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Ann. Rev. Biophys. Bioeng. 1977. 6:445-76

Copyright ® 1977 by Annual Reviews Inc. All rights reserved
ELECTRICAL CONTROLS +9100
OF DEVELOPMENT
Annu. Rev. Biophys. Bioeng. 1977.6:445-476. Downloaded from www.annualreviews.org
by University of North Carolina - Greensboro on 09/25/13. For personal use only.
Lionel F. Jaffe
Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907
Richard Nuccitelli
Physiology Department, Medical School, University of California,
Los Angeles, California 90024
Wonderful as are the laws and phenomena of electricity when made evident to us in
inorganic or dead matter, their interest can bear scarcely any comparison with that which
attaches to the same force when connected with the nervous system and with life.
Michael Faraday, 1839 (I)
. INTRODUCTION
Scope
This review focuses on the natural, relatively steady electrical fields involved in the
control of growth and development of cells and tissues. It is divided into two main
in qu iries. What natural electrical fields are inside or across cells and organisms and
what the consequences are of modifying these fields. The emphasis is on relatively
steady transcellular fields and field effects, as opposed to the transient fields normally
associated with membrane potential changes. Among the important topics not
covered are the following. (a) There are some important cases in which natural
membrane potential changes control develop ment. For example, the l arge reduction
(or even reversal) of membrane potential that follows fertilization of the sea urchin
egg somehow acts as the natural fast block to entry of additional sperm (2). (b)
There is some information bearing on possible piezoelectric effects in bone develop
ment, as well as possible developmental effects of action potentials in animals and
plants. This, too, is beyond our scope here. (c) Finally, we do not discuss the possible
effects of electrostatic fields on organisms, but it appears to us that they involve
entirely different factors than the dynamic fields to be discussed h ere.
445
446 JAFFE & NUCCITELLI
History
As early as 1892, Wilhelm Roux had imposed electrical fields on a wide variety of
animal eggs and observed gross stratifications of the cytoplasm in many cases (3).
However, he found no consistent effects of these fields on development and made
no comment on possible natural cellular electrical fields. The first investigator to
propose that naturallields might influence growth was Albert Mathews in 1903 (4).
He measured potential gradients along the surface of regenerating hydroids, which
seemed to indicate that current entered the regenerating polyp end, and suggested
that this current polarized the protoplasm or cells. However, the most significant
and influential early investigations of developmental fields were surely those of E.

J. Lund (5,6). These studies, begun in about 1921 (5) and culminating in his book
Bioelectric Fields and Growth in 1947 (6), seem to have stimulated a considerable
number of contemporary investigators, such as Spek (7), Went (8), Burr (9), and
Costello (10), and remain as substantial primary sources. However, this. wave of
interest ended in the late 1940s and there was a distinct decline in progress in this
area until quite recently.) The revival of interest and investigation, combined with
the new ideas and advances in electrophysiological techniques over the past 20 years,
is likely to lead to a rapid increase in our knowledge in this area. Therefore we feel
this is an appropriate time to review the older literature from a modern viewpoint,
both to clarify what is already known and to point out the gaps in our understanding
to guide future investigation.
The last general review in this area was done by Crane in 1950 (12), and more
specialized reviews include those of Schrank (13) and Smith (14). Lukiewicz wrote
an historical review (15) and Rosene wrote a comprehensive bibliography of the
early work (see 6).
ENDOGENOUS CURRENTS AND FIELDS

Theory
Theoretical consideration of cytoplasmic fields has been dominated by Ohm's law.
In continuous media this is given by
E= Ip, 1.
where E is field strength, I is current density, and p is resistivity.

This simple formula should be essentially correct as long as the current involved
is of an ion with relatively little tendency to bind to fixed charges in the cytoplasm.
The spectrum of cytoplasmic binding tendencies among the six major inorganic
physiological ions goes from K+ and Cl-, which have almost no tendency to bind
to fixed charges in the cytoplasm, to Ca2+ and H+, which bind very strongly. In
between, but much closer to K + and Cl-, lie Na+ and Mg2+ . So Ohm's law should
yield an excellent approximation for the cytoplasmic fields generated by K+ and
IThis recent revival of interest is indicated by the New York Academy of Sciences Sym
posium of Electrically Mediated Growth Mechanisms in Living Systems (11).
ELECTRICAL CONTROLS OF DEVELOPMENT 447
CI- currents and a reasonably good one for Na+ and perhaps even Mg2+ currents
but a grossly incorrect value for ones generated by Ca2+ and H+ currents. A
theoretical treatment of this problem appears elsewhere (16).
Methods for Measuring Electrical Currents and Fields Through

Developing Systems
There have been three main approaches to the study of steady bioelectric fields and
currents: (a) direct measurements of the field within (or across) the developing
system; (b) measurements of the currents driven through its medium by the system;
and (c) measurements of the voltage gradients along the surface of a system that
has been removed from its natural medium.

(a) In direct field measurements, steady potentials across developing epithelia can
be reliably measured with conventional KCI-filled micropipettes (cfTable 3). A few
efforts have also been made to directly measure fields within the continuous cyto
plasmic phase of cells with such microelectrodes (e.g. see 18). Uncertainties intro
duced by puncture injury currents as well as by tip potentials have generally
rendered such measurements very questionable. An important exception, made
possible by the system's favorable dimensions, is Woodruff & Telfer's apparently
reliable measurements of the potential across the cytoplasmic bridge between the
oocyte and its nurse cell in an insect follicle (19).
Grahm & Hertz determined the field developed across an aerial structure (actu
ally the coleoptile) of growing plant seedlings by measuring the alternating voltage
induced in a capacitor, separated by an air gap from the seedling, and vibrating (at
250 Hz) so as to vary this gap (20, 21). Their "method uses the well-known fact
that if an electric field exists between the plates of a plane condensor and the distance
between the condensor plates is changed (without changing the charge on the
plates), a corresponding change is observed in the voltage across the condensor
because of the change in its capacity." In this elegant way, the serious artifacts
generally produced by contacting an aerial organ were entirely avoided.
(b) External current measurements have the important advantage of minimizing
any disturbance of the system being studied; moreover the current that traverses the
source system can be inferred from the current through its medium. Both static and
vibrating electrodes have been used to measure voltage differences between various
external points. Combined with a measurement of the resistance or resistivity, these
yield the current or current density, respectively, between these points. Examples
of the static method include measurements of the voltages at various points in an
extended medium near a growing root (22) and of the voltage drop across a fine tube
bearing several hundred developing fucoid eggs in series (23). External currents can
also be measured by vibrating a platinum-black microelectrode near a cell or tissue
(at a few hundred hertz) and measuring the voltage difference between the extremes
of the probe's oscillation, with the aid of a lock-in amplifier tuned to the vibration
frequency (24). Such a vibrating probe system has a signal-to-noise ratio about 1000
times that of comparable size static probes; this is because the effects of drift and
of low-frequency noise generated at the probe's surface are filtered out and because
probe impedance (hence thermal or Johnson noise) is radically reduced at the
448 JAFFE & NUCClTELLI
frequency of vibration. A good example of this technique's power is the successful

exploration of the currents around growing pollen tubes (25). Local current densities
as small as 0. 1 ].tAfcm2 were easily measured with a spatial resolution of about 30
].tm.
(c) The third and oldest approach to bioelectric field detection is via surface
potential measurements. This technique measures neither the natural field nor cur
rent values because the electrode contacts the surface of the cell or tissue in a more
or less unphysiological condition. It measures the voltage generated between two
surface points by the extracellular current passing through the surface fluid layer.
This voltage is maximized by maximizing the surface resistance, usually by drying
the cell surface somewhat. Since the surface resistance is usually variable and not
measured, the current magnitude cannot be calculated from these voltages, but only
the current direction can be inferred. The degree of disturbance of the tissue depends
both on the surface drying and the contact fluid in the electrodes. Often an unnatural
contact fluid has been used, such as a strong solution of Nael without the other ions
found in the normal growth medium. Such unnatural contacts could grossly perturb
the electrical behavior of the outer cell membrane beneath it. Examples of good
surface potential technique can be found in Lund's work (6, 26).
In addition to the above, several methods have been used to measure specific ion
currents through developing systems. One uses a vibrating probe to monitor the
immediate effects of rapid changes in specific external ion concentrations on the
current through a system (27). Two others use tracers. Local entry into individual
cells may be detected with low-temperature radioautography of whole (unsectioned)
cells in favorable cases (28), whereas local effluxes from, as well as influxes into, large
populations of cells may be measured using the so-called nickel screen method of
Robinson & Jaffe (29).
Measurements of Currents Through Single Plant Cells
The available measurements of the endogenous currents through single plant cells
are summarized in Table 1 . In all five cases, cellular elongation occurs in a localized
region at one end of the cell, and in each case, with the possible exception of
Acetabularia, current seems to enter the prospective or actual growth region. How
ever, some uncertainties should be noted about the two oldest reports. The first is
taken from Lund (6), who published three measurements of surface potential profiles
along filaments of the fresh water alga, Pithophora. All three show the surface
potential to be highest (i.e. most positive) somewhat behind the tip of the terminal
cell (actually 0.2�.6 mm behind it). These would seem to indicate external current
flow towards the tip from this potential peak and therefore current entry into the
tip. However, it is uncertain whether Pithophora will grow in the tapwater medium
employed or even whether it shows the tip growth, which is typical, though not
universal, in plant filaments. Case 2 is taken from the measurement of Slayman &
Slayman (18), with conventional intracellular electrodes, of an enormous (600
mVfcm) distally positive voltage gradient in the terminal cell of a growing fungal
filament. If reliable, this would indicate intense current flow into the growing tip;
however, it seems possible that the relatively low membrane potentials measured
Table I Natural currents through single plant cells
Inferred current Current Elongation

Measurement Current direction at density rate
Case Cell type method characteristics growth region (IJ.A/cm2) (IJ./hr) Year Ref.
Pithophora green alga extracellular sur- steady inward 1947 6

sp. apical face potential
filament tapwater contact
2 Neurospora fungal intracellular steady inward ?a 3000 1962 18
crassa hypha microelectrode
3 t"I1
Acetabularia green alga extracellular steady variable 1-2 1972 30,31 r<
t"I1
mediterranea posterior current (")
stalk measurement pulses inward 10-20 >-i
�
segment n
4 Pelvetia brown alga extracellular steady inward 2 1974-76 32-34 ;I>
r<
fastigiata germinating vibrating probe pregermination (")
egg 0
4SCa tracer Z
steady inward 0.03 1975 29 >-i
�
pregermination 0
various steady inward 1-2 2 1966-75 23,32 r<
en
postgermination 0
'"r1
extracellular pulse inward 4 1974-75 32,35
t:I
vibrating probe postgermination
5 Lilium lily extra cellular 25
�
steady inward 4 1975 t"I1
r<
longjflorum germinating vibrating probe pregermination 6-10 0
'"tI
pollen postgermination
=:::
grain steady inward 1.3 t"I1
Z
pulse inward 10 ...,
alt is difficult to know how much of the measured potential difference is due to current from a fixed charge gradient or a lower apparent
membrane potential at the relatively fine tip where the penetration injury leak could be greater. If the entire observed 600 mV/cm field t
\C
2
were generated by a current loop, the intrahyphal resistivity of 300 nem would indicate a density of 2000 IJ.A/cm •
near the tip were the result of greater puncture injury there rather than a growth
current.
In the three recently investigated cases, local current entry has been shown to
precede and predict local growth. In both the unilaterally illuminated Pelvetia egg
(29, 32-34) and the lily pollen grain (25) a relatively steady transcellular current
begins hours before germination, i.e. before the initiation of local growth, and such
growth begins at or close to the center of current entry. Similarly, enucleated stalk
segments of Acetabularia may be morphologically depolarized by an appropriate
pretreatment (with light), in the sense that their subsequent regeneration of a cap
occurs with nearly equal frequency at either end. Thirty hours before cap generation
begins in such segments strong loo-sec-Iong current pulses begin to enter one end;
it is this end that then regenerates (30, 31). These facts suggest that local current
entry somehow causes vesicle secretion and local growth, perhaps as a result of the
current's Ca2+ component. There is growing evidence that Ca2+ influx stimulates
vesicle secretion.
This inference is supported by evidence that calcium ions constitute a significant
part of the current through Pelvetia eggs. First, tracer measurements on polarizing
eggs directly indicate a calcium current (29). This current was actually largest when
it could first be measured, at 6 hr after fertilization, and thus 1-2 hr before final
commitment to growth in a particular direction. At this time it was estimated to
be 2 pA per egg. If the current were uniform within the egg it would have had a
density of about 0.03 p.A/cm2, about 5% of the total transcellular current. Second,
despite this relatively small Ca2+ component, the early transcellular current is
remarkably sensitive to the Ca2+ concentration: a threefold increase in external
Ca2+ yields an immediate two- to threefold (and occasionally up to tenfold) increase
in the whole transcellular current (R. Nuccitelli, manuscript in preparation). This
strong Ca2+ dependence suggests that the relatively small calcium current estab
lishes a calcium gradient that in tum acts to induce a steady current of other ions.
Third, direct evidence of a calcium gradient across the ungeminated egg is still
lacking, but low-temperature 4SCa2+ autoradiography indicates that the total Ca2+
concentration is 40% higher in the growing end of the two-celled Pelvetia embryo
than in the opposite end (37). The free calcium concentration may also be propor
tionally higher at this end. Furthermore, vibrating probe and tracer evidence indi
cate that the current pulses that appear after germination are each triggered by
calcium entry (38). Thus, altogether there is little doubt that calcium ions are an
important component of the steady current (as well as the pulsed current) through
Pelvetia eggs.
It may be added that low-temperature 4SCa2+ autoradiography also suggests that
calcium ions are a significant component of the current entering the tips of growing
pollen tubes (28). The relatively immediate responses of the pollen current to
systematic changes in the concentration of various ions in the medium indicate that
the bulk of the steady pollen tube current is carried inwards by K+ ions (and
outwards by H+ ions) (27). Nevertheless, autoradiography shows striking accumula
tions of 4SCa in the growing tips of lily pollen tubes, and this same result is obtained
when the pollen is exposed to labeled medium for times as short as 1 min or as long
as 5 hr. The 1- to 5-hr labeling experiments show that calcium is relatively concen-
_I
trated within the cytoplasm of the growing tips and the 1- to 3-min labeling experi-
ments suggest that calcium may enter the tip faster than it enters other regions.
However, there is still doubt as to whether the short-time accumulations might not
represent calcium absorbed on some special component of the tip wall rather than
calcium that has crossed the tip plasma membrane into its cytoplasm (28).
Measurements of Transcellular Currents and Fields in Animal Eggs
The measurements of transcellular currents and fields made on animal eggs are
summarized in Table 2. In compiling this data we included only those measurements

made on one- or two-celled embryos. We chose not to include measurements on

multicellular embryos at later stages of development (see 43-46) because of the
complexity of these systems and the difficulty of interpreting such data. There are
three very interesting observations apparent from this summary that deserve discus
sion. (a) In all four animal eggs studied between fertilization and first cleavage, a
steady current was found to enter the animal pole while leaving the vegetal pole or
equator. (b) In the silkmoth oocyte-nurse cell complex, or so-called ovarian follicle,
the oocyte cytoplasm is 10 mV more positive than that of the nurse cell cytoplasm
despite their connection by a broad cytoplasmic bridge. Moreover, as discussed
later, there is evidence that maintenance of this remarkable potential difference is
necessary for maintenance of unidirectional transport of macromolecules across the
bridge and into the growing oocyte (19, 41). (c) A steady current enters the prospec
tive cleavage furrow in both frog and sea urchin eggs during the 10 min prior to
cleavage initiation, and about 8 min after initiation this current reverses and leaves
the furrow region.
The most interesting and remarkably consistent result is the steady inward cur
rent at the animal pole of the two fish eggs, Fundulus and Oryzias, as well as the
two amphibians, Hynobius and Rana. This observation by four independent investi
gators using both contact electrodes and the vibrating probe suggests that these
animal-vegetable transcellular currents are both reliable and general. Thus it would
be interesting indeed to learn whether or not these currents play a role in the
determination of the animal-vegetal axis in these eggs. In this regard it is worth
noting that in the ovarian follicles of the Cecropia moth Woodruff & Telfer have
also measured a steady current entering the animal pole (41).
However, in these same follicles, intracellular measurements show a large voltage
difference across the bridge(s) from the oocyte to the nurse cells, which implies
current flow in the opposite direction through the bridge, i.e. towards the animal
pole. We would like to suggest a possible explanation for this apparent paradox
(Figure 1). We suggest, since the whole oocyte-nurse cell complex arises by the
incomplete cleavage of a single precursor cell, that the membrane properties around
each of the cells in this complex changes continuously and in the same way in going
from pole to pole. Hence, current should leave the posterior or vegetal end of each
nurse cell, cross the incomplete cleavage furrow that partially separates it from the
oocyte, and then enter the anterior or animal end of the oocyte. We suggest that
it is such furrow currents that make the animal end of the oocyte electropositive with
regard to the vegetal end of the nurse cell and thus drive a back current through
Table 2 Natural currents (and fields) through developing animal cells
Surface current
Measurement Contact Inferred steady density
Case Cell type method medium current direction (j.lA/cm2) Year Ref.
Unphysiological"
I Chrysamys turtle egg extracellular 0.13 M NaCI leaves animal pole, 1905 39
surface poten- enters vegetal pole
tial cut camel during maturation
hair
Fundulus fish egg extracellular 0.13 M NaCI enters animal pole, 1905 39
surface leaves vegetal pole

potential during 45 min before
I st cleavage
Rana (?) frog egg extracellular insuffi cie nt enters animal pole, 1941
surface description leaves equator be-
potential tween fertilization
and 1 st cleavage
Physiological
4 Hynobius uro dele ex tracellular pond water enters animal pole 1935 40
nebulosus egg surface and sperm entry point,
potential leaves vegetal pole
between fertilization
and lst cleavage
1-5 1977 h
Oryzias fish egg ext racell ular enters an imal pole , -
tatipes vibrating leaves vegetal pole

pro be after fertilization
during cytoplasmic
segregation
6a Hya{ophora silk moth extracellular enters animal pole, 1974 41

cecropia oocyte- voltage across leaves vegetal pole
nurse cell u nk no wn
complex resistance
6b Hya[ophora silk moth intracellular internal current flows -c 1973 19

cecropia oocyte- microelectrode across bridge from
nurSe I;ell oocyte to nurse cells
complex
d
Strongelo- sea urchin extraceUular enters prospective 0.2 1977 -
centrotus egg vibrating cleavage furrow 10
purpuratus probe min before each

cleavage
0.5 1977 -e
Xenopus frog egg extracellular enters as in sea urchin;
faevis vibrating leaves at furrow begin-
probe ing 8 min after
cleavage begins
Rat visual rod extracellular enters outer segment 60 1970 42

voltage gradi- and leaves inner
ent (known segment
resistance)
aWe have not included the measurements of Dorfman (47) in this summary because he used gross 20- to 30-.um
microelectrodes that permanently damaged the egg so that it could not be fertilized.
bR. Nuccitelli, manuscript in preparation.
cThe oocyte cytoplasm was 10 mV more positive than the nurse cell cytoplasm. If this difference was entirely
produced by the steady flow of a highly m o bil e ion, such as K+, it can be estimated to correspond to a density of
the order of 106 JjA/cm2 across the bridge 0) and the order of 1000 IiA/cm2 across the pJasma membrane. To
the extent that it arose from a calcium ion (or relatively immobile ion) flow it could correspond to far lower current
densities.
dR. Nuccitelli and K. R. Robinson, manuscript in preparation.
e Robinson and Nuccitelli, manuscript in preparation.
the bridge. If this explanation is correct, it may indicate the more general develop
ment of back currents across the bridges that join cells that undergo incomplete
cleavage across their axes.
The third interesting observation concerns recently found cleavage currents in
both sea urchin and frog eggs. The first few cleavages in both of these eggs exhibit
a similar current pattern: current enters the prospective cleavage furrow during the
10 min preceding cleavage initiation and then a few minutes after cleavage has begun
there is a 5-10 min interval of reversed current leaving the furrow region. We
suspect that the precleavage inward current may be involved in generating the
contractile ring. The outward furrow current during cleavage may result from the
insertion of new membrane that is highly potassium permeable. Such membrane

insertion has been demonstrated (48, 49) based on membrane hyperpolarization
during cleavage as measured with intracellular microelectrodes inRana and Amby
stoma.
The last case in Table 2 is the dark current through rat visual rods. This is, of
course, a mature, functioning, and highly differentiated cell. However, in an impor
tant sense it is also a developing one. Every 7-9 days, the entire outer segment is
Figure 1 Proposed furrow current to explain Woodruff & Telfer'S (41) observations on an
oocyte-nurse cell complex. A diagrammatic complex containing only one nurse cell (n) plus
the oocyte (0) is shown. The membrane of the complex is postulated to bear a gradient of
properties going from the anterior pole (A), where current has the greatest tendency to enter,
to the posterior pole (P), where it has the greatest tendency to leave. Incomplete furrowing
thus juxtaposes membrane regions with markedly different properties and yields the internal
furrow current loop shown.
apparently replaced by a process of continuing basal growth and apical degeneration

( 50). On the basis of the rod's dark current, shape, and internal conductance, Hagins
et a1 (42) estimated a steady internal voltage drop of about 2 mV along the rod, with
the distal end of the interior positive. However this may be a significant underesti
mate for reasons discussed above.
Potentials Across Developing Epithelia
The most obvious place large steady endogenous potentials exist across metazoan
cells is in epithelia. Various mature, functioning epithelia are well known to
maintain potentials of the order of 30-100 mV or more across themselves (51). To

the extent that the charged components on the lateral membranes of these cells are
free to move, the usual large transepithelia1 potentials would maintain them in a
highly polarized distribution (95). However, one may well ask whether sufficient epi
thelial potentials to efl"ect such redistribution appear while these epithelia are devel
oping; thus, before their structures and functions are so grossly polarized, when the
polarity of the epithelial cells may not yet be fixed, and membrane components may
be more mobile. Table 3 summarizes the limited data available on potentials across
epithelia quite early in development. In all cases the values tabulated were obtained
at the earliest developmental state studied. The four potentials recorded lie between
3 and 8 mY, an order of magnitude lower than those typical of mature epithelia.
Nevertheless they should be large enough to grossly polarize the distribution ofsome
membrane components via laterial electrophoresis. We may add that Morrill et al
(57) claim to find potential differences of about 40 mV (inside negative) across the
frog's blastocoel wall but, within the limits of their measurements (a few millivolts?),
Slack & Warner could find no measurable blastocoel potential in the frog at all ( 58).
In the best studied case, that of the chick chorioallantoic membrane, certain
additional facts may be noted. First, according to Stewart & Terepka (53), during
6- 10 days of inCUbation this potential increases in an exponential way from -3 to
-18 mY. Second, even at 9-10 days this membrane still shows little sign of structural
or functional polarization (53, 59). Third, dissociated and reaggregated cells un
dergo relatively organized development when explanted to this membrane (60), and
Scott (61), who first measured the chorioallantoic potential, raised the interesting
question of whether it helped organize the development of explants. Fourth, as its
Table 3 Potentials aeross developing cpithclia
Number of
Case Epithelium cell layers Pot entiala Year Ref.
Coelenterate (Obelia) body wall 2 -1 to -3 mV 1925 26

2 1- to 2-day fish (Fundulus) gastrula 1 -7 ± 1 mV 1970 52
3 6 -day chick chorioallantoic membrane 2 -3 mV 1969 53,54
4 Early mouse blastocyst wall -5.0 ± 0.5 mV 1973 55
5 5-day rabbit blastocyst wall -7.6 ± 0.6 mV 1969 56
a Negative sign means the inner region is electronegative.

ELECTRICAL CONTROLS OF DEVELOPMENT 4SS
name implies, this membrane is actually a compound structure including two sepa
rate epithelial layers, and a recent study of Kyriakides & Simkiss (54) indicates that,
at least at a somewhat later stage, most of the potential is developed across the outer
chorionic component and is very sensitive to the chloride ion concentration.
Natural Currents and Fields Across Higher Plant Organs

Using noncontacting vibrating electrodes, a Swedish group has shown that corn
coleoptiles, which are stimulated to bend upward by horizontal placement, develop
potential differences of 80 mV or more (1) across themselves, with their lower (and
faster growing) side the positive one. These remarkable geoelectric potentials de
velop after a I S-min latency period and may last for an hour or more. It should be
emphasized that unlike most surface potential measurements these directly indicate
the natural potentials developed across the growing tissue (21, 62; also see 63).
Very similar potentials accompany phototropic responses (64, 65). It seems clear
that these geoelectric and photoelectric potentials are some consequence of a gravi
tation- or light-induced auxin gradient; the tissue becomes electropositive on the
high auxin side. They begin with about the delay known necessary for auxin redistri
bution, are completely dependent upon a supply of auxin within the tissue, and can
be simulated by application of an auxin gradient.
One may ask how an auxin gradient generates a field across the shoot. It
develops rather slowly, with a half time of about 15 min, which might suggest the
slow creation of an extracellular concentration gradient that in turn wou ld generate
a diffusion potential in the extracellular space. However, the final potential differ
ences reached are so high-up to 90 mV-as to make such a mechanism unlikely.
Presumably, cell membranes are more directly involved. There is a growing evidence
that physiological increases in extracellular auxin somehow act to hyperpolarize
plant cell membranes (66-68), though this hyperpolarization may not be a direct
(i.e. electrogenic) effect but rather an indirect consequence of the secretion of
hydrogen ions in turn induced by auxin (69, 70). We suggest that auxin gradients
somehow induce fields by way of this hyperpolarization. This could imaginably
occur in two ways: via the sum of currents loops induced in each of a series of
electrically isolated cells or via one large current loop through a symplasm of
electrically connected cells. The degree and direction of coupling between high plant
cells is stilI quite obscure. (71).
Another piece in this puzzle is recent observations that slow calcium and potas
sium redistributions accompany the tropistic responses of corn coleoptiles (72).
By an hour or two after stimulation, the faster growing side has 10-20% less cal
cium2 and 10-15% more potassium on either a fresh or dry weight basis. The
potassium is presumably largely intracellular and might well be driven in by a higher
membrane potential on the faster growing side; however, the potentially more
interesting calcium difference is harder to interpret since it could easily be either
intracellular or extracellular.
2 Note that the abstract of Goswami & Audus's paper (72) seems to have inadvertently
implied that calcium is higher on the faster growing side.

A consistent pattern of surface potentials has been measured by several investiga

tors on a variety of roots moistened with tap water or other physiological media,
particularly onion roots (73, 74), broad bean and Lupinus a/bus roots (75), and
maize roots (76). It can be inferred from these surface potential patterns that
growing roots generally drive currents through their interiors from base to apex and
that such acropetal currents flow through the terminal centimeter or so of the root.
Longitudinal voltage differences of 30-60 mV were measured in these studies. Since
roots can (and perhaps often do) grow in environments electrically comparable to
those used during the measurements, these surface potential gradients (as well as
the inferred inner fields) may be natural control factors (see Table 8, below).
More recently, Scott & Martin (77) have measured potential gradients along bean
roots growing in more extended media, i.e. fully immersed rather than just moist
ened. This method makes it easier to estimate the size of the currents involved, and
in various media total currents of 0.1-0.4 IJ.A were inferred (77). The exact pattern
of these currents within the roots is unknown; however, considering that their
diameter is about a millimeter, internal longitudinal current densities could easily
reach 10 lJ.a/cm2 or more. However these measurements were made on roots
growing in unnatural, single salt solutions incompatible with extended growth, so
the more or less aberrant current patterns found are of doubtful significance as
natural growth controls. Other aspects of these studies, such as the 5-min oscilla
tions of current density sometimes observed, are reviewed by Scott (78).
Natural Currents Through Regenerating Invertebrates

The literature shows a number of older reports of surface potential measurements
on regenerating annelids and coelenterates. The results of these studies, which
employed physiological contact fluids and are therefore relatively reliable, are sum
marized in Table 4. As an earthworm regenerates posterior segments, its posterior
end remains relatively electropositive indicating that current is emerging from (or
perhaps near) the caudal, regenerating surface (79, 80). Current also tends to emerge
from the regenerating end or ends of a hydroid stem section. In all of these hydroid
studies, it appears that a hydranth (rather than a stolon) was regenerating (81,82).
The voltage measured across a caudally regenerating earthworm rises in propor
tion to the number of new segments rather than the absolute increase in the animal's
length. Moment has interpreted this to mean that segment addition normally stops
when a critical longitudinal voltage difference is reached (79). One difficulty with
this idea is that the animal seems likely to be able to regenerate over a wide range
of surface conductances and to generate a relatively constant current rather than
a constant external voltage as this surface conductance is varied.
Natural Currents Through Regenerating or Healing Vertebrate Limbs,

Skin, and Bone
The surface potential and current measurements made on regenerating vertebrate
limbs as well as healing skin and bone are summarized in Table S.
The pioneering investigation of the natural surface potentials along regenerating
limbs was conducted by Monroy in 1941 (83). Although he used an unphysiological
Table 4 Natural currents through regenerating invertebrates
Source of What is Inferred steady

Group Genus regenerate regenerated current direction Year Ref.
Annelids Eisenia anterior end posterior leaves regenerat- 1949-55 79,80

segments ing end
Hydroids Obelia ste m s ecti on hydra nth leaves regenerat- 192 1-25 5,26,
ing end 81
Tubularia s tern section hydranth variable 1934 82

Eudendrium stem section hydranth leaves both regen- 1934 82

erating ends
Pennaria stem section hydranth leaves regenerat- 1934 82

ing proximal end,
but enters regener-
ating distal e nd
contact solution of 0.8% NaCI, his finding that current leaves the regenerating
stump and enters proximally is in agreement with the two other independent mea
surements, at least during the first 5 days after amputation. Moreover, he found by
surgical denervation of the limb that this current was completely independent of the
nerve supply. After determining that the current also persisted up to 24 hr after
removing the heart, he concluded that the skin must be considered a possible source
of the measured surface potentials. The most recent and reliable studies using the
extracellular vibrating probe indicate that in fact the skin is driving this regeneration
current (Borgens et aI, manuscript in preparation). These first true current measure
ments confirm Monroy's finding that current consistently leaves the regenerating
stump and the probe measures outward current for at least 10 days after amputation.
This current is unaffected (or slightly increased) by surgical denervation and appears
to be generated by the Na+ pump in the skin since the current is greatly reduced
in low Na+ (less than 100 p.M) and greatly increased by an increase in external
Na+ (8 mM), and because it is quickly shut off by application of 0.3 mM amiloride,
a compound that blocks transcutaneous Na+ uptake. Using this more reliable and
physiological vibrating probe method, the 5-day-postamputation current rever
sal reported by Becker (84) was not observed, but rather the current continued to
leave the entire blastema with the largest current in the postaxial region. About
10 days postamputation the current became erratic but did not exhibit a consistent
reversaL
Interestingly, currents have also been measured leaving a lesion in human skin
during healing (86, 87). It is extraordinary that no further investigation into this
extremely intriguing finding has been done. Finally, healing bone fractures in frog,
rabbit, and humans drive current into the periosteum and skin above the fracture
(88, 89).
Table 5 Natural currents through regenerating vertebrate limbs, skin, and bone
Ca se Organism Measuring method Inferred current direction Year Ref.
Triturus regenerating newt su�face potential leaves regenerating end of 1941 83

cristatus limbs and tail 0.8% NaCI contact limb or tail, enters proxi-
mal end throughout re-
generation
2 Triturus regenerating newt surface potential leaves regenerating end of 1961 84

viridescens limb 0.8% NaCl contact limb, enters proximal end
during first 5 days of reo
generation; then reverses

3 Triturus regenera ting newt surface potential enters regenerating end of 1974 85
viridescens limb 10% Holtfreter limb, leaves proximal end
contact 10 to 18 days after am-
putation
2
4 Triturus regenerating newt vibrating probe 30-100 p.A/cm leaves reo 1977 _a
viridescens limb generating end of limb.
enters proximal end and
shoulder throughout re-
generation
5 Human healing skin insufficient data leaves healing skin lesion, 1952 86,87
lesions enters surrounding skin 1972
6 New Zea- healing tibial surface potential enters skin above frac· 1966 88
land white fracture isotonic saline ture during healing
rabbit contact period
7 Human healing tibial surface potential enters skin above frac- 1966 88
fracture isotonic saline ture during healing
contact period
8 Rana healing tibia! surface potential enters intact periosteum 1970 89

pipiens fracture ringer contact above fracture during 7
days after fracture
a Borgens et ai, manuscript in preparation.
EFFECTS OF MODIFYING NATURAL FIELDS

Theory
The direct target ofsteady field action on a single, relatively isodiametric cell should
be the plasma membrane since practically all of the resistance across such cells is
generally across this membrane. Thus, unless field strengths large enough to greatly
reduce membrane resistance are involved, only a very small fraction of a steady
voltage drop across a cell will be exerted across its interior. This point is most easily
illustrated by considering a spherical cell such as an egg cell.
Consider such a cell placed in a conductive medium (e.g. sea water or serum) in
a uniform field, E. If the resistivities of membrane and interior are uniform, then
the field across the interior is known to be uniform, too (90). The voltage drop from
pole to pole across the whole cell is given by
V= 1.5 Ed. 2.
where d is the cells diameter (90). The drop is divided between the resistances (per
unit area) of the plasma membrane (0') at the two poles and that of the cytoplasm
in between. If the cytoplasm has a resistivity, p, then the resistance of a unit
cross-section will be pd. So the fraction of the total voltage drop across the cyto
plasm (c) will be
Ic < pdlO'. 3.
Two relatively well-investigated cases are those of the sea urchin egg and the sea
weed egg. For unfertilized eggs of the sea urchin, d is about 10-2 cm, p is 200
Ocm (17), and 0' is 150 kO cm2 (L.A. Jaffe and K.R. Robinson, manuscript in
preparation); hence, Ic 1 0-5• For fertilized Fucus eggs, dis 8 X 10-3 em, pis 200
=
Oem, and 0' is 1400 Ocm2 (91, 23); hence, Ic = 10-3• We know of no (more or less
isodiametric) cells in which Ic falls outside the range of 10-5-10-3.
Thus the most obvious mode of steady field action on cells is to act across the
plasma membrane, i.e. by introducing a significant gradient in the membrane poten
tial around the target cell. Since the interior will be essentially equipotential, this
gradient in membrane potential will depend only upon the gradient external to the
membrane. For a highly resistant sphere in an otherwise uniform field, the potential
just outside of it is given by
v= 1 .5 D cos 0, 4.
where 0 is the sphere's latitude (90). If the membrane does not actively respond to
the field, the preexisting and imposed potentials will simply add to give
5.
where Vm is the membrane potential before imposition of the field. (Note that
equation 5 does not require membrane resistance to be uniform; it need only be
large compared to that of tIi e cytoplasm to keep the cytoplasm equipotential, as well
as large compared to the medium to make the cell as a whole relatively resistant.)
Provided that relatively small fields are employed (Le. fields evoking essentially
linear responses below the region of inevitable saturation) the degree of growth
polarization to be expected should be about
P = 1.5 Ed/ Vm' 6.
where P is defined as the average cosine of a population of cell outgrowth directions.
The reason for suggesting this simple equation lies in the empirical rule that governs
the polarization of cell growth by vectors (such as light gradients and chemical
concentration gradients) whose size can be meaningfully given by a percentage (i.e.
fraction) of difference per cell: the percentage of growth polarization approximates
the percentage imposed gradient (92). Thus for cells with typical membrane poten
tials of about 50--100 mV, one-tenth maximal growth polarization should be induced
by a potential drop across the target cell of 5-10 mY.
The same gradient in extracellular potential given by equation 4 will exert
forces along the outside of the target cell membrane as well as across it. Many mol
ecules and particles are freer to move along the outer membrane of cells than
out of this membrane (93, 94). Since many of these components are charged, it
follows that application of tangential electrical forces to cells should redistribute
them within the plane of the plasma membrane. This process may be called surface,
lateral, or perhaps perimembrane electrophoresis (95).
The degree of redistribution effected in this way should ultimately be limited by
back diffusion. EventuaJly an equilibrium, or more accurately a steady state between
electrophoresis and back diffusion, may be reached. The time needed to reach a
steady state will be the order of L21 D, where L is the characteristic cell length and
D is the affected components diffusion constant. Such constants have been measured
in a number of cases and generally are in the range from 5 X 10 -II to 5 X 10-9
cm2/sec (93, 94). Most relatively isodiametric target cells will have diameters of
the orders of 10-3-10-2 cm, so steady-state times will generally be of the order
of 102_106 sec. Endogenous developmental fields are likely to be applied to target
cells for the order of 1()4-1Q6 sec, whereas most experiments have employed treat
ments with small fields for the order of l()4-lO' sec. It can be concluded that
steady-state equations may be relevant in many though certainly not all real cases.
In any case, such steady-state equations involve the ratio of the target component's
electrophoretic mobility (m) to its diffusion constant (D), rather than D alone. Since
this mobility-to-diffusion ratio, m iD, can be better estimated than can D itself, the
steady-state equations seem far more useful at this stage of investigation. The
following discussion is condensed from Jaffe (95).
Consider a component in the steady state on the surface of a spherical cell
subjected to a uniform field. It can be shown that the surface concentration, C, is
given by
c = C [E • csch (E)] • exp ( E cos 8), 7.
where C the target particles' average concentration and E = (miD) . V12.

=
For comparison with cell galvanotropic responses (or perhaps other directed
responses) it is helpful to characterize the degree of polarization of such steady-state
distributions by some parameter, </>. Since the tropic responses of cell populations
are commonly characterized by the average cosine of the distribution of outgrowth
angles (96), we define in an analogous way:
</> = C • cos 8 dsl Cds. 8.
Integration of equation 7 yields3
8a.
and for E < 1 is usefully approximated by
</> = (miD) . ( VI6) 8b.
3 Dr. William Hagins has pointed out an interesting fact. This expression is formally
identical with the so-called "Langevin function, L(x)," which indicates the degree of polariza
tion of a population of electrical dipoles at equilibrium in a static electrical field (97).
To apply this equation, we must estimate (m/ D) for the target particles. It can
be shown that this ratio is independent of the hydrodynamic drag they encounter
(95). Hence we can use the values known for particles immersed in the usual one
centipoise, 0.05 M ionic strength, aqueous media. Somewhat, arbitrarily we
assume a mobility value of 3 X 10-4 cm2/sec volt, which is towards the high end
•
of known mobilities, and a particle diameter of 10 nm since the larger particles seen
in freeze-fracture electron micrographs of plasma membranes are of about that
size.4 Particles of that size, etc, have a diffusion constant of about 4 X 10-7 cm2/sec.
So, together these assumptions yield a D/m value for the growth particles of 1.3
mY. Then applying equation 8b with <f> 0. 1, we infer that 1/10 maximal polariza
=
tion should be induced by a voltage drop of only I mV/cell. Note that this character
istic value (for a field effect mediated by perimembrane electrophoresis) is an order

of magnitude smaller than that expected for one mediated by a gradient in mem
brane potential.
A simple but important point to note is that both cross-membrane and perimem
brane effects should usually depend upon the voltage difference across a target cell
rather than the field, i.e. the voltage difference per unit length. In other words one
generally expects larger cells to respond to correspondingly smaller fields.
Strictly speaking, the above theory holds only for spherical cells, and gross
deviations are expected for the responses to fields directed along extremely elongated
cells (such as neurons) in which the cytoplasmic resistance can become large relative
to the membrane resistance. Consider an idealized neurite of radius, a, exposed to
a uniform longitudinal field. The internal field Ei must approach the external one,
Eo, as the neurite length approaches infinity. Por if it did not, the imposed voltage
drop across the membrane would approach infinity and that is obviously false. A
more detailed analysis shows that
Ej = Eo (1 - exp (-x/A» , 9.
where A is the length constant of this cable equal to J au/2p, and x is the distance
from its closed end. In reality, developing neurites have a highly dilated and con
voluted growth cone at their tips. The large surface area of this structure should
make a real neurite even more penetrable by an external field than equation 9
indicates. Altogether, external fields could well effect lateral transport of charged
particles protruding from the inner side of the plasma membrane of growing neu
rites; indeed external fields might even move charged components suspended in the
cytoplasm of such cells.
Finally, in field effects upon tissues (as opposed to single cells) one must consider
extracellular targets. It has been suggested that bioelectric fields may sometimes act
by orienting extracellular, growth-guiding filaments (98). However, studies on elec
trically induced birefringence indicate that fields of the order of 300-3000 V/cm are
• Since mobilities are independent of a particle's radius while diffusion constants vary
inversely with this radius, r, the expected Dim values would be multiplied by 5 nmlr were
r in fact to differ from 5 nm.
generally needed to half-orient populations of various highly elongated biological

particles, e.g. 0.9-p.m-long collagen molecules require 4000 V/cm, and 0.3-p.m
DNA require 500 V/cm (99-101). One exceptionally low value of less than 50 V/cm
has been reported for aggregates of 1- to lO-p.m (?) actin filaments (102). Neverthe
less these fields are the o rder of 100 times or more greater than those either known
or theoretically expected to affect cells more directly, and they similarly exceed the
natural fields that may be expected in the extracellular regions of tissues. So this is
one mechanism that can be generally considered to be highly unlikely. On the other
hand, there is no reason why significant electrophoresis may not sometimes occur
within extracellular regions.
Electrical Activation of Eggs

According to Yamamoto (103), unfertilized eggs of the medaka (a fresh water fish,
Oryzias latipes) can be artificially activated by a field of 2 V/cm or more applied
for 20-60 sec. Activation occurs at both the anodal (i.e. positive) and cathodal poles
but the reaction time is half as great at the anodal pole as at the cathodal one. A
field of 2 V/cm should produce a voltage drop of 360 mV across these 1 .2-mm
diameter eggs. Initially at least this should hyperpolarize the membrane potential
at the anodal pole by 180 mV and depolarize it at the cathodal one by 1 80 mY. An
explosive increase in free cytoplasmic calcium is known to be a central event in the
activation of these eggs (104), so it is reasonable to guess that anodal activation
occurs by driving calcium into the egg; but how is cathodal activation mediated?
Sea urchin eggs can also be anodally activated (at least locally) by a field that should
hyperpolarize the membrane at the positive pole by about 200 mV; however, the field
only seems to require ill few seconds to act in this case (105). Finally, we may note
that hamster ova can a lso be activated by an electrical field, albeit a poorly defined
and apparently extreme one (106).
Direct Evidence of Lateral Electrophoresis
Before reviewing low- and moderate-field effects on cell development, we summarize
what seems to be direct evidence of the predicted phenomenon of lateral or peri
membrane electrophoresis (107). Cultured embryonic Xenopus muscle cells were
exposed to small fields for periods of 0.5 hr to I day. Then they were labeled with
fluorescein-conjugated concanavalin A and examined with a fluorescent microscope.
A striking redistribution of concanavalin A receptors can be seen after sufficient time
in a sufficient field. Thus exposure of the 30-p.m-wide cells to a voltage difference
across them of 12 mV for 2 hr suffices to produce a gross accumulation of con
canavalin A receptors on the negative side of the cell. A tenth this voltage difference
(thus 1.2 mV) or a quarter of this time (thUS 0.5 hr) gives detectible redistributions
in the same direction. These would seem to imply that the receptors have a positive
charge, which is quite surprising. Nevertheless, the facts leave relatively little doubt
that the observed redistribution is brought about directly, by lateral electrophoresis,
rather than some more complex and active mechanism. For one thing, the process
goes at about the rate to be expected of lateral electrophoresis, namely the order of
10-7 em/sec per V/crn, which is several orders of magnitude slower than the rates
ELECfRICAL CONTROLS OF DEVELOPMENT 463
known for the process of active redistribution called capping (l08). For another, a
voltage drop of less than a millivolt per cell gives detectible redistribution. As
discussed above, this easily fits the expectation for lateral electrophoresis but not for
one mediated by a gradient of membrane potential. For yet another, it is not affected
by either high concentrations (5 IJ.M) of colchicine or by reduction of external
calcium from 2 mM to contamination levels of perhaps 1 to 10 IJ.M. Most important,
it is unaffected by treatment with a combination of metabolic inhibitors (10 mM
arsenate, plus 1 mM dinitrophenol, plus 1 mM NaF applied for 2-5 hr) that would
be expected to reduce cytoplasmic ATP to very low levels and is in fact observed
to block the contractile response of these cells to the acetylcholine analogue, carba
chol.
Field and Ion Gradient Effects on Plant Cells
Table 6 [modified from (109)] summarizes the sparse literature on single plant cell
responses to prolonged, steady electrical fields. It shows the recorded responses
falling into two distinct classes: a low-voltage and a medium-voltage group.
The low-voltage responses require about 0.2-0.4 mVfeell (to yield a tenth maxi
mal response) and involve three different cells each no more than 10 p.m in diameter.
The medium-voltage ones require about ten times this voltage, namely about 3-6
mV/cell, and involve six much larger cells, actually cells from 70--10,000 IJ.m in
diameter (or length). The low-voltage responses may well be mediated by perimem-
Table 6 Voltage giving tenth maximal response in known cases of single plant cell galva-
no tropisma
Cell diameter Exposure MiIli·

b
Case (I'm) Genus Cell t ype time (hr) Resp on se Direction volts/cell
� I O,OOOc Acetabularia stalk 45 cap regenerates + a few

segment
2 - 1 ,000 Griffithsill shoot 70 rhizoid starts + -4
3a 100 Pelvetia egg 12 rhizoid starts + 6
3b 1 00 Pelvetia egg 12 rhizoid starts 3
4 70 Fucus egg 10 rhizoid starts 4
5 65 Equisetum spore 10 rhizoid starts + 5
6 - 1 00 Narcissus pollen �5 tube starts -5
grain
7 10 Fucus rhizoid 3 curved growth + -0.3
8 9 U/Vll egg 12 rhizoid starts + 0.4
9 6 Funarill chloronema 3 curved growth -0.2
a In cases 2 , 3 , 4 , and 7 the tenth maximal response is taken as one where the average cosine
of the outgrowth directions is 0. 1 ; in cases 6 and 8, where the outgrowth curved by 1 0° ; and
in cases 1 and 5 , where the response is just detectib le.
bCase 1 is from Novak & Bentrup (30); 2 is from Schechter ( 1 0); 3 from Peng & Jaffe ( 09);
4 , 5 , 7 , and 9 from Bentrup ( 1 1 1 ), Figures 5 , 6 (E. limosum), and 1 2, and Table 4 , respectively ;
6 from Zeijlemaker ( 1 1 2) ; 8 from Sand ( 1 1 3) (wild type).
c Cell len th, not d iameter , in this case.
g
brane electrophoresis, as discussed above. That is, the tangential component of the
electric field. along the periphery of the cell. could move certain mobile. growth
controlling plasma membrane components that have charged heads protruding from
the lipid bilayer. Thus these components would be driven laterally. along the mem
brane. towards one pole or the other.
The medium-voltage responses. on the other hand. involve voltage differences
large enough to act across the plasma membrane, i.e. large enough to introduce a
significant gradient in the membrane potential around each cell. Furthermore.
according to Novak & Bentrup ( 1 1 4). the growth of fucoid eggs can be polarized
by rapidly alternating fields with intensities comparable to the effective direct ones.
It is difficult to imagine how net movement along the membrane could be produced
by rapidly alternating fields, but equal membrane potential displacements in oppo
site directions often fail to have exactly opposite effects. The second field of opposite
polarity may fail to reverse the effect produced by the first one. or it may even
reinforce it. So we tentatively interpret all medium-voltage responses as mediated
by gradients in membrane potential rather than perimembrane electrophoresis.
When placed in a Ca2+ gradient, eggs of the fucoid alga, Pelvetia. tend to grow
towards the end with the highest Ca2+ influx (1 1 5). Furthermore, when these eggs
are cultured in a steady-voltage gradient, most batches tend to grow towards the
positive pole (109), and increases in membrane potential (produced by reductions
in external potassium) are found to yield proportionate increases in the rates of
4�Ca2+ influx (T. Chen and L. F. Jaffe. unpublished data). As discussed above. there .
is good experimental evidence for an early endogenous calcium current through the
polarizing fucoid egg, with calcium entering the future outgrowth pole. Moreover,
there is a substantial theoretical basis for expecting a calcium current to generate
a significant field across the cytoplasm. Put together, these findings indicate that
local calcium entry is part of a natural postive feedback loop that acts to localize
growth in fucoid eggs. and they suggest that calcium entry may act, in part at least,
by way of a cytoplasmic field effect.
Two other curious observations on fucoid eggs seem harder to interpret. First. a
few anomalous batches of Pelvetia eggs responded to steady fields by growing
towards the negative pole (109). Perhaps calcium entry is actually faster at the
negative pole in these anomalous batches because depolarization excites the mem
brane and opens enough extra calcium gates to effect faster entry despite the lower
driving force there. There is some support for this somewhat ad hoc suggestion.
About 20% of the separate egg batches studied by Chen & Jaffe (unpublished data)
responded to a tripling of external potassium (and thus a 30% reduction in mem
brane potential) by an increase in 4SCa influx of about 20%. Second, Fucus zygotes
have been observed to show a fairly strong orientation towards the high potassium
ion side in a potassium ion gradient provided the average potassium is abnormally
high ( 1 1 6). This potassium gradient effect was originally interpreted as a direct effect
of the overall cytoplasmic field produced by the resulting potassium current. How
ever, this interpretation no longer seems plausible in view of the evidence for an
endogenous calcium current and hence a relatively large endogenous field in the bulk
of the cytoplasm. Another explanation might lie in the local modification of the field
within or near the channels that normally carry potassium ions (cf 1 17).
Reduction of the K+ concentration around growing lily pollen tubes effects a
rapid and parallel reduction in both the transcellular current and the tube growth
rate (27). As indicated above, such responses (together with those to other ion
changes) are taken to indicate that most of the current is carried inwards (and indeed
through the interior) by K+. Nevertheless, the field produced by this K+ current is
calculated to be far too small for its stoppage to quickly have much consequence.
Perhaps K+ reduction acts by hyperpolarizing the membrane (which it is known to
do), which in tum closes calcium channels, and it is the resultant blockage of a
calcium current that more directly blocks growth. A growth controlling (and
growth localizing) role for Ca2+ is further supported by the observed curvature of
pollen tubes towards a Ca2+ source in three other species (1 1 8). Since curvature of
a tip-growing system like a pollen tube is mediated by bulging rather than bowing,
this observation means that local application of high Ca2+ favors the initiation of
local expansion. However any direct action of high Ca2+ on cell walls would be
expected to increase their rigidity (by cross-linking acidic polysaccharides) and thus
inhibit local expansion. Hence, local application of high Ca2+ may well act to favor
local expansion of these cells by crossing the plasma membrane to refocus entry of
an endogenous current.
Finally, we may consider Acetabularia. A cap tends to regenerate on that end of
a stalk segment that faces the positive pole of an imposed field (30) or the hyperpola
rized end of an imposed K+ gradient ( 1 1 9), i.e. it regenerates at the end into which
current is forced. The endogenous current pulses that long precede and predict the
cap regenerating end in fact enter this end (30), and together these facts suggest that
endogenous inward current pulses act to favor cap regeneration. Some further
support for this possibility is provided by a report that insulation of one end of a
stalk segment from the other markedly inhibits cap regeneration (120).
Field Effects on Animal Cells

Table 7 summarizes field effects on animal cells. In none of these cases is the target
a single isolated cell. However, all five are relatively simple in cellular terms and it
seems reasonable to interpret their responses at the cell level.
Case 1 refers to Lund's report that regenerating stern sections of a coelenterate
(Obelia) respond to small, steady fields across them by growing towards the positive
pole. A reconsideration of his data indicates that detectible galvanotropic responses
occurred at current densities no higher than 300 ""A/cm2• The sea water used had
a conductivity of 45 Ocm (26) and drawings in this series indicate the stem section
to have a diameter of about 250 ""m. It follows that a detectible response was elicited
by voltage drops as small as 3 . 10--4 A/cm2 X 45 Ocm X 1.5 X 0.025 cm 0.5 mY.
=
[Even this quite low figure is conservative since a replotting of the data (see 121,
Table 2) seems to show substantial responses at less than 70 p,A/cm2 (rather than
3(0) ; however, small responses to the tlow used to keep out electrode products
somewhat confuse interpretation.]
Table 7 Effects of steady fields on animal cells
Case Exposure Field Milli-

(Ref.) Year Organism Cell type time (hr) Response (mV/cm) volts/cell
1 (121) 1924 Obelia stem section 40 grows ;;. 2 0 -0. 1-0.3

(tube with two towards +
cell thick wall)
2 (14, 1 2 1 ) 1970 Perophora stolon (tube with 240 grows 100 .; 1.6"
one cell thick wall) towards +
3 ( 19 , 4 1) 1973-74 Cecropia oocyte-nurse 0.2-0.5 reverse bridge - I ,OOO? '; 0 . 3 a,b

moth cell complex transport
4a (123) 1946 chick embryo medullary neurites 3-29 fibers ;;.500

suppressed
towards +
"
4b chick embryo medullary neurites 3-29 curve ;. 5 00 -0.6c
towards -
4cd 1977 chick embryo dorsal root 24 fiber speeded ;. 500

neurites towards -; slow-
ed towards +
• Smaller fields not tested.

b
Obtained by multiplying applied current of 5 X 10-8 A (19), times a measured resistance of 6 kn (41).
C Across a 10-,um-wide growth cone.
d M . M. Poo and L. F. Jaffe, unpublished data.
The just-effective 0.5 mV or less was exerted across a stem section consisting
essentially of a hollow tube with a wall, two cell layers thick. Thus the voltage was
applied across four cells in series. The conductance of each wall may have been
limited by one of the two layers. It is also possible that pairs of cells in the inner
and outer layers are sufficiently coupled to effectively act as a unit. However, in any
case, the effective voltage drop per responding unit is likely to have been no more
than 0.25 mY, a figure that falls in the low-voltage group and suggests mediation
by perimembrane electrophoresis.
Case 2 refers to a similar galvanotropic response by stolons of a tunicate
(Perophora). In this case, however, only a single field strength was tested and the
smallest one required to elicit a detectible response remains unknown.
Case 3 refers to a report [by Woodruff & Telfer (19)] that injection of current into
an insect oocyte (in an effort to reverse the l O-mV endogenous potential between
it and the nurse cell) is observed to reverse the otherwise unidirectional movement
offiuorescein-conjugated protein across the cytoplasmic bridge from nurse to oocyte
cell. Their actual statement is that "a current of 5 X 10-8 A was then passed in such
a direction as to reverse the normal polarity." However, the electrical arrangements
needed to demonstrate reversal of the voltage across the cytoplasmic bridge were
not employed. Moreover, in a second report, direct measurement of the bridge
resistances seemed to give values of 3-9 kO per bridge, implying that the voltage
drop produced by 5 X I era A was only O. 1 5�.45 mV (41). One possible explanation
is that the IO-mV difference was measured between points towards the middle of
the nurse and oocyte cells, not directly across the bridge, whereas the calculated
back voltage was presumably developed almost entirely across this bridge. If most
of the endogenous potential was not across the bridge-a condition that could occur
if much of it arose from a fixed charge gradient rather than a conventional voltage
drop-then the observed effectiveness of 5 X 10-8 A might be better understood.
Case 4 concerns field effects on nerve growth. The first convincing report of such
effects was that of Marsh & Beams in 1946 (123). They reported that nerve out
growths from explants of embryonic chick medulla were grossly affected by steady
fields of 500 mY/cm or more: the initiation of outgrowths towards the positive
electrode or anode were suppressed while other outgrowths grew in a curved path
towards the cathode.

A careful reinvestigation of field effects on nerve growth has been recently initi
ated by M.m. Poo & L. F. Jaffe (unpublished data). When a steady electrical field
is applied to explants of embryonic chick dorsal root ganglia, neurites that face the
negative electrode or cathode grow faster; anode-facing ones grow slower or even
retract, but no curvature is induced. Altogether, the outgrowths behave as if they
were responding to the longitudinal component of the imposed field but not to the
component across them. The remarkably asymmetric growth pattern that results
can be reversed by reversing the field. This latter study agrees with Marsh & Beams
(123) in finding complete suppression of growth towards the postive pole at about
500 mY/cm. It disagrees in finding no curvature induced in the outgrowths, but
many differences in the experiments might account for this: different source of
neurites, different media, different substrata, as well as the relative absence of glial
cells in the latter study.
In addition to these two clear and positive results, the literature shows a dozen
other attempts to study such field effects, most recently that of Sisken & Smith (124);
however, their meaning is obscured by possible electrode effects, failure to determine
the size of the fields applied, and/or failure to clearly describe the responses. In
particular, objective interpretation of Weiss' negative report is blocked by the last
two lacunae (98).
Field Effects on Multicellular Systems
Table 8 lists what seem to be the more significant developmental effects of imposing
steady fields on multicellular systems. For comparison it also lists measurements of
the corresponding endogenous fields or currents.
Case 1 concerns so-called electrotropic responses of oat (Avena) coleoptiles .
Numerous studies of this phenomenon have been reported (reviewed in 1 3). How
ever, the Wilkes & Lund article (125) is the only one employing relatively low fields
for long periods and thus seems most likely to modulate or simulate a natural field.
(Other studies used currents and hence fields at least a hundred times greater.)
Schrank measured the transverse resistance ofthe coleoptile (between what appears
to have been similar contacts under similar conditions) as about 0.3 MO (1 34).
Therefore, one can estimate (or perhaps we should say guess) that the just effective
(?) voltage drop employed by Wilkes & Lund (125) was about 30 mY. From Avery
& Burkholder's study of the coleoptile's cellular structure (135), we learn that this
voltage drop was expended upon about 100 cells in series, going around the periph-
�
0\
00
....
Table 8 Effects of steady fields on the development of multicellular systems :>
�
tt1
Field or Year Natural external R-
Case System current direction Time Size Response (Ref.) potential gradienta Ref. Z
c::
()
Oat coleoptile across axis 1 hI 0 . 1 p.A= grows faster on neg-
()
-
1 94 7 faster growing sidee 62
'"'l
30 m V ? b ,c ative side ( 1 25) up to 80 mV positive tt1
t""
�O.3 mV/celld t""
-
2 Oat coleoptile base positive 20 m in 0.3 p.Af growth rate doubles 1 937 base ;;" 40 mV positive 1 25 , 1 33
transiently ( 1 26)
base negative 20 min 0 . 3 p.A 15% slower

transiently
base nega tive 1 hI b

2a 0 . 1 p.A rate halved 1 94 7
transiently ( 1 25)
3 Onion root base positive 50 min 3 80 mV/cm b none 1 94 7 base 3 0 - 6 0 m V negative 7 3-76
( 1 2 7)
base negative S O min 3 8 0 mV/cm b growth rate 1 947

halved ( 1 27)
b
3a base negative 3 days 90 mV/cm lateral roots 1 94 7
formed ( 1 28)
Table 8 (Continued)
Field or Year Natural ex ternal

Case S ystem curren t direction Time Size Response (Ref.) potential gradien ta Ref.
4 2-mm-Iong along axis 3 days 90 mV/cm b = stolons at - end and 1921 hydranth positive 81
Obelia stem (either way) 3 2 mY/section hydranths at + end (5)
section
5 10-mm-Iong
Tubularia
along axis
(either way )
1 day 10 mV/cm =
1 5 mY/section
hydranths at + end 1934
( 1 2 9)
variable 82
�
�
stem sections (")
>
f -g
r
6 Stump of frog base to apex 3 wks 0. 1 - 0 . 2 IJ.A regeneration 1967-7 7 base to apex
arm (1 30- 1 32) 8
6a apex to base 3 wks 0.2 p.A destruction 1977
�
( 1 3 0) �
�
a Or internal current direction in case 6. o
"rj
b Minimum current giving detectable response. t:I
c Schrank ( 1 34) gives a value of about 0 .3 Mil for the transverse resistance. �
dAvery & Burkholder (1 35) give the cell structure of the coleoptile . .
eNote that the coleoptile grows by longitudinal extension behind its tip. So it bows away from its faster growing side in response to uni
S
"tl
�
lateral light or gravity.
f
Large response. Minimal stimulus not explored.
gBorgens et aI, unpublished data.
$
ery of the tissue, and on a minimum of 40 going by the shortest path through the
interior. So about 0.3-0.8 mVIcell was applied. It seems likely that the target of such
a small field was in the cell walls or on the outer surface of the cell membranes, not
within the coleoptile cells. Since the geoelectric and photoelectric potentials can be
two to three times larger than 30 mV and have the opposite direction in the
extracellular part of the circuit, this suggests the possibility of negative feedback (i.e.
in the tropistic responses auxin is redistributed, which in tum both induces unequal
growth and a transverse potential gradient; the field then acts back to reduce the
asymmetry in the distribution of auxin and of growth). Such a mechanism could
serve to prevent or limit over-response to tropistic stimuli.

Case 2, and particularly 2a, is opposite in its implications to case 1 . Again, a

substantial growth effect is exerted by as little as 0. 1 ,..,A for 1 hr. Again, the
potential gradient produced by this current along the outer surface. of the tissue is
likely to be comparable to the natural field (although the data suggesting this are
very crude). However, in this case the directions of the effects suggest that extracel
lular potential gradients act back in a positive way. Such a mechanism could help
establish and maintain the longitudinal polarity of plant shoots.
Cases 3 and 3a are harder to assess, for roots do not normally grow in air (though
they can) and certainly can grow in media conductive enough to largely eliminate
any potential gradients along their outer surfaces. So if natural field controls are
present, they are very likely to be along inner surfaces of the root or even inside
chains of root cells, and the degree to which imposed fields get inside the root seems
impossible to even guess at now.
Cases 4 and 5 concern the responses of regenerating stem sections of two closely
related marine coelenterates. These sections consist essentially of a hollow tube,
closed at both ends, whose wall is two epithelial cell layers thick. In both cases,
hydranth regeneration is favored at the positive pole, but whether these imposed
fields modify endogenous ones is obscure. It should be noted that observations of
potential differences along regenerating stem sections placed in moist air for mea
surement purposes (Table 4) need not indicate that significant fields naturally exist
along the outside of such sections. Indeed, the exterior region, as well as the internal
cavity, of such a section are presumably so conductive as to be effectively isopoten
tial under natural conditions; hence, too, the radial potentials (across the animal
from inside to outside) would likewise be effectively equal at all points. However,
these considerations do not preclude a significant natural field along the third
extracellular space, namely the mesogleal space between the ectodermal and endod
ermal layers. Viewed in this way, Lund's observations might suggest that the meso
gleal space of Obelia is normally electropositive at its distal, hydranth-regenerating
end and that an imposed field acts by modifying this natural mesogleal one.
Case 6, the electrical stimulation of frog limb regeneration, is particularly interest
ing. In Smith's pioneering 1967 study (13 1), forelimbs of adult frogs were amputated
and the stumps were implanted with Silver-platinum bimetallic rods until the ani
mals were sacrificed 3 months later. In amphibian Ringers, this primitive battery
generated 200 mV (across a 3-mm gap). In most cases, conical outgrowths were
ELECTRICAL CONTROLS OF DEVELOPMENT 47 1
'
induced. These showed a variable degree or internal structure indicative of regenera
tion, including a thickened terminal epidermis with little dermis beneath it and
isolated columns of lobulated cartilage reminiscent of wrist or hand bones. The
response was somewhat greater when the electronegative silver component was
distal rather than proximal. In his second investigation (1 32) Smith stimulated
amputated frog forelimbs with a commercial battery implanted in the back that
drew about 0. 1 JLA from the stump through a steel electrode for several weeks. The
regenerative response was greatest when the dorsal postaxial part of the stump was
stimulated. Moreover, eight stimulated animals attained "formation of at least one
well-formed movable digit," and in one remarkable case "the regenerated limb is
absolutely indistinguishable from a normal one . . . and the animal used the hand
with what appeared to be normal coordination and range of motion. There was
neither external nor internal indication of the original level of section."
Two criticisms must be made about these remarkable reports. First, it is not clear
from them alone whether regeneration is stimulated by the field imposed upon the
tissue or chemically by electrode products. However, a later study by Borgens et al
shows regeneration to be stimulated by a comparable current provided by means
that clearly avoided electrode products (1 30). Second, and more serious, is the
question of the degree of regeneration claimed. It must be plainly stated that no
significant evidence at all was presented to support the more extreme claims quoted
above: no significant histological sections, no photographs or sketches, and no report
on the course of regeneration. The one section that is shown is of the one perfect
arm, and no evidence is available to show that this perfect arm was in fact regene
rated rather than developing like other frog arms.
Nevertheless, Smith's main claim has been well confirmed by Borgens et al (1 30).
Cathodal (i.e. distally negative) application of 0.2 JLA of current to the postaxial part
of the frog's forelimb stump for several weeks regularly induces partial regeneration.
Such regeneration is indicated externally by substantial growth and internally by
organized extension of: the severed radio-ulna, newly formed muscle; ligament;
isolated, partially segmented cartilage rods; and a thickened terminal epidermis
without an underlying dermis.
Moreover, extraordinarily large amounts of nerve developed in the regenerated
tissue: about 20% of the volume of the distal portions of cathocially stimulated
animals consisted of regenerated nerve trunks, whereas less than 1 % of sham
treated or anodally stimulated animals consisted of nerve. On the other hand,
anode-stimulated animals showed extensive and characteristic degeneration, partic
ularly of the bone and muscle.
Together with the evidence of comparable endogenous currents in naturally
regenerating newts (reviewed above), these findings suggest that these endogenous
currents, and the fields they generate, are normally needed for regeneration. Some
confirmation ofthis important inference is provided by recent evidence that regener
ation of various Urodeles can be prevented by blocking the skin-driven regeneration
current, either by depleting the medium of sodium ions or by treating the stump's
surface with amiloride (Borgens et aI, unpublished data).
Together with evidence that nerve growth is accelerated towards the negative pole
in relatively small fields, these findings suggest that the targets of either endogenous
or imposed fields are the nerve processes within the stump. Moreover, Bodemer
attained a comparable degree of forelimb regeneration in adult frogs by a radically
different kind of electrical stimulation ( 1 36). He simply stimulated the major fore
limb nerve in the scapular region with about 1000, O. l-msec-long, 3OO-mV pulses
delivered during 1- or 2-min periods. The similarity of the regeneration responses
obtained in this way to those obtained by prolonged, very-low-intensity fields may
again suggest the limb nerves as the target of the latter; however, we are definitely
not suggesting that the low-intensity fields act by stimulating action potentials.
Rather, we are led to wonder if action potentials can also stimulate nerve growth;
an interesting question that is logically beyond the stated scope of this review.
Nevertheless, we note that the only evidence bearing directly on this question seems
to be a report of Hoffman (137) that rather violent electrical stimulation of the spinal
cord or the lumbosacral plexus of the rat substantially accelerates the re-innervation
of denervated fibers, by the sprouting of intact axons, in partially denervated
muscles.
Finally, we must note a truly astonishing recent report of electrically stimulated
"partial limb regeneration in rats" (138). We find these claims very difficult to
reconcile with the established information about regeneration. The forelegs of 2 1 -
day-old rats were amputated through the humerus proximal to the epiphyseal plate.
The absolutely astonishing claim is made that 3 days later, in response 3-6 nA of
current, the humerus has been almost perfectly regenerated. Furthermore, unlike
other regenerative events, this did not progress with time. According to the data,
no significant change in the degree of regeneration was attained between 3 and 28
days after amputation. Another astoriishing feature of this report is the claim that
a complete humerus ("supernumerary humerus is well formed") was formed only
7 days after amputation in one case.
Clinical Applications
Brighton et al (144) have recently reviewed the literature on electrically stimulated
healing of bone fractures as well as congenital abnormal bone gaps. It is clear that
clinically significant healing can be induced by implantation of cathodes delivering
direct current of the order of 10 /LA. It is also clear that the new bone forms in the
immediate vicinity of the cathode. These facts suggest that bone healing is mediated
by electrode products rather than by field effects. Other recent papers of Brighton
et al (1 39, 140) show that comparable cathodes in vitro substantially reduce oxygen
and moderately raise the pH in their vicinity, and these papers provide further
evidence indicating that it is these changes, particularly the local reduction in
oxygen, that in fact mediate electrically induced bone formation. No one has investi
gated the effects on fracture healing of direct currents delivered by techniques that
avoid the introduction of electrode products into the tissue. So for the moment there
is little or no reason to believe that direct current electrical fracture healing acts by
directly modifying some endogenous electrical fields. The same may be said for
electrically accelerated healing of wound and skin ulcers (141).
On the other hand, we would like to suggest a genuinely electrical explanation

for two recent and well-documented reports that amputated finger tips in children
may regenerate if surgical closure of the skin is avoided (142, 143). By analogy with
the regeneration of amphibian limbs, we would suggest that skin closure blocks
regeneration, in part at least, by blocking the How of a skin-driven current through
the stump.
SUMMARY
Developing systems generally drive steady ion currents through themselves and thus
produce substantial fields within themselves. Established examples include currents
that enter the prospective and continuing growth point of several tip growing plant
cell s a voltage across the cytoplasmic bridge between an insect oocyte and its nurse
,
cell, current traversing a recently fertilized (and segregating) fish egg from animal
to vegetal pole, early potentials across several embryonic epithelia including the
mammalian blastocyst wall and the chick chorioallantoic membrane, potentials
along the surface of several growing plant organs (particularly the sheath of the
young shoot), and a large current leaving the stump of a regenerating amphibian
limb.
The imposition of small steady fields, or the modification of endogenous ones,
often has profound effects on developing systems. Established examples include the
'control of growth direction in various tip growing plant cells, the control of out
growth extension rate in explanted neurons, the control of growth distribution in
several plant organs, and the control of limb regeneration in amphibians.
One important' mechanism that may mediate these phenomena is electrophoresis
along cell membranes of charged components that Hoat in these membranes. At the
equilibrium between such lateral electrophoresi s and back diffusion, voltage drops
of as little as 1 mV per cell should produce significant redistributions.
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ANNUAL

Ann. Rev. Biophys. Bioeng. 1977. 6:445-76

ELECTRICAL CONTROLS +9100

Michael Faraday, 1839 (I)

and influential early investigations of developmental fields were surely those of E.

ENDOGENOUS CURRENTS AND FIELDS

where E is field strength, I is current density, and p is resistivity.

Methods for Measuring Electrical Currents and Fields Through

has been removed from its natural medium.

frequency of vibration. A good example of this technique's power is the successful

Inferred current Current Elongation

Pithophora green alga extracellular sur- steady inward 1947 6

Measurements of Transcellular Currents and Fields in Animal Eggs

summarized in Table 2. In compiling this data we included only those measurements

made on one- or two-celled embryos. We chose not to include measurements on

Table 2 Natural currents (and fields) through developing animal cells

surface leaves vegetal pole

tatipes vibrating leaves vegetal pole

6a Hya{ophora silk moth extracellular enters animal pole, 1974 41

6b Hya[ophora silk moth intracellular internal current flows -c 1973 19

centrotus egg vibrating cleavage furrow 10

purpuratus probe min before each

Rat visual rod extracellular enters outer segment 60 1970 42

insertion of new membrane that is highly potassium permeable. Such membrane

apparently replaced by a process of continuing basal growth and apical degeneration

maintain potentials of the order of 30-100 mV or more across themselves (51). To

Table 3 Potentials aeross developing cpithclia

Coelenterate (Obelia) body wall 2 -1 to -3 mV 1925 26

a Negative sign means the inner region is electronegative.

Natural Currents and Fields Across Higher Plant Organs

implied that calcium is higher on the faster growing side.

A consistent pattern of surface potentials has been measured by several investiga­

Natural Currents Through Regenerating Invertebrates

Natural Currents Through Regenerating or Healing Vertebrate Limbs,

Table 4 Natural currents through regenerating invertebrates

Source of What is Inferred steady

Annelids Eisenia anterior end posterior leaves regenerat- 1949-55 79,80

Tubularia s tern section hydranth variable 1934 82

Eudendrium stem section hydranth leaves both regen- 1934 82

Pennaria stem section hydranth leaves regenerat- 1934 82

Ca se Organism Measuring method Inferred current direction Year Ref.

Triturus regenerating newt su�face potential leaves regenerating end of 1941 83

2 Triturus regenerating newt surface potential leaves regenerating end of 1961 84

generation; then reverses

8 Rana healing tibia! surface potential enters intact periosteum 1970 89

a Borgens et ai, manuscript in preparation.

EFFECTS OF MODIFYING NATURAL FIELDS

c = C [E • csch (E)] • exp ( E cos 8), 7.

where C the target particles' average concentration and E = (miD) . V12.

</> = C • cos 8 dsl Cds. 8.

Integration of equation 7 yields3

and for E < 1 is usefully approximated by

</> = (miD) . ( VI6) 8b.

istic value (for a field effect mediated by perimembrane electrophoresis) is an order

generally needed to half-orient populations of various highly elongated biological

Electrical Activation of Eggs

Cell diameter Exposure MiIli·

� I O,OOOc Acetabularia stalk 45 cap regenerates + a few

Field Effects on Animal Cells

Table 7 Effects of steady fields on animal cells

A consistent pattern of surface potentials has been measured by several investiga

1. Faraday, M. 1839. Experimental Re 1 1. Liboff, A. R., Rinaldi, R. A. 1974. Ann.