content of the
hypothalamus and midbrain is considerably higher than that of
the cerebra1 cortex or of any other peripheral tissue, these
pineal hormones may influence many different functions of the
brain.
386 B. MESS ET Al.
It is very probable that the pineal hormones influence

pituitary activity mainly through the hypothalamus. While a
direct pineal-pituitary relationship has not been unequivocally
proved, it might have at least minor importance.
The pineal gland acts via the serotoninergic neuron system
to influence the hypothalamo-gonadotrophin axis, thus partici-
pating in the regulation of ovulation and luteinization. A
working hypothesis is presented to explain the mechanism of
action of the pineal gland in modulating the function of the
reproductive system.
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392 B. MESS ET AL.
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HUMORAL INTERRELATIONS OF THE PINEAL GLAND WITH
LATERAL EYES AND ORBITAL GLANDS
W. B. Quay
Neuroendocrine Laboratory, Department of Anatomy

University of Texas Medical Branch
Galveston, Texas, U.S.A.
INTRODUCTION
Three kinds of general interrelationships are currently accep-

ted concerning the pineal gland, the lateral eyes and the glands of
the orbit: (1) The mammalian pineal gland has evolved from eye-like
structures ("median or third eyes") in the pineal complex of lower
vertebrates, and its specialized parenchymal cells, the pinealocytes,
have residual features of photoreceptor cells (Collin, 1969, 1979;
and reviews by Oksche and Kappers in the present volume). (2) The
mammalian pineal gland, starting a few days after birth, receives
its photic input via the lateral eyes, CNS and sympathetic innerva-
tion (Wurtman et al., 1967; Moore et al., 1968; Klein and Moore,
1979). (3) Pineal gland, lateral eye retina, and, to a lesser
degree, the Harderian gland of the orbit, share some special bio-
chemical characteristics or capacities. At the present time, the
best known and most relevant of these shared characteristics is the
presence of the melatonin-forming enzyme, hydroxyindole-O-methyl-
transferase (HIOMT) (Quay, 1965; Quay et al., 1969; Cardinali and
Wurtman, 1972; Nagle et al., 1972a, 1972b, 1974; Cardinali et al.,
1974; Pevet et al., 1978; Suzuki and Yagi, 1978; Balemans et al.,
1980; Pevet et al., 1980a, 1980b, 1981; Ralph, 1980; Vivien-Roels
et al., 1981) and its 5-methoxyindole products (Bubenik et al.,
1974, 1976a, 1976b; Pang et al., 1977, 1980; Reiter et al., 1981;
Yu et al., 1981).
A central theme in the following review is that the synthesis

and possible release of melatonin and related indole derivatives by
pineal, eye and Harderian gland may form the basis for humoral or
hormonal interactions between these organs, or between particular
of their constituent cells. An important, and as yet unresolved,
395
396 w. B. QUAY
question remains about possible tissue sources of circulating mela-

tonin. If the only source of melatonin in the blood of mammals is
the pineal gland, as has been thought by many, then melatonin's
physiological relations as a hormone are more limited in scope and
are less complex for our understanding. However, recent research
presents conflicting results and interpretations of whether there
are extrapineal sources of hormonal or circulating melatonin (Pang
and Ralph, 1975; Lewy et al., 1980; Meyer et al., 1981; Harlow et
al., 1981; Yu et al., 1981; Tetsuo et al., 1982). A tentative and
conservative conclusion concerning this question would place the
burden of proof for extrapineal sources of circulating melatonin on
demonstration of chemical specificity in the assay system used.
Whatever the ultimate answer to this question may be, one conclusion
remains clear: It is possible that endogenous melatonin at levels
below detection by current assay methods, and administered or exo-
genous melatonin, and/or its congeners may possibly participate in
humoral or hormonal interactions within and between these three
organs of concern here - pineal, eyes and Harderian glands.
Our use of the term "humoral" here, refers to chemical mediator

action across intercellular spaces of diverse extent, including those
between adjacent cells within the same tissue as well as between
cells in different tissues. Possible function of melatonin as such
a tissue humor or parahormone can be hypothesized for its actions
within its tissues or organs of origin, namely pineal, eyes and
Harderian glands of present concern, and possibly certain others,
especially in lower vertebrates. Evidence is available, albeit
essentially indirect and incomplete, for melatonin functioning as a
tissue humor or parahormone within the pineal and eyes, but not as
yet within the Harderian gland. The suggestion (Quay, 1974) that the
pineal itself may be a first or direct target for melatonin has been
supported by experiments in which effects of exogenous melatonin on
the pituitary and reproductive organs of rats and hamsters are block-
ed by prior pinealectomy (Reiter et al., 1976; Sackman et al., 1977;
Cardinali et al., 1979; Quay et al., 1982). However, in some as yet
undefined circumstances, administered melatonin has antigonadal
effects in pinealectomized hamsters (Hoffmann and Kliderling, 1977;
Tamarkin et al., 1977; Turek, 1977). The pineal's permissive role
in the elicitation of some of the effects of administered melatonin
may possibly be explained in two ways. Both are consistent with
available information, and either one or both may be true: (1)
Melatonin receptor and/or other mechanisms within the pineal itself
are necessary for melatonin's peripheral actions on some cells in
some circumstances. And (2), a chronic or daily conditioning action
of the pineal on hypothalamic and/or peripheral receptor mechanisms
is necessary for melatonin's actions in some circumstances (Quay et
al., 1982). Direct response of Guinea Pig pineal cells to melatonin
has been demonstrated recently by recording their electrical activity
following microelectrophoretic application of melatonin to them
(Semm et al., 1980).
HUMORAL INTERRELATIONS OF THE PINEAL GLAND 397
Evidence concerning melatonin's possible functioning within the

vertebrate eye is more complex than that concerning its possible
actions within the pineal. Therefore, the next major section of this
review is devoted to evidence for actions by the pineal or by mela-
tonin within the eye. It should be emphasized that the omission of
other pineal products from this discussion is due to the near absence
of information about their occurrences rather than to a belief in
melatonin's being necessarily the only functionally important pineal
product with these functional relations.
The third major section of this review, in an analogous manner,

is concerned with evidence for interactions between the Harderian
and pineal glands of mammals, the only animals in which such possible
relationships have been studied. Here too, much of the information
is about melatonin, and little is known about the possible participa-
tion of other pineal or Harderian gland products. The interpretation
of available experimental results concerning the Harderian gland is
most difficult and leaves us with fundamental questions unanswered.
HUMORAL INTERACTIONS INVOLVING THE EYES
Our attention here is directed chiefly to experimental results

suggesting actions of the pineal, or of melatonin, on the eyes and
their constituent tissues and cells. Although we will assume here
that the specific pineal and melatonin actions to be discussed are
humoral or hormonal in nature, the possibility remains that some of
these actions may either be by way of, or be influenced by, centri-
fugal (non-sensory) innervation of the eye (see Reperant et al.,
1981, for references). This remains an open question. Also remain-
ing outside of our review here are possible interactions with the
autonomic innervation of some of the intraocular cells and tissues
(Cardinali et al., 1981).
There are several general features of melatonin's relations to

the eyes, additional to its synthesis there noted in the Introduction
above. First, uptake of administered labeled melatonin by the iris-
choroid tissues of the eye is second only to that by the pineal it-
self, and is therefore greater than that by the usually considered
target organs of melatonin (Wurtman et al., 1964; Axelrod and Wurt-
man, 1966; Table 1). The possable functional significance of this
will be noted later in this section, in relation to increased fluid
content of hamster eyes following daily injections of melatonin.
Secondly, in experiments concerning melatonin's effects on other
tissues, melatonin has sometimes been injected "intraocularly"
(Pomerantz and Sorrentino, 1973; Pomerantz and Reiter, 1974). It
remains to be determined whether in such experiments intraocular
effects of melatonin have modified its neuroendocrine or more general
systemic effects. Thirdly, if the retina or eye contributes directly
to plasma melatonin or related hormone levels, then experiments
398 W. B. QUAY
Table 1. Tissue Distribution of Radioactivity as 3H-Melatonin

or Metabolites One Hour after Intravenous Injection
of 100 ~c (125 ~g) 3H-Melatonin to Cats (Axelrod and
Wurtman, 1966)
Tissue 3H-Melatonin Content (m~g/g)
Pineal 25.3
Iris-choroid 6.3
Ovary 5.6
Pituitary 2·9
Sympathetic chain 2.6
Peripheral nerve 2.3
Testis 1.9
Thyroid 1.8
Adrenal 1.5
Kidney 1.3
Liver 0·9
Spleen 0.7
Heart 0.6
Plasma 0.6
Skin 0.5
Brain 0.5
Diaphragm 0.3
Fat 0.2
employing blinding by enucleation or removal of the eyes may poss-

ibly have different effects potentially than those in which blinding
was by other means.
A related and important topic that we will not review here is

the similarity in photic and related responses of retinal and pineal
serotonin-N-acetyltransferase activity. Most of this research has
used chick pineal and retina (Binkley et al., 1979, 1980; Hamm and
Menaker, 1980, 1981; Sagar et al., 1982).
The subtopics that will be covered here relate to melatonin's

actions, in what are essentially pharmacological studies, on eyes
and their parts in vivo. These subtopics can be organized in a
variety of ways - all arbitrary. I will follow a plan that is partly
by developmental age and partly by functional relationship.
Early Developmental Effects of Indoleamines
Indole biogenic amines are detectable in early developmental

stages of both invertebrate and vertebrate animals. They appear
to be likely participants in some embryonic inductions and in some
developmentally transient mechanisms and phenomena (Buznikov and
Manukhin, 1961; Buznikov, 1964; Buznikov et al., 1964, 1966; Quay,
1968; Baker and Quay, 1969; Kostellow and Morrill, 1971). Most
studies in this topic area have been concerned chiefly with serotonin
(5-HT) and its precursors. However, in the development of the South
African Clawed Toad (Xenopus Zaevis) melatonin has been shown to have
distinctive stage-related localizations and probable functional rela-
tions (Baker et al., 1965; Baker, 1969; Baker and Quay, 1969). In
Xenopus embryos HIOMT and melatonin are detectable as early as the
gastrula stage. But the first major rise in melatonin content occurs
just before the time of active swimming (stages 41-42). This leads
to a developmental peak in melatonin content, which is located mainly
in the eyes (Baker et al., 1965; Baker, 1969; Baker and Quay, 1969).
At this developmental stage one role of melatonin within the eyes may
be linked to the mediation of retinal photomechanical responses, the
subject of the next subsection.
Melatonin and Photomechanical Responses within the Eye
Photomechanical responses of the eye are responses to illumina-

tion in which morphological changes or physical movements are seen.
When they occur within the retina they are known also as retinomotor
responses. They are ancillary to the primary photosensory reactions
and responses of the retinal photoreceptor cells, and can occur either
within these or other cell types. Some of the more frequently observ-
ed photomechanical responses within the eye are: (1) movement of pig-
ment granules within pigment cells, (2) changes in form or length of
particular kinds of retinal photoreceptor cells or their parts, and
(3) changes in the form and staining reactions of retinal nuclear
layers. Early and classical work on this subject included that by
Arey (1915, 1916a), Detwiler (1943) and Welsh and Osborne (1937).
Later sources are given by Ali (1975, 1981). Although photomechani-
cal responses of the eye are more diverse, higher in amplitude, and
more frequently studied in lower vertebrate eyes, they are known also
in those of some mammals (Detwiler, 1943; Pang et al., 1978). Two
examples of melatonin-activated photomechanical responses will be
noted here. It should be kept in mind, however, that the hypothesis
of melatonin's mediation or activation of these responses is based
upon circumstantial and pharmacological evidence, and is not support-
ed by proof that melatonin is the endogenous mediator.
In Xenopus Zaevis larvae after the developmental stage of the

rise in ocular melatonin content, contraction of the cone myoid seg-
ment occurs as a retinomotor or photomechanical response to light
(Quay and McLeod, 1968). In darkness the lengthening of the cone
400 W. B. QUAY
VI
::J
~ .7
u
::J
C
.E
iii .6
0.
0
-0
.5
'0
/
Q)
c
0
u
.4
E
.g A Rate of
Q)
U .3 V Dark Adaptation-
c
0 Cone Myoid Length
v;
'6 in Xenopus Larvae
.~ .2
0
W
Ir 0 2 3 4
Time (hours)
Fig. 1. Course of dark adaptation in cone myoid elongation of larvae

of Xenopus laevis. Means ± standard errors are plotted for
groups of sibling larvae in separate glass vials. At times
indicated, larvae were killed by the addition of formalin.
Distances within the retinas were measured with a calibrated
ocular micrometer and mounted and stained tissue sections
(Quay, nonpublished data).
myoid is a relatively slow process (Fig. 1). But in either light or

a melatonin-containing solution, the cone myoid begins to contract
within a few seconds and attains an asymptote in about 45 seconds
after exposure (Fig. 2). The near minimal effective concentration of
melatonin in the medium to produce a response greater than that of
control medium is 5 x 10-10 g/ml (Fig. 2). More information on char-
acteristics of these mobile (type 1) cones and technical factors
important for their study can be found in a monograph by Saxen (1954).
Several circumstances still pose problems for answering the question
III
Melatonin Concentration a
'"
OJ
"0
Cone Myoid Contraction
c'"
With Time - Xenopus Larvae
;?
.6
Q;
a.
l?
"0
'0
OJ
c .5
0
u
E --- ---
.g ---
OJ
U
C
~ .4
III
"0
OJ
.~
<;
a; c
a:: .3
o 7 15 45 Time (seconds) 180
~ ~
Standardj 0 .020 .038 .040 .017 0
Errors • . 026 .042 .029 .024 5xIO- IO
af the ... .029 .026 .026 .026 5x 10- 8
Mean. 0 .015 .026.044 .019 5x10- 7
Fig. 2. Comparisons of cone myoid contraction after addition of

melatonin or H20 (control) at "0" time to media contain-
ing dark-adapted sibling Xenopus larvae. Other informa··
tion as in legend of Fig. 1.
whether melatonin is the natural mediator of retinomotor responses

of larval Xenopus cones or whether it is mimicking the action of the
natural mediator. Physical agitation of the medium, including that
produced by gently adding a "control" solution produces a low level
of response (Fig. 2). It might be suggested that this represents a
kind of "stress" response mediated by way of efferent fibers to the
retina (Arey, 1916b; Kline and Pickering, 1975). But at least in
some species retinomotor responses are under local cellular control
within the retina (Easter and Macy, 1978). Retinomotor responses in
lower vertebrates can be visualized as one category of adaptive res-
402 W.B.QUAY
?onse to the peculiarities of photic environment in particular habi-

tats to which the species is adapted (Ali, 1981). Further study of
melatonin's possible involvement as a mediator in these responses
may well shed light on the physiological evolution of melatonin's
roles within the pineal complex of vertebrates.
Melatonin's actions on or in photomechanical responses may also

provide a good system for study of melatonin's molecular mechanisms
within the cytosol of responding cells. It has been suggested that
the mechanism of action of serotonin and some of its congeners, such
as melatonin, may involve actions on changes in microtubular or rela-
ted organelle systems (Quay, 1968). Although melatonin may not be
a general inhibitor of microtubules (Poffenbarger and Fuller, 1976),
melatonin administration or endogenous release of melatonin can affect
at least some cellular mechanisms that depend upon microtubules
(Cardinali, 1980).
Photomechanical responses have been demonstrated also to be in-

fluenced by melatonin in the eyes of a mammal, the Guinea Pig. In
this species light adaptation causes a clumping (lightening) of the
contents of the cells of the retinal pigment epithelium and a disper-
sion (darkening) within the pigment cells of the choroid layer. Dark
adaptation leads to opposite effects in both kinds of cells (Pang et
al., 1978). Melatonin administration leads to pigment aggregation
(lightening) in both (Pang and Yew, 1979).
Melatonin and Light-entrained Disc-shedding by Retinal Rods
Shedding of discs by the rod outer segment and the phagocytosis

of these discs by the retinal pigment epithelium follow light-cued
circadian rhythms (LaVail, 1976a, 1976b; Goldman et al., 1980).
Acute effects of light and darkness on this shedding response in
adult frogs are not affected by pinealectomy (Currie et al., 1978).
Nor does pinealectomy affect shifts in the disc-shedding rhythm
(Goldman,1982). There appear to be both local (intraretinal) and
central (CNS) factors in the regulation of the circadian rhythm in
disc-shedding by rat retinal rods (Teirstein et al., 1980). The
possibility of an intraretinal contribution by melatonin is still to
be tested.
Melatonin's Enhancement of Retinal Damage Induced by Illumination
Prolonged exposure to bright light leads to retinal damage and

photoreceptor degeneration in albino rats (O'Steen, 1970; Q'Steen et
al., 1972, 1974; Reiter and Klein, 1971; Reiter, 1973; Lanum, 1978).
This damage is increased when melatonin (lOO~g/day) is administered
concurrently (Bubenik and Purtill, 1980). In other studies it is re-
ported that the earliest signs of damage by light occur in the retin-
al pigment epithelium (RPE). The RPE cells seem to be unable to
phagocytize outer segment discs and to digest previously ingested

ones as a resul~ of changes in the endoplasmic reticulum and the
phagolysosomal system (Lai et al., 1980). It would be interesting
to determine which if any of these processes are affected by mela-
tonin.
Immunological Relations of Retina with Pinealocytes
Evidence has been presented recently of immunological cross-

reactivity between retinal photoreceptors and pinealocytes (Kalsow
and Wacker, 1978). A kind of experimental allergic uveitis (EAU)
can be elicited in Guinea Pigs by injection of an extract of homolo-
gous retinal homogenate (= "s preparation"). During the development
of the EAU there is involvement of the pineal gland as well. The
multiple changes observed in both tissues suggest that in inflamma-
tory diseases of the retina or pineal, involvement of the other
structure should be assayed. There are several interesting implica-
tions in these findings that merit investigation. One of these is
the possibility that particular populations of pinealocytes might be
immunologically identifiable and might also be immunologically deleted
by appropriate techniques.
Quantitative Effects of Melatonin on the Eye
Effects of pinealectomy, or of melatonin or other pineal hor-

mones, on weights or volumes of mammalian eyes and their parts have
not been reported previously. In recently completed work I have
found that melatonin can have significant effects on the eyes of
Golden Hamsters. These results are from the same set of animals that
was used in study of effects of pinealectomy and melatonin on growth
of endocrine and reproductive organs (Quay et al., 1982), and in
study of effects of melatonin on choroid plexuses (Decker and Quay,
in press). Male Golden Hamsters in an LD 14:10 photoperiod repre-
sented five surgical groups: non-operated, sham-pinealectomized,
sham-pinealectomized with black plastic shielding over the pineal
region of the brain, pinealectomized and pinealectomized with black
plastic shielding over the pineal region of the brain. Starting 4
to 5 days after surgery, animals of each of the 5 surgical groups
were distributed among 3 injection groups: vehicle (= 0.1 ml 0.9%
NaCl 30% ethanol) only, 25 ~g melatonin (Sigma) in 0.1 ml vehicle
and 2500 ~g melatonin in 0.1 ml vehicle. Each animal received a
daily subcutaneous injection at Lll.OO to Ll l.75 in the afternoon of
the daily light phase for 28 days.
At autopsy during the afternoon of the day following the last

injection, the left eyes were removed and fixed. They were later
rinsed, dissected and weighed. None of the surgical procedures had
any effect on the weights of the eyes or their parts. However, mela-
tonin injections did have highly significant effects. There was an
increase in average weight of intact eyes, and this increase was
404 W. B. QUAY
INTACT EYES P<0.002
P<O.OOl
]62
...;
01
:x
'"
~
60
*
Melatonin (~g/day)
Fig. 3. Effect of 28 consecutive daily injections of melatonin on

the weights of intact whole eyes of male Golden Hamsters.
Means ± standard errors are shown for absolute weights of
left eyes (left panel) and left eye weights in relation to
whole animal (body) weights (right panel). Vehicle-inject-
ed animals - clear; 25 ~g melatonin/day - cross hatched;
2500 ~g melatonin/day - solid black. N = 37 or 38 per in-
jection group. * Groups with which other groups are com-
pared in terms of P values based on the Student-Fisher t.
greater in relation to daily melatonin dose level (Fig. 3). This

was not a consequence of differences in overall body weights, as
shown by the equivalence of effects whether absolute or relative eye
weights are analyzed (Fig. 3). After the lens and the fluid content
of the chambers of the eye were removed, the remaining nonlenticular
tissues of the eye showed a lower weight after melatonin (Fig. 4,
right panel). In contrast, the fluid in the chambers was increased
by melatonin (Fig. 4, left panel). Lens weight was also increased
by melatonin, but only with the higher dose level (Fig. 5).
These effects of melatonin on hamster eyes probably represent

two kinds of actions. First, there was a chronic stimulation of lens
growth by a very high dose of melatonin. This is of interest since
most hormones and drugs that are known to affect the lens, are chiefly
inhibitory. It will be of interest to determine whether melatonin
has any effect on the occurrence or progress of degenerative changes
in some kinds of cataract. Second, melatonin's increase of intra-
FLUID IN CHAMBERS NONLENTICULAR TISSUES

26 26
*
*
25 25
P<O.OI
01 01
E E
24 24
o 25 2500
Melatonin (~g/day)
Fig. 4. Effects of 28 consecutive daily injections of melatonin on

weights of fluid in chambers and of nonlenticular tissues
of left eyes of male Golden Hamsters. Additional informa-
tion is the same as that given in the legend for Fig. 3.
LENS WET WGT. LENS DRY WGT.
14.
7.9
14.1
P<O.OOI P<O.OOI
13.
13.7..L-..L-_..I....-..L....<'-L..1..-
o 25 2500 o 25 2500
Melatonin (~g/day)
Fig. 5. Effects of 28 consecutive daily injections of melatonin on

wet and dry weights of lenses from left eyes of male Golden
Hamsters. Additional information is the same as that given
in the legend for Fig. 3.
406 W.B.QUAY
ocular fluid with decreased nonlenticular tissue (Fig. 4) could have

resulted through a single mechanism, such as increased intraocular
secretion or diminished outflow of aqueous humor, thence possibly
leading to increased intraocular pressure (lOP). This effect of
melatonin is of particular interest in relation to the possibility
that it is relevant to some type(s) of glaucoma, or defects in cont-
rol of lOP. Whether or not this is true, it is a possibility that
should be considered a caveat to those who experimentally administer
melatonin to humans.
It may be physiologically significant that the "iris-choroid"

has been shown to have an uptake of melatonin second only to that of
the pineal (Wurtman et al., 1964; Axelrod and Wurtman, 1966)(Table 1).
This suggests that melatonin in the systemic circulation, whether
exogenous or derived from the pineal, may have an action on iris-
choroid tissue. Since the ciliary epithelium was presumably included
in the "iris-choroid" (Table 1) anatomical fraction of the eye, it
may have been involved in this melatonin uptake activity. This is
worthy of investigation since the eye's aqueous humor is usually
thought to be secreted by the ciliary epithelium. Nevertheless, we
can not deny that there are alternatives still to be tested for the
mechanism of melatonin's actions in the eye. There remains as well
the question whether retinal melatonin could possibly reach the
ciliary epithelium and thereby modify secretion rate of aqueous humor.
More needs to be determined about rhythmic and physiological

changes in melatonin formation, release, levels and actions within
mammalian and human eyes. Twenty-four-hour rhythmic changes in
diverse vertebrate species have been reported in retinal contents of
melatonin and of enzymic activities involved in melatonin's biosyn-
thesis ('Nagle et al., 1972; Pang et al., 1977; Gern et al., 1978;
Joss, 19(8). However, in male Golden Hamsters little 24-hour change
has been found in retinal HIOMT activity, possibly for technical
reasons (Pevet et al., 1980a, 1980b). In rats in a LD 14:10 photo-
period the daily peak in retinal HIOMT activity has been reported to
occur near the end of the light phase (Nagle et al., 19(2). But
retinal melatonin levels in the rat have been reported to be minimal
at this time and to peak near the onset of light (Pang et al., 1980).
This more closely matches the timing of the daily cycle in retinal
N-acetylserotonin in this species (Pang et al., 1981), suggesting
that serotonin-N-acetyltransferase rather than HIOMT activity may be
rate-limiting for retinal synthesis of melatonin. This is similar
to what has been demonstrated for rat pineal synthesis of melatonin.
rop in man follows a 24-hour rhythm. Although there is some

disagreement or discrepancy concerning both mean pressures and the
daily timing of peak pressures, peaks and acrophases are generally
attributed to the morning (deVenecia and Davis, 1963; Follmann,
1977; Ferrario et al., 1982), and this is true in the glaucoma popu-
lation also (Worthen, 1978). This timing, at least superficially,
resembles that of the daily melatonin peak in the rat eye. Informa-
tion on ocular or retinal melatonin rhythms in man is yet to be re-
ported. Nevertheless, it may be of interest in the evaluation of
humoral contributions to changes in lOP. This is most important in
trying to improve understanding of mechanisms in the etiology, and
the pharmacologic approaches for control, of different types of glau-
coma.
HUMORAL INTERACTIONS INVOLVING THE HARDERIAN GLANDS
Harderian glands are members of the complex of glands occupying

the orbit and adjacent regions, and whose secretions bathe the cornea
and conjunctiva. These secretions also can drain to the nasal chamb-
ers via the nasolacrimal canal. The relevance of the Harderian
glands to pineal physiology and pharmacology stems from four kinds of
information: (1) Harderian glands of at least some mammals contain
HIOMT, serotonin-N-acetyltransferase and melatonin (Cardinali and
Wurtman, 1972; Bubenik et al., 1976a, 1976b, 1978; Pang et al., 1977;
Brammer et al., 1978; Pevet et al., 1980a, 198ob; Balemans et al.,
1980; Reiter et al., 1981). Removal of the Harderian glands in rat
and hamster leads.to changes in the pineal and reproductive organs
(Wetterberg et al., 1970a, 1970b; Reiter and Klein, 1971; Panke et
al., 1979). (3) Castration of male hamsters produces Harderian
glands of the female type, and studies combining pinealectomy with
other procedures have been interpreted as suggesting a pineal invol-
vement in the mediation of such changes (Hoffman, 1971; Clabough and
Norvell, 1973). (4) Injection of melatonin modifies the porphyrin
content of rat Harderian glands (Jo6 and Kahan, 1975). (5) Continu-
ous light and blinding each have effects on the Harderian glands and
their endocrine relations (Hoffman, 1971; Reiter and Klein, 1971;
Reiter, 1973; Clabough and Norvell, 1973; O'Steen et al., 1978).
The microscopic structure of the Harderian glands is that of

a merocrine exocrine gland. But the above noted research reports
have sometimes interpreted their results to suggest photosensory
and/or endocrine activities as well. These still need to be provided
with necessary supporting information. Abundant experimental evi-
dence based upon rodents indicates that at least in this animai
group, Harderian glands have complex relations with the endocrine
system. This is most strongly indicated by: (1) sexual dimorphism,
(2) responsiveness to steroid hormones, and (3) contributions to
pheromonal mechanisms which in turn may have secondary impacts on
diverse aspects of physiology and behavior (Thiessen et al., 1976,
1977; Thiessen, 1977; Kittrell and Thiessen, 1981; Lin and Nadakavu-
karen, 1981; Shirama et al., 1981). The currently confusing picture
of Harderian gland function in mammals results partly from the fact
that basic information, concepts and experimental results used and
obtained by specialists in one biological or biomedical discipline
are either ignored by, or unknown to, specialists in other topics.
408 W. B.QUAY
Another and more fundamental problem is that the homologies and

exact identity or derivation of the Harderian gland, among orbital
glands of vertebrates, are not reliably known (Sakai, 1981). The
location within a particular part of the orbit, and the gross and
microscopic anatomy, of the Harderian glands differ among the major
Orders of the Class Mammalia. Biochemical Harderian gland markers,
such as lipid and porphyrin contents, have been studied in very few
taxa.
Harderian glands in mammals are reportedly relatively largest in

lagomorphs and rodents, and to be lacking in primates (Sakai, 1981).
This circumstance of itself might be taken as reason enough for con-
sidering the Harderian gland of little significance in human biology.
However, this conclusion may be premature, since it rests upon
faulty and deficient information. This view is offered on the basis
of: (1) The homologies and exact identities of the various orbital
glands in man and other mammals are poorly known. Therefore, it may
be premature to assume that either a homologue or a functional ana-
logue of the Harderian gland does not exist in humankind. (2) Har-
derian and certain other orbital glands are present and of large size
in some marine mammals, including cetaceans and pinnipeds, suggest-
ing that their functional importance is not limited to bathing the
exterior surfaces. (3) Endocrine and neuroendocrine studies are
either lacking or present conflicting and confusing results for the
various named orbital glands. Thus, much more information is needed
before confident conclusions can be made about presence or absence
of neuroendocrine relations of such glands in man.
CONCLUSIONS
Experimental evidence has been summarized suggesting that some

of melatonin's actions may occur in, or by way of, pineal tissue it-
self, rather than always or necessarily directly upon central, neuro-
endocrine or peripheral targets.
Melatonin is also both a product of, and a likely chemical medi-

ator within, the lateral eyes of vertebrates including mammals. New
findings on melatonin's effects on hamster and Guinea Pig eyes show
that at least pharmacologically it can cause: (1) movement of melanin
granules in retinal pigment epithelium and choroidal melanocytes, (2)
increase in lens material, and (3) increased fluid content of ocular
chambers with associated decreased weight of nonlenticular tissues.
The latter result suggests that melatonin may be capable of increas-
ing lOP. For this reason its chronic administration at high dosage
should not be undertaken without ocular tonometry or e~uivalent moni-
toring of lOP.
The high uptake of 3H-melatonin from blood in vivo by the iris-

choroid fraction of the eye may possibly be related to the increased
fluid content of ocular chambers after systemic melatonin administra-
tion. This suggestion follows from the fact that the intraocular
fluid with the most rapid turnover, the aqueous humor, is secreted by
the ciliary epithelium covering a part of this anatomical fraction of
the eye. A hypothesis to explain these findings can be offered -
melatonin may have a stimulatory action on the secretory activity of
the ciliary epithelium of the eye, leading to increased production of
aqueous humor and increased intraocular pressure (lOP) in some condi-
tions or circumstances. This may be analogous to melatonin's stimu-
latory action on the choroidal ependymal epithelial cells described
in another report in this volume. In the latter case, increased
secretion of CSF is a possible result. Other kinds of experimental
studies are needed, with the objective of testing these and alterna-
tive hypotheses.
Harderian glands of some rodents contain melatonin and the

enzymes for its biosynthesis. Although results of experiments
suggest complex interrelations involving Harderian, pineal and cer-
tain of the endocrine glands in rodents, the mechanisms and biologi-
cal significances of these relationships remain to be demonstrated
or clarified.
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MODIFICATION OF HYPOTHALAMIC ELECTRICAL ACTIVITY BY PINEAL INDOLES
C. Demaine
Department of Physiology
Chelsea College
London University
London, England
INTRODUCTION
In a number of mammalian species, the pineal gland has been

shown to mediate the inhibitory effects of short photoperiods on
the secretion of several pituitary hormones. Although most studies
have centred on gonadotropin secretion, in the last twenty five years
there have been reports which have provided evidence for pineal-
mediated inhibition of the production and release of each of the
adenohypophyseal hormones (see review Mess et al., 1979). The Syrian
hamster (Mesocricetus auratus) is a highly photosensitive species, in
which the pineal can readily be shown to exert a dramatic and potent
influence on the release of luteinising hormone (~H), follicle
stimulating hormone (FSH) and prolactin (Reiter, 1980). Light
restriction, obtained by blinding hamsters or placing them in con-
tinuous darkness or short photoperiod, results in reproductive
involution. In males, after 50 days of light restriction, Lhe testes
are reduced to less than 20% of their original weight and do not
produce spermatozoa (Hoffman and Reiter, 1965). These changes are
associated with a reduction in the pituitary content of both LH and
FSH (Reiter and Johnson, 1974al, and of prolactin (Reiter et al.,
1975). Turek et al. (1976) found that serum LH· and FSH levels in
animals exposed to short photoperiods were lower than in hamsters
maintained in a L:D, 14:10 lighting cycle. In light restricted
females, the oestrous cycles are interrupted and the uteri become
infantile {Johnson and Reiter, 1978}. Although the ovaries tend to
increase in weight under these conditions, there are pronounced
changes in ovarian morphology. There is extensive proliferation of
interstitial tissue, a small number of antral follicles and a distinct
lack of corpora lutea (Vaughan et al., 1981). The relationship
between reproductive collapse and pituitary gonadotropin secretion
417
418 C. DEMAINE
is more complicated in female hamsters. In fact, LH levels actually

appear to rise when animals are blinded (Reiter and Johnson, 1974b).
Presumably the occurrence of daily surges in both LH and FSH in female
hamsters exposed to short days (Seegal and Goldman, 1975) underlies
the disruption of normal reproductive function.
Evidence for an inhibitory role of the pineal gland on the

pituitary-thyroid axis of the hamster has recently been reported
(Vriend et al., 1977J. These workers found that blinding male
hamsters resulted in a depression of plasma thyroxin. The effect
was found to be abolished by surgical removal of the pineal gland or
by superior cervical ganglionectomy, an operation which interrUpts the
neural pathways by which photic information from the retina is trans-
mitted to the pineal gland (Moore, 1978]. In the rat at least, the
pineal gland appears to exert its influence on the thyroid gland via
a reduction of pituitary thyrotropin (TSH) output (Niles et al.,
1979a). Thus in animals kept under short photoperiods, pinealectomy
was associated with a highly significant increase in serum TSH.
Similar data on TSH levels in hamsters are not currently available.
Numerous investigators have attempted to determine the pineal

factor(s) responsible for the effects of the pineal gland on the
neuroendocrine system, with by far the greatest attention being paid
to the pineal methoxyindoleamine, melatonin. In the Syrian hamster,
daily injections of this indole can induce both gonadal atrophy and
thyroid suppression similar to that obtained by light restriction
(Vriend and Reiter, 1977; Vriend et al., 1979}, although the effect
depends on the time of day of administration (Reiter et al., 1976;
Tamarkin et al., 1976; Vriend and Reiter, 1977). Despite the large
number of investigations of the effects of melatonin, the site(s) of
its action on the hypothalamo-hypophyseal-endocrine axes have yet to
be firmly established. These sites may include one or a variety of
tissues such as the gonad (Ellis, 1972; Cohen et al., 1978J, the
pituitary gland (Martin and Sattler, 1979) or the central nervous
system (CNSJ (Fraschini et al., 1968; Trentini et al., 1979; Chen
et al., 1980; Reiter et al., 1981). Most recently, the hypothalamus
has been regarded as the most likely target tissue (Reiter, 1980}.
Melatonin is known to be taken up (Anton-Tay and WUrtman, 1969) and
specifically bound (Niles et al., 1979b; Vacas and Cardinali, 1979)
in the hypothalamus of the laboratory rat. Using fluorescence histo-
chemistry, Bubenik et al. (19761 were able to localise an affinity
for melatonin in the hypothalamic region. Fraschini et al. (1971)
have shown that implants of various pineal indoles, including mela-
tonin, into the hypothalamus of female rats significantly reduced the
level of gonadotropins in the anterior pituitary. In addition, the
injection of melatonin into the lateral ventricles of the brain just
before the 'critical period' on the day of proestrus, either blocked
ovulation or reduced the number of ova shed. Kamberi et al. (1970)
were also able to show a significant reduction in the output of
MODIFICATION OF HYPOTHALAMIC ELECTRICAL ACTIVITY 419
pituitary LH following the injection of melatonin into the third

ventricle of the brain in male rats. Vriend (198l} has recently
arrived at the conclusion that a single CNS neuroendocrine mechanism
is involved in both pineal-thyroid and pineal-gonadal effects in the
Syrian hamster, since the parallels between the action of melatonin
on the two systems are so striking.
POSSIBLE SITES OF ACTION OF PINEAL INDOLES IN THE BRAIN
In order to modify the hypothalamic control of anterior pituitary

secretion, pineal hormones must ultimately influence the secretion of
releasing hormones into the hypophyseal portal system. At least three
possible mechanisms of action may be considered. Firstly, melatonin
could induce increased release of an inhibitory transmitter, e.g.
serotonin, by acting on neurones with axons terminating in the hypo-
thalamus. The parts of th.e brain most likely to be target sites for
the indoleamine would then be the amygdala, the hippocampus and the
midbrain monoaminergic neurone system. Secondly, pineal hormones
could increase the sensitivity of steroid (or thyroid) hormone
negative feedback sites in the hypothalamus itself, and thirdly,
they could themselves have inhibitory 'transmitter-like' or neuro-
modulatory effects on hypothalamic'neurones involved in releasing
hormone secretion.
Trentini et al. (1979) have suggested that serotonin actually

mimics some of the effects which are known to follow melatonin
administration, and that, in general, the action exerted by the pineal
gland on CNS activity is similar to that induced by variations in
brain serotonin levels. This neurotransmitter has been particularly
implicated in the control of pituitary growth hormone secretion (Collu
et al. 1972; Smythe and Lazarus, 1973; Smythe et al., 1973) and the
effects of pineal melatonin on this neuroendocrine axis may well
operate via the serotonergic neuronal system. Similarly, Yates and
Herbert (1976) reported that melatonin given to ferrets kept in long
photoperiods caused serotonin rhythms, characteristic of animals
exposed to short photoperiods, in both the pineal. gland and the
hypothalamus. The role of serotonin in the hypothalamic control of
pitUitary gonadotropin, prolactin and TSH secretion is extremely
ambiguous. Although many authors have described evidence for an
inhibitory effect of increased serotonin turnover on gonadotropin
release and OVUlation, equal numbers of reports have indicated a
separate positive action of the transmitter on the cyclic release of
gonadotropins and on prolactin secretion (see review Trentini, 1979).
However, electrical stimulation of the medial raphe nucleus was found
to be inhibitory to ovulation and led to decreased plasma LH levels
tCarrer Taleisnik, 19721. Pazo (1979) showed that melatonin had
prediminantly inhibitory effects on the spontaneous neuronal elec-
trical activity of the mesencephalic reticular formation. He
420 C. DEMAINE
suggests that this effect may contribute to the changes in the sleep-
wakefulness cycle and anticonvulsant action attributed to the pineal
hormone. It is clear that if this is the main influence of melatonin
on midbrain serotonergic neurones,then their release of this neuro-
transmitter would be reduced.
Brain regions usually considered to have a more important

influence on the activity of gonadotropin releasing hormone (GnRH)
neurones are the amygdala and the hippocampus (Komisurak et al.,
1981). Although most studies have shown the amygdala capable of
stimulating LH secretion, electrochemical stimulation of the baso-
lateral amygdala in the freely moving rat resulted in a delayed and
diminished LH surge (Carrillo et al., 1977). The findings of Brown
et al. (1981) indicate that melatonin binds with high affinity in
limbic brain areas including the hippocampus. Th~s region has been
shown to be predominantly inhibitory with respect to LH secretion,
thus electrochemical stimulation results in blockade of the LH surge
during proestrus in the freely moving rat (Carillo et al., 1977).
However, the effects of manipulations of the hippocampus on FSH
secretion are contradictory (Komisurak et al., 1981).
AHA Anterior Hypothalamic Area
ARC Arcuate Nucleus

DMH Dorsomedial Hypothalamus
OC Optic Chiasma
POA Preoptic Area
SC Suprachiasmatic Nucleus
VMH Ventromedial Hypothalamus
~ TRH
GnRH&TRH
Fig. 1. Localisation of some releasing hormones in the

preoptic-hypothalamic region of the brain.
Although these inputs to the hypothalamic releasing hormone

neurones must undoubtedly be considered as potential sites for pineal
indoles, their influence on the general levels of GnRH release,
particularly in males, has not been demonstrated. A more likely site
of action for the active principles of the pineal are the hypothalamic
regions themselves. In the rat brain, the distribution of the
endogenous melatonin has been demonstrated immunohistochemically,
particularly in the suprachiasmatic nuclei (SCNI and the ventral
region of the anterior hypothalamus (Bubenik et al., 19761.
Fig. 1 shows the localisation of some of the releasing hormone

neurones within the hypothalamic nuclei. If pineal indoles act on
these cells to alter their sensitivity to gonadal or thyroid hormone
negative feedback, or if they themselves can influence neuronal
electrical activity, then these areas must be considered as prime
target sites. Melatonin has been shown to have significant effects
on microtubule protein content of the hypothalamus (Cardinali and
Freire, 1975; Cardinali et al., 1975) and to specifically bind in
the mediobasal hypothalamus of bovine brain (Cardinali et al., 1979)
and in the hypothalamus of the rat (Niles et al., 1979b). An inverse
relationship between diurnal variations in pineal melatonin and
GnRH content of the preoptic-suprachiasmatic area and of the medial
basal hypothalamus has been demonstrated (Brown et al., 1977). The
pineal indoleamine content was found to be low throughout the light
phase, when GnRH levels were significantly higher while the reverse
was observed throughout the dark period. Of the neural pathways
influencing GnRH release, attention may be focussed on the preoptic-
suprachiasmatic area rather than the mediobasal hypothalamus following
experiments in which the latter region was surgically isolated from
the rest of the hypothalamus in hamsters (Sorrentino and Reiter,
1971; Reiter and Sorrentino, 1972). These investigators found that
this type of surgical isolation prevents the gonadal atrophy that
usually follows as a consequence of light restriction. Since the
lesions were unlikely to interrupt the pathways which serve to
transmit photic information to the pineal gland, it is reasonable
to conclude that the site of action of pineal antigonadotropic
hormones may lie anterior to the cut. More recently, Reiter et al.
(1981) have shown that th.e inhibitory action of exogenously applied
melatonin on reproduction in male pinealectomised hamsters depends
on intact neural pathways in the anterior hypothalamus (AHA). The
authors suggest that one possible interpretation of this finding is
that melatonin may modify the functioning of the hypothalamo-
pituitary axis by working on neurones which influence the synthesis
and secretion of hypothalamic releasing hormones.
The suprachiasmatic nuclei in the hypothalmus are involved in

the generation and entrainment of circadian rhythms (Rusak and
Zucker, 1979). These rhythms appear to be entrained to the photo-
period via the action of the pineal gland (Elliott and Goldman,
1981), in a number of mammalian species. Thus the SCN have been
422 C.DEMAINE
considered as possible target sites for pineal indoles (Brown et al.,

1981). The suggestion has been endorsed by immunohistological
studies which have localised melatonin in the rat seN OBubenik et
al., 1976) and the finding that the nuclei have the ability to
concentrate the indoleamine from the peripheral circulation {Reiter,
1980}. Bittman et al. (1979) have recently studied the potential
action of melatonin in hamster SCN in relation to the antigonado-
tropiC properties of the pineal indole. Male hamsters were housed
under a typical lighting schedule (L:D, 14:10). Groups of animals
were pinealectomised and some were subjected to bilateral lesions
of the SCN. The animals were injected with melatonin C25Ug dose)
either once a day or three times daily. Thrice daily melatonin
injections caused testicular atrophy within six weeks in the
pinealectomised hamsters whether the SCN had been completely
lesioned or left intact. In hamsters with intact pineal glands,
reproductive collapse in response to thrice daily melatonin
injections only occurred in animals with complete SCN lesions.
Thus electrolytic destruction of the SCN does not interfere with
the ability of melatonin to induce gonadal involution when applied
in this manner. Bittman et al. (1979) conclude that the SCN are
not essential target tissues for the effects of exogenously
administered melatonin on the reproductive system.. In fact their
results demonstrate that seN lesions in hamsters with intact pineal
glands have the same effects on the sensitivity of the reproductive
system to exogenously applied melatonin as do pinealectomy or superior
cervical ganglionectomy. The SCN are intimately involved with the
regulation of pineal secretory activity since bilateral lesions of
these structures abolish the darkness induced peak of pineal N-
acetyl transferase activity and therefore the normal production of
melatonin in the gland (Moore, 1978).
In the rat, there is considerable evidence for the occurrence

of a population of GnRH neurone cell bodies in the medial preoptic
area (POA) of the rostral hypothalamus (Fig. 1), immediatelyover-
lying the optic chiasma. Not only can GnRH be extracted from this
region, but lesions of the axons of these neurones which project
to the mediobasal hypothalamus are followed by a decline in the
content of GnRH stored in the median eminence as measured by
subsequent assays. Such a loss would be consistent with the
degeneration of preoptico-tuberal GnRH neurones and loss of the
stored GnRH in their axon terminals (.McCann, 1981). In the case
of thyrotropin releasing hormone (TRH), activity is also found in
the POA, particularly in the region of the interstitial nucleus
of the stria terminalis. The activity is present in an area extending
caudally from this region to the dorsomedial nucleus (DMNl where a
higher activity is present (Fig. 11. McCann (1981) postulates that
cell bodies of TRH neurones are likely to be present in the POA, the
activity of the DMN representing the caudal projection of their axons
or the cell bodies of a second population of TRH neurones. Thus a
considerable proportion of both GnRH and TRH neuronal cell bodies
can be localised in the POA. Moreover, there is also a large amount
of evidence to indicate that elements in the POA may also be involved

in the generation of circadian rhythms ORusak and Zucker, 1979), al-
though unlike the SCN, POA neurones do not appear to playa part in
the neural pathways by which retinal photic information is trans-
mitted to the pineal gland (Moore, 1978).
POSSIBLE MECHANISMS OF ACTION OF PINEAL HORMONES IN THE ROSTRAL

HYPOTHALAMUS
One of the possible mechanisms for the effects of pineal

indoles in the hypothalamus is the alteration of the sensitivity
of releasing hormone neurones to the negative feedback signals
provided by the final products of the various neuroendocrine axes
which are influenced by the pineal gland. However, decreases in
serum prolactin levels in light restricted male hamsters occur even
in castrated animals and prolactin levels are not affected by exo-
genous androgen. Castration also does not alter serum prolactin
concentrations in males kept on a long photoperiod. Thus photoperiod-
induced changes in prolactin secretion do not result from alterations
in the effectiveness of the feedback signal provided by the levels of
circulating testicular hormones in this species (Bex et al., .1978;
Elliott and Goldman, 1981J. By contrast, in the ewe, Goldman (1981)
postulates that seasonal changes in LH secretion are mediated by
alterations in sensitivity to the negative feedback effect of
oestrogen. OVariectomised ewes maintain high serum LH levels
throughout the year, but when ovariectomised anbmals were admini-
stered a constant dose of oestrodiol, they showed pronounced
seasonal fluctuations in serum LH concentrations, with the highest
levels coinciding with the normal breeding season (Legan et al.,
1977). On the other hand, in the ram, changes in LH secretion occur
in castrated animals in relation to alterations in photoperiod
(Pelletier and Ortavant, 1976). Moreover, in castrated Soay rams,
exposure to long days after a period of short days resulted in a
decline in the plasma levels of both LH and FSH despite the fact
that the levels were always much above the normal range for the
intact animal (Lincoln and Short, 1980J. These latter results are
consistent with the idea that pineal-mediated effects of the photo-
period occur directly on the hypothalamus regulating the release of
GnRH independently of androgen feedback.
To return to the situation in the Sy'rian hamster, the decrease

in serum gonadotropins in light restricted males appears to be at
least partly caused by an increased sensitivity of the hypothalamo-
pituitary axis to the feedback effects of androgens (Tamarkin et
al., 1976; Turek, 1977J. However, there is evidence for an androgen-
independent suppression of gonadotropins in short photoperiods in
the male (Ellis and Turek, 1979), and the pattern of LH secretion in
light restricted female hamsters shows little or no change even after
combined ovariectomy and adrenalectomy (Bittman and Goldman, 1979).
424 c. DEMAINE
Melatonin
""" t~".'11 i'llh~PIII~ill"'~~~~'I~II".l!/i"""IP I,'A-

I
30nA
5-Methoxytryptophol
30nA
5-Hydroxytryptophol
35nA
L--l
I sec
Fig. 2. Responses of rostral hypothalamic neurones to the

application of pineal indoles.
Effects of Pineal Indoles on the Electrical Activity of Rostral

HypothalamiC Neurones
In a search for an alternative mechanism for the action of the

pineal gland on hypothalamic releasing hormone neurones we have shown
that the putative pineal hormones, melatonin, S-methoxytryptophol
(SMTPH) and S-hydroxytryptophol (SHTPH) can influence the rate of
discharge of neurones in the rostral hypothalamus in both rats and
Syrian hamsters (Demaine and Kann, 19791. By contrast, neurones
randomly tested in the cerebellum and cerebral cortex showed their
electrical activity to be unaffected by the application of these
pineal indoles. Recordings of extracellular unit activity were made
by means of a multibarrelled micropipette stereotaxically inserted
in either the preoptic or anterior hypothalamic areas ~OA/AHA) of
males anaesthetised with urethane. In some experiments melatonin
was expelled in picolitre quantities from the tip of the micro-
pipette by means of a thermal expansion device (Demaine et al.,
1977). Otherwise the drugs were all expelled from the pipette
by passing cationic currents of 30-40 nA for periods of 10 seconds.
In this first series of experiments, recordings were made

from fifty POA/AHA neurones in hamsters kept under normal animal
house conditions. The lighting schedule was 14 hours of light and
10 hours of darkness (i.e. long photoperiods - L:D, 14:10), and
recordings made in the first seven hours of the light phase.
Under these conditions, melatonin, SMTPH and SHTPH all modified
the spontaneous electrical activity of some rostral hypothalamic
neurones. Fig. 2 shows typical actions of these three indoles on
single POA/AHA neurones in the hamster, while Fig. 3 illustrates
the proportions of neurone cEill bodies in which spontaneous activity
was either increased, reduced or unaffected by local application of
the three indoles. Melatonin increased the rate of discharge of
spontaneously active neurones by between 10 and 187% in twenty-nine
out of forty cells tested, while equal numbers of excitatory and
inhibitory responses to SMTPH were observed. By contrast, of the
forty-one neurones tested with SHTPH, thirty-two were inhibited by
between 8 and 60%. The remaining neurones were unresponsive to the
application of these indoles. When cells were tested with the
neurotransmitter, dopamine, in the presence of the pineal indoles,
the inhibitory responses to standard doses of the catecholamine
were modified. SHTPH enhanced the inhibitory responses of thirty-
four neurones to the application of the transmitter. However, both
melatonin and SMTPH caused a reduction of dopamine-induced inhibition
in the majority of cells tested in this way. In seven neurones the
inhibitory response to the catecholamine was effectively abolished
when melatonin was present.
426 c. DEMAINE
100
-
CI
z
Q
z -
...0
III
w 50
-
a:
-
-
III
..J
..J
W
U
...
..
0
-
-
n o o o
+ + +
MELATONIN 5- M T P H 5-HTPH
Fig. 3. Responses of rostral hypothalamic neurones to the appli-

cation of melatonin, 5-methoxytryptophol (5MTPH) and 5HTPH,
expressed as percentages of the total numbers of cells
tested with each substance.
+, excitation; -, inhibition; 0, no response
The influence of Light ~estriction on the Electrical Activity of

Hypothalamic NeUrones which respond to Pineal rndoles
Although striking endocrinological changes mediated by the

pineal gland occur in Syrian hamsters exposed to short photoperiods,
little is known about the effects of long periods of darkness on the
activity of hypothalamic neurones involved in the control of neuro-
hormone release. The sexually quiescent phase in the seasonal
reproductive capability of the hamster is known to require an
intact, fully innervated, pineal gland (Reiter, 1987; 1980al.
Thus during this phase the target site for antigonadotropic pineal
hormones must be sensitive at least for a substantial part of each
day. Since the overall output of pineal melatonin is not markedly
increased during short day exposure (Tamarkin et al., 1979), it is
likely that receptor sensitivity, may be greater under these conditions
than in animals housed in long days.
Having established that, using electrophysiological techniques,

pineal indoles may influence the hypothalamo-pitu±tary neuroendocrine
system by acting on neurones in the rostral hypothalamus which may
be implicated in either GnRff or TRff release, we went on to study the
effect of housing hamsters in short photoperiods (L:D, 2:22) on
POA/AHA neurones. Again the electrical responses of these cells to
the application of dopamine and the same three pineal indoles were
investigated. Male hamsters were housed in short ph6toperiods for
six to eleven weeks, during which complete testicular regression
occurred, and the results of pineal indole application compared with
those previously described. In complete contrast to animals housed
in L:D, 14:10, very few rostral hypothalamic neurones in animals kept
in L:D, 2:22 exhibited spontaneous electrical activity, although when
active units were foun~ the iontophoretic application of dopamine
caused characteristic inhibition. Table 1 gives the proportions of
POA/AHA cells responding to or unaffected by application of pineal
indoles in the two groups of hamsters. Whereas in the long day
hamsters melatonin caused excitation in 70% of the cells tested and
never induced inhibition, in th.e short day animals a considerable
proportion of neurones were inhibited by the indole. Similarly, the
proportion of inhibitory responses to 5MTPH was substantially greater
in the dark-accommodated animals. Excitatory responses to 5HTPH were
only recorded in hamsters which had been kept in short photoperiods.
Finally, in the hamsters kept in short days, dopamine-induced
inhibitory responses appeared to be unaffected by the presence of
all three pineal indoles. Thus the results indicate that exposure
to short photoperiods caused marked changes in the electrical beha-
viour of these rostral hypothalamic neurones, particularly in their
response to pineal indoles.
Table 1. Responses of rostral hypothalamic neurones,

in hamsters housed in long or short photo-
periods, to the application of pineal
indoles expressed as percentages of the
total number of cells tested with each
substance
Melatonin 5MTPH 5HTPH

0 + 0 + 0 +
LONG DAY
70 0 30 20 20 60 0 40 60
L:D, 14:10
SHORT DAY
52 32 16 20 60 20 19 50 31
L:D, 2.22
Key: +, excitation; inhibition; 0, no response

428 C. DEMAINE
Effect of Pinealectomy on responses of Rostral Hypothalamic NeUrones

to Pineal Indoles
Early reports of the ability of melatonin to induce testicular

atrophy when administered by daily subcutaneous injection to male
Syrian hamsters clearly showed that the effects were markedly
different in pinealectomised animals (Reiter et al., 1976; Tamarkin
et al., 1976). Both groups of investigators showed that daily late
afternoon or evening injections only induced reproductive collapse
in hamsters with intact, fully innervated pineal glands. The results
were consistent with, although not proof of, the idea that the pineal
gland itself may be a target organ, at least for exogenously admini-
stered melatonin, as suggested by the biochemical investigations of
Freire and Cardinali (1975). However, Tamarkin et al. (1977) were
subsequently able to show that thrice daily injections of melatonin,
during the night, resulted in testicular regression in ~oth pinealec-
tomised and intact hamsters. Their data clearly show that the action
of melatonin in inducing gonadal quiescence need not involve the
stimulation or inhibition of the release of another pineal product.
One consistent finding from all investigations of pineal-

mediated effects of light restriction in the hamster is that the
action of exogenously administered melatonin on the neuroendocrine
system is particularly dependent on the time of day of adminis-
tration (Reiter et al., 1976; Tamarkin et al., 1976; Vriend and
Reiter, 1977). Thus in intact hamsters exposed to long photoperiods,
even thrice daily injections have been shown to be ineffective in
inducing testicular regression when administration is made in the
first half of the light phase. In pinealectomised hamsters, Tamarkin
and Goldman (1978) found that thrice daily injections resulted in
testicular atrophy regardless of whether the injections were given
during the day or the night. This finding indicates that the target
tissue for melatonin is sensitive throughout the light phase in
pinealectomised hamsters. Accordingly, a series of experiments
was designed in order to assess the influence of an intact pineal
organ on the electrical responses of POA/AHA neurones (Demaine and
Kann, 1981). The actions of pineal indoles on nerve cells in
hamsters which had undergone pinealectomy were compared with those
occurring in animals subjected to the appropriate sham operation.
All th.e animals were housed in long photoperiods (L:D, 14:10) and
recordings made during the first half of the light phase of the L:D
cycle.
The results of these experiments are summarised in Table 2.

Hypothalamic neurones in the pinealectomised hamsters showed marked
differences in electrical behaviour from that of cells in sham
operated animals. Relatively more units appeared to be spon-
taneously active in the pinealectomised group, but, in most
instances, th.e activity of a given neurone did not persist for
more than a few seconds. Thus the proportion of active cells
which could be tested with the drugs was quite small. The neuro-
transmitter dopamine inhibited the units in both groups of animals.
By contrast, marked differences in the responses to the application
of the methoxyindoles, melatonin and 5MTPH were observed when
recordings were made in pinealectomised hamsters. Whereas all the
units responding to melatonin in the sham operated animals showed
excitatory responses, only inhibitory responses to the indoleamine
were recorded when cells in pinealectomised animals were tested.
In a similar way, a significantly greater proportion of units were
inhibited by 5MTPH in the pinealectomised animals. There were no
significant differences, however, in the effects of 5HTPH appli-
cation between the two groups of animals.
Table 2. Responses of rostral hypothalamic neurones,

in pinealectomised or sham operated hamsters,
to the application of pineal indoles ex-
pressed as percentages of the total'number
of cells tested with each substance.

+ 0 + 0 + 0
Pinealectomised
0 72 28 14 48 38 I) 53 47
hamsters
Sham operated
69 0 31 40 26 34 0 67 33
hamsters
Key: +, excitation; ,inhibition; 0, no response
The results of these experiments are clearly in line with the

concept that the action of melatonin on the hypothalamus of intact
hamsters is dependent on the activity of the pineal gland. The
administration of exogenous melatonin the the light phase is only
effective iii inducing testicular regression in hamsters in which
the pineal has either been removed or deprived of normal neural
inputs (Tamarkin et al., 1977; Bittman et al., 1979J. The findings
embodied in Table 2, therefore, suggest that melatonin inhibition
of the electrical acrivity of rostral hypothalamic neurones may
reflect the antigonadotropic or anthithyrotropic action of the
compound.
430 C.DEMAINE
Circadian Variation in the Electrical Re~ponses of Rostral

Hypothalamic NeuronestoPineal Methoxyindoles
As described in the previous section, the antigonadotropic and

anti thyrotropic actions of exogenously applied melatonin in Syrian
hamsters are dependent on the time of day of administration, sug-
gesting that the target tissue of this putative pineal hormone is
less sensitive or responds differently at different times of day.
In an effort to further strengthen the hypothes'is, that the in-
hibitory responses of POA/AHA neurones to pineal methoxyindoles may
reflect their abilities to suppress hypothalamic releasing hormone
neurones, experiments were conducted in which the effects of pineal
indoles on rostral hypothalamic neurones were assessed in hamsters
kept in long photoperiods, the recordings being additionally made
during the dark phase of the L:D cycle (Demaine and Stoughton, 1982).
When recordings made in the dark phase were compared with those
obtained in the light, there were significant differences between
the proportion of neurones excited or inhibited by the methoxyindoles.
The proportions of neurones responding to or unaffected by application
of pineal indoles are given in Table 3. The predominant response to
melatonin in the L phase was once again excitation, while of the
units tested in the D phase the majority were inhibited by the
indoleamine. There were similar findings in the responses to 5MTPH,
the proportion of cells inhibited being significantly increased.
In contrast, neuronal responses to 5HTPH appeared to be unaffected
bv the L:D cycle.
These results demonstrate. that the responses 6f neurones which
may control releasing hormone secretion, to both melatonin and 5MTPH,
Table 3. Responses of POA/AHA neurones to the application of

pineal indoles expressed as percentages of the total
number of cells tested with each substance a

+ o + o + o
LIGHT PHASE 70 0 30 24 22 54 o 45 55
DARK PHASE 12 54 34 8 56 36 o 60 40
Key: +, excitation; , inhibition; 0, no response.
~ecordings were made either in the light phase or in the

dark phase of the L:D cycle.
are clearly dependent on the time of day at which they are applied to
the cells.
CONCLUSIONS
In a number of photoperiod-sensitive mammalian species there is

now good evidence that the action of the pineal gland is mediated
through specific receptive sites located in the hypothalamus. In
the Syrian hamster, we have found that under conditions when the exo-
genous administration of melatonin causes involution of the gonads,
the indoleamine appears predominantly to inhibit POA/AHA neurones.
These results suggest that the rostral hypothalamus is one of the
target sites for pineal methoxyindoles, and that th.eir possible
mechanism of action is a direct modification of the rate of elec-
trical discharge of target neurones and consequently of their trans-
mitter (or neurohormone I release. Vacas and Cardinali (1979} have
located receptors with high affinity binding of melatonin in the
membrane fraction of the hypothalamus of both rats and hamsters.
Since melatonin is lipid soluble, however, it seems reasonable to
assume that it exerts its effect on electrical activity as it
crosses the neuronal membrane without the necessity of combining
with. membrane receptors. Similar rapid modification of the
electrical activity of excitable cells by other lipid soluble
substances, including steroids and thyroxin, have been observed
(Pfaff, 1981; Semm et al., 1981a; 1981b1.
-
The effects exerted by-p-lneal methoxyindoles on the firing rate
of POA/AHA neurones is clear:ly not always the same, and thus the
interactions between the cell membranes of the neurones and these
indoles must be altered by other factors. There is some evidence
that the pineal gland itself affects the responsiveness of neurones
in this region of the brain, perhaps by its endogenous rhythm of
methoxyindole secretion. It is possible that the down-regulation
hypothesis of the melatonin receptor (Reiter, 1980b) could be tested
on these neurones by studying the effects of repeated applications
of melatonin onto the same cell over a long period.
ACKNOWLEDGEMENTS
I would like to thank Hazel Kann and Roger Stoughton for

providing much of the data presented here, and Diane Newman for
secretarial help. Some of the work reported here was financially
supported by the Medical Research Council.
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Tamarkin, L., Westrom, W.K., Hamill, A.I. and Goldman, B.D., 1976b,
Effect of melatonin on the reproductive systems of male and
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Pineal-brain relationship, Progr. in Brain Res., 52:329
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NEUROBIOLOGICAL INVESTIGATIONS OF THE PINEAL GLAND AND
ITS HORMONE MELATONIN
P. Semm
J.W.Goethe University, Dept. of Zoology
Siesmayerstr.70,6000 FRANKFURT, FRG
Since very recently an extensive review of the results of

electrophysiological experiments in the pineal gland of
rodents and birds has been published(Semm,1981), in this
article I will focus on four important neurobiological
aspects of the pineal in guinea-pigs,rats and homing pi-
geons: 1. circadian rhythmicity,as shown by long-term re-
cordings and the use of microelectrophoresis, 2. electro-
physiological and morphological studies on the central
innervation of the gland 3. the transmitter-like activi-
ty of melatonin and its precursors in the pineal and
other brain areas and 4. as a major point investigations
of the magnetic sensitivity of the pineal, including single
unit recordings, biochemical measurements of melatonin
synthesis during and following earth-strength magnetic
stimulation and the metabolic mapping of brain areas,
which are sensitive for magnetic stimuli in the range
of the natural field. The latter results may be of spe-
cial interest for pineal researchers as most of the brain
areas exhibiting magnetic sensitivity, as shown by the
437
438 P. SEMM
(14C)2-deoxyglucose-method,also contain or synthesize

N-acetylalkylamines or melatonin respectively.
CIRCADIAN RHYTHMICITY
Long-term recordings from the guinea-pig pineal

(Semm and Vollrath,1980;Semm,1982) have shown that three
types of intrinsic cells can be distinguished: (a) Cells
showing constant firing rates over a period of up to 24h.
(b) Cells that are highly active during the day and show
a low firing rate during the night(light activated cells).
Artificial darkness during the day depresses the activity
of these cells gradually, but it seems that this is an
effective stimulus only until 16.00 h(Semm,1982). Further-
more, artificial illumination during the night had no mea-
surable effect.(c) Cells that exhibit low activity during
the day and enhanced activity during the night(darkness
activated cells). Artificial light given for 1 min during
the night leads, in contrast to the effect of artificial
darkness given to light-activated cells during the day,
to an abrupt decrease of activity followed by a gradual
renewed increase in firing rates.Artificial darkness
during the day had no measurable effect on these cells.
Both the light-and darkness-activated cells showed an
oscillatory pattern in maintained activity, which close-
ly followed season-dependent differences in day-and night-
lengths.Darkness-activated cells were discussed as being
involved'in melatonin secretion(Semm and Vollrath,1979)
and in measuring different night-lengths, while light-
activated cells may serve to measure day lengths.Both
these cells together may represent a complex time-keep-
ing device in which continuously oscillating mechanisms
may play an important role. The constantly firing cells,
which show no oscillating spontaneous activity, could serve
NEUROBIOLOGICAL INVESTIGATIONS OF THE PINEAL GLAND 439
as a reference principle, related to the amount of acti-

vation of the two other cell types(see also Fig.1).
It could be shown that some of the constantly firing cells
are under the influence of the CNS(Semm,1982),whilst the
other two cell types cease firing following sympathecto-
my.
When long-term recordings in the pineal gland were made
from animals kept under continuous illumination for more
than six weeks, no darkness activated cells could be de-
monstrated.Light-activated cells exhibited an altered
rhythmicity when compared with cells from animals kept
under natural lighting conditions.
Using the technique of microelectrophoresis and thus ap-
plying melatonin, noradrenaline and acetylcholine during
the day to single pinealocytes,most of the cells were
excited by melatonin and noradrenaline, the remaining cells
being inhibited or showing no response. Acetylcholine caused
in most of the cells either an inhibition or no response.
When the responses to melatonin and noradrenaline during
the day were compared with those occuring at night, the
numbers of excitatory/inhibitory effects were markedly
different. The majority of responses that were recorded
after 19.00 hours were inhibitory.No rhythmicity could
be observed in the responses to the application of ace-
tylcholine.Cells that could be excited by electrical stim-
ulation of the lateral habenular nuclei showed also an
excitation to the application of acetylcholine(Semm et
al.,1981b).
In another study(Semm et al.,1981c),the responses to thy-
roxine showed a similar circadian pattern as observed with
melatonin, calcitonin exhibiting a predominantly inhibito-
ry and parathormone a predominantly excitatory action on
the activity of pinealocytes.
Surprisingly, also the administration of a B-blocking agent
440 P. SEMM
uooo
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,
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'000
1000
_ OAy.,,""e -------+---- "IG",·TlM! ---~
>--_ _ _ _ _ D A r - T l M E - - - - - + - - - - NIGHT-TIN! - - - - - + - - O A Y - J I N e -
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.... <D
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e<
.,
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,,-~~~-~,~,-~,~'-~'~r~.-~'-~~~~~~.~-~,~-~.~-~.~-~'r~"~-
DAY_TINE MIGHT-TINE ---~ o"'_t'ME-----I
c
Fig. 1. Long-term recordings from guinea-pig pineal cells,
showing three different patterns of activity.
a. Three darkness-activated cells, showing low
electrical activity during the day and higher
activity during the night.
Note the close correspondence between the length
of the night at different seasons(1,autumn;2,win-
teri3,summer)and the nocturnal activation.
b.Two light-activated cells, the activity of which
is high during the day and diminuished during
nighttime.
c.Four constantly firing cellsinote the absence of
circadian rhythmicity.
(propranolol) elicited a circadian rhythmicity in the re-

sponsiveness of single pineal cells.The majority of re-
sponses that were recorded during daytime were excitato-
ry,whilst most of the cells responded with an inhibition
during the night. This implicates that a higher intrinsic
activity of pinealocytes is depressed by the low sympathi-
cotonus during daytime (Semm, unpublished observation).
However, the direct proof for this assumption has to come
from recordings of cultured pinealocytes lacking associa-
tions with nervous elements.
Using the same technique(Semm et al.,1981d),oestrone appli-
cation in pregnant animals lead to an increase in firing
frequency of most of the cells, while in males the majori-
ty of cells were inhibited. After administration of testo-
sterone in females the pineal cells were usually inhibi-
ted, while in males the cells were often excited. Whereas
in males a large majority of cells responded with exci-
tation between 14.00 and 22.00 hours, in the morning the
predominant effect was one of inhibition.
CENTRAL INNERVATION
A number of neuroanatomical and electrophysiologi-

cal reports(see Ueck,1979 and Semm,1981 for the pertinent
literature)have demonstrated a clear neural connection
between the brain and the pineal gland. Some of the neural
pathways are pinealofugal,while others are clearly pinealo-
petal. Some recent findings from our laboratory may be
briefly discussed here.
In order to evaluate the possible influence of central
structures on the electrical activity of single pineal
cells, in the rat stimulation electrodes were inserted
into the hippocampal formation. In the guinea-pig,the Nc.
para ventricularis and the inferior and superior colliculi
442 P. SEMM
were stimulated electrically(Reuss and Semm,1982).

Stimulation of the hippocampus elicited a gradual augmen-
tation of spontaneous electrical activity in some of the
rat pineal cells. This· excitation usually lasted for some
minutes following stimulation(Fig.2). All stimulated pineal
cells met the criteria of orthodromic stimulation,when com-
pared with usual central neuron properties. The latencies
of these cells were variable and in the range from 4.0 to
6.0 msec. In contrast to earlier findings in sympatheti-
cally influenced cells(Semm and Vollrath,1979a,b) and
similar to other centrally influenced cells(Semm et al.,
1981a) no particular pattern in the firing of these cells
was detected. As projections from the hippocampus to the
lateral habenular nucleus have been studied both anatomi-
cally and electrophysiologically(literature reviewed in
Mok and Mogenson,1974), it may well be,that information
from the hippocampus reaches the pineal via the habenu-
lar nuclei. Furthermore,as both the habenular nuclei and
the hippocampus contain melatonin(Brown et al.,1981),it
can be speculated, that the pineal may influence these
structures via melatonin and that in turn the melatonin-
induced alterations are fed 'back via nervous pathways.
Electrical stimulation of the Nc.paraventricularis
in the guinea-pig elicited both orthodromic and antidromic
responses in the pineal gland, thus confirming again that
both efferent and afferent elements are present in the
gland.
Recently central fibres coming probably from and/or via
the lateral habenular nucleus could be identified as
being neurosecretory in nature in the guinea-pig(Schneider
et al.,1981).Moreover,a direct projection from the Nc.
para ventricularis to the posterior part of the pineal
gland could be demonstrated,using the horseradish peroxi-
dase method(Korf and Wagner,1980).
-
u
W·
III
I II
W
::.::
a:::
III
20
,. .•
10
o 2 4 6 8
TIME (MIN)
Fig. 2. Peri-stimulus time histogram of a rat pineal

cell, the activity of which was gradually aug-
mented following a train of electrical stimu-
li applied to the hippocampus(indicated by
black circles)
Another central route of innervation via the posterior

commissure is indicated by pineal responses following
stimulation of the superior and inferior colliculi.Ag~in,
both orthodromic and antidromic responses could be obtai-

ned.Since the posterior commissure is known as a structure,
444 P. SEMM
which receives fibres from the colliculi and the tegmen-

tum and short-term photic and acoustic stimuli can influ-
ence the electrical activity of some pineal cells in the
rat(McClung and Dafny,1975) and in the guinea-pig(Semm,
1978), this second central innervation appears likely.
However,in the rat the superior colliculus, the pretectum
and the midbrain central gray receive inputs from the la-
teral habenular nuclei (Mok and Mogenson, 1974). Further
electrophysiological studies have to be undertaken to show
whether some of these structures can also be influenced
by the pineal gland.
MELATONIN AS A TRANSMITTER IN THE PINEAL, VISUAL SYSTEM

AND CEREBELLUM
It has been hypothetized that one of the target
organs for melatonin and/or 5-methoxyindoles could be
the pineal gland itself, where indoles could possibly
function as chemical messengers(for review see Vollrath,
1981). It is therefore not suprising that in addition to
the previously demonstrated neurophysiological action of
melatonin(Semm et al.,1981b) 5-methoxytryptophol and also
5-hydroxytryptophol influence the electrical activity of
pineal cells(Semm and Vollrath,1982).
5-Methoxytryptophol caused on average a similar distri-
bution of excitatory and inhibitory responses of the cells
recorded,as did melatonin. However, only 29% of the single
cells showed the same reactions to melatonin and the
methoxyindole when applied successively. It is therefore
not unlikely that both substances exhibit on most of the
pineal cells a different intrinsic action,possibly influ-
encing intracellularly or extracellularly the formation,
storage, and/or release of different peptidergic or other
effector compounds(see Quay,1974;Benson,1977).
Very recently it could be demonstrated that the delta

sleep-inducing peptide(DSIP),a peptide which induces slow-
wave EEG(sleep) (for review see Schoenenberger et al.,1978),
is highly concentrated in the pineal after i.v.injection
when compared with 9 other different regions in the brain
(Graf et al.,1981a). Injections of DSIP at different times
of the day(7.00 and 18.00 h) produced significant changes
in the 24-h rhythmicity of 5-HT,dopamine and noradrenaline
in the rat brain and of prolactin,growth hormone and cor-
ticosterone levels (Graf et al., 1981 b). This strongly sug-
gests (a) that DSIP acts on a higher level of coordina-
tion as a "programmer" for the circadian rhythmicity,the
sleep inducing property appearing as a subset and (b) as
melatonin shows a similar overall effect,that there is
a functional relationship between the two substances.
Microelectrophoretical studies have been made to deter-
mine the interaction of DSIP and melatonin on the single
cell level in the pineal and the cerebellum(Semm and Reuss,
1982) •
Recordings in the visual system of the guinea-pig reveal
that melatonin and 5-methoxytryptophol can alter the spon-
taneous and light evoked or depressed activity in the reti-
na,optic chiasma and geniculate body, but not in the supe-
rior colliclus (Semm and Vollrath, 1982). Suprisingly the dif-
ferent cell classes in the visual system responded identi-
cally with an augmentation of activity to the application
of the substances, the methoxyindole exhibiting in most of
the cases a more pronounced effect than melatonin. Since
melatonin plays a role in the pigment aggregation of the
retinal pigment epithelium in the guinea-pig(Pang and Yew,
1979) it may together with its excitatory action in the
retinal ganglion cells act as a substance which makes the
visual cells more sensitive to light.
446 P. SEMM
In the cerebellum of the homing pigeon and the guinea-

pig,melatonin and two of its precursoTS caused clear al-
terations in the electrical activity of Purkinje cells
and other cell types(granule cells, Golgi cells).
In the pigeon,an equal number of Purkinje cells was in-
hibited and excited during day time by melatonin and
5-methoxytryptophol, whilst the responses to 5-hydroxytryp-
tophol were predominantly inhibitory. The responses to the
indoles varied significantly depending on whether the cells
were tested during the day or at night(p>0.001).90% of the
cells were inhibited by noradrenaline independently of the
time of recording.
In the guinea-pig,most of the Purkinje cells were inhibi-
ted by the application of melatonin and 5-methoxytrypto-
phol,while 5-hydroxytryptophol caused no response in most
of the cells. In this species no significant circadian chan-
ges in sensitivity to the application of the indoles could
be observed(Fig.3).
Purkinje cell responses to microelectrophoretically applied
indoles were examined before and during noradrenaline ap-
lication. This substance was found to augment indole exci-
tation and inhibition in all the cases investigated, sup-
porting the hypothesis that noradrenaline and thereby the
indoles act on postsynaptic processes to increase the re-
sponsiveness of the Purkinje cells to afferent inputs.
Most of the other cerebellar units in the pigeon were in-
hibited by melatonin and 5-methoxytryptophol,whilst 5-hy-
droxytryptophol exhibited an excitation in most of the
cells investigated. In the guinea-pig the responses of
these cells were more complicated. Melatonin caused an ex-
citation in most of the units, while the predominant respon-
se caused by 5-methoxytryptophol was one of inhibition.
Only 30% of these units responded to 5-hydroxytryptophol,
the remaining cells showing no measurable response.
A B
Fig.3 Purkinje cell responses to the application of me-

latonin(A;guinea-pig),5-methoxytryptophol(B;pigeon),
5-hydroxytryptophol(C;pigeon) and noradrenaline
(D;guinea-pig).
A:the left part of the picture shows spontaneous
activity, which is strongly inhibited during appli-
cation of melatonin. The downward deflection of
the oscilloscope beam indicates the current arti-
fact caused by electrophoresis. The discharge of
the simple spikes is completely inhibited,while
the complex spike is apparently not influenced
(indicated by white circles).
B:The left part shows spontaneous activity, which
is clearly augmented by 5-methoxytryptophol.
C:spontaneous activity of another Purkinje cell,
which is inhibited by 5-hydroxytryptophol.During
application time(indicated by the two current
artifacts) background activity remains unaffected.
448 P. SEMM
D:Purkinje cell electrical activity is completely

inhibited by the application of noradrenaline.
Following application time simple spike activity
remains depressed, complex spike activity shows
ongoing activity.
Calibrations A:O.5sec 2mV;B:O.5sec 2mV;C:O.5sec
1mV;D:O.5sec 1mV.
It is apparent from these studies that(1) pineal indoles

can bring about direct changes in the firing frequency of
Purkinje cells and other cells in the cerebellum of two
different species and (2) that their action and thereby
their influence on locomotor activity may be different
in the two species investigated and (3) that these sub-
stances although primarily regarded as hormones represent
new types of neurotransmitters or neuromodulators.
The results of the present study, showing a predominantly
inhibitory action of melatonin on Purktnje cell activity
at night, when levels of circulating melatonin are higher
than during the day, implicate that melatonin causes a
disinhibition of the inhibitory influences of the cere-
bellum on other brain areas, which are involved in the
regulation of locomotor activity. Whether or not the re-
sult of this influence is a depression of overall loco-
motor activity remains to be elucidated(Semm and Voll-
rath,1982)
NEUROBIOLOGICAL INVESTIGATIONS ON THE MAGNETIC SENSITI-

VITY OF THE PINEAL GLAND IN RODENTS AND PIGEONS
In the past 15 years growing evidence has accumulated that

the geomagnetic field can influence the behavior andorien-
tation of a variety of organisms from bacteria through
vertebrates, including man (for review see Wiltschko,
1980). From this body of experimental work it seem impor-

tant to note, that most behavioral responses generally
fall into two categories: either they involve magnetic
compass orientation or, in addition, imply some form of
physiological sensitivity to weak fluctuations in natural
magnetic intensity.
Some of the characteristics of the bird's magnetic com-
pass, its dependence on a rather narrow intensity range
centred aroupd the actual intensity of the natural field,
its plasticity and its working as an inclination compass
are well documented(Wiltschko,1980). On the other hand,
there is a number of pathological phenomena,the temporal
occurence of which can be correlated with geomagnetic
activity(Gerisch and Becker,1979;Malin and Srivastava,
1979).
These results imply the existence of some sort of senso-
ry receptors for sensing and transducing the magnetic
field to the central nervous system. However, it is not
known,where in vertebrates the magnetoreceptor is loca-
ted.As the initial orientation of individual birds can
be altered by changes in the direction of the magnetic
field around their heads,it was assumed that the magne-
tic sensitive system is located in the head(Walcott,
1977) •
From a theoretical point of view the pineal gland was
considered as being a part of the postulated magnetic
system(Semm et al.,1980),and testing this hypothesis
by means of electrophysiological, biochemical and auto-
radiographic methods, it could be shown,that this struc-
ture,probably beneath others,is indeed involved in sen-
sing magnetic variations in the natural environment.
This chapter summarizes the very new findings on the
magnetic sensitivity of the pineal gland of rodents and
pigeons and of other parts of the pigeon's brain.
450 P. SEMM
1.Electrophysiology
a Guinea pig
About 20% of single cells in the guinea-pig pineal gland

showed a marked effect to earth strength magnetic stimu-
lation(Semm et a~.,1980). Spontaneous electrical activity
during daytime could be diminuished by more than 50% by
an induced field,generated by Helmholtz-coils(the verti-
cal component was added to the natural field), and resto-
red when the magnetic field was inverted. Both, the latency
of depression and renewed activation,was in the range of
about 2 min. After turning off the inverted magnetic field
the cells showed their normal spontaneous activity which
could be depressed again with a low magnetic stimulus.
Some cells responded in the range of msec, but they were
infrequently found.
In addition, brain regions in the neighbourhood of the pi-
neal gland were tested under identical experimental con-
ditions.A comparable response(activation of the sponta-
neous activity) could be obtained in the molecular layer
of the cerebellum,a finding which could be made valid
indirectly later by means of the deoxyglucose method.
·Some of the magnetical sensitive pineal cells in the
guinea-pig showed an oscillatory electrical activity with
a pattern characteristic for that of "darkness-activated
cells." Thus, it was suggested that these cells may combine
the abilities of time measurement,sensing variations in
the natural magnetic field and in lighting conditions,
and the synthesis of hormones,probably melatonin(Semm,
1982) •
From this point of view it appears likely that these cells
belong to the magnetic system which is sensitive to the
daily fluctuations of the magnetic field rather than
being involved in orientation within the magnetic field.
Because the daily heating and cooling of the atmosphere

displaces the jet streams north and south,a more or less
regular circadian variation of the magnetic field is ob-
served on the ground. This furthermore implies that this
rhythmicity of the magnetic field may have a "Zeitgeber"
function,a hypothesis,which could be confirmed in ham-
sters ( Brown and Scow, 19 7 8) and in birds (Bliss and Hepp-
ner,1976).On the other hand,the normal fluctuations in
the earth's magnetic field can influence pigeon orienta-
tion (Keeton et al., 1974). Since mos t of the magnetic fluc-
tuations in these behavioral experiments were less than
70 gamma,it was concluded that the sensitivity of pigeons
to magnetic cues probably approaches that already demon-
strated for insects.
b Homing pigeon
As to our knowledge nothing is known about effects of

magnetic fields on the behavior of guinea-pigs,we tur-
ned to homing pigeons,which are known to use the natu-
ral magnetic field for directional information, especial-
ly under total overcast(Walcott,1977).
As only a few recordings had been made from the pigeon
pineal gland(Oksche et al.,1969) we checked the proper-
ties of the pigeon's pinealocytes, especially their sen-
sitivity to light,given either only to the pineal follo-
wing enucleation or to both the eyes and the pineal(Semm
and Vollrath,1982~ Immediately after penetration of the
pineal capsule action potentials could be observed exhibi-
ting a wide range of firing rates from 2 to more than
90 actionpotentials/sec.Even after enucleation some pineal
cells responded to light, mostly in a gradual manner
(Fig,4).
452 P. SEMM
~ "0"'
Fig.4 Single unit recordings in the pineal of

enucleated pigeons (Averager Histograms, lsec/bin)
A: Spontaneous electrical activity of a pineal
cell, the activity of which is depressed by light
and renewed gradually by artificial darkness (B).
C: Response of another pineal cell, which is gradu~
ally excited by darkness.

D: Activity of another pineal cell, the reaction of
which starts immediately with the presentation
of a light stimulus. The augmentation of activity
during the light stimulus is followed by an atten~
uated activity for some seconds.

The electrical activity of about 30% of cells in the

upper part of the pigeon's pineal gland could be altered
by changing the strength of the local magnetic field and
the direction of its vertical or horizontal component,
using two pairs of Helmholtz coils(Semm et al.,1982).
In contrast to the most cells in the guinea-pig, such cells
responded to a rapid and/or a gradual change in the mag-
netic field with either excitation or inhibition of elec-
trical activity with a latency in the range of msec.
A considerable number of cells showed responses to slow
or fast changes in the magnetic field,but the reactions
could not be repeated within a time of 30 min. although
recordings had a good quality and the experimental situ-
ation was unchanged. It may well be that these cells are
due to unknown reasons refractory to a second magnetic
stimulus within a certain period.
At present it is yet not possible to decide whether the
magnetical sensitive cells react primarily to changes in
the direction or to changes in the intensity of the mag-
netic field. As some of the cells responded as well to a
complete rapid inversion of one component(i.e. a diffe-
rent direction but the same intensity as in the natural
magnetic field) as to a gradual change(i.e. a distinct
range of a different direction and an altered intensi-
ty), it may be possible that some of the cells are only
direction selective and others can encode both direction
and intensity changes by different amounts of excitation
or inhibition of activity.
Comparing the electrophysiological results under magne-
tic stimulation in the guinea-pig and the pigeon one
may have the impression that pigeon pineal cells are more
sensitive and complicated in response to alterations in
the magnetic field.However, the participation of these ele-
ments in magnetic orientation remains to be elucidated.
454 P. SEMM
2.Biochemistry
The magnetic sensitivity of single pineal cells poses the

question whether the secretory activity of the gland and
thereby the synthesis of melatonin is also affected by an
artificial magnetic field in the strength of that of the
earth.
To test this hypothesis,the effects of a magnetic field
on pineal serotonih-N-acetyltransferase(NAT) activity
and the melatonin content in male rats were investigated
(Welker et al.1982a+b).
Experimental inversion of the horizontal component of the
natural magnetic field,periormed at different time points
during the night,led to a significant decrease of both
parameters investigated,although the animals were free
moving within a "globus," consisting of three Helmholtz-
coils in north-south, east-west and vertical direction
(Semm,1982).During day-time,when the secretory activity
of the gland is normally low,this effect was less conspi-
cuous. Inversion of the horizontal component was follo-
wed by a reduced pineal melatonin formation,for about 2
hours,when the magnetic stimulation was perrormed at
24.00 hours. When the experiments .rere carried out at
22.00 hours,a clear depression was still noticeable after
2 hours.
In this context it is relevant to note that exposure of
rats to 1 min of light during the night leads to a stri-
king decrease of pineal melatonin,the synthesis of which
is activated again, when the light puIs is given at 22.00
hours.After midnight,in the second half of the scotophase
a further increase is no longer detectable(Illnerova et
al.,1979).As this effect is discussed as indicating a
circadian rhythmicity in photosensitivity,a similar cir-
cadian rhythmicity in magnetosensitivity may also be
present in the pineal, thus giving further evidence for

a functional relationship between the 24 h cycle and the
magnetic field.
After 24 hours exposure to the inverted horizontal compo-
nent,return to the natural condition was followed by a re-
newed clear depression of pineal NAT activity and melato-
nin content, indicating that the change of the magnetic
field is the stimulus. Changing the inclination of the lo-
cal magnetic field alao decreased the secretory activity
of the rat pineal gland.
In pigeons a similar response of the nocturnal melatonin
synthesis in the pineal gland could be demonstrated, but
due to unknown reasons, the results were not as clear as
in rats. There were much more individual differences in
the experimental group,whilst the melatonin values of the
controls were in the normal range.
In conclusion,as the alteration in the ambient magnetic
field rather than a movement of the head of a free mo-
ving animal causes a depression of melatonin synthesis,
it appears likely that there is a compass system in the
brain calibrated either on the polarity or the intensity
of the natural field.In turn, after staying for 24 hours
in the inverted magnetic field, the compass system is
adaptet and calibrat€d to the new polarity.
Very recently also the melatonin synthesis in the retina
of the rat could be demonstrated as being sensitive to
earth-strength magnetic stimuli(Cremer-Bartels and Krause,
pers. communication).
Th~se results pose the question whether the pineal gland
itself is sensitive to changes in the magnetic field or
whether these effects are neuronally mediated,either by
the sympathetic nerve fibres or the central commissural
fibres. Since the sympathetic nervous system as a whole is
sensitive to magnetic alterations(Sakharova,1976),the
456 P. SEMM
sympathetic innervation of the pineal and perhaps also of

the retina may be responsible for the magnetic sensitivi-
ty.A further explanation could be that the magnetic field
is capable of influencing the enzymes themselves,which
are involved in melatonin synthesis. It was pointed out by
Beischer(1963) that hydrogen nuclei and other cell consti-
tuents briefly precess with frequencies according to their
mechanical and magnetic moments when the body turns about
in the earth's magnetic field.It was suggested,that such
an interaction may provide living matter with spatial
cues.
In general,what effect the magnetically induced short
term decrease in melatonin synthesis has for the func-
tioning of the organism as a whole remains to be deter-
mined. From the current status of knowledge it may be spe-
culated. that also other hormones which are functionally
coupled to melatonin(prolactin, growth hormone, sex hor-
mones,thyroxine) and other brain regions,which either
contain melatonin or are affected by the hormone,are sen-
sitive to changes in the magnetic environment. Possibly
this concept explains the phenomena which have been attri-
buted to extremely low intensity magnetic fields(Busby,
1968;Gerisch and Becker,1979;Ossenkopp and Nobrega,1979).
Some observations indicate that interactions bet-
ween organisms and the magnetic field have,during evolu-
tion, become a necessity for developing and maintaining
normal functional integrity of higher organisms,the mag-
netic field possibly serving as a governing force that
controls the rate of cellular growth and proliferation
(Ossenkopp et al.,1972;Beck,unpublished observations).
The relative importance of the pineal gland and its hor-
mone melatonin in overall orientation will be tested in
pinealectomized and/or melatonin treated pigeons and mi-
gratory birds under magnetic and non magnetic conditions.
It may well be that the pineal,independently from its mag-

netic sensitivity,plays a role in the time compensated sun
compass orientation,and that in turn the time compensation
of this compass can be affected by magnetic fields,which
influence melatonin secretion.In comparison, when tiger
salamanders are trained to select a special direction
(Adler and Taylor,1980), they cannot orient themselves
under continuous light or darkness, but when melatonin,but
not serotonin and thyroxin are given,the animals were again
capable of orientation,but move in a 90 0 clockwise shifted
direction.
3. Deoxyglucose-technique
Since the electrophysiological and biochemical studies

have made it probable that the pineal gland may be part
of the postulated magneto-sensitive system,an attempt was
made to find further areas of the brain related to detec-
tion and transformation of magnetic stimuli from the na-
tural environment,using the (1 4C)2-deoxyglucose technique.
This method has been shown to provide auto radiographic
maps of regional glucose consumption under several experi-
mental conditions,in particular during sensory stimula-
tion (Sokoloff et a1., 1977; Streit et a1., 1980).
It could be shown that the functional uptake of deoxyglu-
cose induced by magnetic field(inversion of the horizontal
component) takes place in different brain regions in the
pigeon(Mai and Semm,1982). Those regions exhibiting exten-
sive glucose uptake as compared with controls included the
molecular layer of the cerebellum,the optic tectum,the
parvocellular component of the isthmic nuclei,the inter-
collicular nuclei,the trapezoid body, the habenular nuclei
and the pineal gland.
Even in experiments during daytime and at night after
458 P. SEMM
presentation of light the pineal was well labelled, a fact,

which speaks in favor for specific magnetic sensitivity
within the gland, since secretory activity is low during
daytime and depressed by light during daytime. The label-
ling of the habenular nuclei, however, can also be explai-
ned by their involvement in the transmission of light to
the pineal, as demonstrated earlier in the quail(Herbute
and Bayle,1977).
In view of the fact, that the rat and pigeon pineal gland
melatonin synthesis is influenced by magnetic fields,it
appears meaningful,that some brain areas,which contain
and/or synthesize melatonin(cerebellum,visual system,brain
stem) are also responding to magnetic stimuli,as shown by
the incorporation of (1 4C)deoxyglucoe. Since electrophy-
siological studies have shown that these areas probably
contain receptors for melatonin(Semm and Vollrath,1982a+b;
Pazo,1979),it may well be that a magnetically induced al-
teration in blood melatonin concentration influences these
areas.
Specific magnetic sensitivity in the optic tectum in ani-
mals sti~ulated in darkness during the night supports the
proposal of Leask(1978) that magnetic field detp.r.tion
could take place in the retina, mediated by rhodopsin. In
this context it is interesting to note,that(a) retinal
ganglion cells can respond to magnetic stimuli by a spe-
cific alteration in their by light induced activity(Lov-
sund et al.,1981),(b) that in humans the scotopic criti-
cal flickerfusion frequency shows a tendency to diminuish
gradually during exposure to a magnetic field with reduced
intensity(Beischer et al.,1967) and (c) that behavioral
studies have made it likely(Wiltschko and Wiltschko,1981)
that light is an important prerequisite for sampling mag-
netic information during an outward journey of young unex-
perienced pigeons. Furthermore, rhodopsin is also present
in the pineal gland(Deguchi,1981) and may have contributed

to the magnetic sensitivity in this area.
However, the direct proof for these assumptions supporting
the proposal of Leask (1978) has to come from experiments
in total darkness showing that for example the spontaneous
activity of single cells in magnetic sensitive areas can-
not be influenced by a magnetic field
In general,I believe that all these results have opened
a new door for the understanding of the complex orientation
behavior of vertebrates and that the multiple implications
of geomagnetism on behavior can now be investigated also
in the brain itself. We have done only the first steps in
this direction and we are full of hope,that the combina-
tion of neurobiological and behavioral methods will allow
further insight into the old enigma of brain-magnetic
field interrelationships.
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INFLUENCE OF THE PINEAL GLAND ON TUMOR GROWTH
IN MAMMALIANS: A REAPPRAISAL FROM A BIOCHEMICAL
POI NT OF VlEW
Maria E. Feriol i, Giuseppe Scalabrino, and

Franco Fraschini
Institute of General Pathology and C.N.R. Center
for Research in Cel I Pathology (M.E.F., G.S.)
and lnstitute of Pharmacology, Chair of Chemo-
therapy (F.F.), University of Milan, 20133
MILANO, Italy
In nature infinite book of secrecy

A I ittle I can read
W. Shakespeare, Antony and Cleo-
patra, Act I, Scene 2.
I NTRODUCTI ON
The regulation of the growth of mammatian tumors
by different hormones remains an intriguing mystery.
The claim that the pineal gland is involved in control-
ling neoplastic growth is one segment of that large area.
Experimental and cl inical reports summarized by
Bartsch and Bartsch (1981) and by Lapin and Ebels (1981)
indicate that there is some I ink between cancer develop-
ment and pineal function, although the data so far avail-
able are often contradictory. The I ink was detected when
the effects on neoplastic growth of pinealectomy and/or
administration of melatonin were studied. This approach
has been complemented and supplemented by identification
of other active pineal substances with anti-tumor ac-
tivity and by the study of histological changes in the
pineal glands of humans and animals that have died of
cancer.
467
468 M. E. FERIOLI ET AL.
It has been demonstrated that pinealectomy acceler-

ates the growth of transplanted melanoma in hamsters
{Das Gupta and Terz, 1967a,b}, of transplanted Walker
256 carcinoma in rats {Rodin, 1963; Barone and Das Gupta,
1970} and of transplanted Yoshida sarcoma in rats {Lapin,
1975; Lapin and Frowein, 1981}. Similarly, removal of the
pineal gland enhanced the induction of mammary adenocar-
cinoma in the rat by 7,12-dimethylbenz{a}-anthracene
{DMBA}, particularly when the carcinogen was administered
at low doses {Tamarkin et al., 1981}. But, when DMBA was
administered at high doses, there was little if any dif-
ference in the occurrence of mammary neop.lasms in pineal-
ectomized and sham-pinealectomized rats (Lapin, 1978;
Aubert et al., 1980).
The administration of the pineal indole hormone
melatonin to pinealectomized hamsters abol ished the ac-
celerating effect of pinealectomy on the growth of mela-
noma implants {EI-Domeiri and Das Gupta, 1973}. Anal-
ogously, tumor development was slowed down and tumor in-
cidence decreased by exogenous melatonin administration
to female rats in which mammary adenocarcinomas were
being induced by DMBA {Aubert et al., 1980; Tamarkin et
al., 1981}. The only opposite finding was reported by
Hamilton {1969}. Furthermore, melatonin was shown to re-
duce the incidence of methylcholanthrene-induced sarcoma
in mice {Lapin and Ebels, 1976}. Interestingly enough,
it was demonstrated by Bartsch and Bartsch {1981} that
the inhibitory effect of melatonin on the growth of fi-
brosarcoma ascites cells or of Ehrl ich ascites cells
injected into the peritoneal cavities of mice was de-
pendent on the photoperiod and the time of day of admin-
istration. Melatonin administration also slowed down the
development of Yoshida sarcoma cells transplanted into
rats {Lapin and Ebels, 1976; Lapin and Frowein, 1981},
but for this type of tumor, too, an opposite finding
has been reported {Huxley and Tapp, 1972a}. The inhibi-
tory effect of melatonin on tumor growth was also demon-
strated on a transplantable leukemia in mice {Buswell,
1975} and a transplantable mammary tumor in female rats
{Karmali et al., 1978}. Unexpectedly, melatonin reduced
the survival time of mice bearing Lewis lung carcinoma
{Lapin and Ebels, 1976}.
Melatonin does not have any in vitro anti-tumor ac-
tivity on KB cells {Bindoni et al., 1976}, but there is
some substance in sheep pineal gland that has antimitotic
activity on that cell strain. It has been partially puri-
fied {Bindoni et al., 1976}. The bovine pineal gland
also contains a polypeptide substance with anti-tumor
PINEAL GLAND ON TUMOR GROWTH 469
activity on different transplantable mouse tumors and

chemically induced rat mammary adenocarcinomas (Di Iman
et al., 1979).
From this survey of the I iterature, it is quite
clear that so far no studies using the rei iable putative
biochemical markers of neoplastic growth have been done
to evaluate whether or not pinealectomy accelerates neo-
plastic development or whether or not exogeneous mela-
tonin slows down tumor growth. A large body of exper-
imental evidence demonstrates that the physiologically
occurring di- and poly-amines accumulate during al I cel I
growth processes, including growth of experimental
animal tumors and human tumors (Scalabrino and Feriol i,
1981). Polyamines appear to be tightly I inked to bio-
chemical processes crucial for cell ~rowth and prol ifer-
ation, although the precise functionts) of these cat-
ionic substances are not fully understood. L-Ornithine
qecarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17)
(DOC) catalyzes the conversion of ornithine to putrescine
and is widely reported to be the rate-I imiting enzyme
in the biosynthetic pathway of polyamines. Besides being
the first enzyme in the polyamine biosynthetic pathway,
DOC is easily inducibile and to a considerable degree
by a great variety of stimul i. Its half-I ife is the
shortest known for any enzyme and, last but not least,
it carries out the only known reaction for putrescine
biosrnthesis in mammal ian cells (Scalabrino and Feriol i,
1981). Moreover, induction of ODC activity is an early
and marked event when eukaryotic cells are stimulated
to grow, in al I systems studied to date (Scalabrino and
Feriol i, 1981). With this in mind, we monitored the
levels of ODC activity sequentially in the I ivers of
pinealectomized rats and of sham-pinealectomized rats,
both in the process of chemical hepatocarcinogenesis
induced by 3 '-methyl-4-(di methyl ami no)-azobenzene (3 '-Me-
-DAB). Additionally, we compared the levels of ODC ac-
tivity in the mammary glands of pinealectomized and of
sham-pinealectomized rats, both in the process of DMBA
chemical carcinogenesis, since we hypothesize that the
influence, if any, of the epiphysis on neoplastic growth
and development might be more easily detected in a neo-
plasm whose growth is under hormonal control.
We did not consider it necessary to perform any,
histological or biochemical studies of the pineal glands
of our rats with tumors, since the relevance of that
would be marginal and inconclusive. Very few studies
have included the weights and the morphology of the
pineal glands of human who died with cancer. The pineal
470 M. E. FERIOLI ET Al.
weights were heavier than those in patients who died of

nonmal ignant diseases in two studies (Rodin and Overall,
1967i Tapp, 1980a), but just the opposite has also been
reported (Tapp and Blumenfield, 1970). Pineal morpho-
logical abnormalities (Hajdu et al., 1972) and calcifi-
cation (Cohen et al., 1978) have also been described.
In experimental animals the I ipid content of pineal
glands from rats with c~emical Iy induced fibrosarcomas
was significantly greater than that of glands from the
controls (Huxley and Tapp, 1972bi Tapp, 1980b).
MATERIALS AND METHODS
Chemicals
L_[1_14C] Ornithine monohydrochloride (SA 58 mCi/-
mmol) was purchased from the Ra~iochemical Centre
(Amersham, U.K.). The volatile 4C02 contamination of
labeled L-ornithine was removed before use (J~nne and
Williams-Ashman, 1971). The source of 3'-methyl-DAB
was Koch Light (Colnbrook, U.K.) and that of DMBA was
Sigma, Chemical Co., St. Louis, Mo., U.S.A .• All of the
other chemicals were the highest grades commercially
avai lable.
Animals
Adult rats of both sexes (Sprague-Dawley strain)
were obtained from Charles River (Cal co, Italy). AI I
rats were rigidly standardized, starting 12 days before
pinealectomy or sham-pinealectomy and continuing until
they were kil led. They were maintained in a cl imate-con-
trol led room (23°C), artificially illuminated with a
I ight-d~rk cycle of 12:12 h daily (I ights on, 06:00-
18:00 h). During both the habituation period and exper-
iment, the nocturnal feeding habit of the rats was re-
inforced by removal of their food when the lights came
on and its replacement just before the I ights were
turned off. The composition of the semi~ynthetic basal
diet for the controls was essentially that reported by
Sneider and Potter (1969). The diet oncogenic for the
I iver was identical with the basal diet except that
0.06% 3'-Me-DAB was added and this was given to male
rats only. 8.0 Mg of DMBA in peanut oil was given by
i.g. intubation to 60-day-old female rats, which had
been pinealectomized 35 days earl ier. Pinealectomy was
performed under Nembutal anesthesia by the method of
Hoffman and Reiter (1965). In sham-pinealectomized rats,

the forceps were introduced into the vicinity of the
pineal gland without injuring the gland or neighboring
vascular or brain tissues. Each animal was checked for
successful pinealectomy at zooscopy. The operations were
always performed between 08:00-11:00. The overal I mor-
tal ity of rats, on either of the oncogenic treatments,
was 5%. In order to minimize the diurnal changes of ODC
activity levels in the organs of the rats, the rats were
always ki lied between 14:00 and 16:00 h.
Preparation of Orsan Extracts

At fixed times the rats were kil led by decapitation.
The I ivers were quickly removed from the rats on the
diet containing 3'-Me-DAB and were processed individu-
ally. The mammary glands were quickly removed from the
rats treated with DMBA and were also processed individu-
ally. The homogenizing medium was that previously de-
scribed by Scalabrino et al. (1979). The I ivers were
homogenized in 2 vol and the mammary glands in 4 vol of
medium. The 20,000 x g supernatant fraction was used
immediately for assay of the ODC activity.
Enzyme Assay
The ODC activity was assayed essentiallr as pre-
viously described by Scalabrino et al. (1979). The
assays were always done in dupl icate. The concentration
of L-ornithine (labeled plus unlabeled) used in the
routine assay was saturating (final concentration, 2 mM).
Protein Determination
Protein content was estimated, with bovine serum
albumin as the standard, as described by Geiger and
Bessman (1972).
RESULTS AND DISCUSSION

Table 1 shows the levels of ODC activity in livers
of the control male rats, both sham-pinealectomized and
pinealectomized, fed the basal diet and of rats, both
sham-pinealectomized and pinealectomized, fed the diet
for hepatocarcinogenesis. It appears that pinealectomy
does not modify the levels of hepatic ODC activity in
Table 1. ODe Activity in Livers of Sham-pinealectomized

or Pinealectomized male Rats during 3'-Me-DAB
Hepatocarcinogenesis
Rats Diet Time of ODe Activity

diet
(months) (pmol 14e02/mg protein/30 min)
Sham-PLX Basal 1 70.4 + 12.15 (6)

PLX Basal 1 65.9 + 9.84
Sham-PLX 3'-Me-DAB 1 194.4 + 25.16 ~~~
PLX 3'-Me-DAB 1 195.3 + 23.42 (6)
Sham-PLX Basal 3 16.6 + 2.26 (5)
PLX
Sham-PLX
PLX
Basal
3'-Me-DAB
3'-Me-DAB
3
3
3
11.8
47.6
38.0
+
+"
+
1.15
3.72 fB
2.34 (5)
The results are expressed as mean values + E.S. Number

of animals in parentheses. Sham-PLX=Sham-pinealectomized
rats. PLX=Pinealectomized rats.
rats fed the basal diet throughout the experimentation

period, since sham-pinealectomized and pinealectomized
animals nearly always had the same I iver enzyme ac-
tivity. Furthermore, again from Table 1, it appears
that pinealectomy does not accelerate hepatocarcino-
genesis when the ODe activity is the biochemical par-
ameter for evaluating tumor growth. In fact, the in-
crease in hepatic ODe activity, which was observed at
the end of the first month, caused by the carcinogen
(Scalabrino et al., 1978), is of the same order of mag-
nitude in both sham-pinealectomized and in pinealecto-
mized rats. Simi larly, at the end of the third month,
the hepatic levels of ODe activity in pinealectomized
and in sham-pinealectomized rats did not significantly
differ.ln keeping with an earl ier study (Scalabrino et
al., 1978, at this time we observed a marked fall in
hepatic levels of ODe activity.
Table 2 shows the levels of ODe activity in the
mammary ~Iands of intact untreated rats and of sham-pin-
ealectomlzed and pinealectomized rats 2 months after
i .g. intubation of a low dosage of DMBA. This low dosage
of DMBA was chosen because Tamarkin et al., (1981) dem-
onstrated that pinealectomy accelerated tumor development
Table 2. ODC Activity In Mammary Glands of Sham-pineal-

ectomized or Pinealectomized Female Rats
Treated with DMBA
Rats Time after i. g. o D C Act i vi ty

DMBA intubation
(months) (pmol 14C02/mg protein/30 min)
Intact o 2.1 + 0.94 !4j

Sham-PLX 2 5.7 +" 0.71 4
PLX 2 22.8 ± 2.15 4
The results are expressed as mean values + E.S. Number

of animals in parentheses. Sham-PLX=Sham-pinealectomized
rats. PLX=Pinealectomized rats.
in mammary glands only when DMBA was given at a low

dosage. From Table 2 it appears that the treatment with
DMBA enhances ODC levels, as other carcinogens do in
their target tissues. High ODC activity was found by
Andersson et al., (197~ in fully developed rat mammary
tumors induced by DMBA. But, what is more interesting,
is that the enhancement of ODC activity induced by DMBA
2 months after i.g. intubation was much greater in pin-
ealectomized rats than in sham-pinealectomized rats.
This demonstrates that the removal of pineal gland fa-
ci I itated tumor development in mammary glands~ This
conclusion is in agreement with Tamarkin et al., (1981),
but must be taken with caution, since our investigation
is still in progress. However, we can state unequivo-
cally at this time that the pineal gland does not at
al I influence the development and growth of hepatomas
induced chemically in the rat.
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gren, E., and Sundler, F., 1976, Diamines
and Polyamines in DMBA-Induced Breast Carci-
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pound 48/80, Agents and Actions, 6:577.
Aubert, C., Janiaud, P., and Lecalvez, J., 1980, Effect
of Pinealectomy and Melatonin on Mammar~ Tumor
Growth in Sprague-Dawley Rats Under Different
Conditions of Lighting, J. Neural Transm.,
47:121.
Barone, R. M., and Das Gupta, T. K., 1970, Role of Pin-
ealectomy on Walker 256 Carcinoma in Rats, 1.
Surs. Oncol., 2:313.
Bartsch, H., and Bartsch, C., 1981, Effect of Melatonin
on Experimental Tumors Under Different Photope-
riods and Times of Administration, J. Neural
Transm., 52:269.
Bindoni, M. Jutisz, M., and Ribot, G., 1976, Character-
ization and Partial Purification of a Substance
in the Pineal Gland Which Inhibits Cel I Multi-
pi ication In Vitro, Biochim. Biophys. Acta,
437:577.
Buswel I, R. S., 1975, The Pineal and Neoplasia, Lancet,
i : 34.
Cohen, M., Lippman, M., and Chabner, B., 1978, Role of
Pineal Gland in Aetiology and Treatment of
Breast Cancer, Lancet, i i : 814.
Das Gupta, T. K., and Terz, J., 1967a, Influence of
Pineal Body on Melanoma of Hamsters, Nature,
213: 1038.
Das Gupta, T. K., and Terz, J., 1967b, Influence of
Pi nea I Gland· on the Growth and Spreeld uf Me I anoma
in the Hamster, Cancer Res., 27:1306.
Dilman, V. M., Anisimov, V. N., Ostroumova, M. N.,
Morosov, V. G., Khavinson, V. K., and Azarova,
M. A., 1979, Study of the Anti-Tumor Effect of
Polypeptide Pineal Extract, Oncolosy, 36:274.
EI-Domeiri, A. A. H., and Das Gupta, T. K., 1973, Rever-
sal by Melatonin of the Effect of Pinealectomy
on Tumor Growth, Cancer Res., 33:2830.
Geiger, P. J., and Bessman, S. P., 1972, Protein Deter-
mination by Lowry's Method in the Presence of
Sulfhydryl Reagents, Anal. Biochem., 49:467.
Hajdu, S. I., Porro, R. S., Lieberman, P. H., and Foote,
F. W., Jr., 1972, Degeneration of the Pineal
Gland of Patients with Cancer, Cancer, 29:706.
Hamilton, T., 1969, Influence of Environmental Light and
Melatonin Upon Mammary Tumour Induction, Brit.
J. Surs., 56:764.
Hoffman, R. A., and Reiter, R. J., 1965, Rapid Pineal-
ectomy in Hamsters and Other Smal I Rodents,
Anat. Rec., 153:19.
Huxley, M., and Tapp, E., 1972a, Effects of Biogenic
Amines on tbe Growth of Rat Tumors, Life Sci.,
11 (Part 11):19.
Huxley, M., and Tapp, E., 1972b, The Histochemical
Measurements of the Lipid Content of the Pineal
Gland in Rats Suffering from Mal ignancy, Brain
Res., 37:123.
J~nne, J., and Wi II iams-Ashman, H. G., 1971, On the Puri-

fication of Ornithine Decarboxylase from Rat
Ventral Prostate and Effects of Thiol Compounds
on the Enzyme, J. Bioi. Chem., 246:1725.
Karmali, R. A., Horrobin, D. F., and Ghayur, T., 1978,
Role of Pineal Gland in Aetiology and Treatment
of Breast Cancer, lancet, i i : 1001.
Lap i n, V., 1975, The Pi nea I and Neop I as i a, Lancet, i: 341.
Lapin, V., 1978, Effects of Reserpine on the Incidence
of 9,10-Dimethyl-l,2-benzanthracene-induced Tumors
in Pinealectomised and Thymectomised Rats,
Oncoloay, 35:132.
Lapin, V., an Ebel s, I., 1976, Effects of Some Low Mol-
ecular Weight Sheep Pineal Fractions and Melatonin
on Different Tumors in Rats and Mice, Oncol09Y,
33: 11 O.
Lapin, V., and Ebels, I., 1981, The Role of the Pineal
Gland in Neuroendocrine Control Mechanisms of
Neoplastic Growth, J. Neural Transm., 50:275.
Lapin, V., and Frowein, A., 1981, Effects of Growing
Tumours on Pineal Melatonin Levels In Male Rats,
Rodin, A. E., 1963, The Growth and Spread of Walker 256
Carcinoma in Pineal~ctomized Rats, Cancer Res.,
23:1545.
R~din, A. E., and Overal I, J., 1967, Statistical Re-
lationships of Weight of the Human Pineal to A~e
and Mal i gnancy, Cancer, 20: 1203.
Scalabrino G., and Fer-ioli, M. L, 1981, Polyamines In
Mammal ian Tumors, Part I, Adv. Cancer Res.,
35: 151.
Scalabrino, G., Ferioli, M. E., Nebuloni, R., and Fra-
schini, F., 1979, Effects of Pinealectomy on the
Circadian Rhythms of the Activities of Polyamine
Biosynthetic Decarboxylases and Tyrosine Amino-
transferase in Different Organs of the Rat,
. EndocrinoI09~' 104:377.
Scalabrino, G., pBs , H~, HBltt~, E., Hannonen, P.,
Kall io, A., and J~nne, J., 1978, Synthesis and
Accumulation of Polyamines in Rat Liver During
Chemical Carcinogenesis, Int. J. Cancer, 21:239.
Sneider, T. W., and Potter, V. R., 1969, Deoxycytidylate
Deaminase and Related Enzymes of Thymidine Tri-
phosphate Metabol ism in Hepatomas and Precan-
cerous Rat Liver, Adv. Enzyme Re9ul ., 7:375.
Tamarkin, L., Cohen, M., Roselle, D., Reichert, C.,
Lippman, M., and Chabner, B., 1981, Melatonin
Inhibition and Pinealectomy Enhancement of 7,12-
-Dimethylbenz(a)anthracene-induced Mammary
Tumors in the Rat, Cancer Res., 41:4432.
Tapp, E., and Blumfield, M., 1970, The Weight of the

Pineal Gland in Mal ignancy, Brit. J. Cancer,
24:67.
Tapp, E., 1980a, The Human Pineal Gland In Mal ignancy,
Tapp, E., 1980b, Pineal Gland in Rats Suffering from
Mal ignancy, J. Neural Transm., 48:131.
INTERACTIONS BETWEEN PINEAL AND NONREPRODUCTIVE ENDOCRINE
GLANDS
B. Mess
Department of Anatomy, Univ. Mad. School, Pecs
Hungar,y
INTRODUCTION
It has not been too long a time that the endocrine nature of
the pineal gland has been doubted. In the light of the research
work perfonned world-wi.de in the last few decades, it has be-
came evident and generally accepted that the pineal gland fonns
an integrate part of the endocrine system. However, the role of
the pineal does not fit simply into the general scheme of the
hypothalamo-pituitar,y-peripheral target organ axis.
The most widely investigated and most firmly sUbstantiated
endocrine function of the pineal gland is ~elated to the repro-
ductive system. Since, in the last few years, I have had several
opportunities to summarize the poss1ble function and significance
of the pineal-reproductive interaotion (Mess et al., 1978; 1979;
1980; 1981), and that Dr. Reiter spoke yesterday in detail on
this subj ect, I shall omit discussing this problem. Furthennore,
it is also my task to summarize the question of pineal-hypotha-
lamic interactions, and consequently the relationship between
the pineal and the hypothalamo-pi tui tar,y unit. Therefore, in
477
478 B. MESS
the present paper I shall restrict my subject to the problem

genera.11y tenned as "interglanduJ.ar interplays". i.e. to the
direct or indirect functional interrelationships between the
pineal and the peripheral endocrine glands.
Since pineal-endocrine organ interglanduJ.ar interplay is a
fairly complicated and ambiguous question. I do not wish to in-
crease the confusion b,y dealing with different phylogenetic
orders of animals. Therefore, I shall restrict my talk mainly
to mammal s. with an occasional reference to birds.
II. PINEAL-THYROID INTERRELATIONSHIPS
The investigation of the functional relationship between

the pineal and thyroid gland, and vice versa, has not been a
popuJ.ar subject of pineal research. However, a short monograph
(Peschke, 1980-81) and a sh~rt, comprehensive chapter (Vollrath
1981) have appeared in the last year dealing with this subject.
The first sentence of Vollrath's survey states " ••• pineal
usually exerts an inhibitory influence on thyroid function,
though some studies have pointed to stimulatory effects". May
I add that severa.1 reports (Yamada, 1961; Mess, 1969; Naber et
al., 1969; Rowe et al., 1970; Brammer et al., 1979; Mattila,
1981) have indicated no effect of the pineal gland on the dif-
ferent parameters of thyroid activity that were investigated.
A similar dubious position was stated b,y Reiter et al., (1975):
"Ambivalent resuJ.ts concerning the connections between the
pineal gland and the thyroid are characteristic".
Omitting chronological order and the older data, we shall
focus on the literature of recent pineal research and to the
data obtained with the more sophisticated and reliable newer
methods of thyroid investigation.
The possible explanations for the contradictions, consis-
PINEAL AND NONREPRODUCTIVE ENDOCRINE GLANDS 479
tently found in the pineal-thyroid literature, were summarized

by Peschke (1980) as follows:
1., Differences in the species of experimental anjmaJs,
and within the species, sex differences might also be
responsible.
2., Age differences of the employed animals
.3., Circadian and circannual rhythms, and divergent light-
ing schedules used by the different investigators
4., Duration of the experiments; different intervals
between experimental manipulation and observation of
the results
5., Further factors, difficult to standardize, such as:
ambient temperature, constituents of the diet, olfacto-
ry and acoustic stimuli, different kinds of stress
situations etc.
One can easily agree with the arguments of Peschke (1980).
To provide on1.y a few characteristic examples supporting each
argument, Singh and Turner (1972) found that the thyroid secre-
tion rate (TSH) responded with a maximal decrease after the
injection of a lower dose of melatonin (50 ~g/lOO g body weight)
in the female hamster, while a double dose of melatonin was
required to obtain the same degree of inhibition in the male
rat. According to these data, the TSH-thyroid system of the
hamster is more sensitive to melatonin than that of the rat.
At the same time, the sex difference also could be taken into
account in this experiment. Wurtman (1971) discussed in greater
detail the role of species differences in his summary of the
pineal colloquium of the Ciba Foundation in London. He pointed
out that some species of animals show greater responses to
light/dark effects Or to melatonin than others, e.g. the
hamster Showed much greater changes in gonad weight in response
to environmental lighting and to removal of the pineal than the
-I'>
(Xl
o
Table 1. Factors influencing pineal reactivity
Factors Exp. results References

Species difference Hamsters B.re more sensi ti ve to Singh and Turner, 1972
melatonin than rats
Hamsters are more sensitive to Wurtman, 1971., Hoffmann 1975
lip:ht chanp:es than rats
Sex difference Female animals are more sensi- Singh and Turner, 1972
tive to melatonin than males
Age or duration of expo Pineal regulation on TSH secretion Panda and Turner, 1968
is more marked in imma. ture animals
than in adults
Pineal exerts short-lived inhibition Relkin, 1972
on the thyroid
Pineal has relatively long-term Vriend et al., 1977
iMibition_on thyroid
Ambient temper. Pineal N-acetyl-transferase activity Ulrich et al., 1974
is highest et 22 0 C ambient temperature
Pineal reactivity is affected by cold Miline et al •• 1970
Acoustic stimuli Acustic stress results in involution Miline et al., 1969
of the I)'.inelil-J._in__the_l1:l._berna.ting b.a t
Olfactory stimuli Experimental anosmia inhibits compen- Reiter, 1969; 1972 OJ
sa toryhyj)e~tro]:)hyofdifferent organs
5:
m
CJl
CJl
albino rats (Hoffmann and Reiter, 1965; Hoffmann 1975).

Investigating changes in blood and pituitary TSH concent-
rations of melatonin-treated rats of different age~Panda and
Turner (1968) concluded that the regulatory effect of the
hypophyseal-pineal axis is more marked in immature animals than
in the adul ts.On the other hand, Relkin (1972) demonstrated that
pinealectomy performed on the 21st day of life of male rats and
which were exposed to constant light or constant darkness,
changes in thyroid activity (plasma and pituitary TSH level,
protein bound iodine (PBI) blood level) were characteristic
when investigated on the 25th day of life, but all these para-
meters were unchanged on the 28th day of life. These findings
might be due to age differences, but more probably to the dif-
ferencein the duration of the experimental period. The author
interpretes his results by stating: "the pineal gland appears
to be capable of exerting a significant short-lived inhibitory
influence over the pituitary-thyroid axis of prepuberal male
rats ••• " Vriend et al., (1977) found that blinding of young
male hamsters led to a depression of the plasma free thyroxine
index after an 8 week period, an effect that was reversed either
by pinealectomy or by removal of the ganglion cervicale superi-
us. However, no significant effects were observed 2 weeks after
treatment. These latter data, in contrast to Relkin's results,
seem to indicate that the pineal gland has a relatively chronic
inhibitory effect on thyroid function.
The transference of environmental lighting impulses towards
the endocrine system is one of' the most obviously proved func-
tions of the pineal gland. Since this organ is an important re-
gulator of different biorhythms (circadian, circannual) and
termed "a biological clock" by Axelrod and Wurtmann (1966), this
position will be discussed later in detail.
Changes in ambient temperature have been proposed to influ-
482 B. MESS
ence pineal activity. According to the investigations of Ulrich

et a1. (1974), intact suckling rats exposed to constant dark at
7, 22 and 34°C ambient temperature, produces a significantly
higher activity of pineal N-acety1 transferase in the group kept
at 22°C than in any other group. It was concluded that light
and temperature both regulate pineal function probably by dif-
ferent mechanisms. S1m11ar1y,M11ine et a1. (1970) showed that
pineal gland behaviour is sever1y affected by cold. Other fac-
tors, such as olfactory and acoustic stimuli,a1so were demon-
strated to influence pineal function. Vigorous acoustic stimuli,
applied twice daily each time for 1 hour, caused characteristic
involution of the pineal gland in the hibernating bat (Mi1ine
et al., 1969). Olfactory impulses also play an important role
in the regulation of pineal function. As it was shown by Reiter
(1972), experimental anosmia considerably influences the com-
pensatory hypertrophy of different organs (gonads, adrenals,
kidneys). This effect of olfactory bulbectomy was proved to be
mediated by the pineal gland, thus demonstrating clearcut rela-
tionship between pineal function and olfaction (Reiter, 1969).
According to my knowledge, no direct data have yet to prove the
existence of an olfactory-pineal-thyroid circuit. Based on the
analogy to the above mentioned olfactory-pineal-reproductive
interrelationship, this former possibility is a consideration.
The role of different stress situations and the pineal-adrenal
relationships will be the next point of this report.
As was briefly shown, all factors, mentioned by Peschke
(1981), or their different combinations might be responsible
for the modifications of the experimental results obtained con-
cerning the role of the pineal gland in the regulation of
thyroid function.
If one critically surveys the recent data reported in the
literature dealing with this subject (besides the differences
in the above mentioned factors), an even more important common

feature becomes evident. Positive results demonstrating that
the pineal can influence thyroid function have been obtained,
when basic thyroid function was not the object of investigation,
but rather was focused on finer modifications, under more sophis-
ticated experimental conditions. This character of pineal func-
tion -was for.mulated in respect to pineal-gonadal interplay in
the last paragraph of our monograph (Mess et al., 1918) as
follows: "This point stresses the ~odulator" role of the pineal
gland plays in the regulation of gonadotrophin secretion, and
the importance of employing an appropriate experimental model
to be able to demonstrate the true physiological nature of the
pineal." I am convinced that this ~odulator" character of
pineal function is true also in its influence exerted on the
TSH-thyroid axis, I would like to provide support for this as-
sumption by presenting selected data reported by outstanding
pinealologists in the last decade.
l31I uptake was significantly elevated after pinealecto~
plus bilateral enucleation, but not following simple pineal-
ectomy (Seibel and Schweisthal, 1973) indicating that the pineal
modifies the light-induced changes in thyroid function rather
than influencing the basic mechanism of iodide uptake.
The elegant experiment of Relkin (1918) furnished convinc-
ing evidence that the pineal mediates the changes in lighting
conditions that influences not only the reproductive system,
but also pituitary TSH secretion. It was shown (Table 2.) that
pinealectomy or constant illumination decreased pituitary TSH
and increased serum TSH and T4 levels. On the contrary, constant
darkness roughly decreased all three of these parameters. This
latter effect, however, could be counteracted by pinealecto~.
- Since animals of each experimental group equally responded
to exogenous TRH with TSH release, the conclusion was drawn
484 B. MESS
Table 2. Changes in pituitary and serum TSH levels in the con-

stant dark exposed, intact and pinealectomized rat
CRelkin, 1978)
Exp. Group Number of Pit. TSH Plasma TSH PBI

animals mU/mg mU/lOO ml ~g/100 ml
Diurnal Sh. 13 + + +
4,7-0,2
47,a.:.5,4 37-5,5
Diurnal Px. 12 + + +
15,6-3,7 54-6,2 7,1-0,3
Dark Sh. 12 +
17,5-'-3,0 1]!2,7 2,~0,1
+
Dark Px. 11 +
22,0-3,9 + +
7,6-0,2
51-6,5
that the inhibitory effect of the pineal is exerted at the

level of hypothalamic secretion of THH. - On surveying these
resul ts, all the values are moderately significant, and are
shifted around control values. The most pronounced effect of
pinealectomy was observed in the dark-exposed group. - The ex-
periments of Niles et ale Cl979) also suggest a role of the
pineal gland in the light-regulated diurnal secretion of TSH.
Fig.l. demonstrates that nearly constant darkness ClL:23D)
does not alter plasma levels of TSH (in contrast to Relkin's
data, 1978) compared to those of controls maintained under nor-
mal C12L:12D) lighting conditions. Under this short photoperiod,
pinealectomy increased the average level and shifted the peak
of TSH to 6 AM. It was concluded that the pineal may have a
role in entraining the TSH rhythm to environmental lighting.
In various instances, it is not only the complicated ex-
perimental schedule, but also the nature of the investigated
parameter, representing finer variations in thyroid activity,
tha t favour the concept of a "modulator" role for the pineal
gland. The thyroid hormone secretion rate (TSR) CSingh and
500
400
300
06.00 12.00 18.00 2400 06.00

Hour in cycle
Fig.l. The effects of pinealectomy or immunization against mela-

tonin and NAS on the diurnal plasma TSH rhythm of animals
exposed to short daily photoperiods.e--. =Control
lL:23D; 0----0 =pinealectomized lL:23D; A-.~ =NA3-innnuniz-
ed lL:23D. (Niles et al., 1979).
Turner,1972) and the free thyroxine index (Vriend et al., 1977)

both are slowly-reacting indica tors of thyroid activity. The
free thyroxin index shows a slight but significant decrease
after exposure to continuous darkness for an 8 week period.
Pinealectomy, or superior cervical ganglionectomy, reverse this
effect. TSR was also slightly depressed by daily melatonin
treatment continued for over a week. If one takes into account
that even these slow reacting parameters of thyroid function
486 B. MESS
show only moderate changes, and very often only under sophisti-
cated experimental conditions, the primary or "vi tal" role of
the pineal gland upon thyroid function can be refuted. The same
resul ts verify, however, the "modulatory" role of the pineal
gland with respect to its regulation of the TRH-TSH-thyroid
system.
Another widely discussed question in this field is the
mechanism of aotion of melatonin by whioh it influenoes thyroid
aotivity. This latter question will be mentioned here only very
briefly. Basohieri et ala (1963) were the first to describe the
inhibitory role of melatonin on thyroid function. They showed
that melatonin prevents thiouraoil-induced hyperplasia, and
oauses reduced thyroidal l31I_uptake. The oonolusion was drawn
that melatonin acts either on TSH seoretion or directly on the
thyroid gland. A controversial finding has been reported by
Thieblot et ala (1966) who showed that administration of mela-
tonin produced marked hyperaotivity of the thyroid as evidenoed
by histological changes.
This controversy could not be resolved t since subsequent
data demonstrated a "goitrogenic-like" effect of melatonin.
Panda and Turner (1968) observed an inorease in blood TSH levels
and a deorease in pi tui tary TSH content with concomitant hype:r--
plasia and hypertrophy of the thyroid gland equally in the
melatonin- and in the goitrogen(tapazole) treated rat. They
ooncluded that "melatonin is similar in its action to that of
goitrogen (antithyroid agent) and in some way inhibits thyroid
hormone synthesis". Similar findings and oonclusions were re-
ported later by De Fronzo and Roth (1972). A proportional in-
crease in thyroid DNA and RNA content with significantly in-
creased l31I_uptake and decreased PBI level were observed in
chronically (four weeks) melatonin-treated rat. These findings
led to the conclusion that melatonin has a true goitrogenic
effect, which occurs at a point beyond the thyroid trapping

mechanism. The results of Singh and Turner (1972), showing a
decreased TSR in melatonin-treated rats and hamsters, also
appear to support this assumption.
However, there are findings which contradict the proposed
goitrogenic effect of melatonin. Seibel and Schweisthal (1973)
found a significant decline in thyroidal l31I_uptake after
melatonin treatment. They stressed that "our results are in
essential agreement with Baschieri et ale (1963) ••• ". Inves-
tigating an other characteristic parameter, TSH blood levels
also were decreased in melatonin-treated rats compared to saline
treated controls. The authors concluded that melatonin might
act at the hypothalamic level instead of the proposed peripheral
level.
This evident controversy hardly can be explained simply by
the different species or ages of animals, or by the differences
in the experimental design. The possibility cannot be excluded
that melatonin might actually have a direct peripheral action
on the thyroid gland thus inhibiting the biochemical mechanisms
of thyroid honnone secretion. At the same time, melatonin might
act-analogous to that observed for the gonadotrophin system -
also at the level of the central nervous system, influencing
the function of the serotoninergic neurons of the brain stem
and inhibiting TRH release.
III. PINEAIr-ADRENAL INTERRELATIONSHIPS
In 1959 when Lerner described the structure of melatonin,

Farrel (1959) also proposed the existence of a pineal hormone,
adrenoglomerulotropin. No wonder that .much enthUsiasm was
generated by these findings towards research into pineal-
-adrenal interrelationships. Many investigators, including
488 B. MESS
ourselves (Fraschini et al., 1968), have shown that pinealec-

tomy provokes an increase in the activity of the adrenal gland.
Changes in several different parameters of adrenal function
were then reported following pineal gland interventions. Lommer
(1966) found that pineal extracts inhibit S -hydroxylation
within the adrenal cortex, thereby inhibiting production of
corticoids. Similarly, Trentini et ale (1965) described altered
enzymatic activities and lipid content in the adrenal cortex
follOwing pinealectomy. A decreased corticosterone production
also was reported following administration of pineal principles
(Gromova et al., 1967). These collective findings suggest a
peripheral action of pineal hormones exerted directly on adre-
nocortical tissue. - On the other hand, data indicating a
central effect of the pineal gland also appeared during this
period. An enhanced ACTH secretion was reported by Jouan (1963)
following ablation of the pineal gland. Motta et al. (1971)
found significantly decreased plasma corticosterone levels aft-
er intraventricular injection of different pineal indoles both
under resting conditions and in cold-exposed animals. In cont-
rast, an enhanced aldosterone secretion was reported by Giordano
and Balestreri (1963) under in vitro conditions in which adrenal
tissue was incubated with pineal extract. Kinson and Singer
(1967) stated that pinealectomy increases the basal secretion
rate of aldosterone, but does not influence the adrenal response
to dietary sodium deficiency. The most important data of this
period have been summarized in our previous review articles
(Mess, 1968; Mess et al., 1979).
Publications dealing with pineal-adrenal interrelationships
have become relatively scarce in recent years, indicating that
this subject is considered to have only a minor importance
among the many functions of the pineal gland.
Partly negative findings were reported in the last decade.
De Proposto and Hurley (1971) found no appreciable change in

the weight of the adrenal gland following intraventricular in-
jection of melatonin in rats. Similarly, no weight changes were
obtained in pinealectomized, blinded, or blinded and pineal-
ectomized hamsters. Exogenous melatonin also failed to influence
adrenal weight in this species (Seibel and Schweisthal 1973).
Even under more sophisticated experimental conditions, Vermes
and Te1egdy (1979) reported negative results. Pinealectomy or
superior cervical sympathetic ganglionectomy neither altered
the hypothalamic 5-hydroxytryptamine content, nor the diurnal
adrenal rhythm or the adrenal stress response.
Some discrepancies are evident even among the positive
results, obtained with more advanced techniques. Ogle and Kitay
(1977) stated that melatonin (but not aqueous pineal extract)
enhances ~ -reductase activity in the adrenal cortex. This
effect also was elicited in hypophysectomized animals. This
indicates that melatonin acts directly on the adrenocortical
cells augmenting steroid hormone production. In contrast, mela-
tonin was shown (De Fronzo and Roth, 1972) to inhibit, while
pinealectomy to stimulate, adrenal activity.
However, the main trend in research of pineal-adrenal re-
lations is the investigation of either the rhythmic adrenal
secretion influenced by the pineal gland, or of the finer de-
tails of the regulatory mechanism of the adrenal cortex.
Thurley et ale (1975) presented data which indicated that
the diurnal rhythm of cortisol production was abolished by
pinealectomy. Accordingly, Barrel and Lapwood (1978) found that
pinealectomy inhibits the morning rise in cortisol secretion
in the ~. They concluded that the pineal mediates not only
the long (seasonal), but also the short (daily) changes in
photoperiods affecting the neuroendocrine system. - Not only
is the rhythmic secretion of adrenocortical steroid influenced
490 B. MESS
by the pineal gland, but changes in the rhythm of the adreno-

medullary system also were reported. Kachi et ale (1979; 1980)
distinguished two types of synaptic vesicles in the chromaffin
cells using quantitative electronmicroscopy. The number of the
small light vesicles showed two peaks in normal animals, in the
middle of both the light- and dark phase of the day. Pinealectomy
abol:ishes the peak in the dark phase. The so-called big granular
vesicles showed the same two peaks, however, to a considerably
less pronounced degree. Pinealectomy diminished the smaller
peak at the begining of the dark phase. These investigators
concluded that both the adrenalin and noradrenalin secretory
rhythm is influenced by the pineal gland, however, in a somewhat
different way.
Regarding the influence of the pineal gland on the stress-
-inducing activation of the adrenocortical system, interesting
data were reported by Develerski (1979) and tiline (1980). On
the basis of histological and histochemical investigations,
they stated that reactive changes in the adrenal cortex under
stress conditions are more pronounced in pinealectomized rats
than in unoperated animals. It was concluded that pinealecto-
mized animals are more sensitive to stress caused by hyper-
thermia. - Even more sophisticated experimental models were
applied to approach the finer details of the mechanism of the
pineal-adrenal interplay. Vaughan et ale (1980) reported that
blinding and anosmia, termed "sensory deprivation", caused a
moderate increase in ACTa secretion, which was reversed by
pinealectomy. The unilateral adrenalectomy-induced increase in
ACTa blood levels, however, was not influenced by pinealectomy
alone, whereas blinding and anosmia+pinealectomy elicited more
than a two-fold increase, compared either to sham operated, to
simply pinealectomized or to simply blinded anosmic rats
(Fig.2. ).
SPBBSPBB
hx-\AhxAA
P P
x X
N=II 10 12 12111102110
non-alb ad.~
Fig.2. Influence of sham-pinealectomy (SH), pinealectomy (Px),

blinding-anosmia (BA) combined BAh, and bilateral
adrenalectomy (adx) on plasma ACTH (mean!SE). The number
of rats per group (n) is indicated below the group
symbols near the bottom of the figure. The inset in the
ACTH graph, labelled CORT, represents corticosterone
concentrations in ~g/dl. Significance levels are indi-
cated above the bars: a, p < 0,001 vs Sh; b, P < 0,01 VB
Sh; c, P < 0,05 VB Sh; d, P < 0,001 vs BA; f, p < 0,05 vs

BA, comparisons being between groups with like adrenal
status (non-adx or adx) (Vaughan et al., 1980).
Compensatory adrenal hypertrophy following unilateral

adrenalectomy was not inhibited by pinealectomy. Removal of the
pineal gland only also failed to influence the dopamine /3-hyd-
roxylase (DBH) activity of the adrenal cortex. However, pineal-
ectomy combined with unilateral adrenalectomy significantly in-
hibited the unilateral adrenalectomy-induced rise in this
enzyme activity of the remaining and hypertrophied adrenal
gland (Banjeri and Qlaay, 1981).
492 B. MESS
MELATONIN ON DAY OF BIRTH (MALES)

12 15 10
9
5
4
~
'"
E
...J
~
w
a:
~ 4
f-
u.
W
...J
C 250,..9 C SHAM UA SHAM UA +

MEL UA UA+ MEL
MEL
Fig.3. Weight of the left adrenal (mg/100 g body weight) remov-

ed at the time of unilateral adrenalectomy is statistical-
ly reduced in male mice receiving a single subcutaneous
injection of melatonin at the time of birth (stippled
bar on left). The weight of the right adrenal (mg/IOO g
body weight), removed 5 days later, is statistically
reduced both in sham and unilaterally adrenalectomized
male mice receiving melatonin on the day of birth
(stippled bars on right). Compensatory adrenal hyper-
trophy did not occur in these melatonin-treated males.
Vertical lines indicate standard errors. Parentheses
signify the number of animals in each group (Vaughan
and Vaughan, 1974).
The pineal hormones also appear to influence the differ-

entiating adrenal and gonadal/central neural control mechanisms
during the neonatal critical period. A single subcutaneous
injection of melatonin into newborn rats or mice prevented the

compensatory adrenal- and ovarian hypertrophy following unilat-
eral extirpation of the corresponding endocrine organ during
the adult stages (Vaughan and Vaughan, 1974) (Fig.J.).
In summary, similar conclUsions can be drawn concerning
the role and mechanism of pineal influences exerted upon the
adrenal gland, as have been proposed with respect to the pineal-
-thyroid interplay. The pineal might act as a "modulator" rath-
er than as a basic regulator in influencing the function and
responses of the adrenal gland.
IV. INFLUENCE OF THE PINEAL GLAND ON OTHER TROPHIC HORMONES OR

ENDOCRINE ORGANS
Sporadic and less finnly-proved data have appeared in the

Ii tera ture suggesting a regula tory role of the pineal gland on
other endocrine functions. These reports will be mentioned here
only very briefly.
Prolactin (PRL) belongs, in a wider sense, to the gona-
dotrophins but will be mentioned here. Kuew-Hsiung Lu and
Meites (1973) reported that brain serotonin, its precursors
(tryptophan and 5-hydroxytryptamine) and melatonin may have a
role in stimulating PRL release. Pinealectomy was found to abolish
the early morning rise of plasma PRL levels in the male rat;
superior cervical ganglionectomy had a similar effect (Ronne-
kleiv and MoCann, 1973). Shino et ale (1974) have shown that
pinealectomy prevents the blinding and anosmia-induced rise in
serum PRL levels. They ooncluded that the pineal gland inhibits
the secretion of prolactin inhibitory factor (PIF), thereby
promoting PRL release. In contrast, Blask and Reiter (1978)
proposed that the pineal gland exerts its regulatory influence
on PRL release via the proposed hypothalamic prolactin releasing
494 B. MESS
factor (PRF). In an in vitro system, medial-basal hypothalamic

extracts of pinealectomized or ganglionectomized and blinded
anosmic animals provoked a significantly higher degree of PRL
release than that of the solely blinded anosmic rats.
Acidic extracts of sheep pineal (protein fractions) inhib-
i ted the release of PRL in pi tui tary monolayer culture (Demou-
lin et al., If577J. This finding demonstrates that protein sub-
stances of the pineal gland might play an inhibitory role on
PRL release. On the other hand, these data suggest a direct
action of pineal ho~ones on adenohypophyseal cells. According
to the results of Pavel et al. (1f575J, the effect of pinealectomy
exerted on PRL release can be counteracted by minute amounts of
arginine vasotocin (AVT). These data also suggest that hor-
mones other than the indoleamines might play a role in the
mechanism whereby pineal gland influences PRL secretion. Vaughan
et al. (1976) proposed AVT to be a "potential pineal PRL releas-
ing factor". In agreement with these latter findings, but in
contrast to the data of Kuew-Hsiung Lu and Mei tes (1f573J, it
was shown that melatonin and its derivatives were without effect
on PRL release from bovine pituitary tissue culture. Serotonin
strongly inhibited, while AVT stimulated, PRL output (Padmanab-
han et al., 1979).
It would be diffioult to formulate a uniform picture re-
garding the mechanism of pineal-PRL interplay. The only conclu-
sion that can be drawn with reasonable certaini ty is that the
pineal gland, in contrast to the other two gonadotrophins (FSH
and LH), stimulates PRL secretion.
Even less data are available with respect to the role of
the pineal gland in the regulation of Growth Ho~one (GH) se-
cretion. Melatonin was shown to blook, while serotonin to stim-
ulate, GH release in the rat (Collu et al., If572; Smythe and
Lazarus, If573 aJ. Furthermore, the stimulating effect of seroto-
n-14
100
E
~
c
.
:z:
"..• 50
a::
E
~
•
1/1
n-IO n-13
10 n-6
CONTROLS 5-HTP 5-HTP 5-HTP

+ +
MEL Cyp
Fig.4. Changes in GH blood levels of the rat following simple

serotonin or simultaneous serotonin and melatonin treat-
ment (Smythe and Lazarus, 1973 b).
nin could be prevented by simultaneous melatonin treatment

(Smythe and Lazarus, 1973 b) (Fig •••• ).
In man, however, melatonin treatment had an opposite effect in
which serum GH levels were elevated (Smythe and Lazarus, 1974).
The effeot of pinealeotomy on GH seoretion was investigat-
ed mainly in experiments that combined pinealectomy with envi-
ronmental light-changes, food restriotion or sensory deprivati-
on. Osman et al. (1972) desoribed subnormal body growth under
conditions of constant darkness, but only in animals with an
intact pineal gland. Sinoe the delay in vaginal opening did
496 B. MESS
not show parel1e1ism with the retardation of body growth under

the different experimental circumstances, these investigators
concluded that the anti-gonadotrophic and anti-growth propel'-
ties of the pineal gland are separate physiological entities.
In the same year, Relkin (1972) reported that constant darkness
suppresses GH blood levels, but that effect of light deprivation
was counteracted by pinea1ectomy. It was concluded that the
darkness-induced increase in pineal activity inhibits the secre-
tion of GH releasing factor resulting in a decreased release of
pituitary GR. Similar results were obtained by Sorrentino et al.
(1971; 1972); blinding and anosmia provoked marked decrease in
~ody growth and GR levels. These phenomena only occur in the
'presence of the pineal gland (Fig.5.). These findings were later
corroborated by Shino et ale (1974).
320 • I NORMAL
• m BlIN[H'INX
c Jr IIiND-ANOI PiNX
280 .. ][ BliND
" Ilr IIiND-ANOI
240
"'
...
~ 200
't:
~ 160
~
..
~ 120
80
40
33 41 49 57 65
AGE IN DAYS
Fig.5. Growth rates of rats as evaluated by body weights.

Asterisks indicate groups different from respective
pinealectomized group (pc(O,Ol) (Sorrentino et al.,
1971).
The role of the pineal gland in the maintenance of the

diurnal rhythm of GH secretion also was studied. Pinealectomy
significantly reduced GH release of rats during the light period,
but did not significantly reduce further GH release during the
night (Ronnekleiv and McCann, 1978). In contrast, Niles et ale
(1979) found no difference between the plasma Glf levels of rats
kept in a nonnal (12 L:12 D) or a short photoperiod (1 L:23 D)
environment. Neither pinealectomy, nor immunization against
melatonin, influenced the rhythmic pattern of GH release. A
discussion of the possible explanations for this discrepancy
is beyond the scope of this presentation.
Finally, an interesting but somewhat vague concept should
be mentioned. On the basis of light- and electronmicroscopic
investigations, Csaba and Barath (1974) demonstrated that the
parafollicular cells of the thyroid gland increase both in their
number and granulosi ty following pinealectomy. Pinealectomy
also was shown to decrease b100 d sugar levels under resting
conditions. However, the alloxan-induced increase of the blood
sugar level was significantly more dramatic in the pinealectomiz-
ed than in the unoperated animals (Csaba and Barath, 1971).
Neonatal pinealectomy provoked a wasting disease and tetany in
the majority of the chickens suggesting that the parathyroid
gland also might be regulated by the pineal (Csaba et al.,
1972); furthermore, they suggested the existence of a negative
feed back mechanism. Adrenaline, noradrenaline and insulin
(hormones, which generally are not regulated by pituitary
trophic hormones) strongly increased the serotonin fluorescence
in pineal tissue culture. It was presumed that the inhibitory
effect of the three administered hormones on the conversion or
elimination of serotonin could playa role in a negative feed
back mechanism (Csaba and Bernad, 1972).
On the basis of these and of a series of other investiga-
498 B. MESS
tions, it was proposed that the pineal gland might regulate

those endocrine organs which are of ecto- and endodermal origin
Cthyroid, parathyroid, adrenomedulla, Langerhans islets), and
those of mesodermal origin Cadrenocortical tissue, gonads) that
are under pituitary influence CCsaba and Bernad, 1972; Csaba
and Barath 1971).
It sufficies to state that the most extensively investi-
gated, confirmed and generally accepted endocrine effect of the
pineal gland is its regulatory in:f'luence on the reproductive
system. Since the gonads, and especially their endocrine targets,
are derivatives of the coeloma Cmesoderma), the conoept of
Csaba and associates is without foundation.
V. PINEAL REACTIONS TO CHANGES IN THE ACTIVITY OF THE ENDOCRINE

ORGANS
The main task of this talk was to demonstrate the regula-

tory influence of the pineal gland on the fUnotion of the dif-
ferent endocrine systems. The reverse aspect, i.e. the survey
of the morphological and fUnctional changes in the pineal gland
observed following increased or decreased hormone secretion of
one or the other endocrine organs, will be outlined here only
very superficially.
Hypophysectomy causes a marked decrease in pineal volume
and an atrophy of the pinealocytes CIto and Matsushima, 1968).
All ultrastructural signs of inactivation in the pinealocytes
also were shown following hypophysectomy CSatodate et al.,
1970; Karasek, 1971). HIOMT activity also decreased in the
pineal of hypophysectomized animals CUrry et ale 1976).
Adrenalectomy provoked an increase in the number of
pinealocytic prooesses with a concomitant increase in HIOMT
activity (Deussen Schmitter et al., 1976). On the other hand,
increased pineal activity was registered following thyroxine

administration. In in vitro studies, Nir and Hirschmann (1978)
found an increase in the melatonin and N-acetyl serotonin con-
tent of pineal tissue after adding thyroxin (but not TSH) to
the organ culture. They proposed the existence of a direct
positive feedback between the thyroid and the pineal gland.
It would exceed the possibilities of the present paper to
enter into a discussion of the fascinating question of how
pineal gland responded to its own secretory products. Thus,
melatonin might enhance the release of AVT (Pavel, 1973), and
conversely, AVT might decrease melatonin secretion (Sratin et
al., 1978). Benson (1977) proposed in bis comprehensive paper
that melatonin might exert its antigonadotropbic activity by
influencing the synthesis and/or the release of the as yet
unknown "pineal antigonadotrophins (PAG)" of purported peptidic
character.
SUMMARY
The survey of numerous, and sometimes contradictory data,

have led to the conclusion that the pineal gland modulates
rather than basically influences the main neuroendocrine cir-
cuits regulating thyroid and adrenocortical activities. Mainly,
changes in external environmental conditions (photiC, osmic or
thermal stimuli) are mediated by the pineal gland to the re-
leasing honnoneC s )..pi tu1 tary trophin( s )..target gland( s) pri-
mary regulatory system.
Pineal influences exerted upon the secretion a PRL and GH
also are briefly surveyed and the relationship between the
pineal and endocrine glands of ecto- and endodennal origin is
briefly discussed. Finally, data dealing with the effects of
different hormones upon the pineal gland are summarized.
500 B. MESS
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502 B. MESS
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506 B. MESS
Rowe J.W., Richert J.R., Klein D.C., Reichlin S., 1970,

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THE PINEAL GLAND AND SEXUAL FUNCTION IN MAN
Merrily Poth, Sadayoshi Higa, and Sanford Markey
Department of Pediatrics, USUHS, and Laboratory Clinical

Clinical Science, NIMH
Bethesda, MD 20814
INTRODUCTION
The pineal gland has potent effects on the pituitary - gonadal

system in many different species (1). In some situations it seems
clear that these effects are mediated by melatonin secreted by the
pineal gland (2).
Humans share several aspects of this system, that is, they

have a pineal gland which episodically secretes melatonin in a 24
hour "circadian rhythm," and they have a pituitary-gonadal system,
immature at birth, which after sexual motivation exhibits clear
rhythmicity in the female although not in the male. However, at
every point of potential action and interaction of the neuroendo-
crine and endocrine systems there is question and even active
controversy as to whether or not the pineal gland and specifically
melatonin has any effect on human reproductive function.
The major problem in dissecting out the function and relevance

of the pineal in man has been the relative inacessibility of the
gland and its products in situ. In this chapter we will not con-
sider the question of pineal factors or hormones other than melato-
nin but will concentrate on melatonin as the active pineal hormone.
We will extrapolate to man from the biochemistry and pharmacology
of rat and hamster pineal glands which have been well studied. We
will assume that in man a rhythm-entraining stimulus communicates
with the pineal by a pathway including the superior cervical gan-
glion and involving S-adrenergic neurotransmission, that specific
circadian enzyme change occur and that these enzyme changes are
reflected in a circadian rhythm of melatonin production.
509
510 M. POTH ET AL.
This chapter will restate some of the critical questions re-

garding the function of the pineal in humans, review the evidence,
circumstantial and direct from the literature and from our own
data, and attempt to formulate a kind of progress, (or lack of
progress) report of the area. It will be obvious that this re-
search is in its infancy and that much remains to be done.
METHODOLOGY
Most studies of melatonin in humans have involved the assay

of melatonin by radioimmunoassay or by gas chromatography and
mass spectrography of serum samples. Ideally this entails nearly
constant or integrated blood sampling techniques and a sensitive
and highly specific method for melatonin assay. The radioimmuno-
assays used appear to be sensitive enough for the purpose in that
day/night differences are seen clearly using these assays. How-
ever, some controversy exists regarding the specificity of the
substances measured (3). In addition, obvious problems exist in
measuring frequent (30-90 min) serum melatonin samples in a humans,
and particularly children. This mitigates against long-term
sequential studies.
Melatonin in blood is rapidly metabolized to 6-hydroxymela-

tonin and excreted in the urine, mostly as the glucuronide or sul-
fate conjugates (4). We have developed a sensitive, very specific
and highly reproducible assay for these compounds in urine (5) and
we have used this assay to study human pineal function.
Twenty four hour urines were collected from subjects under

normal conditions of work, acitivity, diet, and sleep. The urine
was refrigerated (4°C) during the collection period and measured
aliquots were frozen at - 20°C until analyzed.
The 6-hydroxymelatonin content of the urine was determined

by gas chromatography negative chemical ionization mass spectrom-
etry. Briefly, the assay utilizes tetradeuterated 6-hydroxymela-
tonin sulfate as an internal standard added to a 3 ml urine ali-
quot, followed by enzymatic (glusulase) hydrolysis, extraction of
free 6-hydroxymelatonin into dichloromethane, reaction to form a
stable t-butyldimethylsilylpentafluoropropionyl derivative, column
chromatography on silica gel, and quantitation by selected ion
recording to two specific and intense fragment ions (endogenous
m/z 470 and 450; internal standard m/z 473,453).
The use of this assay allows us to monitor the daily output

of melatonin from the pineal gland in both children and adults
during normal life activities and to correlate their pineal output
with other endocrine events of their life.
THE PINEAL GLAND AND SEXUAL FUNCTION IN MAN 511
We make some important underlying assumptions in interpreting

our data. Some of these have been proven and some have been taken
for granted and await direct proof in the future.
The assumption is made that the 6-hydroxymelatonin measured

in human urine comes from the pineal gland. Pinealectomized humans
are not available for study. However, in the Rhesus monkey we
measured urinary 6-hydroxymelatonin excretion before and after
pinealectomy. This data shows clearly that in the absence of
pineal gland )96% of the urinary 6-hydroxymelatonin rapidly
disappears (6).
Next, it would be optimal if the urinary excretion of 6-hy-

droxymelatonin reflected the rhythmic secretion of pineal melatonin
allowing both quantitative and temporal measurement of pineal
activity. This is shown to be the case in studies of normal adult
males where 70-95% of total 24 hour urinary 6-hydroxymelatonin is
excreted in the period of time between midnight an 8 am (7). In
Rhesus monkey, exposure to constant light abolished the urinary
6-hydroxymelatonin rhythm (6). Dr. Lewey discusses the effects
of light on pineal rhythms in man much more fully in his chapter.
Patients with defective autonomic nervous systems (patients

with Shy-Drager Syndrome or idiopathic orthostatic hypotension)
did not exhibit normal circadian patterns of 6-hydroxymelatonin
excretion (8). This implies that an intact sympathetic nervous
system is necessaary for normal pineal rhythmicity (7).
The last major assumption upon which clear interpretation of

this data depends is that hepatic metabolism and urinary excretion
of melatonin and its 6-hydroxymetabolites does not vary signifi-
cantly with the other parameters under study. Studies have not
been performed to confirm this. We assume that these factors
remain relatively constant and do not lead to significant changes
in urinary 6-hydroxymelatonin. Because of these unknown factors
which might be expected to lead to small changes in excretions,
we do not consider changes of less than 30% to be of any signifi-
cance in analysis of our data.
SPECIFIC QUESTIONS REGARDING PINEAL FUNCTION IN HUMANS
Development of melatonin rhythms in children:
It is known that infants synchronize to external stimuli and

exhibit circadian rhythms of temperature and cortisol excretion
by two weeks of age (9). However, it is not known at what age the
pineal has rhythmic function and what effects this pineal rhythm
has on other physiologic parameters in children.
512 M. POTH ET AL.
We studied 24 hour urinary total 6-hydroxymelatonin excretion

in 101 normal children between the ages of 3 and 17 years. We
collected urines in 3 aliquots; the first (Aliquot r) from bedtime
through the first morning void, the second (Aliquot II) from then
through 3 pm and the last (Aliquot III) from 3 pm to bedtime. In
most of the children two consecutive 24 hour timed excretions
periods were measured.
The results are seen in Figures I & II.

The total amount of 6-hydroxymelatonin excreted was not dif-
ferent for children as a group when compared to adults. All normal
children studied had clear circadian excretion of 6-hydroxymela-
tonin, with 75-90% of that excretion in period I and <10% of
excretion during period III. Again, children did not differ from
adults in this rhythm.
40
35
> 30
oCt
c
.....
Cl
:l.
z 25
Z
0
S
w
20
~
>
x
0 15
a:
C
>
J:
c&,
10
3 4 5 6 7 8 9 10 11 12 13 14 15 16 ADULTS
AGE
Fig. I Total amount of 6-hydroxymelatonin excreted in urine

in 24 hours by normal girls and women.
40
35
:ll •
>
~
c......
CI
::t.
25
z
Z •
0
5
w
20
•
~
> • •
X
0
II:
C
15 . . ..
> I
X • •
··•-
rD 10
• .•
5
3 4 5 6 7 8 9 10 11 12 13 14 15 16 ADULTS
AGE
Fig. II Total amount of 6-hydroxymelatonin excreted in urine

in 24 hours by normal boys and men.
QUESTION OF INTERACTION OF MELATONIN WITH PUBERTY
There is considerable data both positive and negative in the

literature regarding the possibility of pineal puberty interaction
in children. Tumors in the area of the pineal gland have been
associated with both acceleration and delay of puberty in children
(10). This has been explained by hypothesizing that some of these
tumors secrete melatonin and hence can suppress gonadal maturation
and result in delayed puberty. Other tumors or lesions results in
destruction of the normally inhibitory pineal gland and by re-
leasing this suppression result in precocious sexual development.
However, no data is available to correlate changes in melatonin se-
cretion with either early or late puberty and pineal lesions. In
a recent study Silman assayed serum melatonin in late morning or
514 M. POTH ET Al.
early afternoon in normal school children (11). He found a marked

decline in serum melatonin in boys in Tanner II stage of puberty
and hypothesized that this decrease in the inhibiting substance,
melatonin allowed puberty to progreSs. Another study by Tamarkin
et al., (12) used integrated 24 hour blood sampling techniques and
assayed serum melatonin by RIA and failed to confirm that pre pu-
bescent boys had higher se~m melatonin levels.
We studied this question in normal children using two consec-

utive 24 hour urines assayed for 6-hydroxymelatonin. Each child
had a complete history and physical examination, including pubertal
staging. This was done for boys using Tanner criteria for public
hair and estimation of testicular volume with Prader beads as
reference. Girls were staged according to Tanner criteria for
breast development and for pubic hair (13).
BOYS GIRLS
30
>
II
......
"1:1
aI
::s.
i
~
z
0
II ....
~w 20
:i! ..· :
~
i• ·· 1
·I
f
0
i I
a:
Q
> ·• II
..:
::E:
·•
.;, 10 ~
II III IV V II III IV V
TANNER STAGE
Fig. III 6-hydroxymelatonin excretion in normal children during

puberty. Boys were staged by Tanner criteria for pubic
hair and girls for breast development.
No change was seen in amount of 6-hydroxymelatonin excreted

at any stage in pubertal development in the normal boys (Data is
shown in Fig. III).
Surprisingly, girls who were identified as Tanner Stage II

for breast development were higher when compared with boys at any
stage or girls at any other stage of development. The girls who
were at Tanner Stage II of puberty by breast configuration were at
various stages of puberty by pubic hair determination, that is
they had Tanner Stage I, II or III pubic hair. Pubic hair is a
manifestation of adrenarche in girls and is not mediated by pitu-
itary gonadotropins.
INTERACTION OF MENSTRUAL CYCLE AND PINEAL GLAND
There are several reports of measurement of pineal function

during the normal menstrual cycle in women. Some of these using
plasma melatonin measured by RIA have reported changes coincidental
with the cycle (14).
20 400
w
z
I
0 m
Ul
~E
Ul--
w OJ
10 200
r1J~
co:D
--0
30
(!)c
-j;
0 r
II:
0-
0 0
Ul 20
II:
I
~OJ
"-
~
z
I
0
f-
::s lO
w
2
0
x>-
II:
0
>-
I
cD 0
10 30 50 70
DAYS
Fig. IV & V 6-hydroxymelatonin excretion during menstrual cycle

in normal women, urinary 6-hydroxymelatonin excretion
was measured in 24 hour urine for 65 consecutive days.
Serum estradiol and progesterone were assayed three
times per week.
516 M. POTH ET AL.
20 400
w
z m
0 300
r
(fl
ffi'EI
1- ___
(flO) 10 200
c.o ~
U :IJ
---:t>
We
(!)
~~
0
0
a:
100 r
a..
0 0
25
(fl
a:
J:
20
~
---:s..
0)
z
Z 15
I
0
I-
::s
W
::?! 10
>-
><
0
a:
0
>-
J:
5
cD
0
10 30 50 70
DAYS
Fig. IV & V (continued)
We are studying women during normal menstrual cycles. Each

women is asked to collect 24 hour urines on 65 consecutive days.
Results of 2 women followed through 2 consecutive normal cycles are
shown in Figs. IV & V. Each had normal ovulatory cycles as mea-
sured by morning temperatures and by serum estrogen and proges-
terone levels measured three times per week. Completeness of 24
hour collection was verified by measurement of urinary creatinine.
The women showed marked day to day variation in the amount of
6-hydroxymelatonin excreted. A specific relationship between
the components and hormonal changes of the menstrual cycle can not
be ascertained from the data, but a 3-4 day rhythm appears to
exist in each of these women.
This marked variability in daily 6-hydroxymelatonin excretion

in cycling women was in marked contrast to the relatively constant
daily excretion rates seen in the prepubertal and early pubertal
girls. These girls were studied on 2 consecutive 24 hours - every
2 months in a 14 month time period, data is shown in Fig. VI,
note that variability is only in the order of 10-20%.
As a fourth "control" for the normally cycling women we are

studying young adult men. Each collects IS daily 24 hour urine
samples which are assayed for 6-hydroxymelatonin. It is clear
that there is less variability in 6-hydroxymelatonin in these men
than seen in the women (data not shown).
In conclusion we are using a sensitive, specific and highly
reproducible gas - chromatographic - mass spectrometric assay for
the measurement of the major metabolites of melatonin in human
urine. This method allows the non-invasive study of pineal func-
en ,a.. ..
a:
I " "
40
_--..a' tf1" .... ,-0
....... . . . . -<:>---- --- ...0----

(i!j
---::1.
Cl
Z
..c>-------O"..
--
Z 30 ,_,P
-- .
.t:? ______
0 .,.,
I-
.........,.' ,0
~ ........ __ A........ ()" ~:;o
w ...... -- _____ -.::. $ --
____ ().. . . . . -0- ---"
-.... -- .. ....-... '--g
~ ~< ~
~ 20 -~-.--
>-
X
:::--~!~~----
--- =~- . . --~-~-~--~
~
--~. -:~~::-
--- ,~
0
a:
0
-___ 0--:_---:.=: ---. . .
-~-
.:~=s::.:.:.~~--------:.B
. . . . . . _-0-: _-----0.--_ ----0-___
........
>- 10 0- --'>-----0
I
ch
0
0 2 4 6 8 10 12
MONTHS
Fig. VI Longitudinal study of 6-hydroxymelatonin excretion in

preovulatory girls. Each point is mean of excretion in
two consecutive 24 hour urines. Each line is individual
girl.
518 M. POTH ET Al.
tion in humans. Quantitation of the absolute amount of 6-hydroxy-

melatonin excreted as well as temporal relationships in the time
of excretion are able to be determine on both adults and children
during their normal daily activities.
Using this assay we find that children at least by age three,

have normal circadian rhythms of pineal function. We find no
changes in pineal secretion of melatonin in boys during sexual
maturation. Girls in breast Tanner Stage II of puberty excrete
greater amounts of 6-hydroxymelatonin during the normal night time
peak time with no change in the normally low day time excretion
rate. This increase is seen when compared to girls at any other
stage of puberty and with boys of any age.
Women monitored during normal ovulatory cycles show marked

day to day variation in the amount of 6-hydroxymelatonin excreted
compared to preovulatory girls and to males. It is not clear
what specific relationships hold between pineal, pituitary and
various function in these women. Greater numbers of women are
being studied and results will be subjected to more sophisticated
statistical treatment to try to ascertain the specific relation-
ships between the hormonal events of the menstrual cycle and
pineal activity.
REFERENCES
1. R.J. Reiter, The pineal and its hormones in the control of

reproduction in mammals, Endocr Rev 1:109-131 (1980).
2. L. Tamarkin, C.W. Hollister, N.G. Lefebvre, and B.D. Goldman,
Melatonin induction of gonadal quiescence in pinealectomized
Syrian hamsters, Science 198:935-936 (1977).
3. S.P. Markey, Quantitative mass spectrometry, Biomedical Mass
Spectrometry 8:426-430 (1981).
4. I.J. Kopin, C.M.B. Pare, J. Axelrod, and H. Weissbach, The
fate of melatonin in animals, J BioI Chem 236:3072-3075 (1961).
5. M. Tetsuo, S.P. Markey, R.W. Colburn, and I.J. Kopin, Quantita-
tive analysis of 6-hydroxymelatonin in human urine by gas
chromatography-negative chemical ionization mass spectrometry,
Analyt Biochem 109:208-215 (1981).
6. M. Tetsuo, M.J. Perlow, M. Mishkin, and S.P. Markey, Light
exposure reduces and pinealectomy virtually stop urinary
excretion of 6-hydroxymelatonin by Rhesus monkey, Endocrinology
110:997-1001 (1982).
7. M. Tetsuo, S.P. Markey, R.W. Colburn, and I.J. Kopin, Measure-
ment of 6-hydroxymelatonin in human urine and its diurnal
variations, Life Sciences 27:105-109 (1980).
8. M. Tetsuo, R.J. Polinsky, S.P. Markey, and I.J. Kopin, Urinary
6-hydroxymelatonin excretion in patients with orthostatic
hypotension, J Clin Endocr Metab 53:607-610 (1981).
9. R.C. Franks, Diurnal variation of plasma l7-hydroxycortico-

steroids in children, J Clin Endocr Metab 27:75-78 (1967).
10. J.I. Kitay, Pineal lesions and precocious puberty: a review,
J Clin Endocr Metab 14:622-625 (1954).
°11. R.E. Silman, R.M. Leone, R.M. Hooper, and M.A. Preece, Mela-
tonin, the pineal gland and human puberty, Nature, Lond
282:301-303 (1979).
12. J.R.L. Ehrenkranz, L. Tamarkin, F. Comite, R.E. Johnsonbaugh,
D.E. Bybee, D.L. Lorianx, and G.B. Cutler, Jr., Daily rhythm
of plasma melatonin in normal and precocious puberty. J Clin
Endocr Metab 55:307-310 (1982).
13. J.M. Tanner, "Foetus into Man," Harvard University Press,
Cambridge (1978).
14. L. Wetterburg, J. Arendt, L. Pamnier, P.C. Sizenenko, w.
VanDonselaar and T. Heyden, Human serum melatonin changes
during the menstrual cycle, J Clin Endocr Metab 42:752-764
(1976).
MELATONIN SECRETION -- A BIOLOGICAL MARKER
FOR HUMAN PINEAL ADRENERGIC FUNCTION
Al fred J. Lewy
Departments of Psychiatry, Pharmacology and Ophthalmology

Oregon Health Sciences University
Portland, Oregon, U.S.A.
INTRODUCTION
Spanning the las·t 20 years, hundreds of anatomical and pharmaco-

logical studies have indicated that melatonin secretion by the mam-
malian pineal gland is an ideal "biological marker" for adrenergic
function, the endogenous circadian pacemaker, and the effects of
light. Nighttime beta-adrenergic stimulation of melatonin synthesis
provides the theoretical basis for using the pineal as a model of
adrenergic function. The biochemical steps resulting in melatonin
synthesis appear to be intricately coordinated.
In recent years, several controversial areas have been clari-

fied; many of the remaining inconsistencies are in the process of
being resolved. One such area, for example, had been the apparent
inverse day/night activity rhythm of pineal cyclic AMP; however, it
is now known that pineal cyclic AMP levels are greatest during the
night (Mikuni et al., 1981).
Another controversial area concerns extrapineal melatonin.

There are two issues here. One is whether or not melatonin is syn-
thesized in other organs than the pineal. The other is whether or
not plasma melatonin is derived from such sources, if they exist.
We have addressed the second issue, using the highly accurate and
sensitive gas chromatographic-negative chemical ionization mass
spectrometric (neg CI GCMS) technique for measuring plasma melatonin.
GCMS ASSAYS
There seems to be a growing consensus that the neg CI GCMS assay
521
522 A. J. LEWY
is the most accurate of the human plasma melatonin assays (Arendt,

1981; Rollag, 1981). However, GCMS assays require expensive instru-
mentation that is difficult to maintain. Therefore, a principle use
of this assay is for validation of other assays and for answering
certain questions that require a high degree of specificity.
To compare an assay to the neg CI GCMS assay, plasma levels of

melatonin should be 2-10 pg/ml during the day and 25-100 pg/ml at
night. As with all assays, the lower the values, the more specific
the assay. However, the dynamic range is probably more informa-
tive: with the mass spectral assay there is a ten to fifty-fold in-
crease from day to night. Values that are similar to those of the
GCMS assay at night but higher than the GCMS values during the day
probably indicate the presence of a consistent level of a contami-
nant that appears to be relatively greater during the day than at
night. Ideally, values should be corrected for recovery, since
artifactually lower results could occur due to low or variable
recovery. (The values of the neg CI GCMS assay are corrected for
recovery which is therefore 100 percent.)
GCMS is considered to be theoretically highly specific, since

compounds are identified by molecular weight as well as by retention
time as they are quantified. However, not all GCMS assays are opti-
mally specific. A deuterated internal standard should be used. The
deuterated internal standard is chemically similar to endogenous
melatonin and melatonin of the external standard curve, except that
it has a slightly higher molecular weight, permitting separate iden-
tification and quantification by the mass spectrometer. When added
to the plasma at the initiation of the assay, the deuterated inter-
nal standard coextracts, coderivatizes, cochromatographs, and
cofragments with the nondeuterated (endogenous) melatonin. Measure-
ment of two fragment ions at a characteristic intensity ratio is the
mass spectral "fingerprint" for the parent molecule. Retention
times are known exactly for each injection by comparing the reten-
tion time of the plasma sample to that of the internal standard.
Interfering substances can be visually noted, because they are char-
acterized by shoulders or humps preceding or following the melatonin
peak. A recovery is calculated for each sample: thus each point of
the external standard curve and each plasma sample is corrected for
any losses that might occur during the assay.
Adequate sensitivity has been a problem for GCMS. The neg CI

GCMS assay (Lewy and Markey, 1978) has the requisite sensitivity for
measuring plasma melatbnin (minimal detectable concentration <1
pg/ml) and is among the most sensitive of the human plasma melatonin
assays (Rollag, 1981). Since it uses a deuterated internal
standard, this assay also has a high degree of specificity and
precision.
MELATONIN SECRETION-A BIOLOGICAL MARKER 523
Measurement of urinary 6-hydroxymelatonin sulfate has also been

performed with mass spectral assays using deuterated internal stan-
dards (Fellenberg et al., 1980~ Tetsuo et al., 1980). The day/night
dynamic range in the concentration of this urinary metabolite is
similar to that of plasma melatonin as measured by the neg CI GCMS
assay. Extremely low levels of 6-hydroxymelatonin are excreted
during the day, indicating that marked daytime pulsatile secretion
of melatonin does not occur (this is also in agreement with the
plasma melatonin data as measured by the neg CI GeMS assay, because
it is rare that plasma levels above 10 pg/ml are found in healthy
sighted humans during the day). The urinary 6-hydroxymelatonin
assay is the method of choice for measuring amplitude of secretion.
Measurement of plasma melatonin requires many time points, not only
for determination of onset, maximum, or offset of secretion, but
also for description of a curve of values under which an area can be
calculated, whereas urinary excretion of 6-hydroxymelatonin usually
involves only a few data points for each subject. The plasma mela-
tonin assay is the best suited for evaluating timing of secretion.
urine is difficult to collect in shorter than six-hour intervals~
consequently, only a gross estimate of the day/night rhythm can be
obtained using 6-hydroxymelatonin.
EXTRAPINEAL MELATONIN?
After pinealectomy, rats have very little melatonin during the

day or night, or after administration of isoproterenol as measured
by the neg CI GCMS assay (minimal detectable concentration <1 pg/ml)
(Lewy et al., 1980) (see Figure 1). Markey's group has recently
confirmed these findings measuring 6-hydroxymelatonin in the rat
(Markey and Buell, 1982) and in the monkey (Tetsuo et al., 1982).
Pelham found similar results in the chicken using a bioassay (Pelham
et al., 1972~ pelham, 1975). Arendt showed that only after complete
pinealectomy could sheep plasma melatonin be abolished (Arendt et
al., 1980). It is not known as yet exactly how low are the "true"
levels of melatonin and 6-hydroxymelatonin after pinealectomy,
because these levels are close to, or less than, the minimal detec-
table concentration of the most sensitive assays. Measurement of
substantial amounts of melatonin in the circulation after pineal-
ectomy most likely indicates an assay specificity problem.
Reduction of melatonin at night in humans after administration of
propranolol (Vaughan et al., 1976~ Hanssen et aI, 1977, author's
unpublished data) suggests that most of human plasma melatonin is
derived from the pineal gland, because it is unlikely that other
sources of melatonin would be under beta-adrenergic regulation. We
(Lewy and Neuwelt, in preparation a ) have recently used the neg CI
524 A. J. LEWY·
DAY DAY NIGHT

(Saline) (Isoproterenol, C
3 mg/kg s.c.) N = 10
60
E
C,
50
..e,
z 40 C
Z N =6
§w
30
:i:
< 20
:i:
en
:5
a. 10 C PX PX PX
N =5 N=3 N =9 N =7
rl~
N.D. N.D. N.D.
Fig. 1. Sham-operated control (e) rats showed expected low daytime

and high nighttime plasma melatonin values. Rats injected with 3
mg/kg l-isoproterenol-d-bitartrate during the day had substantial
melatonin levels. Pinealectomized (PX) rats had nondetectable
(N.D.) plasma melatonin under all three conditions.
From Lewy et al., 1980.
GCMS assay to measure plasma melatonin in a patient whose malignant

pineal gland was surgically removed: a normal day/night pattern was
present prior to extirpation, whereas no melatonin could be detected
following pinealectomy.
Whether or not there exists extrapineal synthesis of melatonin

is still controversial. Future studies should be carried out in
pinealectomized animals to rule out the pineal as a source for mela-
tonin. Identification of melatonin by immunohistological techniques
using antibodies whose specificity has not been absolutely assured
leaves unanswered the question of the true identity of "immuno-
reactive melatonin" in extrapineal tissue. Demonstration of mela-
tonin production in tissue culture after adding serotonin is not
adequate proof of extrapineal production in vivo: identification of
extrapineal N-acetyltransferase and HIOMT activity should also in-
clude demonstration of serotonin in those organs at adequate concen-
trations. If extrapineal synthesis of melatonin exists, it is
unlikely that these sites contribute significantly to plasma or
urine·levels.
Abolition of plasma melatonin after pinealectomy validates the

use of circulating levels for estimating pineal secretion of this
hormone and provides the basis for further work with plasma melato-
nin as a biological marker for adrenergic activity as well as for
the circadian system.
ADRENERGIC REGULATION OF MELATONIN PRODUCTION
The pineal is unique in its sympathetic innervation. Most endo-

crine glands are regulated by blood-born substances usually of
pituitary origin. (Some exocrine glands are innervated by peri-
pheral postganglionic neurons.) The adrenal medulla (which is
considered to be similar to a postganglionic sympathetic neuron,
because it synthesizes and secretes catecholamines) is innervated by
preganglionic fibers from splanchic nerves. The pineal is inner-
vated by postganglionic neurons from the superior cervical ganglia.
What is also remarkable (and has important implications) is that
these neurons do not respond to stress (Klein and Parfitt, 1976;
Parfitt and Klein, 1977). The "fight or flight" response typical of
the sympathetic nervous system does not include the sympathetic
nerves of the pineal (which are discretely regulated by an endog-
enous circadian pacemaker) •
The pineal is also unusual in that melatonin production is not

compensatorily regulated by parasympathetic nerves. Cholinergic
control (Wartman et al., 1979) is in doubt, because choline acetyl-
transferase appears not to be present in the rat pineal (Schrier and
Klein, 1974). Similarly, reports of parasympathetic innervation of
the rabbit pineal (Romijn,1973) are confounded by the fact that cho-
line acetyl transferase is also not present in the pineal of this
species (Schrier and Klein, 1974). Acetyl cholinesterase has ap-
parently been measured in the sympathetic fibers innervating the
pineal (Eranko et al., 1970a; Eranko et al., 1970b; Manocha, 1970;
Trueman and Herbert, 1970) and ganglionectomy (Machado and Lemos,
1971) and 6-hydroxydopamine (Snyder et al., 1965) has apparently
diminished it, but again, this work must be evaluated in the light
that there may be no choline acetyl transferase present in the pineal
(Schrier and Klein, 1974).
There are a few studies, however, that suggest at least in some

species the existence of efferent innervation to the pineal whose
function is as yet unknown (David and Herbert, 1973; Nielsen and
Moller, 1975; Moller, 1978; Ronnekleiv et al., 1980), but at this
point it seems safe to assume that neuroregulation of melatonin pro-
duction is exclusively by sympathetic nerves (Ariens Kappers, 1960;
Ariens Kappers, 1979; Ariens Kappers et al., 1979;). The pineal
apparently also has presynaptic alpha-2 autoadrenoceptors (Pelayo et
al., 1977). (Postsynaptic pineal alpha-adrenergic receptors appear
526 A. J. LEWY
to regulate phosopholipid metabolism in the pineal (Nijjar et al.,

1980) •
Since the pineal is innervated by peripheral postganglionic sym-

pathetic neurons that appear to be exclusively involved in the regu-
lation of melatonin production, this gland has advantages as an
experimental model for evaluation of adrenergic function. It is
particularly useful, because synthesis of melatonin occurs at night,
and because stress (which increases the levels of circulating cate-
cholamines) does not influence melatonin production due to highly
active uptake mechanisms (Klein and Parfitt, 1976; Parfitt and
Klein, 1977). Catechol-O-methyltransferase activity is greatest
during the day, which perhaps affords another mechanism that mini-
mizes daytime production of melatonin (Backstrom and Wetterberg,
1972). (Since melatonin production is not affected by stress,
measurement of melatonin is not a "marker" for stress.) It is also
important that the pineal is a peripheral, and not a central, organ
(its beta-adrenergic receptors are outside of the blood-brain
barrier) •
In animal experiments the pineal gland facilitates several ways·

in which to evaluate adrenergic function. Since the nerves of the
pineal are quiescent during the day (and firing rate can be suppres-
sed at any time by light), neuronal firing can be reduced without
the use of drugs. During the day postsynaptic function can be
selectively evaluated (because the presynaptic nerves are quies-
cent), by administration of an agonist, such as isoproterenol (see
Figure 2, top). Uptake mechanisms can also be evaluated by compar-
ing the melatonin produced in response to isoproterenol (which is
not taken up presynaptically) to exogenous administration of nore-
pinephrine (which is). During the night the sympathetic nerves
release endogenous norepinephrine that stimulates melatonin produc-
tion. Measurement of nighttime melatonin secretion evaluates both
pre- and postsynaptic function (see Figure 2, bottom).
A hypothetical experiment would be as follows. Suppose a drug

caused a 50% reduction of melatonin production at night and a 50%
decrease in the response to isoproterenol during the day. This
would indicate that the drug had primarily postsynaptic activity and
relatively little presynaptic activity. On the other hand, suppose
the drug caused only 10% reduction in response to isoproterenol
during the day but a 50% reduction at night. Then the drug probably
had primarily presynaptic effects.
HUMAN STUDIES
As in all mammals studied to date, human melatonin appears to be

under adrenergic control. Patients with transection of the cervical
I soprot.,tno!
Fig. 2. postsynaptic function can be selectively evaluted by.

measuring melatonin production in response to administration of a
beta-adrenergic agonist during the day (top); total pre- and
postsynaptic adrenergic activity can be evaluated by measuring
nighttime melatonin production stimulated by endogenous release of
norepinephrine (NE) from the pineal's sympathetic neurons (bottom).
spinal cord have disturbed melatonin secretory rhythms (Kneisley et

al., 1978). Patients with Shy-Drager Syndrome (multiple system
atrophy) and idiopathic orthostatic hypotention (diseases which are
manifested by central and peripheral sympathetic lesions) have re-
duced levels of plasma melatonin (Vaughan et al., 1979) and urinary
6-hydroxymelatonin (Tetsuo et al., 1981). These data suggest that
the human pineal is regulated by sympathetic adrenergic neurons.
Vaughan originally demonstrated in one healthy subject (Vaughan

et al., 1976) that propranolol blocks nighttime melatonin secre-
tion. This was later confirmed in a psychiatric patient (Hanssen et
al., 1977) and in several human subjects (author's unpublished
528 A.J.LEVVY
data). Propranolol, in doses of 120-140 mg po administered at

night, can completely reduce melatonin secretion to daytime levels
or below (author's unpublished data). This drug may therefore be
used to help evaluate adrenergic function of the human pineal in
vivo. The amount of propranolol that reduces melatonin levels may
be related to the functional number of beta-adrenergic receptors in
the pineal. Propranolol may also have a use in the treatment of
melatonin hypersecretion. Administration of propranolol also
affords the opportunity to understand the relationship between mela-
tonin secretion and secretion of other endocrine substances. After
reducing melatonin secretion with propranolol, changes in the levels
of other hormones could then be assessed. On a subsequent night,
administration of the same dose of propranolol in addition to an
infusion of physiologic levels of melatonin would control for any
beta-adrenergic and/or nonspecific effects of propranolol; thus hor-
monal changes due to the reduction of melatonin levels could be
isolated.
Clonidine also reduces human melatonin secretion (Lewy et al.,

in preparation b ). This alpha-adrenergic agonist is primarily
selective for presynaptic alpha-2 adrenergic autoreceptors but
stimulates postsynaptic alpha-l and alpha-2 adrenergic receptors as
well.
Clonidine passes through the blood-brain barrier and has been

used to evaluate alpha-adrenergic receptors in a variety of dis-
orders, including affective disorders (Siever et al., 1981).
Clonidine's effects have been evaluated by measuring growth hormone
or blood pressure. However, these physiological variables are regu-
lated by more than one neurotransmitter. Also, compensatory auto-
nomic mechanisms can confound the cardiovascular effects of cloni-
dine. Consequently, measurement of melatonin secretion in response
to clonidine may provide a more specific index of alpha-adrenergic
receptor function.
One hour after intravenous administration of clonidine, mela-

tonin levels are reduced in a linear dose-response relationship
(Lewy et al., in preparation b ). Oral clonidine is also effective
(this may be a more practical route of administration). After oral
administration there is a lag time and a longer duration of action.
It may also be possible to measure urinary 6-hydroxyrnelatonin
sulfate in an overnight collection in order to quantify melatonin
excretion.
Measurement of nighttime melatonin is the simplest way of eval-

uating pineal adrenergic activity. In a preliminary study, we
measured plasma melatonin in five manic-depressive patients, in both
manic and depressed states (Lewy et al., in preparationC ).
Variables that might confound the results, such as age, sex, drug
regimen, or time of year, were controlled, in that patients were

compared to themselves; they were on the same drug regimen and were
studied at the same time of year in each mood state. Melatonin
levels were increased in mania compared to depression, which is com-
patible with increased adrenergic activity in mania compared to
depression.
Wetter berg et al. (1979) also found that unipolar patients have
decreased melatonin levels when depressed, compared to when they are
not depressed. Jimerson et al. (1977) studied bipolar depressed
patients and found no difference in urinary melatonin; however, they
compared patients to healthy subjects. Since it is not known if
phase of the menstrual cycle, age, sex, and time of year, affect
melatonin secretion, it is recommended that these variables be ap-
propriately controlled. Either the area under the curve of plasma
melatonin levels or an overnight (or 24-hour) urinary 6-hydroxy-
melatonin sulfate level are the optimal methods for measuring pineal
adrenergic activity.
Although the human pineal is probably regulated by sympathetic

adrenergic neurons, stimulation of these beta-adrenergic receptors
during the day in humans has been difficult, because the dose of
agonist that would be necessary would cause untoward cardiovascular
effects. At least one hour of continuous stimulation is necessary
to provide sufficient time for the induction of N-acetyl-
transferase. This problem may have been avoided in a recent study
of volunteers who exercised for a length of time on a bicycle,
resulting in a two-fold increase in melatonin levels (Carr et al.,
1981). This increase, however, is considerably less than the
response seen in experimental animals with isoproterenol. Assay
specificity may have contributed to this apparent difference in
response: using the neg CI GCMS assay (Lewy and Markey, 1978),
doses of isoproterenol, tyramine, I-dopa, and dopamine (which caused
a 50% increase in heart rate for one hour) were insufficient to
raise melatonin levels significantly (author's unpublished data).
Negative results in attempting to stimulate melatonin production
during the day in some species (such as man) is probably a problem
of dosage.
SUMMARY
The amplitude of plasma melatonin levels or urinary 6-hydroxy-

melatonin sulfate levels appears to be a useful biological marker
for adrenergic activity of the pineal gland in humans. Perhaps in
the future, we will develop the appropriate methods to be able to
stimulate human melatonin production with a safe dose of a beta-
adrenergic agonist. until then, reduction of nighttime secretion
with clonidine or propranolol remain the best tools for specific
530 A. J.lEWY
evaluation of presynaptic and postsynaptic adrenergic activity in

the pineal gland.
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HUMAN MELATONIN SECRETION, ITS ENDOGENOUS CIRCADIAN PACEMAKER
AND THE EFFECTS OF LIGHT
Alfred J. Lewy
Departments of Psychiatry, pharmacology and Opthalmology

Oregon Health Sciences university
Portland, Oregon, U.S.A.
INTRODUCTION
Elucidation of pineal physiology must be based on an under-

standing of the circadian as well as adrenergic regulation of
melatonin production. For more than 50 years it has been known that
the endogenous circadian pacemaker is located in the brain {Richter,
1965: Richter, 1967}. In many species of animals, this pacemaker is
thought to be located in the suprachiasmatic nucleus {SCN} of the
hypothalamus {Moore and Eichler, 1972: Stephan and Zucker, 1972:
Ibuka and Kawamura, 1975: Rusak, 1977}. There may be more than one
pacemaker: however, with regard to melatonin production, there is
increasing agreement that there is one pacemaker located in the SCN.
Lesioning of the SCN results in a decrease of N-acety1-

transferase activity {Moore and Klein, 1974: Klein and Moore,
1979}. It was for this reason that it was thought that the SCN
"turns on" the sympathetic stimulation of the pineal. In other
words, when the seN is "on," the pineal is "on." Recent studies may
have challenged this view, since stimulation of some seN neurons
causes a decrease in sympathetic activity (Nishino et al., 1976).
Also, both inhibitory and excitatory responses have been recorded in
response to light {Nishino et al., 1976: Groos and Mason, 1980}.
However, these neurophysiological studies are difficult to interpret
and do not necessarily rule out that seN activity results in an "on"
signal. Studies of uptake of 2-deoxy-D-{C14}glucose show in-
creased uptake during the day compared to night {Schwartz and
Gainer, 1977: Schwartz et a1., 1980}. {Should the seN prove to be
"off" at night, then there must be a center of sympathetic activity
caudal to the SCN, that is inhibited by the SCN during the day}.
535
536 A. J. LEWY
Future studies are necessary to determine with certainty whether the

SCN is "on" or "off" at night, with respect to sympathetic stimu-
lation of the pineal.
THREE BIOLOGICAL EFFECTS OF LIGHT
Light has three effects on melatonin production. One, light

suppresses melatonin production. TWo, the light-dark cycle entrains
the endogenous pacemaker that drives the melatonin production
rhythm. Three, the changing lengths of daylight and darkness regu-
late the annual (seasonal) patterns of melatonin production.
Suppression of Melatonin Production
The suppressant effect of light appears to be unique to mela-

tonin production by the pineal gland. It is thought that the
retinohypothalamic tract (RHT) mediates both the entrainment effect
of light and the suppressant effect of light (Klein and Moore,
1979). Consequently, the suppressant effect may be a means of
assessing the properties of light important for the entrainment
effect.
In experimental animals very dim light is sufficient to suppress

melatonin production. As little as 1-2 lux will suppress N-acetyl-
transferase activity by 50% in the rat (Minneman et al., 1974).
Less than 500 lux will completely suppress N-acetyltransferase
activity in the sheep and the monkey (Rollag and Niswender, 1976;
Perlow et al., 1981). Suppression occurs immediately, and the
decline in melatonin levels corresponds to its half-life in plasma
or CSF (Reppert et al., 1979). upon return to darkness, N-acetyl-
transferase and melatonin levels resume their pretreatment values
within a few minutes. However, in the rat, melatonin production
resumes slowly, and only if the light pulse occurs during the first
half of the night (Deguchi and Aelrod, 1972; Illnerova and vanecek,
1979). One minute of light is sufficient for suppression of mela-
tonin production in the rat or the hamster (Illnerova and Vanecek,
1979; Hoffman et al., 1981).
Previous investigators have had difficulty in attempting to

demonstrate effects of light in man (Vaughan et al., 1976; Jimerson
et al., 1977; Lynch et al., 1977; Arendt, 1978; weitzman et al.,
1978; Wetterberg, 1978; Akerstedt et al., 1979; Vaughan et al.,
1979) leading many to speculate that humans are quantitatively, and
perhaps qualitatively, different in their response to light compared
to all other warm-blooded species tested, including nonhuman pri-
mates (Perlow et al., 1980; Reppert and Klein, 1980). Recently,
however, we have shown that light suppresses human melatonin
HUMAN MELATONIN SECRETION 537
secretion (Lewy et a1., 1980); humans apparently require bright

artificial light or sunlight for suppression of melatonin secretion
that is accomplished with much lower intensities of light in other
species.
First, sunlight was tested (Lewy et a1., 1980). The idea that
sunlight might be effective in suppressing human melatonin secretion
(though ordinary room light was ineffective) carne from an instance
of self-experimentation: shortly after returning to Washington,
D.C., from a two-week stay in Sydney, Australia, the author's
morning plasma melatonin level was quite low. (It should have been
at a higher level, based on the phase of the author's endogenous
pacemaker. )
The sleep/wake cycles of two healthy volunteer subjects were

then shifted in the laboratory; that is, they slept between 3 and 11
a.m. for several days. After seven days melatonin secretion was
maximal during the late morning (between 9 and 10 a.m.). On the
eighth day of the study, the volunteers were awakened at 7 a.m. and
exposed to sunlight. Melatonin concentrations declined precipi-
tously. It appeared that sunlight was indeed capable of suppressing
melatonin secretion in man.
Why was sunlight (and not ordinary artificial light) effective?

Since sunlight is more intense than ordinary room light (at all wave
lengths), intensity was studied next. The intensity of sunlight on
a sunny afternoon is 100,000 lux; ordinary room light is rarely
above 500 lux and is usually 200-300 lux (Thorington, 1980). By
increasing intensity from 500 to 2500 lux-, the intensity at practi-
cally each wavelength will increase, despite a change in light
spectra.
Six volunteers were studied under differen~ light intensities

during an interruption of sleep between 2 and 4 a.m. Five hundred
lux fluorescent light had little or no effect in reducing melatonin
concentrations, but 2500 lux incandescent light profoundly reduced
melatonin secretion (Figure 1, left). (Fluorescent light [Vita-
Lite] of this intensity is also effective, produces less glare and
heat, and is currently being used in these studies.) TWO of these
subjects who were exposed to 1500 lux appeared to have a 50% reduc-
tion of melatonin secretion at this intensity, thus suggesting a
dose-response relationship between light intensity and suppression
of melatonin secretion (Figure 1, right). This relationship had
been previously shown in the rat with respect to N-acetyltransferase
activity (Minneman et al., 1974).
These findings have several implications. First, the human

pineal appears to be regulated in the same way as in other species.
(The SCN has been identified in man as well as in other primates
538 A. J.lEWY
1.r_J
I!
60 \
E
1- \1
--
OJ
"-
" I
40
~
Z
0
....
~
w
~
0100 0200 0300 0400 0500 0100 0200 0300 0400 0500
TIME OF DAY (hr.)
Fig. 1 (left). Effect of light on melatonin secretion. Each point

represents the mean concentration of melatonin (± standard error)
for six subjects. A paired t-test, comparing exposure to 500 lux
with exposure to 2500 lux, was performed for each data point. A
two-way analysis of variance with repeated measures and the Newrnan-
Keuls statistic for the comparison of means showed significant dif-
ferences between 2:30 a.m. and 4 a.m. (*, P < .05; **, P < .01».
Fig. 1 (right). Effect of different light intensities on mela-
tonin secretion. The averaged values for two subjects are shown.
Symbols: (0) 500 lux; (X) 2500 lux; (e) 1500 lux; and (0) asleep in
the dark.
From Lewy et al., 1980. Copyright, 1980, AAAS.
(Moore, 1979; Lydic et a1., 1980).) These results also suggest that
the human pineal may function in a similar way as in other mammals.
The second implication is that (perhaps on the basis of inten-

sity) humans have adapted to ordinary room light, yet may still re-
main sensitive to the natural (brighter) sunlight/dark cycle. Thus,
the changing length of the natural photoperiod might affect humans
in similar ways in which other animal species are affected. These
results may stimulate more epidemiological studies of seasonal

rhythms in humans.
The third implication of these findings is that a "bright

light/dim light" paradigm may be a useful research strategy for
investigating the effects of light in man. Most previous studies of
the effects of light in man have resulted in negative or ambiguous
findings (Hollwich, 1979). Previous studies that used insuf-
ficiently intense light should be repeated with bright artificial
light or sunlight.
Why does man require brighter light? It is unlikely that it is

evolutionary, because the discovery of fire was not such a long time
ago. A more likely explanation is that short-term adaptation occurs
in the retina, retinohypothalamic tract, or SCN. Recent evidence
suggests that there may be an annual rhythm in light sensitivity
(author's unpublished data): if a person is exposed to 100,000 lux
during a sunny summer day and is tested that night with 500 lux, he
will have a relatively subsensitive response than after having been
exposed to 20,000 lux during a winter day. This type of short-term
adaptation may be responsible for apparent species differences. To
confirm this, laboratory animals should be tested who have been
reared outdoors. Either a toxic effect of light or a type of
short-term adaptation has recently been demonstrated in the rat:
dim light permitted melatonin production, when it was alternated
with bright light (Lynch et al., 1981~ Rivest et al., 1981).
Entrainment by the Light-Dark Cycle
The second effect of light on melatonin production is entrain-

ment of its circadian secretory rhythm. (Circadian means "about a
day" or about 24 hours [Halberg, 1959]). The effect of the light-
dark cycle on the circadian rhythm of melatonin production is so
profound that at first it was thought that the light-dark cycle
directly regulated melatonin production, i.e., that melatonin pro-
duction passively followed environmental conditions of light and
dark. Later, it was discovered that the rhythm persisted in blinded
animals or animals kept in constant darkness (Klein and Weller,
1970~ Klein et al., 1971~ Ralph et al., 1971). Identification of
the RHT and the SCN led scientists to conclude that the light-dark
cycle regulates melatonin production through its effects on an
endogenous hypothalamic circadian pacemaker.
The activity rhythm and the melatonin production rhythm are

"h ands of the clock" and are not actually the "clock" itself (wh ich
is presumedly the SCN). When an animal is no longer entrained to
the light-dark cycle, it displays a circadian rhythm of approxi-
mately, but not precisely, 24 hours~ this is termed "free-running."
540 A. J. LEWY
In constant darkness the melatonin production rhythm free-runs with

the activity/rest cycle (melatonin production occurs during the
activity phase of nocturnal animals or during the rest phase of
diurnal animals) (Ralph et; al., 1971; pohl and Gibbs, 1978; Tamarkin
et al., 1978).
The period ("'l-, or tau) of the free-running endogenous pacemaker

is a characteristic of an individual animal (and usually of a par-
ticular species). Some animals have endogenous rhythms that are
greater than 24 hours (1">24), some less than 24 hours ("l" <24)
(Aschoff, 1960; Aschoff, 1969). In general, the former is more com-
mon in diurnal animals, the latter in nocturnal animals (Aschoff,
1960; Aschoff, 1969). Why is the endogenous free-running period
("'l-) of the biological clock not precisely 24 hours? Perhaps be-
cause a biological clock can never be as precise as a physical
clock. Perhaps being slightly different than 24 hours permits
greater flexibility for accommodation to the changes in the times of
dawn and dusk throughout the year. Pittendrigh has reasoned that
internal phase relationships would be unstable if the period of the
endogenous pacemaker were to be exactly 24 hours (Pittendrigh, 1981).
When endogenous rhythms are synchronized by an environmental

time cue, such as the 24-hour light-dark cycle, they are
"entrained." The period of the environmental cycle must be close to
the endogenous period (,..) for entrainment to occur. The "range of
entrainment" is the range in the periods of the environmental cycle
that are capable of synchronizing the endogenous pacemaker.
The interaction of light as a zeitgeber (literally, time giver)

with the endogenous pacemaker can be described and predicted by a
phase response curve (PRC). The PRC was first discovered by expos-
ing an animal free-running in constant darkness to a short
(IS-minute) pulse of light. The animal responded with an advance or
a delay in phase, depending on when the light pulse occurred
(Pittendrigh, 1981). Because the animal is in constant darkness,
the term "subjective day" is used to indicate circadian time repre-
sented by behavior typical during the day (rest for nocturnal ani-
mals, activity for diurnal animals). Subjective night indicates
circadian time represented by behavior typical during the night
(rest for diurnal animals, activity for nocturnal animals). (Sub-
jective dawn and dusk, therefore, refer to the transitions between
activity and rest in constant dark.)
If the light pulse occurs during the animal's subjective night,

the ani~al will respond with either an advance or a delay; if the
light pulse occurs during the animal's subjective day, phase shifts
will be minimal. (This is true for both diurnal and nocturnal ani-
mals.) During subjective night, the closer the pulse is to subjec-
tive dawn, the more likely the animal will respond with an advance
in phase; the closer to dusk, the more likely there will be a delay
in phase. In the middle of the night there is an inflection point
where an advance is separated from a delay by only a few minutes.
In general, the closer the pulse is to the middle of the night the
greater the magnitude of the response -- in either direction. Thus,
the middle of the night is a time when the maximum phase advance is
separated from the maximum phase delay by only a few minutes (Figure
2). Since the animal is in constant darkness, it continues to
free-run throughout this procedure, relative to the transient phase
shift due to the light pulse. PRCs have recently been described
PHASE RESPONSE CURVE

"PRe"
Figure 2. Schematicized phase response curves (PRCs) for animals

with endogenous periods equal to (top), greater than (middle), and
less than (bottom) 24 hours; these curves may help to explain how
animals use light to maintain stable steady-state entrainment to the
24-hour day.
542 A. J. LEWY
measuring chicken (Binkley et al., 1981) and rat (Illnerova and

Vanecek, 1982) N-acetyltransferase activity. In the rat, a light
pulse as short as one minute can shift the phase of the N-acetyl-
transferase activity rhythm (Vanecek and Illnerova, 1979).
What happens under entrained conditions? Another experimental
paradigm for evaluating the PRe is the skeleton photoperiod. This
is accomplished by two IS-minute pulses of light, several hours
apart. The pulse that occurs near the phase-advance portion of the
PRe functions as dawn, the pulse that occurs close to the phase-
delay part of the PRe functions as dusk. The relative magnitude of
the phase advance compared to the phase delay produces the net
effect.
In an animal with't" > 24 hours, each day the light-dark cycle

must advance the endogenous pacemaker one hour for stable steady-
state entrainment to 24 hours. The net effect from the PRe must
also include a phase advance to compensate for any phase delay that
might result each day from the action of light on the phase-delay
portion of the PRC (see Figure 2, middle). If "r < 24 hours, then
the animal must delay more than advance each day for entrainment to
a 24-hour period (see Figure 2, bottom). A major factor in deter-
mining the shape of the PRe is whether an animal's endogenous period
is greater than, or less than, 24 hours (see Figure 2).
In order to evaluate the entrainment effects of light in humans,

we studied totally blind subjects. Previous work on blind subjects
has produced suggestive, but not conclusive, data (Hollwich, 1979).
This might have resulted because most studies were not done longi-
tudinally. For example, in an initial study of melatonin patterns
in ten blind subjects, subjects were studied for only one day (Lewy
and Newsome, in preparation). "phase dispersion" was found in this
group. Melatonin onset occurred in these blind subjects at all
times of the evening or early morning. (In sighted subjects, mela-
tonin secretion begins regularly between 10 p.m. and 1 a.m.)
More information data was obtained, however, by studying two of

these subjects longitudinally once a week for four weeks. Studied
in this way these two subjects displayed markedly unusual circadian
melatonin secretory rhythms (Lewy and Newsome, in preparation). One
subject appeared to be entrained to a 24-hour period but was more
than 120 0 phase-delayed from normal. The other subject was
"free-running" wi th a rhythm of approximately 24.7 hour s.
Although only two subjects were studied, these data suggest that
light is important in the entrainment of the human melatonin cir-
cadian secretory rhythm. Many researchers have thought that, unlike
other animals, light has little or no effect in the entrainment of
human circadian rhythms. Perhaps past negative results were due to
the use of insufficiently intense light. Bright artificial light or
sunlight should be used in future experiments.
There are some past data that suggest that sunlight is more ef-
fective than ordinary-intensity artificial light, particularly in
entraining human circadian rhythms •. Sunlight deprivation may
increase the number of days necessary for reentrainment of circadian
rhythms after air travel across several time zones (Klein and
Wegmann, 1974). Field studies, compared to laboratory simulation,
of travel across time zones have been noted to produce different
results (Wever, 1980). perhaps these differences are due to the
fact that the laboratory simulations were conducted with ordinary
room light. In a few subjects studed under isolated conditions, the
light-dark cycle has been shown to be able to entrain the tempera-
ture rhythm, while the activity/rest rhythm continued to free-run
(Aschoff and Wever, 1981). In other studies, the range of entrain-
ment of the human temperature rhythm appears to be greater under the
natural light of the Arctic summer (Lewis and Lobban, 1959) than
under ordinary room light (Aschoff and Wever, 1981). Finally, the
light-dark cycle appears to be capable of entraining at least part
of the cortisol circadian secretory rhythm (Orth and Island, 1969).
Photoperiodism
There are a few epidemiological studies of seasonal rhythms in

human physiology and disease (Aschoff, 1981). Nonetheless, human
seasonal rhythms have generally not been attributed to changes in
the photoperiod, probably because previous data on effects of light
in humans has been unimpressive. It is hoped that recent melatonin
research demonstrating suppression of secretion by sunlight and
bright artificial light will stimulate more epidemiological studies
of seasonal rhythms in humans, as well as stimulate more testing of
possible photoperiodic responses. Again, the bright light/dim light
paradigm may be useful here.
In a recent study of one individual, photoperiodic effects of

light were evaluated (Lewy et al., 1982). A manic-depressive
patient with a 13-year history of winter depressions and springtime
remissions was exposed to bright artificial light between 6 and 9
a.m. and between 4 and 7 p.m. during the first week of December,
1980. After four days of exposure to a spring photoperiod using
bright full spectrum light (Vita-Lite), he "switched" out of his
typical winter depression (which typically happens to him in the
spring) •
MELATONIN AS A "BIOLOGICAL MARKER"
Melatonin secretion is unique in that its active phase occurs at

night, completely confined between dawn and dusk. (The peaks and
troughs of the circadian rhythms of cortisol and thyroid stimulating
544 A. J. LEWY
hormone secretion and core body temperature straddle the dawn and
dusk transitions [Aschoff, 1979]). Melatonin secretion is also
unique among circadian rhythms in that its active phase occurs at
night in both diurnal or nocturnal species of animals. (Other cir-
cadian rhythms have a 180 0 phase-angle difference when diurnal
animals are compared to nocturnal animals [Aschoff, 1979].) For
these reasons, melatonin has been thought to be a highly useful
marker for the phase and period of its endogenous pacemaker, and
perhaps functions as part of the endogenous biological clock as well.
One of the major methodological problems concerning circadian

rhythm research has been that of "masking. n For example, motor
activity causes an increase in core body temperature7 inactivity
(and sleep) decrease temperature (Weitzman et al., 1979). Stress
increases cortisol secretion (Czeisler et al., 1979). However,
melatonin secretion appears to be free from these types of masking
effects in that acute changes in the activit--/rest cycle do not seem
to affect the rhythm of melatonin secretion (Jimerson et al., 19777
Lynch et al., 1977)7 that is, sleep deprivation has no effect
(Jimerson et al., 19777 Akerstedt et al., 1979). It is also likely
that diet does not affect the rhythm of melatonin secretion (Arendt,
19797 Herbert and Reiter, 1981), nor does stress (Lynch et al.,
19737 Illnerova, 19767 Klein and Parfitt, 19767 Parfitt and Klein,
19777 Vaughan et al., 1978 a 7 vaughan et al., 1979 b ). Melatonin
secretion is not related to the stages of sleep (Vaughan et al.,
1978a7 Weinberg et al., 19797 author's unpublished data). Aside
from specific drugs which can affect the amplitude of melatonin
secretion, melatonin secretion is masked only by light. By reducing
light intensity below threshold (in human research a fairly high
intensity is permissible), this masking effect can be avoided.
The study of manic-depressive patients provides an opportunity

to examine "abnormal" conditions of melatonin secretion. Many of
these patients are also thought to have phase-advanced circadian
rhythms in cortisol secretion, core body temperature, and other phy-
siological variables (Wehr and Goodwin, 1981). The circadian rhythm
of melatonin secretion appears to be consistent with the "phase-
advance hypothesis," in that it appears to have an earlier timing in
mania compared to depression and in depression compared to healthy
subjects (Lewy et al., in preparation a ).
Recently, light suppression of melatonin secretion was studied

in a group of manic-depressive patients compared to a group of con-
trols matched for age but not sex (Lewy et al., 1981). Patients
were more sensitive to light than the control subjects7 that is,
melatonin secretion was suppressed to twice the extent in the
patient group compared to the control group when exposed to 500 or
1500 lux. A group of euthymic (i.e., neither manic nor depressed)
patients also appeared to be supersensitive to light, suggesting
that this finding might possibly be a "trait marker," and might

possibly explain the phase-advanced circadian rhythms observed in
these patients (Lewy et al., in preparation b ).
SUMMARY
Melatonin appears to be a useful biological marker, not only for

adrenergic activity in the pineal, but also for the endogenous cir-
cadian pacemaker that regulates the nighttime increase in melatonin
production and for the effects of light on this system. These phy-
siologic processes are potentially important in human health and
disease. The study of the effects of light on melatonin production
has also increased our interest in the possible effects of light on
human physiology and behavior and in seasonal rhythms in humans.
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THE SECRETION AND EFFECTS OF MELATONIN IN HUMANS
Richard J. Wurtman, Franz Waldhauser** and Harris R.

Lieberman*
Laboratory of Neuroendocrine Regulation

Department of Nutrition and Food Science
and *Department of Psychology
Massachusetts Institute of Technology
Cambridge, MA 02139
** Present address: Univ. - Kinderklinik

Waehringer Guertel 74-76
A-1090 Wien 9 Austria
I. INTRODUCTION
Although it has been well established for two decades that

the human pineal gland produces and secretes melatonin, and
although investigators began describing effects of exogenous mela-
tonin on humans (principally on sleepiness and subjective vigor)
soon ,after its discovery, relatively little information has been
available until recently about the normal range of melatonin
concentrations in human plasma and other body fluids, and even
less about the effects of exogenous melatonin or its roles in
human physiological processes.
In part this state of affairs can be ascribed to the lack of

sensitive, specific, and reasonably convenient assays for melato-
nin until the mid-1970's; in part it probably also reflects the
fact that, until recently, the cost of synthetic melatonin was so
great as to preclude its administration in large doses to more
than relatively few subjects. Good assays for melatonin do now
exist based on radioimmunoassay and GCMS, and these are in routine
use within a growing number of laboratories; moreover the cost of
synthetic melatonin has decreased considerably. Hence it appears
likely that information about melatonin's levels in, and effects
on, humans will accumulate rapidly during the next few years.
551
Table 1. Blood Melatonin Values in Adult Humans
DaytIme NIghttIme Subject Reference

S"mple X .!. SE pg/m I X .!. SE pg/m I 'k>. Sex
BIOASSAY:
PLASMA NOH - 12*

-
49.6 + 10.5 5 M Pelhm et al. 1973
162 .!. 41
PLASMA
-
258 + 44 3
2
M
F
Arendt at a I. 1975
PLASMA ND** 20 - 150* 16 M Vaughan at al. 1976
RADIOIMMUNOASSAY:
PLASMA 132+ 10
- 188 + 15 14 M Arendt et a I • 1975
SERUM 47 - 481* --- 5 F Wetterberg et al. 1976
SERUM 63 + 22t --- 8 M Arendt et a I. 1977"

100 -; 45 t
- --- 7 F
PLASMA <20 50 - 100* 2 M Kannaway et a I. 1977
SERUM
-
20 + 3
-
78 + 13 5 M SmIth et al. 1977
SERUM ~ 12-14 sprIng & autumn 5 M Arendt et a I • 1977b

~ 20-24 wInter & summer
PLASMA 14.61. 0 • 9
-
49.1 + 3.8 47 M Arendt .& Wllk I nson 1978
13.9 + 0.5
-
66.1 + 5.3 50 F
PLASMA 23 1. 7
-
97 + 33 2 M Lynch at "I. 1978
PLASMA
-
32 + 8
-
179 + 26 5 M
Mult. samp Ie
Vaughan at a I. 1978
.,
74 + 26 t
SERUM
-
19 + 7t
- 7 SmIth et al. 1979
12 PM 4 AM
PLASMA --- 36+14
-
51+11 7 M SI zo nerl< 0 at a I. 1979
PLASMA 50 - 180*** 150 - 450*** 5 M weInberg et al. 1979
SERUM 11-21* --- 18 Sllmanatal. 1979
SERUM
-
24+1.6
-
79+8.6 8
15
M
F
Wa Idhauser et a I. 1981
THE SECRETION AND EFFECTS OF MELATONIN IN HUMANS 553
Table 1. (continued)
Daytime NI ghttlme SUbJect Reference

Sa"" Ie X.:. SE pg/m I X':' SE pg/m I No. Sex
GCMS:
PLASMA 25 - 40* 125 - 440* 3 Wilson et al. 1977
PLASMA 10 - 55* 5
PLASMA 1.5 - 4.9* 19.4 - 42.6* 2 M Lewy & Mar1<.ey 1978

2 F
** NO = nondetectable
t SO
*** range of mean values from 5 Individuals obtained by sampling every 15 min over 24 h.
* range
PL plasma
SE serum
This article summarizes our present knowledge about melatonin

levels in body fluids from normal humans and from people with par-
ticular diseases. It also describes the little information
available about melatonin's physiologic roles and the consequences
of its administration to humans.
II. MELATONIN LEVELS IN BODY FLUIDS
A. Presence and Levels in Blood, Urine and CSF
Melatonin levels apparently are similar in plasma and serum

(Wetterberg, 1978), and values obtained using RIA are in good
agreement with :those based on bioassays or GC-MS, i.e., up to
about 40 pg/m1 during daytime and 40-150 pg/m1 during nighttime
(Table I). No sex difference was noted in plasma melatonin levels
(Arendt et a1., 1975). Measurements of melatonin levels in human
urine have been difficult to make because of the abundance of
unknown cross-reacting substances. However, available data
(Table II) do indicate that it is present, that it exhibits a
substantial daily rhythm, and that a very good correlation exists
between blood and urinary melatonin levels at times when these
levels are changing (Lynch et al., 1978b; Lang et al., 1981). At
present, CSF melatonin levels have been measured mainly in
patients with particular diseases, and only in samples obtained
Table 2. Urine Melatonin Values in Adult Humans
Daytime Nighttime ,SubJect Reference

x.:. SE ng/4 h X':' SE ng/4 h No. Sex
BIOASS"Y:
NO - 1.3* 0.55 - 13.4* 2 M Lynch et al. 1975b

4 F
RADIOIMMUNo,o.SS"Y:
1.3 + 0.1 4.3 + 0.4 4 M Lynch et Ill. 197511
38.2 + 7.B· 3 M Wetterberg 1979

31.7 + 9.5' 3 F
10.1 + 1.7 23.2 + 2.4 4 M Lllng et al. 19B1

10 F
67.75 + 29! 12 Lerna I tra at II I. 19BI11
• on basis of first morning urine sample

! on basis of II 24-h urine collection
* range
Table 3. Melatonin Content in Human CSF

(All samples except those Indicated otherwise were obtained by lumber puncture)
Daytime NI ghttlme SUbJect Reference

X.!. SE pg/ml X.!. SE pg/ml No. Sex
GC/MS:
55 - 80* --- 2 Wilson et al. 1977
RAD IOIMMUNOASSAY:
59 + :nt 8 M Arendt et al. 1977a

57! 28t 7 F
<10 - 30* 10 M Vaughan et al. 197Bb

<10 - 23* 5 F
1.7 - 68.7* 12 M Brown et al. 1979

2.2 - 25.2* 7 F
<2.5 - 41.8*. 15 M 19
1.6 - 35.7*·:1: 11 F
39 + 15t 3 Smith et al. 1979
64 + 11.5**
~
4 Tan and Khoo 1981
41 + 4.6** 4
t SO
* range
** CSF obta I ned by I umbar puncture
* CSF obtained by cisternal puncture
** CSF obtained by ventricular cailicles?

during the daytime (Table III). These levels are apparently simi-
lar to (Vaughan et al., 1978b) or slightly lower than (Arendt et
al., 1977aj Tan and Khoo, 1981) those in blood. In an early study
(Smith et al., 1976a) based on samples from children with leuke-
mia, the opposite conclusion was drawn: serum melatonin levels
averaged 14 pg/ml while melatonin concentrations in CSF samples
drawn simultaneously averaged 98 pg/ml.
B. Rhythms in Melatonin Concentrations
1. Day-night rhythm: Day-night rhythms in melatonin levels

of human blood (Table I) and urine (Table II) have been
demonstrated by all investigators who have looked for them.
Highest blood levels have been noted in samples taken between mid-
night and 2 AM (Arendt et al., 1977a; Smith et al., 1977), and
lowest levels between noon and 2 PM (Vaughan et al., 1978a).
Expressed in cosinor terminology, the acrophase of the melatonin
rhythm in nine healthy subjects was -357 0 from mid-sleep, and its
amplitude was 65% from the mesor (Scheving et al., 1981). Similar
results were reported by Birkeland et al. (1980) and Fevre-Montange
et al. (1981). Tbe physiologic basis of the day-night rhythm in
melatonin secretion was originally thought to be an endogenous
oscillator, whose effects could be amplified or diminished by
light and darkness but didn't completely depend on the lighting
environment. Consistent with this hypothesis, Vaughan et al.
(1976) found that a plasma melatonin rhythm persisted among sub-
jects kept under continuous illumination (12 ft-candles) for 60 h.
Jimerson et al. (1977) and Akerstedt et al. (1979) also described
persistent urinary melatonin rhythms among subjects exposed to
continuous illumination. Furthermore, some investigators have
found a clear dissociation between the time of onset of darkness
and the time of melatonin secretion: Arendt et al. (1977a) and
others (Vaughan et al., 1978a) observed that melatonin secretion
can precede dark onset, While Weinberg et al. (1979) observed a
considerable delay between dark onset and the nocturnal rise in
melatonin secretion. If light or darkness per se did not trigger
the day-night changes in melatonin secretio~what then did?
Apparently not sleep stage (Vaughan et al., 1978a), even though,
in one very preliminary study (Sizonenko et al., 1979), a correla-
tion was noted between the nocturnal plasma melatonin peak and the
number of preceding REM sleep periods.
More recent observations have again focused on light as the

phase-setter for the melatonin rhythm. Lynch et al. (1978b) found
that an artificial phase shift of 180 0 in the light/dark regime
did cause a corresponding shift in plasma and urinary melatonin
rhythms after 5-7 days. A similar delay was observed after a
natural phase shift, i.e., a transatlantic flight (Fevre-Montange

et al., 1981; Wetterberg, 1978). Blindness was also found to
affect melatonin secretion from the human pineal: among two of four
blind subjects studied by Lynch et al. (1975a), highest urinary
melatonin levels were found in the morning or at noon. Serum
melatonin contents in four blind subjects sampled at nighttime
(11 PM) and during the day (2 PM) were higher at daytime (Smith &
O'Hara, 1981). This may indicate a free-running diurnal melatonin
cycle in blind humans.
Most compelling, Lewy et al. (1980a) found that light of

sufficient intensity could cause serum melatonin levels to fall
rapidly to daytime levels, among normal volunteers awakened at 2
AM and then exposed to light for 2 hours. If subjects were then
returned to darkness, melatonin levels again rose within 30 minutes.
A light intensity of 500 lux had no effect on melatonin levels,
while light intensity of 2500 lux was extremely effective (2500
lux is about 3 times the intensity normally present in an artifi-
cially lit office, but only 3-5% of the intensity present out of
doors on a sunny day.)
Hence the only difference between humans and the laboratory

rodents usually used as experimental animals, in terms of pineal
responses, may be their different sensitivities to light. In
retrospect it should not be surprising, that humans -- who normally
work outside in daylight -- are less sensitive to light than
albino rats -- which normally spend daylight hours sleeping in a
completely dark burrow.
2) Menstrual rhythms: Wetterberg et al. (1976) measured

morning serum melatonin levels in five healthy women at 2-3 day
intervals. Melatonin was lowest at the time of ovulation,
increased during the following days, and -- after a small decrease
during the late follicular phase -- exhibited a second peak at the
time of menstruation. A 4.5-fold increase was found between ovu-
lation and menstruation. Later, using a more specific antibody,
Arendt (1978a) confirmed this relationship.
3) Annual rhythms: By estimating morning (8 AM) serum mela-

tonin levels monthly for one year, Arendt et al. (1977b) found a
significant seasonal variation in melatonin among five men, with
peak values in winter and summer and lower values in spring and
autumn. In a later study (Arendt et al., 1979) the same pattern
was observed among females, using either midday or midnight mela-
tonin levels. The latter finding was taken as evidence that the
annual rhythm is real, and not simply due to a shift in the day-
night rhythm. Wetterberg et al. (1981b) confirmed this interpre-
tation by showing no seasonal alteration in the acrophase of the
24-hour urinary melatonin excretion among 13 normal volunteers.
4) Pulsatile secretion: Three groups of investigators

(Vaughan et al., 1978aj Weitzman et al., 1978; Mullen et al., 1981)
have sampled melatonin levels in blood with sufficient frequency
(i.e., at intervals of 30 minutes or less) to look for - and affirm -
pulsatile secretion of the hormone (like LH, prolactin, and ACTH).
All of the groups observed evidence for such pulses and concluded
that this pattern is superimposed on the day-night melatonin
secretory rhythm. The frequency of pulses appears to be about one
per hour (Weinberg et al., 1979), and their amplitude sufficient
to raise plasma levels by as much as 200%. In addition to these
hourly pulses, melatonin may also be secreted in even more frequent
pulses: when Vaughan et al. (1979b, 1979a) measured melatonin
levels in blood samples at intervals of 2.5 min, they observed
nighttime secretory bursts with frequencies of 8.25 and 12.14
minutes, and amplitudes of 35 and 33% (nadir to peak). These
amplitudes are not much greater than melatonin's usual interassay
variancej thus the bursts, if real, aren't very robust. Arendt et
al., 1982, measuring daytime serum levels every 60 minutes, did
not find evidence for pulsatile secretion. This is in agreement
with preliminary observations by the present authors who could not
find evidence for melatonin pulses in 7 male volunteers sampled
every 30 minutes between 7 AM and 11 AM.
C. Changes With Age
That age in general, and the stage of puberty in particular,

are factors which influence pineal activity has been suggested
since the first publications concerning the pineal (Marburg, 1909j
Kitay & Altschule, 1954).
Several studies have addressed the question of changes in

melatonin levels with puberty. Arendt (1978a) estimated serum
midday and midnight levels in six prepubertal and three pubertal
children: In all nine, noontime levels were less than 14 pgj in
the postpubertal children midnight levels averaged 128 pg/m1, and
in the prepubertal subjects, 162 pg/ml. Thus, in this small
sample no significant differences in melatonin levels were noted.
Lenko et al. (1982) recorded daytime plasma and 24-hour urinary
melatonin values among children aged 7 years and more. Plasma
levels (n=116) did not change with pubertal stage. Urine levels
(n=43) averaged 42.1 ng/24 h/m 2 for prepubertal and 30.5 ng/24
h/m 2 for pubertal children (Tanner Stage III - V). These did not
differ significantly. Similar results were obtained by Willig et
al. (1979), who measured daytime and nighttime plasma melatonin
levels.
Opposite results were recorded by other groups. Silman et

al. (1979) measured daytime serum melatonin levels by GC/MS in a
school class of 51 healthy boys and girls aged 111/2 -14 years.
Melatonin concentrations in girls ranged from less than 5 to 280
pg/ml, and didn't change with puberty. However, levels in boys
ranged from less than 10 to 2,300 pg/ml, and were significantly
higher during Tanner Stage I than during other stages. Waldhauser
et al. (1981) performed a cross-sectional study throughout
childhood by measuring daytime and nighttime melatonin levels in
101 children and 25 adults. They found highest concentrations in
the group aged 1-3 years and a tendency to decreasing values in
both daytime and nighttime during childhood. Melatonin levels
were lower in infancy than at ages 2-6. Lemaitre et al. (198lb),
who estimated morning blood samples in 26 male infants, found
undetectable or low values during the first 3 months of life and
thereafter values up to 1508 pg/ml.
These data suggest that melatonin levels exhibit an inverse

pattern to that of LH, which is high during the first months of
life and then suppressed until the onset of puberty (Winter et
al.,1975).
The view that melatonin production is elevated in children is

supported by observations on urinary melatonin and 6 OH-melatonin,
its main metabolite. Among 58 male subjects, Lemaitre et al.
(1981a) found significantly higher melatonin excretions among
children aged 4-15 years than in adults. Tetsuo et al. (1981),
measuring 6 OH-melatonin by GC/MS in 101 children and 20 adults,
observed that the total quantities of 6 OH-melatonin excreted per
day were similar in children and adults. Unfortunately, results
were not analyzed with regard to the body weights of the children.
However, if small children excrete the same amount of 6 OH-
melatonin as adults, it can be assumed that relative excretions
per bodyweight are greater in the children.
The available results are conflicting. However, it should be

noted that in all studies except one involving a large subject
population studied throughout childhood, decreases of serum and
urinary melatonin were observed during childhood. Even when dif-
ferent RIA and GC/MS methods were used a wide range of serum mela-
tonin values was noted, from undetectable levels up to more than
2000 pg/ml.
The extent to which increasing pineal calcification during

aging affects melatonin secretion is uncertain. Increasing age
was not associated with changes in the activity of various pineal
enzymes (Wurtman et al., 1964c), but was associated with reduced
melatonin concentration in CSF (Brown et al., 1979), and blood
(Touitou et al., 1981).
D. Effects of Various Treatments
1) Stimulation: It would be very useful to identify an

innocuous treatment capable of stimulating melatonin secretion in
humans. Such a treatment could serve as the basis of a pineal
function test. Since the pineal is controlled by norepinephrine
released from its sympathetic nerves and since administation of
L-Dopa, a norepinephrine precursor, increases melatonin synthesis
in rats (Deguchi & Axelrod, 1972; Lynch et al., 1973) L-Dopa seemed
a reasonable candidate as a pineal stimulator. Unfortunately,
L-Dopa was without effect on melatonin secretion in humans
(Arendt, 1978a; Wetterberg, 1978; Moore et al., 1979; Vaughan et
a1., 1979c). Infusions of B-receptor agonists like isoproterenol
(Vaughan et al., 1976) or orciprenaline (Moore et al., 1979) also
failed to increase melatonin secretion. One possible explanation
for this lack of action, i.e., an inhibitory effect of daylight or
a diminished number of pineal-B-receptors during daytime, was
excluded by the observation that L-Dopa and orciprenaline given in
the evening also failed to increase blood melatonin levels (Moore
et al., 1979).
Psychosocial stress (Akerstedt et al., 1979) and stress due

to insulin administration (Vaughan et al., 1979c; Wetterberg,
1979), pneumoencephalography (Vaughan et al., 1978b), electrocon-
vulsive therapy (Wetterberg, 1978) or sprinting (Vaughan et al.,
1979c), also failed to elevate blood melatonin. However, Carr et
al. (1981) did find a 100-200% increase in blood levels among
seven healthy women after 1 h of exercise on a bicycle ergometer
during daytime. If this preliminary report can be confirmed it
might provide the basis for a pineal function test.
In very preliminary studies, such other neuroactive substances

as scopolamine (Vaughan et al., 1976), amphetamine, TRH, LHRH and
TRH, desaminocys-D-arg-vasopressin (Wetterberg, 1978; Wetterberg,
1979) also failed to affect melatonin secretion.
2) Suppression: As described above, bright light (2500 lux)

can suppress nocturnal melatonin secretion in normal humans (Lewy
et al., 1980a). The nocturnal release of melatonin can also be
prevented by administration of propanolol, a B-receptor blocker
(Vaughan et al., 1976; Wetterberg 1978; Moore et al., 1979; Lewy
et al., 1981). This effect can be produced by as little as 40 mg
propanolol, given p.o. at 8 PM (Moore et al., 1979). It is not
clear why B-receptor blockers prevent nocturnal melatonin secre-
tion while B-receptor agonists fail to elicit its secretion in the
daytime. Perhaps a particular B-1 or B-2 receptor is involved.
Preliminary reports suggest that clonidine (Lewy et al.,

1981) and dexamethasone (Wetterberg, 1979) can inhibit melatonin
secretion.
III. MELATONIN LEVELS ASSOCIATED WITH DISEASE STATES
In the last few years much speculation has been offered

suggesting relationships between melatonin secretion and various
disease states. Unfortunately, very little supporting evidence
seems to be available.
A. Neurological Diseases
To our knowledge, melatonin secretory patterns have been

described in seven patients with pineal tumors, unfortunately most
with inadequate clinical and histological data (Barber et al.,
1978; Arendt, 1978b; Tapp, 1978; Kennaway et al., 1979; Vaughan
et al., 1979c). Kennaway et al. (1979) described two males, one
with a histologically unidentified pineal tumor, the other with a
pineoblastoma. Both had undetectable serum melatonin levels on
several occasions. Arendt (1978b) described two males, one with
low and the other with unusually high melatonin levels. Interest-
ingly, Barber et al. (1978) found elevated serum melatonin levels
in one patient whose pinealoma showed much lower HIOMT activity
than pineal tissue itself. A similar case with a pineal germinoma
associated with high serum melatonin levels was reported by Tapp
(1978). Thus it seems possible that high .or low serum melatonin
levels can occur in association with pineal tumors, and perhaps
circumstances are more complex than Marburg (1909) and Kitay &
Altschule (1954) supposed in suggesting that parenchymatous pineal
tumors produce an excess of an antigonadal hormone, while pineal-
destroying tumors cause a lack of the hormone.
Kneisley et al. (1978) demonstrated abolition of the day-night

urinary melatonin rhythm among six patients with clinical evidence
of transected cervical spinal cords. This constitutes evidence
that similar neural pathways mediate the central control of pineal
function in humans and rats.
B. Psychiatric Diseases
Since depression and perhaps other psychiatric diseases can

be associated with alterations in neuroendocrine rhythmicity
(Bunney et al., 1977) and since these disorders often have a
seasonal pattern of exacerbations and remissions, it is not
surprising that psychiatrists have become interested in examining
disease-related rhythms in melatonin secretion.
Wetterberg et al. (1979) described a single depressed woman

in whom nocturnal melatonin levels were lower and cortisol levels
higher during depression than during remission. Later similar
patterns were found in four out of twelve depressive patients
(Wetterberg et al., 1981a), and in a number of schizophrenics
(Ferrier et al., 1982). Mendlewicz et ale (1979) described dimin-

ished circadian melatonin rhythms in four depressed patients,
during both the depressive and recovery phases.
Lewy et ale (1979) described significantly higher melatonin

levels in four manic patients than in healthy controls. However,
possible effects of medication were apparently not ruled out and
may have been involved: chlorpromazine has been reported to ele-
vate melatonin levels in rats (Ozaki et al., 1976) and humans
(Smith et al., 1979) (by impairing its hepatic metabolism), and
certain antidepressive drugs have similar effects in rats
(Wirz-Justice et al., 1980).
Very recently Lewy et ale (1981) proposed that depressive

patients may be considerably more sensitive than normal controls
to the photic inhibition of nocturnal melatonin secretion. Failure
of other investigators to take light intensities into con-
sideration may explain why, for example, Jimerson et ale (1977)
found no difference in melatonin excretion between controls and
depressive patients. At this point, perhaps the best lead relating
depression to melatonin and pineal function seems to be the possi-
bility of altered (i.e., enhanced) light sensitivity. It will be
important to determine how general this alteration is, and whether
or-not its presence is correlated with such other neuroendocrine
disturbances as abnormal dexamethasone-suppression tests.
C. Carcinoma
In several animal models, melatonin administration reportedly
diminished the growth of tumors and their metastases (Lapin, 1979;
Karmali et al., 1978). Cohen et ale (1978b) proposed that dimin-
ished melatonin production might be a factor in the etiology of
breast cancer. And the same group (Tamarkin et al., 1982) sub-
sequently reported lower nocturnal melatonin levels among 10 women
with estrogen-receptor-positive breast cancers than among control
subjects, or women whose cancers lacked estrogen receptors. Tapp
et ale (1980) found no differences in serum melatonin levels among
patients with benign or malignant tumors.
D. Other Disorders
Birau & Schloot (1979) reported plasma melatonin levels to be

elevated in spina bifida and sarcoidosis and diminished in
psoriasis vulgaris, Turner's Syndrome and Klinefelter's Syndrome.
IV. EFFECTS OF EXOGENOUS MELATONIN ON HUMANS
A. Physiological Disposition
The fate of small doses of exogenous melatonin has been

studied in rats (Wurtman et al., 1968). The indole enters all
tissues, especially the pineal and certain endocrine organs
(Wurtman et al., 1964b); it disappears rapidly from the circulation
because of its metabolism within the liver (by 6-hydroxylation
followed by conjugation); its major urinary metabolites are thus
6-hydroxymelatonin sulfate and glucuronide (Kopin et al., 1961).
Few if any data are available on the fate of exogenous mela-

tonin in humans, and, to our knowledge, none of the disposition of
large doses such as might be useful to produce pharmacological and
behavioral effects. We have thus initiated studies on the levels
of melatonin in serum before and after subjects receive single
large (80 mg) oral doses of melatonin, by capsule, at 11:00 AM.
Among the three young male subjects described in Fig. 1, daytime
serum melatonin levels rose after one hour to levels 500 to 8000-
fold higher then peak nocturnal values in untreated subjects. The
hormone subsequently disappeared from the circulation with an
apparent half-life of 45-60 minutes. Among individual subjects,
those with highest peak serum melatonin levels also had greatest
urinary secretions. In all cases less than 0.5% of the administered
melatonin was present unchanged in urine samples collected during
the next 18 hours. Among our three subjects receiving the same
melatonin dose, we observed at least 20-fold differences in peak
serum melatonin levels. The sources of this variability await
characterization. It will b~ interesting to determine whether
people with highest nocturnal serum melatonin levels do or do not
also attain highest peak levels after melatonin administration.
B. Pharmacological Effects
Published reports describe approximately 100 subjects who

have received melatonin (Lerner & Nordlund, 1978). Its only con-
sistent effect (among those sought) in the doses given has been to
cause mild sedation and analgesia, probably by acting on the CNS
(Cramer et al., 1974; Wetterberg, 1977; Vollrath et a1., 1981).
No effect, or in some cases an exacerbation of clinical findings,
has been observed after its administration to people with schi-
zophrenia (Lerner & Nordlund, 1978), parkinsonism (Papavasilion
et at., 1972), and depression (Carman et at., 1976). Its
influence on epi1ipsy is not clear, but justifies further explora-
tion (Anton-Tay et al., 1971).
Very few studies have been performed on melatonin's gonadal

effects in humans. Apparently melatonin does not suppress gonado-
100 1000
10 100
E
"-
CJ"I
C
c
c
.2
o
~ 0.1
E ~
::J
o
c
-MS.
-~-- R.T
-.- D. f'
001
8 12 4 8 12 4 8 12 4 8
AM Nooo PM
Clock Hours
Fig. 1. Effect of exogenous melatonin on serum and urinary mela-

tonin levels. Three young male subjects provided serum
and urine samples for 15 hours before, and 22 hours
after, receiving melatonin-containing capsules (80 mg)
orally at 11:00 AM. Subjects were exposed to darkness
between 10 PM and 7 AM. Horizontal bars represent total
mealtonin present in urine samples collected during a 3
or 6 hour interval, divided by the number of hours in the
collection period.
tropin secretion caused by giving LHRH (Fideloff et al., 1976;

Weinberg et al., 1980); gonadotropin values in people receiving
only melatonin were reported to be unaffected (Fideloff et al.,
1976) or blunted (Nordlund & Lerner, 1977). Acute melatonin admi-
nistration reportedly reduces the rise in plasma growth hormone
after insulin (Smythe & Lazarus, 1974) or L-tryptophan (Kolu &
Lammintausta, 1979) administration. Melatonin did not modify the
increase in plasma growth hormone after apomorphine or L-Dopa
(Kolu & Lammintausta, 1979).
No side effects of melatonin have been described except for

mild headache or abdominal cramps, even when as much as 6.6 g were
administered orally each day for 35 days (Lerner & Nordlund,
1978). Melatonin is thus probably a highly non-toxic substance,
and deserves considerable further study in humans. In mice even
800 mg/kg failed to cause death; the LD50 could not be estimated
because solubility limitations precluded testing higher doses
(Barchas et al., 1967).
C. Behavioral Effects of Exogenous Melatonin
Several published studies suggest that melatonin's secretion

may have significant behavioral sequence: In man, melatonin
reportedly acts as a potent sleep-inducing agent. Thus, Cramer
et al. (1974) found that administration of 50 mg (I.V.) of mela-
tonin in the daytime induced sleep within 15-40 minutes, which
lasted 26-60 minutes, reachng stages 3 and 4. When administered
at night, 50 mg of melatonin (I.V.) decreased sleep onset time
significantly, but did not alter total sleep time nor time spent
in particular sleep stages. They also reported that melatonin
administration may have decreased anxiety in their more anxious
patients. In a more recent study, Vollrath et al. (1981) observed
similar effects of a small intranasal dose of melatonin (1.7 mg)
on healthy volunteers. Most of their subjects (70%) fell asleep
within 40-60 minutes, and others reported feelings of mild seda-
tion or mild tiredness. All subjects but one, who described
depression, reported feelings of well-being and felt emotionally
well-balanced after melatonin-induced sleep. The potent effect of
such a small dose of melatonin was attributed to its intranasal
route of administration. Weinberg et al. (1980) found that small
amounts of slowly-infused melatonin (30 ~g/min, total dose of 7.2
mg) had no readily observed behavioral effect. This study should
not be considered as contradicting the Cramer et al. (1974) and
Vollrath et al. (1981) studies in light of its lower doses and
very slow rate of infusion.
Related investigations carried out on rats suggest that mela-

tonin can reduce certain behavioral concomitants of emotional
stress. For example, melatonin administration facilitates extinc-
tien in a passive shock avoidance paradigm, and decreases defeca-

tion induced by this aversive situation (Datta and King, 1977).
Golus et al. (1979) reported that melatonin significantly atten-
tuated the normal fear responses of rats given a novel drinking
solution. Melatonin also ,significantly increased open field acti-
vity in rats (Golus and King, 1981). These observations are con-
sistent with recently-obtained biochemical evidence suggesting
that melatonin itself, or one of its brain metabolites, may be an
endogenous ligand for benzodiazepine receptors (Marangos et al.,
1981).
Since melatonin release occurs primarily at night, its beha-

vioral effects should be considered with respect to this fact:
perhaps the pineal, by secreting melatonin, helps to synchronize
certain diurnal behavioral rhythms to changes in the external
lighting environment. It can be speculated that cyclically-
secreted melatonin may act on the human brain to modulate beha-
viors that vary with 24-hour cycles. Since the nocturnal release
of melatonin in humans is immediately suppresed by exposing them
to bright light (Lewy et al., 1980), it is clear that environmental
lighting also controls melatonin secretion in humans.
We are examining the hypothesis that melatonin may synchronize

central human behavioral rhythms to diurnal changes in lighting,
specifically by reducing levels of arousal, general responsiveness,
and sensitivity to the environment, at night. The behavioral
studies cited above, which indicate that melatonin can induce
sleep and reduce anxiety, would tend to support this hypothesized
role. We are thus studying the effects of exogenous melatonin on
numerous indices of behavioral and perceptual responsiveness, many
of which are known to vary diurnally in man (Colquhoun, 1971).
We administer oral melatonin (80 mg in 1 or 3 doses) using a

double-blind placebo-controlled crossover design, to healthy males
aged 18-36. The tests we have selected measure sensation, motor
performance, mood and memory. To evaluate sensory sensitivity,
visual critical flicker fusion threshold (CFF) is measured. CFF is
known to vary diurnally (Musumeci and Misiak, 1974). Performance
is assessed using a variety of different tasks. Wilkinson 4-choice
simple reaction time (RT) and simple auditory RT are not difficult
tasks but require sustained attention for optimal performance.
The Digit Symbol Substitution Te'st (DSST) is more intellectually
demanding, but lasts for only 90 seconds. Finally, the Grooved
Pegboard Test requires sustained performance and considerable
motor coordination.
Mood is assessed using 3 self-reporting questionnaires. The

Profile of Mood States (POMS) yields 6 factor analytically-derived
scales: tension, depression, anger, vigor, fatigue and confusion.
The Visual Analog Mood Scale (VAMS) yields 3 scales: vigor, calm,
and sad. The Stanford Sleepiness Scale (SSS) quantifies the

progressive steps in sleepiness. Finally, memory, which also
varies diurnally (Colquhoun, 1971) is measured via a category list
recall test. In addition, we are monitoring core temperature and
collecting blood and urine so that we can relate melatonin levels
to any effects we may observe.
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Wetterberg, L., Arendt, J., Paunier, L., Sizonenko, P. C., van
Donselaar, W., and Heyden, T., 1976, Human serum melatonin
changes during the menstrual cycle, J. Clin. Endocrinol.
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Wetterberg, L., Beck-Friis, J., Aperia, B., and Petterson, U.,

1979, Melatonin/cortisol ratio in depression, Lancet ii, 1361.
Wetterberg, L., Aperia, B., Beck-Friis, J., Kjellman, B. F.,
Ljunggren, J. G., Petterson, U., Sjolin, A., Tham, A., and
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Regulation of the Brain," K. Fuxe,J. A. Gustafson, and L.
Wetterberg, eds., Pergamon Press, Oxford.
Wetterberg, L., Halberg, F., Haus, E., Kawasaki, T., Uezono, K.,
Ueno, M., and Oma, T., 1981b, Circadian rhythmic urinary
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Wetterberg, L., Aperia, B., Beck-Friss, J., Kjellman, B. F.,
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Circadian variation in urinary melatonin in clinically
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Reyes, F. I., 1975, Pituitary-gonadal relations in infancy.
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Endocrinol. Metab., 40:545-551.
Wirz-Justice, A., Arendt, J., and Marston, A., 1980, Antidepressant
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MELATONIN AS A CHRONOBIOLOGICAL MARKER IN HEALTH AND DISEASE
Lennart Wetterberg
Karolinska Institute, Department of Psychiatry

St Goran~s Hospital, Box 125 00
S-112 81 Stockholm, Sweden
INTRODUCTION
The psychiatric literature has reported possible relationship

between mental illness and time of day, season of birth and even
lunar cycle. Many psychiatric patients e.g. with affective disord-
ers cycle with more or less regularity. The modern life style has
led scientists to study body rhythms in shift-workers. air-tra-
vellers across time zones and astronauts. The influence of rapid
shifts in light-dark cycles and magnetic fields, social isolations
and even weightlessness has been examined. In order to understand
the effects of these changes there is a need for new markers for
endogenous rhythms. Hormonal variations may be associated with
boredom, withdrawl and irritability in healthy persons. Desynchro-
nization, upsetting the circadian, circaannual and other rhythms
may also cause fatigue, decreased performance, insomnia, anxiety,
depression and other psychiatric and somatic symptoms.
The objective of this review is to summarize the results of

the recent and ongoing studies in which the pineal gland hormone
melatonin has been used as a chronobiological marker in health
and disease in man. Melatonin (5-methoxy-N-acetyl-tryptamine) is
synthesized from tryptophan via 5-hydroxytryptophan, serotonin
and acetyl serotonin and this pathway is illustrated in Fig. 1.
Measurement of melatonin
The current status of assay methods of melatonin was recently

summarized by Arendt (1981). In her review tadpole bioassay, gas-
chromatography-mass spectrometry and radioimmunoassay (RIA) were
575
576 L. WETTERBERG
HO -cc:r
~
~
I
~2~
I I c: - ~ - NH2
C.O
I
OH
- O:T
~
~ I I
N
H
~~
C-C-NH2
~.O
I
OH
H
5 - Hydroxy tryptophan Tryptophan
! H2 H2
HO~C-~-"'2
I
H
Serotonin
I I
H H
N -Acetylserotonln Melatonin
Fig. 1. Metabolic pathway of melatonin from tryptophan
discussed. Several RIA~ethods were compared in an international

collaborative study (Wetterberg and Eriksson, 1981). Most of the
12 participating laboratories reported comparable results. The
most sensitive and specific melatonin antibody reported so far has
been used in more than 30 studies and is now commercially available
in a RIA melatonin kit (WHB, Box 19018, S-16l42 Bromma, Sweden).
Using this assay melatonin has so far been found in amniotic fluid
cerebrospinal fluid, serum, plasma, lymph, saliva, tears, urine
and pineal gland tissue (Wetterberg, 1979).
Melatonin as a marker for adrenergic B-receptor blockade and

stimulation
Melatonin in serum or plasma and urine from healthy controls

on a regular sleep-dark cycle displays a diurnal rhythm with the
l~west concentrations during the day and peak levels during the
night. This nocturnal peak of melatonin occurs in most people
around 02 hr when they are sleeping in the dark (Fig. 2).
MELATONIN AS A CHRONOBIOLOGICAL MARKER 577
FEMALE 26y
M.I~tonin
nM
0.6
0.3
08 12 16 20 24 04 08
Tim. (hr)
Fig. 2. Serum melatonin levels in a person with normal night/day

pattern studied twice, one month apart. The woman was
sleeping in the dark between 23 and 07 hr. The dotted
horizontal line indicates the low normal range of nocturnal
peak level of serum melatonin.
The human pineal is thought to be innervated by sympathetic

neurons. Melatonin in serum decreases following treatment with
adrenergic S-receptor blocking agents (Vaughan et al., 1976;
Hanssen et al., 1980; Moore et al., 1979). This blockade occurs
even at moderate doses of 60 mg propranolol per 24 hr which in-
dicate that melatonin production in humans may also be under nor-
adrenergic control.
However, administration of adrenergic S-receptor-stimulating

agents have not been effective in stimulating melatonin produc-
tion (Vaughan et al., 1976; Moore et al., 1979). A shortcoming
in the human studies of the pineal function is that there is,
as yet no reliable methodology for a stimulatory test of the me-
latonin production.
Effect of light on melatonin ~n healthy humans
In an early report by Wetterberg (1978) bright light from

day light tubes suppressed blood melatonin at night (Fig. 3),
although short-time (30 min) exposure of a 40 W light bulb did
not cause a significant decrease.
Urinary excretion of melatonin was measured during 64 hours

of sleep deprivation in 12 healthy males when the subjects were
exposed to artificial light in the laboratory. The light was kept
578 L. WETTERBERG
nM
.50
.40
.30
.20
.10
20 22 24 02 04 06 08 hours
Fig. 3. Plasma melatonin concentration in a 25 year old

healthy male during the night sleeping in dark-
ness (. .) and awake in bright light
(0 - - - 0). (From Wetterberg, 1978).
constant and consisted of five light bulbs in the range 40-100 W

(Akerstedt et al., 1979). In this experiment the 'urinary excre-
tion of melatonin followed a circadian pattern even though the
subjects were exposed to light of the same intensity which rapid-
ly suppressed melatonin in rat serum and pineal gland (Illnerova
et al., 1978). The practical conclusion of these studies for blood
sampling of melatonin at night is that dim light does not supp-
ress melatonin ip humans but bright light should be avoided when
the physiological status is examined.
Humans are however under certain conditions sens~t~ve to

alteration of the light/dark cycle. In four healthy subjects ex-
posed to an extended dark period of four hours in the morning
for ten days the melatonin rhythm was shifted with peak levels
later in the night (Wetterberg, 1978). This result was inter-
preted "that the rhythm does not seem to be regulated only by the
time of onset of darkness and sleep but rather dependent on the
total length of the period of darkness".
In a series of experiments Lewy et ale (1980) further studied

the effects of bright light and confirmed its suppressive effect
in healthy humans. Although the qualitative effect of light on
serum melatonin in human is similar to that of other mammals, in-
cluding monkeys, the quantitative difference is marked (Reppert
et al., 1981). The results of these experiments indicate that the
differences between man and other mammals may be due to the fact
that man has been exposed to artifical light at night for many
hundred thousand years through this use of fire. The important
difference in sensitivity to light between man, and animal however,
has been revealed using melatonin as a marker for circadian rhythms.
Melatonin as a marker for circadian rhythm disorder m affective

disease.
Halberg (1968) proposed that manic-depressive cycles could

result from desynchronization of two biological clock systems.
Experimental shifts in the timing of sleep cause dysphoric mood
and poor performance (Kripke et al., 1970). A circadian covaria-
tion of fatigue and urinary melatonin has been reported by Aker-
stedt et ale (1982). In 12 healthy male subjects, vigilance per-
formance and self-rated sleepiness showed a correlation to melato-
nin. High levels of the hormone was associated with reduced per-
formance and increased sleepiness. The relation between melatonin
and fatigue was comparable with the suggestion that the pineal
hormone might be involved in the physiological sleep-wake regula-
tion.
The occurrence of mood change and disturbed 24 hr rhythm

in depressed patients (Sachar et al., 1973) has prompted studies
of melatonin secretion in affective disorders. Both the hypothes-
is of a low S-adrenergic activity and estimations of time shift
for the evaluation of the phase advance hypothesis in depression
(Wehr et al., 1982) may be tested using melatonin as a marker for
amplitude and rhythm.
A possible relationship between high cortisol levels found

in many patients with depression (Gibbons, 1964; Sachar et al.,
1970) and melatonin has recently been reported (Beck-Friis et al.,
1981; Wetterberg et al., 1979a; 1981a; 1982a,b,c,d). The hypo-
thesis that the dexamethasone non-suppressors may have a dysfunc-
tion in the pineal gland was also investigated. These studies com-
prise so far more than 50 patients with affective disorder and
30 healthy control subjects.
Melatonin and cortisol was assayed in blood samples drawn

every four hr between 08 - 20 hr and every two hr during the
night. The patients were sleeping in the dark between 23 and 07
hr. A dexamethasone suppression test (DST) was included in the
580 L. WETTERBERG
Cortisol
nM o Melatonin
nM pg/ml
400 0.40
75
200 0.20 50
25
Control 05T- 05T· Uni Bi

(22) (15) (17) (12) (12)
CIM 0.65 0.86 2.11 1.38 1.71
Ratio !0.17 !0.t9 !0.38 !0.43 !0.71
Fig. 4. Cortisol (C) and melatonin (M) in serum at 02 hr

(mean ± SEM) in apparently healthy controls and
patients with acute major depressive disorder.
DST - = normal and DST + = abnormal dexamethasone
suppression test. Uni = unipolar and Bi = bipolar
affective disorder. For details see Wetterberg
et al., 1982d.
test protocol using one mg of oral dexamethasone according to

Carroll (1982). The main results of the studies from our labora-
tory are summarized in Fig. 4 adapted from a recent report by
Wetterberg et ale (1982d). The 32 patients with acute major
depressive disorder have been divided into two groups according
to their response to DST. It is seen that the patients with an
abnormal DST had significantly higher cortisol to melatonin
ratio than did the patients with normal DST. The 24 patients with
with unipolar and bipolar affective disorder in Fig. 4 all had
a severe disease history but were studied in a state free from
acute clinical manifestations. The patients had significantly
lower melatonin levels than the 22 healthy controls. Results
showing significantly higher hereditary factors for major de-
pressive disorders among probands with low melatonin levels 1n

serum compared to probands with higher levels points to the possi-
bility that low melatonin production may be associated with the
vulnerability of depression (Wetterberg et al., 1982d). Melatonin
may thus be a genetic trait marker of the gene for affective dis-
order. The identification of persons at risk for depression may be
helpful in prophylaxis and choice of therapy.
Lewy et al. (1981) reported that manic-depressive patients

might be supersensitive to light and Kripke (1981) used bright
early morning light to palliate depressive symptom. Weller and
Jauhar (1981) found that changing of time zone by two hr or more
cause a significant excess of affectively ill persons compared to
passengers flying within the same time zone. No such correlation
was found for schizophrenic break-downs. It will be important to
further study the melatonin levels in persons at risk for depres-
sion. The rapid change of circadian light ratio between day/night
in the spring and fall in several regions have been associated
with depressive disorders (see Parker and Walter, 1982). These
authors have also discussed a hypothetical link between depression
and a pineal gland dysfunction.
Urinary melatonin rhythms have been studied in depressed pa-

tients and normals during sleep deprivation which is used as treat-
ment for acute depressive episodes (Jimerson et al., 1977). Lynch
et al. (1978) used melatonin measurement as marker for entrain-
ment of rhythmic secretion in man in an experiment when the light-
dark cycle was shifted 12 hr in phase.
Melatonin and pituitary and pineal tumours
A relationship between melatonin and pituitary insufficiencies

has been reported by Werner et al. (1981) who found that six pa-
tients with pituitary tumours had low melatonin levels in serum.
In ten patients with Cushing's disease six had low melatonin at
02 hr. This could possibly be interpreted as indicating a subgroup
of patients with Cushing's disease who have a suprapituitary defect
as part of their dysfunction. Brismar et al. (1982) have proposed
that Cushing's disease, in some cases, might possibly be associat-
ed with a defect in the pineal gland. Such a defect has been hypo-
thesized to be a decrease of a corticotropin-releasing factor-
inhibiting factor (CRF-IF) which in its turn may be related to
melatonin (Wetterberg et al., 1981a).
Young (1981) has reported abnormally high melatonin levels

in three of four patients with Cushing's disease. Obviously, more
studies with \arger samples of patients are needed to clarify the
possible relationship between the pineal and pituitary glands in
Cushing's disease.
582 L.VVETTERBERG
Melatonin may be a marker for melatonin-producing tumours in

the pineal gland but it is also clear that some pinealoma do not
have excess production of melatonin (Arendt, 1978; Barbet et al.,
1978; Tapp, 1978, Vaughan et al., 1978; Wetterberg, 1979).
Melatonin and breast cancer
A possible relationship between breast cancer and melatonin

has been discussed by Cohen et al. (1978) and Lapin (1978). Pineal
extracts reportedly counteracts malignancy and pinealectomy in
rats is associated with accelerated tumour growth. In a study by
Wetterberg et al. (1979b) testing the possible association between
melatonin levels and risk for breast cancer it was found that
Japanese women had lower melatonin levels than American women,
although Japanese women are presumably at lower risk than white
Americans. The melatonin levels have to be followed in longitudi-
nal studies to test the hypothesis that the brake to carcinogenes-
1S is diagnosed as a case of chronopathology, a rhythm alteration.
Some indications that chronopathology indeed may occur in

breast cancer has been provided by Bartsch et al. (1981) who mea-
sured urinary melatonin in ten Indian women suffering from advanc-
ed stages of breast cancer and in nine controls. The 24 hr urinary
melatonin excretion in the cancer patients was on the average 31 %
decreased and the cancer patients showed a dyssynchronization of
their melatonin rhythm compared to controls.
A recent report by Tamarkin et al. (1982) showed decreased

plasma melatonin in patients with breast cancer. The authors sugg-
est that "low nocturnal melatonin concentrations may indicate the
presence of estrogen receptor positive breast cancer and could
conceivably have etiologic significance".
Monthly and yearly rhythms of melatonin
A melatonin rhythm over the menstrual cycle has been reported

(Wetterberg et al., 1976; Birau et al., 1981). However, Matthews
et al. (1981) did not confirm these changes using 6-hydroxymela-
tonin sulphate measurement of urinary excretion during a normal
menstrual cycle. More studies of both the phase and amplitude of
melatonin secretion are needed in relation to the menstrual cycle
in women.
Seasonal variations in the melatonin rhythm was reported by

Arendt et al. (1977) in healthy persons from middle Europe. The
circadian rhythmic urinary melatonin excretion over four seasons
in Kyushu was also studied in clinically healthy Japanese subjects
(Wetterberg et al., 1981b). In this report the average urine mela-
tonin exhibited remarkably stable acrophase in a group assessment
throughout the seasons.
Concluding comments
Melatonin as a marker in relation to reproduction has been

reviewed at this meeting by Reiter. The reader is also referred
to each original paper for methodological details and to earlier
reviews about the clinical aspects of the pineal gland (eg. Wet-
terberg, 1981a).
Although the normal function of melatonin is not yet known,

the hormone has, during the last few years, found its place as a
useful chronobiological marker. One reason for this is the pro-
nounced rhythmic day/night fashion in which it is produced.
Another is the close relationship between melatonin and the re-
gulation of the pituitary-adrenal axis, as well as its correlation
to behavioural states in man. Melatonin is an additional tool in
chronobiology which allows us to look into the neuropsycho-
endocrine window for causes of suprapituitary desynchronization,
a major disturbance in many psychiatric conditions.
Acknowledgements
This review ~s based upon studies supported by grants from

the Swedish Medical Research Council no 3371, Karolinska Institute
and St. Goransfonden for psykiatrisk forskning. The secretarial
assistance of Elsa Rylander is acknowledged.
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Wetterberg, L., Aperia, B., Beck.Friis, J., Kjellman, B.F.,
1junggren, J .-G., Petterson, U., Sjolin, A., Tham, A., and
Unden, F., 1982a, Hormonal changes in affective disorders,
in: Brain Peptides and Hormones, R. Collu, J.R. Ducharme,
A: Barbeau, G. Tolis, eds., p. 257, Raven Press, New York.
Ljunggren, J.-G., Nilsonne, A., Petterson, U., Tham, A.,
and Unden, F., 1982b, Melatonin and cortisol levels in
pschiatric illness, Lancet, July 10, 100.
Ljunggren, J .-G., Nilsonne" A., Petterson, U., Tham, A.,
and Unden, F., 1982c, The relationship between cortisol and
melatonin serum levels in patients with affective disorders
and in healthy subjects, in: Proceedings of XV Int.Conf. of
the Int. Soc. Chronobiol.~Minneapolis, Minn.
Wetterberg, L., Beck-Friis, J., Kjellman, B.F., and Ljunggren,
J.-G., 1982d, Circadian rhythms in melatonin and cortisol
secretion in depression, in: Frontiers in biochemical and
pharmacological research in depression, F. Sjoqvist, M.
Asberg, E. Usdin, eds., p. 000, Raven Press, New York.
Wetterberg, L., Beck~Friis, J., Aperia, B., and Petterson, U.,

1979a, Melatonin/cortisol ratio in depression, Lancet, ii, 1361.
Wetterberg, L., and Eriksson, 0., 1981, Melatonin in human serum -
A collaborative study of current radioimmunoassays, in:
Melatonin: Current status and perspectives, N. Birau-,-
W. Schloot, eds., p. 15, Pergamon Press Ltd, Oxford.
Wetterberg, L., Halberg, F., Haus, E., Kawasaki, T., Uezono, K.,
Ueno, M., and Omae, T., 1981b, Circadian rhythmic urinary
melatonin excretion in four seasons by clinically healthy
Japanese subjects in Kyushu, Chronobiologia, VIII: 188.
Wetterberg, L., Halberg, F., Tarquini, B., Cagnoni, M., Haus, E.,
Griffith, K., Kawasaki, T., Wallach, L.-A., Ueno, M.,
Uezo, K., Matsuoka, M., Kuzel, M., Halberg, E., and Omae,T.,
1979b, Circadian variation in urinary melatonin in clinically
healthy women in Japan and the United States of America,
Experientia, 35: 416.
Young, I.M., 1981, The pineal indole horrr.ones in Cushing's
disease and acromegaly, in: Pineal Function, C.D. Matthews,
R.F. Seamark, eds., p. 7~Elsevier/North-Holland, Biomedical
Press, Amsterdam.
INDEX
N-Acetylserotonin Adenohypophysis, effects of pineal

brain. differential hormones at 368-372
localization, 257-276 Adrenal-pineal interrelation-
binding studies, 268-271 ships, 487-493
immunoreactive, 260-271 ACTH secretion, 488, 490
cerebellar, 266-267 cortisol production, 489-490
effect of age, 265-266 melatonin, 489
hippocampal, development, pinealectomy, effects, 490-493
264-266 with adrenalectomy, 491-493
localization, 263-264 stress-inducing activation, 490
mapping, 260-262 Adrenalectomy
relationship to serotonin effect on pineal, 498-499
and melatonin, 262-263 and pinealectomy, effects, 490,
specific antisera studies, 491-493
259-260 Adrenergic blocking agents,
serum, 24-hour pattern, 243-256 a and S
development of antisera, control, 525-529
248-251 effect on melatonin circadian
radioimmunoassay, 246 rhythms, 5 (see aZso
regulation studies, 248, Circadian rhythms)
251-253 melatonin as marker, 576-577
specific antisera, regulation, 6-8 (see aZso
development, 248-251 N-Acetyltransferase
Na-p-carboxybenzyl, 250-251 and specific headings)
N-Acetyl-transferase activity, Adrenoglomerulotropin, 487
4-5, 305, 535, 536 Affective disease, melatonin as
adrenergic regulation, 6 marker, 579-581
and pineal S-adrenergic Age
receptor, 8-11 effect on melatonin metabolism.
serotonin, light and melatonin 235-237, 281, 558-559
rhythm, 221, 233, 234 and hippocampal immunoreactive
super sensitivity and N-acetylserotonin, 265-266
subsensitivity, 8-9 Amphetamine, effect on melatonin
24-hour period, 318-319 secretion, 560
ACTH, identification and Amphibians, neurosecretory cells,
characterization, 115, 88-92
122 investigation of types, 92
589
590 INDEX
Amphibians, neurosecretory cells Blood (continued)

(continued) supply, pineal gland, 43-45
photoreceptor cells, 92 Body fluids, melatonin, 553-556
pineal tract, 93-94 (see aLso specific
Anatomy, comparative, pineal fluids)
gland, 61-70 Bovine pineal glands
classification, 62-63 aqueous-acetone extraction,
cytology, 65-67 method, 187
histology, 63-65 comparison of proteic/peptidic
size of gland, 62 fractions, 189-192
Angiotensins (I and II) isolation of threonylseryl-
identification and lysine, 187
characterization, 115 partially purified fraction,
and norepinephrine release, effects, 188-189
207-208 Bradypus, pineal gland anatomy,
Annual levels, melatonin, 557, 62
582 Brain, melatonin levels, 364
Anterior pituitaries, bioassay, differential localization,
181 257-276
Antigonadotropic fractions, N-acetylserotonin, differential
effect on fertility and localization, 257-276
ovulation, 186 binding, 268-271
Avian pineal organ, 27-29 inhibition, 269-270
cells, 95-98 regional distribution, 269
AVP see Vasopressin structural characteristics,
AVT see Vasotocin 269-271
immunoreactive, hippocampal,
Baboon see Papio ursinus 264-266
Behavioral effects of exogenous cerebellar, 266-267
melatonin, 565-567 and pineal indoles, possible
Beta-adrenergic receptors, sites of action, 419-423
pineal, super and sand, 47
subsensitivity, 8-11 Breast cancer and melatonin, 562,
effect of circadian rhythm, 582
9-11
Biopterin cAMP levels, pineal, circadian
and gonadotropic activity, 182 rhythm, 521
isolation from sheep pineals, Canary, sensory nerve cell
155-168 bodies, 95
isobutanol extraction, 158- Na-p-Carboxybenzyl as specific
159 antiserum, 250
methods used, 152-154 Carcinoma
retention time (GLC), 158,160 breast, effect on melatonin
HPLC, 168, 169 levels, 562, 582
Birds, afferent and efferent effect of melatonin
fibers, 95-98 administration, 562 (see
chemical composition, 97 aLso Tumors)
Blood Central nervous system, pineal
melatonin levels, 363, 365, and gonadotrophin ~ircuit
553-556 (see aLso 372-385
~necific headings) Centrioles of pinealocyte, 49
INDEX 591
Cerebrospinal fluid, and Clonidine, effect on melatonin

melatonin, 349-360 secretion, 528, 560
human, 553-556 Cobalt chloride iontophoresis
levels, 363, 365, 553-556 technique, 91
pineal gland actions, 73, Cone myoid elongation and
349-360 contraction, course of
Children, 6-hydroxymelatonin, dark adaptation, 399-402
urine levels, 511-513 Connective tissue, pineal, 48
(see also Puberty) Constant estrous anovulatory
Chlorpromazine effect on syndrome, 372-376
melatonin levels, 562 light induced, 375-378
Choroid Corpora arenacea (acervuli), 47
iris, uptake of melatonin, 406 Cortisol levels, and melatonin
plexus, melatonin levels, 365 secretion, 544, 561-562
Choroidal epithelial cells, affective disease, 579-581
effects of melatonin, Cushing's disease, melatonin
351-355 secretion, 581
Cilia of pinealocyte, 49 Cysts, pineal, 47-48
Circadian rhythm cytology, comparative, pineal
a and S adrenergic receptors gland, 65
and, 5, 9-11 Cytoplasm, endothelial cells, 45
biosynthesis, 1-2 Cytosol
and breeding habits, 304-313 and membrane melatonin binding
(see also Reproductive sites, 283-284
events seasonal) estrogen receptor levels,
disorder, affective disease, 208-210
melatonin, 579-581
and melatonin, 221-226, 227-241 Darkness see Circadian rhythm
brain, 285-287 Dasypus spp. pineal gland
neurobiological anatomy, 62
investigations, 438-441 Daylength, photoperiodic response
metabolism, 1-2 and breeding, 304-305
pinealocytes, dark cells, 77 (see also Circadian
light cells, 77 rhythm: Light: Reproduc-
role of ribbons and tive events, seasonal)
spherules, 74 Dense-core vesicles, 66
stereological studies, 78-79 Deoxyglucose technique induced by
regulation of indoleamines, magnetic field, 457-459
1-11 Depression, melatonin levels,
synthesis in pineal organ 528-529, 561-562, 563,
culture, 4-6 581
variation in electrical Desaminocys-D-arg-vasopressin,
responses, rostal effect on melatonin
hypothalamic neurones, secretion, 560
methoxyindoles, 430 (see Dexamethasone
also Light) effect on melatonin secretion,
Circannual levels 560
melatonin, 557, 582 suppression test, 579-581
pineal gland, stereological 5 a-Dihydrotestosterone
studies, 79-80 receptors, pineal,
Circulating hormones, 121-122 208-213. 214
592 INDEX
5-Dihydroxytryptamine and Evolution, pineal organ

electrolytic lesions, (continued)
381-383 early work to 1970, 17-18
Dopamine, effect on melatonin 1970-1982, 18-22
levels, 529 intrinsic neuronal circuitry,
Down regulation hypothesis, 23-24
melatonin action, 323-325 pinealofugal and pinealopetal
neuronal connections,
Electrolytic lesions CNS, in CEA 24-26
syndrome, 380-385 vascular pattern, 26-27
Electron microscopy, pineal Eyes
gland, mammalian, 71-85 early developmental effects of
quantitative, 72-76 indoleamines, 399
dense-core vesicles, 75-76 humoral interactions involving,
synaptic ribbons and 397-407
spherules, 73-75 melatonin, and photomechanical
Electrophysiology responses within, 399-403
guinea pig, 450-451 cone myoid elongation and
homing pigeon, 451-453 contraction, 399-402
light studies, 454-457 enhancement of retinal
Embryology, pineal gland, damage, 402-403
mammalian, 37-39 immunological relations of
a-Endorphins, characterization retina with pinealocytes,
and identification, 115 403
Enkephalins, characterization and and light entrained disc
identification, 115 shedding by retinal rods,
Epilepsy, effect of melatonin, 402
563 quantitative effects, 403-407
6-L-Erythrobiopterin isolation neural connection, 228-230
from sheep pineals,
152-155 Falck-Rillarp technique, 91
gonadotropic activity, 182 Fishes, neurosecretory cells,
methods used, 152-154 88-92
radioimmunoassay, 174, 175 histochemical technique, 90
reduced, 154 Fluorescent histochemical obser-
retention time, 158, 160, 163 vations, pinealocytes.
Rf values, 158, 160, 162 123
Esox lucius, catecholaminergic 6-Fluoromelatonin, effect on cAMP
green fluorescent fibers, levels in MBR, 291-292
91 Follicle stimulating hormone
Estradiol circulating levels, 305, 307
effect on norepinephrine turn- hypothalamus role, 420, 423
over, 213-214
receptors, pineal, 208-213 Gas chromatographic-negative
Estrogen effect on pineal chemical ionization mass
estrogen receptor, 208- spectrometric technique,
211 melatonin, 521-523
Evolution, pineal organ, 15-35 extrapineal, 523-525
avian, 27-29 6-hydroxymelatonin, 521-523
comparative, cytology, 22-23 Glial cell, 53-54
submammalian systems, 17-22 Glis glis, pineal gland anatomy,
conclusions, 29-30 63
INDEX 593
Gonadal Hormones (continued)

involution, 309-310 effects on pineal, binding
pineal interrelations, 509-519 (continued)
Gonadotropic hormones, endocrine-endocrine trans-
regulation, 133-134 duction, 208-213
Gonadotropins endocrine-neural trans-
central nervous system and duction, 213-214
pineal circuit 372-385 pineal, actions on eNS, 364-367
effect on norepinephrine at level of adenohypo-
turnover, 214 physis, 368-372
inhibition by melatonin, 319- pineal-eNS-gonadotropic
321 circuit, 372-385
circadian rhythm, 320 action on rostral
counteractions, 321-323 hypothalamus, 423
and photoperiodic conditions, Humoral interrelations of pineal
306 gland with lateral eyes
releasing hormone localization, and orbital glands,
brain, 420-423 395-416
in rostral hypothalamus action, Harderian glands, 407-408
423 interactions involving, 497-498
Granular vesicles Hydroxyindole-O-methyltransferase
influence of noradrenalin, (HIOMT)
130-132 day/night rhythm, 172
influence of sexual hormones, effect of indomethacin, 202
132-133, 134 effect of light, 171
melatonin and 5-methoxyindoles influence of pteridines on,
effects, 134, 135 170-174
Growth hormone effect of substrate specificity, 2-3
melatonin, 494 6-Hydroxymelatonin
Guinea pigs, innervation, pineal effect on cAMP levels in MBH,
gland, 102-103 291-292
GV see Granular vesicles measurement, by GeMS, 521-522
by mass spectral assays, 523
Harderian glands, humoral urine levels, 510-518
interactions involving, children, 511-513
407-408 methods, 510-511
Hedgehog, pinealocytes, 20 puberty, 513-515
fluorescent histochemical 5-Hydroxytryptophol
studies, 123, 133 effects on anterior
Hibernation and gonadal hypothalamus, 367, 368
hypertrophy, 30-38 effect on electrical activity,
Hippocampus, immunoreactive pineal cells, 444-448
N-acetylserotonin, rostral hypothalamus neurones,
263-266 425
development, 264-266 circadian variation, 430
localization, 263-264 and light restriction,
Histology, comparative, pineal 426-427
gland, 63 pinealectomy, 428-429
Hormones Hypophysectomy effect on pineal,
effects on pineal, binding, 498
208-209
594 INDEX
Hypophysial-hypothalamic-gonadal Indoles, pineal (continued)

hormones in pineal, rostral neurones (continued)
179-198 circadian variation, 430
anterior pituitaries, bioassay, light restriction, 426-427
181-183 pinealectomy, 428-429
RIA, and detection of metabolism, influence of
prolactin, 183-184 pteridines, 168-174
anti-ovulatory properties, 180 general remarks, 173-175 (see
Hypothalamus (ic) aLso Melatonin and
activity, 121-122 specific headings)
electrical activity, pineal Indolealkylamines, immunohis-
indoles, 417-436 tology, 257-258
releasing hormones, 419-423 N-acetylserotonin antisera
rostral, action of pineal studies, 259-260
hormones, 423-425 mapping, 260-262
site of action, 419-423 melatonin antiserum studies,
hormones, identification and 258-259
characterization, 114-115 specificity, 260
releasing hormone Indoleamines
supplemental source, 123 circadian rhythms, regulation,
interactions with Pineal, and 1-11
monoaminergic neurone effect of light, 2-3
system, 361-385 relationship with
melatonin activity, 287-288 protein/peptide secretion,
regulation of GRF by pineal 134-136
factors, 184 Indomethacin
isobutanol extraction method, effect on pineal, 201-202, 205
185 and melatonin, similarities
releasing hormones between, 288
secretion, influence of Innervation, pineal gland
pineal indoles, 361-385 comparative, 64-65
evolution, 24-26
Immunocytochemical studies vertebrate, 87-112
MSH, 125-126 central neural pathway, 101
LHRH, 123-124 circadian rhythm, 100
perspectives, 129-130 habenular and posterior
somatostatin, 125-126 commissure course,
TRH, 125-126 101-102
ultrastructural, 130-136 mammalian, 98-105
hormone, sexual, influence, non-mammalian, 87-98
132-134 orthosympathetic, 98-101
and indoleamines, 134-136 parasympathetic, 103-105
noradrenaline influence, pinealofugal (efferent),
130-132 90-98, 103 .
vasotocin 126-128 pinealopetal (afferent),
Indoles, pineal 90-98, 101, 103
brain, sites of action, 419-423 Intraocular pressure, effect of
and hypothalamic electrical melatonin, 406-407
activity, 417-436 Iodine, protein bound, effect of
rostral neurones, 425-426 pinealectomy, 481
INDEX 595
Iris-choroid, uptake of Light and lighting conditions

melatonin, 406 (continued)
Isobutanol extraction, 158-159, environmental, melatonin rhythm
166-167 response (continued)
Isoproterenol, 9 and endocrine influence, 232
effect on melatonin levels, 529 intensity, 222-224, 232
effect on pineal cytosol ER neural connection between
levels, 210 eyes and pineal, 228-230
Isoxanthopterin influence on production rise, 230-232
HIOMT activities, 171 nocturnal, 230
serum, 24-hour pattern, 243-
Karnovsky-Roots histochemical 256
technique, 95 sunlight spectrum, 222
Klinefelter's syndrome, melatonin wavelength influence, 235
levels, 562 and gonadotrophic activity,
320-232
Lacerta sicula neuronal humans, 577-579
connections, 25 and magnetic field, 454-457
Lampetra planeri, pinealocytes, pineal/thyroid interrelation-
19 ships, 483-487
Levadopa, effect on melatonin restriction, influence on
levels, 529 hypothalamic neurones,
LHRH see Luteinising hormone 426-427
releasing hormone Lizards, pinealocytes, 20
Light and lighting conditions, Location, pineal gland, 39
2-3, 221-226, 227-241 Luteinization and pinealectomy,
day length and reproductive 372-375
system, 304-305 (see also Luteinizing hormone
Circadian rhythm: Repro- circulating levels, 305, 307
ductive events, seasonal) and effect of melatonin,
day-night rhythm, 557-558 368-372
effect on indoleamine in rostral hippocampus, action,
metabolism, 2-3 423
effect on melatonin, 535-550, Luteinizing hormone releasing
576-577 hormone (LHRH) pineal
biological marker, 521-533, and effect of melatonin,
543-545 368-372, 560
entrainment of light-dark identification and character-
cycle, 539-543 ization, 114, 119-120,
photoperiodism, 543 122
sunlight, 537-539 immunocytochemical studies,
suppression of production, 123-124
536-539 influence of sexual hormones,
effect on pteridine effect on 133
indole metabolism, 171
electron microscopy, 454-457 Magnetic system
environmental, melatonin rhythm biochemistry, 454-457
response, 221-226, 227- deoxyglucose-technique, 457-459
241 electrophysiology, 450-453
guinea pig, 450-451
homing pigeon, 451-453
596 INDEX
Mannich antigen, melatonin, Melatonin (continued)

244-246 compared with 5-methoxyindoles
formaldehyde reaction, 244-245, in effect on sexual
249 axis, 331-348
Mannich antisera to melatonin and down regulation hypothesis,
N-acetylserotonin, 243- 323-325
244, 249 effect on adrenal gland, 489
Melanocyte stimulating hormone effect on electrical activity,
identification and character- rostral hypothalamus
ization, 115, 121, 122 neurones, 425
immunoreactivity, 125-126, 129 circadian variation, 430
release inhibiting factor, and light restriction,
identification and 426-427
characterization, 114 pinealectomy, 428-429
Melatonin effect on growth hormone, 494
action on CSF secretion, endogenous, human, effects of
349-360 light, 535-550
adrenergic regulation, 525 exogenous, effects on humans,
antigonadotrophic activity, 563-565
319-321 behavioral effects, 565-567
circadian rhythm, 320-323 pharmacological disposition,
counter actions, 321-323 563-565
as biological marker for pineal physiological disposition,
adrenergic function, 563
521-533, 543-545 extrapineal, GOMS assay, 521,
blood levels, 278-279 523-525
human, 553-556 and eyes see Eyes
brain, differential goitrogenic effect, 484-487
localization, 257-276 human studies, 526-529,
age-related studies, 281 536-539, 551-573
binding sites, 283-285 and indomethacin, similarities
daily rhythm, 285 between 288, 289
immunohistology, 257-258 labelled, uptake, 397-398
indolealkylamines, 257 measurement, assays, 575-576
mechanism of action, 287-294 metabolic pathway, 575, 576
analogues, 291-292 neurobiological investigations,
in MBH, 290-294 437-465
metabolic fate, 278-279 central innervation, 441-444
neuroendocrine activity circadian rhythmicity,
studies, 280-282, 361-385 438-441
receptors, 283-287 magnetic sensitivity, pineal,
specific antiserum studies, 448-457
258-259 biochemistry, 454-457
cerebrospinal fluid levels, deoxyglucose technique,
human, 553-556 (see aLso 457-459
Cerebrospinal fluid) electrophysiology, 450-453
chronobiological marker, as transmitter, 444-448
575-587 and neuroendocrine-reproductive
circadian rhythm see Circadian axis, hamster, 317-330
rhythm: Light
INDEX 597
Melatonin (continued) Mesocricetus auratus, pineal

and pineal-hypothalamic gland anatomy, 63
interaction, 361-385 (see Methoxyindole(s)
also Hypothalamus) circadian variation, 430
plasma, assays, 522-523 5-methoxyindoles (5-MTL),
pre/post-synaptic function, 526 different from melatonin,
production in pineal, hamster, effect on sexual axis,
318-319 331-348
receptors, pineal, 208-213 and protein/peptide
relationship to immunoreactive secretion, 135
N-acetylserotonin, 262-263 5-methoxyindole-3-acetic acid
release, effects of inhibitors, (5-MIAA), physiological
204-206 properties, 338-340
retina, 284 content in different mammals,
rhythm, response to light, 339
221-226, 227-241 5-Methoxytryptamine
serum, 24-hour pattern, and LH release, 336
243-256 physiological properties,
Mannich antigen, 244-246 335-338
Mannich antisera, 243-244 and protein/peptide secretion,
radioimmunoassay, 246 135
regulation studies, 247-248 effect on sexual axis, 336-337
(see also Circadian 5-Methoxytryptophan (5-MTP),
rhythm) physiological properties,
side effects, 565 340-341
stimulatory effects on 5-Methoxytryptophol
choroidal epithelial effects on anterior hypo-
cells, 351-357 thalamus, 367
animals and procedures, effect on cAMP levels in MBG,
351-352 291-292
cellular changes, 355 effect on electrical activity,
discussion, 355-357 pineal cells, 444-448
intracranial pressure, 354, rostral hypothalamus
356 neurones, 425
observations, 352-355 circadian variations, 430
ventricular secretion, 353, and light restriction,
356 426-427
and tumor growth and pinealectomy, 428-429
pinealectmy, 468 physiological properties,
effect on KB cells, 468 (see 332-335
also Tumor growth) effect on sexual development,
urinary levels, assays, 522-523 332
human, 553-556 mammals, 333-335
metabolites, 510-511 6-Methyltetrahydropterin, brain
specific findings, 511-518 content, 174
children, 511-513 Microtubular sheaves, 67
menstrual cycle, 515-517 comparative studies, 67
puberty, 513-515 Microtus agrestis, pineal gland
Menstrual cycle cytology, 66
6-hydroxymelatonin, urine Mole see Talpa europaea
levels, 515-517
melatonin levels, 557
598 INDEX
Monoamine oxidase activity, Neuroendocrine system (continued)

pineal, 202 integrative processes, pineal,
pinealectomy, 366 molecular aspects,
Monoaminergic neurone system, and 199-220
pineal-hypothalamic a-adrenergic effects of
interactions 361-386 norepinephrine, 200
Monthly levels, melatonin, 582 arachidonic acid metabolites,
Morphology, pineal gland, role, 200-201
mammalian, 37-59 biochemical mechanisms, 200
activity, 40 blockade of PG synthesis,
blood supply, 43-45 205-208
cysts, 47 endocrine-endocrine
cytoplasm, 45 transduction, 208-214
embryology, 37-39 endocrine-neural
glial cell, 53 transduction, 213-214
location, 39 norepinephrine, effect on PG
ontogenetic development, 39 release, 202-204
perivascular spaces, 45-46 pineal organ cultures, 204
size, 40-42, 62 SNAT activity, 204-205
striated muscle fibers, 48 reproductive axis, melatonin
suprapineal (suprahabenular) activity, hamster, 317-330
recess, 42-43 (see also (see also Melatonin:
Pinealocytes) Reproductive system)
Site and mechanism of action of
Natrix, pineal afferent and pineal on 361-385
efferent fibers, 95 Neurohypophysial hormones, pineal
Neopterin, isolation from sheep identification and
pineals, 155-168 characterization, 114,
mass spectrum, 161 117-121
reduced, influence on Neurophysins, pineal, identifi-
5-methoxyindoles, 171 cation and character-
retention time, 158, 163 ization, 114, 119
Rf value, 158, 160, 164 Neurosecretory nerve fibers,
Nerve cells, pineal gland, mammalian pineal gland,
extramural, 104 104-105
non-mammalian, 92-98 Neurosensory cells, pineal non-
Neurobiological investigations mammalian, 88-92
central innervation, 441-444 afferent (pinealofugal) tract,
magnetic sensitivity, 448-450 90-98
biochemistry, 454-457 development, 88
deoxyglucose-technique, efferent (pinealopetal) tract,
457-459 90-98
electrophysiology, 450-453 functions, 88
melatonin, circadian intercalated, 89
rhythmicity, 438-441 photoreceptor, types, 89
as transmitter, 444-448 sensory cells, 89
Neuroendocrine system Neurotoxin lesions, CNS, in CEA
activity, melatonin, central or syndrome, 380-385
peripheral, 280-282
INDEX 599
Noradrenaline Parkinsonism (continued)

circadian rhythms, 4 and pteridine metabolism, 173
via adrenergic receptors, 5 Passer domesticus, pineal tract,
effect on pineal gland 25, 27-29
processes, 199 Peptides, pineal
and angiotensin II, 208 identification and character-
and indomethacin, 201-202 ization, 113-117 table,
modulation of adrenergic 114-116
neurotransmission, 206 first class, 117-121
negative feedback control, not identified, 115
206 partially identified, 115
prostaglandin release, second class, 121-123
202-204 third class, 123-136
adrenoceptor involved, 203 Peptidic substances
influence on protein/peptide comparison with proteic,
secretion, 130-132 189-192
and granular vesicles, different classes, pineal
130-132 gland, 113-149
sympathetic nerves, hormones isolation, 180
effecting, 213-214 pineal, and hypophysial-
hypothalamic-gonadal
Ontogenetic axis, 179-198
development, pineal gland, Perivascular spaces, 45-46
mammalian, 39-40 autonomic nerve fibers, 45-46
origin of pinealocyte, 49 tissue layers, 46
Opossum, pinealocytes, 20 Petaurus breviceps and P.
Orthosympathetic innervation, norfolcensis, pineal
pineal gland, 98 gland anatomy, 62
OT see Oxytocin Petromyzon planeri, secretory
Ovarian hypertrophy, compensa- cells, 89
tory, effects of pineal Phenylketonuria and pteridine
extracts, 185-187 metabolism 173
Ovaries, weight, and daylength, Photoneuroendocrine cells, 23
321,322 Photoperiod, pineal, and seasonal
Ovulation reproduction, 307-312
influence of peptidic substance Photoreceptor cells, non-
in pineal, 180 mammalian, 89
and pinealectomy, 372-375 mammalian cells, 92
Oxytocin, pineal, identification Phoxinus phoxinus
and characterization, 114 pinealocytes, 20, 21
mammalian, 117-121 pineal tract, 91
Papio ursinus, pinealocytes, 78 Pigeon, homing, magnetic field,
Parafollicular cells, thyroid, 451-453
after pinealectomy, 497 Pike, pinealocytes, 19
Parasympathetic pineal Pineal gland
innervation, 103-105 adrenal interrelationships,
Parkinsonism 487-493 (see also
effect of melatonin, 563 Adrenal-pineal inter-
relationships)
600 INDEX
Pineal gland (continued) Pinealoctyes

comparative anatomy, 61-70 centrioles, 49
(see also Anatomy, cilia, 49
comparative) comparative analysis, 18-22
evolution, 15-35 (see also comparative cytology, 22-23
Evolution) dark, 51
hormones see Hormones, pineal dense-core vesicles, 66
and specific names different types, 51
hypothalamic interactions, role early work, comparative, 17-22
of monoaminergic neuron histology and cytology, 48-49
system, 361-393 light, 51
interactions with non- nucleus, 50
reproductive endocrine phylogenetic and ontogenetic
glands, 477-508 origin, 49
mammalian, 98-105 and sex organs, 51
embryology, 37-39 -specific organelles, 67
morphology, 37-39 (see also ultrastructure, 71-72 (see also
Morphology) Nerve cells: Neuro-
reactivity, factors secretory cells)
influencing, 480 Pinealofugal and pinealopetal
secretion, route, 362-364 neuronal connections,
thyroid interrelationships, 24-26
478-487 (see also Pinealoma, effect on melatonin
Thyroid-pineal inter- secretion, 561, 581-582
relationships) Progesterone receptors, pineal,
Pinealectomy 208-213
effect on cortisol production, Prolactin
489 circulating levels, 305,
effect on growth hormone 306-307
levels, 495-497 detection, radioimmunoassay and
effect on hypothalamus, 372-374 anterior pituitaries, 183
induction of ovulation and pituitary levels, 188
luteinization, 375 effect of ganglionectomy, 493
effect on parafollicular cells effect on norepinephrine turn-
of thyroid, 497 over, 214
effect on prolactin levels, 493 effect on pineal and SCG
effect on protein-bound iodine, tyrosinase hydroxylase
481 activity, 213
effect on responses of rostral effect of pinealectomy, 493
hypothalamic neurones to receptors, pineal, 208-213
indoles, 428-429 Propranolol
effect on TSH levels, 483-484 effect on night-time melatonin
and MAO activity, 366 secretion, 527-528
and melanoma growth increase, effect on pineal cytosol ER
468 (see also Tumor levels, 210
growth) Prostaglandins
quantitative effects on the effect of norepinephrine on
eye, 403-404 release from pineal,
rabbit, 42 202-203
'Pinealin', 179 adrenoceptor involved, 203
INDEX 601
Prostaglandins (continued) Pteridines, effect on indole

effect of norepinephrine on metabolism, pineal
release from pineal (continued)
(continued) effect of light, 171
effect on SNAT activity, 204 HPLC experiments, 168, 169
pre/post-synaptic effect, identification, 151
206-208 influence on hydroxyindole-O-
phospholipids, role, 203 methyl transferase,
radioligand binding studies, 170-173
203-204 isolation from sheep pineals,
inhibition by melatonin, 152-168
288-290 6-L-erythrobiopterin, 152-155
Proteic substances biopterin, 155-168
comparison with peptidic, neopterin, 155-168
189-192 pterin-6-carboxylic acid,
different classes, pineal 155-168
gland, 113-149 (see aLso methods used, 157, 166-167
specific substances) synthetic and LMW pineal
hypophysial-hypothalamic- fractions, 168-173
gonadal axis, 179-198 Pterin-6-carboxylic acid
in vitro, 181-185 isolation from sheep
bioassay, 181-183, 184-185 pineals, 155-168
isolation, 179-180 isobutanol extraction, 158-159
radioimmunoassay, 183-184 mass spectrum, 164, 165
in vivo, 185-187 methods used, 152-154
bovine glands, 187 retention time (GLC), 158, 160,
compensatory ovarian 163
hypertrophy, 185 HPLC, 168-169
partially purified bovine Rf values, 158, 160, 162
pineal fraction E5, Puberty
188-189 6-hydroxymelatonin, urine
Proteins, pineal, identification levels, 513-515
and characterization, melatonin levels, 558-559
113-117 tabLe, 114-116 Pulsatile secretion, melatonin,
first class, 117-121 558
not identified, 116 Puntius sophore, afferent neural
partially identified, 115 pathway, 91
second class, 121-123
third class, 123-136 Quail, Japanese, nerve cell
Psoriasis vulgaris, melatonin bodies, 95
levels, 562
Psychiatric diseases, effect on Rabbit, pinealocytes, fluorescent
melatonin secretion, 561, histochemical studies,
579-581 123
Pteridines, effect on indole Radioimmunoassay
metabolism, pineal, for detection of prolactin, 183
151-179 of 6-L-erythrobiopterin, 175
BH4 (tetrahydrobiopterin) role, melatonin, 246, 553, 576
151-152 N-acetylserotonin, 246
determination aspects, 174 specific, 248-251
diseases affected, 173-174
602 INDEX
Rana esculenta, pinealoctyes, Sexual function and pineal gland,

18-20 509-519 (see also
neuronal connections, 24-26 Reproductive events)
Reproductive events, seasonal Shy-Drager syndrome,
303-316 6-hydroxymelatonin excretion, 511
changes induced by photoperiod, Size, pineal gland, 40-42, 62
305-307 and nocturnal/diurnal life, 41
day length measurement, 304-305 variations, 40-42
gonadal hypertrophy and Somatostatin, identification and
hibernation, 308, 309-310 characterization, 115,
melatonin role, 325-326 120
photoperiod, pineal and immunoreactivity, 125-126, 129
seasonal reproduction, Sparrow, sensory nerve cell
307-312 bodies, 95-96
gonadotrophic activity, Spherules, pinealocytes, 73-75
320-323 Spina bifida, melatonin levels,
inhibition phase, 308-310 562
melatonin role, 325-326 Spinal cord, cervical,
refractoriness, 311, 312, 325 transected, effect on
restoration phase, 310-311 melatonin secretion, 561
sexually active phase, Stalk, pineal, 42
311-312 Stereology, pineal gland,
sexually quiescent phase, 310 mammalian, 76-85
related to pineal gland, circadian rhythms, 78-79
hamster, 303-316 circannual rhythms, 79, 80
testicular weights, 308 composition of tissue, 76-78
Reptiles, pineal tract, 94 Stimulation and melatonin, 560,
Retina 576-577
immunological relations with Stress and melatonin secretion,
pinealocytes, 403 544, 560
melatonin binding site, 284 Striated muscle fibers, pineal,
rods, disc shedding, and 48
melatonin, 402 Substance P, identification and
Ribbons synaptic pinealocytes, characterization, 115,
73-75, 92 119
Sunlight, effect on melatonin,
Salmo, afferent neural pathway, 222, 537-539
pineal, 91 Suppression of melatonin
Sarcoidosis, melatonin levels, secretion, 560
562 Suprachiasmatic nucleus activity,
Schizophrenia and melatonin 535, 537
secretion, 561-562, 563 Suprapineal (suprahabenular)
Serotonin recess, 42-43
circadian rhythms, 3-4
via adrenergic receptors,S Talpa europaea pinealocyte
inhibition studies, 269-271 sub-types, 72
relationship to immunoreactive Testicular weight, and day
N-acetylserotonin, length, 321, 322
262-263 Testosterone
and site of indole action in receptors, pineal, 208-213
brain, 419-420
INDEX 603
Testosterone (continued) Tumor (continued)

receptors, pineal (continued) ornithine decarboxylase
effect on castrated rats, 213 monitoring of levels
post-synaptic level, 213 (continued)
Tetrahydrobiopterin (BH4) role, preparation of organ
151-152 extracts, 471
in dopaminergic nerve protein determination, 471
terminals, 174 results and discussion,
rat brain content, 173 471-473
Threonylserinyllysine (TSL) pineal, and melatonin
identification and secretion, 561, 581
characterization, 115 and pteridine metabolism, 173
isolation from bovine pineal Turner's syndrome, melatonin
gland, 187 levels, 562
TSL see Threonylserinyllysine Tyramine, effect on melatonin
Thyroid-pineal interrelation- levels, 529
ships, 478-487
melatonin influence on TSH Urine levels, melatonin, human,
rhythm, 484-487 553-556
pineal influence on thyroid,
483-487 Vascular pattern, pineal organ,
pineal reactivity, factors 26-27
influencing, 480 Vasoactive intestinal peptide
pinealectomy, 481 (VIP), identification and
temperature changes, 481-482 characterization, 115,
thyroid secretion rate, 479 119
Thyrotropin releasing hormone Vasopressin, pineal, 560
effect on melatonin secretion, identification and
560 characterization, 114
identification and character- mammalian, 117-121
ization, 115 immunocytochemistry, 129
immunoreactivity, 125-126 Vasotocin, pineal
Tissue, pineal gland, antibody against, 128, 129
composition, 76-78 content pineal gland, 188-189
Trout, pineal organ, innervation, 'controversy', 126-128
24-25 identification and
Tryptophan metabolism in pineal, characterization, 114
319, 331 mammalian, 119
TSH levels in constant dark, non-mammalian, 119, 120
intact and pinealectomy, as peptide, 126
483-484 physiology, 127-128
Tumor radioimmunoactivity, 127
growth, pineal gland influence, Vesicles
467-476 dense-core, quantitative
biochemical markers, 469 electron microscopy,
effect of melatonin, 468 75-76
ornithine decarboxylase granular see Granular vesicles
monitoring of levels, Volume, pineal, 40
469-473
enzyme assay, 471 Yearly rhythms melatonin, 557,
materials and methods, 582
470-471
604 R. J. REITER
ODE TO THE ERICE MEETING
Erice was the venue which NATO did pick

We avoided the water, so as not to get sick.
Our "clocks" were confused, when first we met
This caused many to sleep, directly on the set.
The speakers were good, the students were great

Discussion was ample, and we never ran late
All matters were discussed, well into the night
And we did it all, without a fist fight.
Axelrod began, the meeting with skill

While Wurtman stayed only, briefly on the hill
Oksche from Giessen gave a fine talk
He rarely used slides, and never used chalk.
Vollrath and Kappers considered the gland

Many items were mentioned, even brain sand
The biochemistry was investigated by Cardinali's group
When he finished his talk, it was alphabet soup.
Bob Moore described retinohypothalamic way

By which light tells the SCN exactly what to say
Lewy and Poth, looked at the human in depth
Urine and blood were collected while the subjects slept.
Wetterberg and Brown, they did it with class

While the Frenchman Pevet, was with his lass
Semm from Frankfurt, had a new thought
I expected some doubt but no one fought.
From Milano, Calabrino did not come

But Demaine and Ebels, provided the fun
Mess and Quay, each spoke twice
While the discussants themselves, got their slice.
On a visit to Marsala to see a ship

A pig in the sea was the hit of the trip
On the beach in San Vito, we all turned red
While to Segesta and Selinunte we were kindly led.
To Fraschini and Velo, we bid you adieu

We wish success, to you and your crew
Reiter is concerned about the short days of fall
As a result he says, arrivederci to all.
Russel J Reiter

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NATO Advanced Science Institutes Series

Pineal Gland and

This series is published by an international board of publishers in conjunction with

A Life Sciences Plenum Publishing Corporation

C Mathematical and D. Reidel Publishing Company

0 Behavioral and Martinus Nijhoff Publishers

F Computer and Springer Verlag

Recent Volumes in Series A: Life Sciences

Springer Science+Business Media, LLC

Library of Congress Cataloging in Publication Data

© 1983 Springer Science+Business Media New York

The pineal gland has been a subject of interest and speculation

Within a few years the pathway for the biosynthesis of melatonin

For this study group, a limited number of investigators in anato-

points of view. There was an informative and stimulating exchange

The organizers and participants are grateful to NATO for their

J. Axelrod, F. Fraschini and G.P. Velo

Regulation of Circadian Rhythms of Indoleamines in

Aspects of Evolution of the Pineal Organ. 15

Comparative Gross and Fine Morphology of the Mammalian

Perspectives of Comparative Anatomy of the Mammalian

The Use of Electron Microscopy and Stereology in the

Innervation of the Vertebrate Pineal Organ. . . . . . . . . . 87

The Different Classes of Proteic and Peptidic Substances

Pteridines in the Pineal and Effects of These Substances

Peptidic and Proteic Substances Isolated from Pineals

Molecular Aspects of Neuroendocrine Integrative Processes

The Responses of Melatonin Rhythms to Environmental

The Role of Light and Age in Determining Melatonin

Differential Regulation of the 24 Hour Pattern of Serum

Differential Localization of Melatonin and N-Acetylserotonin

Melatonin Action: Sites and Possible Mechanisms in Brain. 277

Seasonal Reproductive Events Related to the Pineal Gland . . , 303

Melatonin as the Hormone Which Mediates the Effects of the

The 5-Methoxyindoles Different from Melatonin: Their

Actions of the Pineal Gland and Melatonin on the

Pineal-Hypothalamic Interactions: Possible Role of the

Humoral Interrelations of the Pineal Gland With Lateral

Modification of Hypothalamic Electrical Activity by

Neurobiological Investigations of the Pineal Gland

Influence of the Pineal Gland on Tumor Growth in Mammalians:

Interactions Between Pineal and Non-reproductive Endocrine

The Pineal Gland and Sexual Function in Man. . . . . . . . . . 509

Melatonin Secretion -- A Biological Marker for Human

Human Melatonin Secretion, Its Endogenous Circadian Pace-

The Secretion and Effects of Melatonin in Humans . . . . . . . 551

Melatonin as a Chronobiological Marker in Health and

Index. . '. . . . . . . . . . . . . . . . . . . . . . . . . . . 589

Table 1. Substrate Specificity of Hydroxyindole-O-Methyltransferase

Substrate Relative Activity

Purified enzyme obtained from bovine pineal was incubated with

the best substrate for this enzyme which we named hydroxyindole-

that of the ovaries. Pineal extracts were then found to decrease

in organ culture can synthesize [14C]melatonin from [14C]tryptophan.

....._-_._ ... _- .........

Fig. 1. Circadian rhythms of serotonin, N-acetyltransferase and

At about the same time Klein and Berg (1970) demonstrated a

Fig. 2. Effect of adrenergic drugs and other manipulations on

elevation N-acetyltransferase. Thus indicating that the pacemaker