The perivascular space of the rat pineal gland is known to contain phagocytic cells that are immu... more The perivascular space of the rat pineal gland is known to contain phagocytic cells that are immunoreactive for leukocyte antigens, and thus they appear to belong to the macrophage/microglial cell line. These cells also contain MHC class II proteins. We investigated this cell type in the pineal gland of mice. Actively phagocytosing cells with a prominent lysosomal system were found in the pericapillary spaces of the mouse pineal gland following intravenous injection of horseradish peroxidase. The cells also exhibited strong acid phosphatase activity. Perivascular cells were immunopositive for MHC class II protein and for CD68, a marker of monocytes/ phagocytes. This study verifies that perivascular phagocytes with antigen-presenting properties are present in the mouse pineal gland.
The vertebrate retina contains a circadian clock participating in adaptations to day and night vi... more The vertebrate retina contains a circadian clock participating in adaptations to day and night vision. This peripheral clock is independent of the master clock in the suprachiasmatic nucleus (SCN). The retinal clock is located in several cell types, including the photoreceptors. To investigate the role of the circadian clock of the photoreceptor cells in regulation of retinal protein rhythms, we analysed diurnal protein expression in the photoreceptor-deficient cone-rod homeobox knockout mouse (Crx(-/-) ) and the 129/Sv mouse. 2D gels were made from retinal homogenates of 129/Sv and Crx(-/-) mice killed at midday and midnight. Stained gels were analysed by use of PDQuest 2D gel analysis software. After trypsin digestion of differential expressed spots, the proteins were identified by LC-MS/MS using a nano-liquid chromatograph connected to a Q-TOF Premier mass spectrometer. These data were used to search the SWISS-PROT database. Both the retinae of the control and the Crx(-/-) mice e...
Journal of the Medical Association of Thailand Chotmaihet Thangphaet, Jul 1, 2005
The human pineal gland secretes melatonin in a circadian rhythm manner. The rhythm of melatonin s... more The human pineal gland secretes melatonin in a circadian rhythm manner. The rhythm of melatonin synthesis is primarily controlled by the noradrenergic sympathetic system originating from the superior cervical ganglion. Several neurotranmitters/neuropeptides have been reported to influence the production of melatonin in the pineal glands of many mammalian species. Both opioid peptide, a pain suppressing peptide and substance P, a pain inducing peptide were also reported to be present in the pineal gland of several kinds of mammals. However, few studies have been demonstrated in humans. Therefore, in the present study, the immunohistochemical investigation was performed in the human pineal gland by using antisera raised against leu-enkephalin, met-enkephalin and beta-endorphin to demonstrate an opioidergic system; and antisera raised against substance P, neurokinin A, and neurokinin B to study a tachykinin system. A high amount of leu-and met-enkephalin immunoreactivities were observed in intrapineal neuronal-like cells while very few were presented in nerve fibers. This result suggests a local regulatory function or paracrine opioidergic control in human pineal. Substance P-and neurokinin A-immunoreactivities, but not neurokinin B were observed in the human pineal gland. They are located mostly in nerve fibers but a few in neuronal-like cells. The tachykininergic control of human pineal is mainly from the nerve fibers which have their perikaryal origin outside the gland. Some of the nerve fibers might originate from neurons in the brain and/or from a peripheral ganglion.
The retinohypothalamic tract (RHT) relays photic information from the eyes to the suprachiasmatic... more The retinohypothalamic tract (RHT) relays photic information from the eyes to the suprachiasmatic nucleus (SCN). Activation of this pathway plays a role in adjusting circadian timing to the light/dark environment. Two transmitters, glutamate and pituitary adenylate cyclase activating polypeptide (PACAP) having phase shifting capacity during the night and day, respectively, are located in the RHT. Using double staining immunohistochemistry at the light and electron microscopic level, we showed that PACAP was co-stored with glutamate in a subset of retinal ganglion cells and in nerve terminals in the retino-recipient area of the SCN. These findings provide an anatomical basis for the recent demonstration of the interaction between these two transmitters on the SCN phase response at night.
The parenchymal cells of the mammalian pineal gland are the hormone-producing pinealocytes and th... more The parenchymal cells of the mammalian pineal gland are the hormone-producing pinealocytes and the interstitial cells. In addition, perivascular phagocytes are present. The phagocytes share antigenic properties with microglial and antigen-presenting cells. In certain species, the pineal gland also contains neurons and/or neuron-like peptidergic cells. The peptidergic cells might influence the pinealocyte by a paracrine secretion of the peptide. Nerve fibers innervating the mammalian pineal gland originate from perikarya located in the sympathetic superior cervical ganglion and the parasympathetic sphenopalatine and otic ganglia. The sympathetic nerve fibers contain norepinephrine and neuropeptide Y as neurotransmitters. The parasympathetic nerve fibers contain vasoactive intestinal peptide and peptide histidine isoleucine. Recently, neurons in the trigeminal ganglion, containing substance P, calcitonin gene-related peptide, and pituitary adenylate cyclaseactivating peptide, have been shown to project to the mammalian pineal gland. Finally, nerve fibers originating from perikarya located in the brain containing, for example, GABA, orexin, serotonin, histamine, oxytocin, and vasopressin innervate the pineal gland directly via the pineal stalk. Biochemical studies have demonstrated numerous receptors on the pinealocyte cell membrane, which are able to bind the neurotransmitters located in the pinealopetal nerve fibers. These findings indicate that the mammalian pinealocyte can be influenced by a plethora of neurotransmitters.
Serial frontal and sagittal sections through the organum vasculosum laminae terminalis (OVLT) of ... more Serial frontal and sagittal sections through the organum vasculosum laminae terminalis (OVLT) of the male Mongolian gerbil (Meriones unguiculatus) were incubated with an antibody against substance P and stained by the indirect peroxidase anti-peroxidase technique. A high density of nerve fibres with large-sized terminals exhibiting substance P-immunoreactivity was observed in the OVLT in close relation to the blood vessels both in the internal and the external zone of the organ. In addition, a few fibres penetrated into the ependyma covering the organ. These results indicate that substance P is released into the bloodstream from the OVLT and in addition might influence the ependymal cells bordering the organ towards the third ventricle.
Succinate dehydrogenase activity was investigated histochemically in the rat pineal gland. The in... more Succinate dehydrogenase activity was investigated histochemically in the rat pineal gland. The influence of fixation on the activity pattern, the possible diffusion of enzyme, the nothing dehydrogenase reaction, and the substantivity of the tetrazolium salts and formazans were investigated in control experiments. In rats maintained on a 17/7 h light/dark schedule a distinct circadian rhythm of the succinate dehydrogenase was demonstrated in the pineal gland. Activity was lowest during the day and highest during the night. The dorsocaudal part of the gland showed the highest activity and within the same part of the gland the activity varied between individual pinealocytes. A relative lack of endogenous coenzyme Q, as well as a circadian rhythm of this coenzyme, highly influenced the activity of succinate dehydrogenase. It is concluded that succinate dehydrogenase activity in the pineal gland of the rat is regulated by changing the concentration of the active enzyme itself as well as the level of the endogenous coenzyme Q. Whether this is caused by a circadian rhythm in the synthesis or in the catabolism of the enzyme and the coenzyme was not revealed by the present study .
Otx2 and Crx are vertebrate orthologs of the orthodenticle family of homeobox genes, which are in... more Otx2 and Crx are vertebrate orthologs of the orthodenticle family of homeobox genes, which are involved in retinal development. In this study, the temporal expression patterns of Otx2 and Crx in the rat retina during embryonic and postnatal stages of development were analyzed in detail. This confirmed the presence of Otx2 mRNA in both the embryonic retinal pigment epithelium and the developing neural retina. During development, the expression of Otx2 persists in the pigment epithelium, whereas Otx2 expression of the neural retina becomes progressively restricted to the outer nuclear layer and the outer part of the inner nuclear layer. Immunohistochemistry revealed that Otx2 protein is also present in cell bodies of the ganglion cell layer, which does not contain the Otx2 transcript, suggesting that Otx2 protein is synthesized in cell bodies of the bipolar neurons and then transported to and taken up by cells in the ganglion cell layer. Crx is also highly expressed in the outer nuclear layer starting at E17 and postnatally in the inner nuclear layer. The onset of expression of Crx lags behind that of Otx2 consistent with evidence that Otx2 activates Crx transcription. These expression patterns are consistent with evidence that Otx2 and Crx function during retinal development and extend the period of probable functionality to the adult. In this regard, these results provide an enhanced and expanded temporal and spatial framework for understanding the multiple roles of Otx2 and Crx in the developing and mature mammalian retina.
An immunohistochemical investigation of the mink pineal gland was performed by use of antibodies ... more An immunohistochemical investigation of the mink pineal gland was performed by use of antibodies raised in rabbits against neuropeptide Y (NPY) and Cys-NPY(32-36)-amide recognizing neuropeptide Y with an amidation at position 36 (NPYamide). NPY-immunoreactive nerve fibers were located predominantly in the rostral part of the pineal gland and in the pineal stalk. Immunoreactive nerve fibers were found throughout the pineal gland, but the number of fibers in the caudal part of the gland was low. The fibers were present both in the perivascular spaces and between the pinealocytes. Many NPY-immunoreactive fibers were also located in the posterior and habenular commissures; some of these fibers were connected with the fibers in the rostral part of the mink pineal gland, indicating that at least some of the NPY-immunoreactive nerve fibers are of central origin. The nerve fibers immunoreactive to amidated NPY were distributed in a similar manner. However, the number of fibers immunoreactive to NPYamide was lower than the number of fibers immunoreactive to NPY itself. After removal of the superior cervical ganglia bilaterally 22 days or 12 months before sacrifice, NPY-immunoreactive nerve fibers remained in the gland. This immunohistochemical study of the mink pineal gland therefore shows that the NPY/NPYamide-immunoreactive nerve fibers innervating the pineal gland in this species are a component of the central innervation or originate from extracerebral parasympathetic ganglia.
Light and electron microscope autoradiography indicated that 3H-labelled inulin was taken up by n... more Light and electron microscope autoradiography indicated that 3H-labelled inulin was taken up by neurons and glia cells of rat brain cortex in vitro. The mechanism, by which inulin passed the cell membranes, was studied by comparing the transport of inulin (molecular weight 5000) with the transport of dextran (molecular weight 75000). The half-time for the cellular in-and efflux for the two molecules was the same although their diffusion coefficients differed by a factor of 4-5. The transport mechanism was therefore interpreted as bulk transport, and vesicular transport is suggested. Efflux of inulin from brain cortex exposed to inulin in vivo indicated that cellular uptake of inulin also occured in vivo.
The ultrastructure of the pineal gland of 18 human fetuses (crown-rump lengths 30-178 mm) was inv... more The ultrastructure of the pineal gland of 18 human fetuses (crown-rump lengths 30-178 mm) was investigated. The pineal gland exhibits a pyramidal shape and consists of an anterior and posterior lobe. Only one parenehymal cell type, the pinealocyte, was observed. Few neuroblasts were seen between the pinealoeytes and in the extended perivascular space. The pinealocytes possess all the organelles necessary for hormone synthesis. No specific secretory granule could be observed. The organ is abundantly vascularized and richly innervated. The morphology of the capillaries indicates the existence of a blood-brain barrier. The ultrastructure of the human fetal pineal gland suggests that the gland has a secretory function in early intrauterine life.
The rat pineal gland has been extensively used in studies of melatonin synthesis. However, the ce... more The rat pineal gland has been extensively used in studies of melatonin synthesis. However, the cellular localization of melatonin synthesis in this species has not been investigated. Here we focus on the localization of melatonin synthesis using immunohistochemical methods to detect the last enzyme in melatonin synthesis, acetylserotonin O-methyltransferase (ASMT), and in situ hybridization techniques to study transcripts encoding ASMT and two other enzymes in melatonin synthesis, tryptophan hydroxylase 1 (TPH1) and aralkylamine N-acetyltransferase (AANAT). In sections of the rat pineal gland, marked cell-to-cell differences were found in ASMT immunostaining intensity and in the abundance of Tph1, Aanat and Asmt transcripts. ASMT immunoreactivity was localized to the cytoplasm in pinealocytes in the parenchyma of the superficial pineal gland, and immunopositive pinealocytes were also detected in the pineal stalk and in the deep pineal gland. ASMT was found to inconsistently colocali...
Silver-impregnated series of cat and monkey (Cereopithecus aethiops) brains were studied in an at... more Silver-impregnated series of cat and monkey (Cereopithecus aethiops) brains were studied in an attempt to demonstrate the existence of nervous connections between the brain and the pineal gland via the pineal stalk (central pineal connections). The presence of such connections between both the pineal gland and the habenular area, and between the pineal gland and the posterior commissure was verified in this study. A well defined median nerve tract between the pineal gland and the posterior commissure is described.
With some exceptions, in most of the mammals the pituitary pars tuberalis and the hypothalamic su... more With some exceptions, in most of the mammals the pituitary pars tuberalis and the hypothalamic suprachiasmatic nuclei are reportedly the main targets for the pineal hormone melatonin. However, it is not known if the conspicuous diversity in the distribution pattern of melatonin binding sites in these areas depicts differences in reproductive behavior observed in the seasonally breeding species in the temperate zones. We explored the distribution and the characteristics of melatonin binding sites in the hypothalamus and pituitary of three species (bovine, horse, and donkey) different in terms of seasonal reproductive competence. The topographical localization, investigated by in vitro autoradiography, revealed 2-[1251]iodomelatonin binding sites only in the pituitary gland in all three species, primarily in the pars tuberalis (PT), but also in the pars distalis (PD) and pars intermedia (PI). Kinetic, inhibition, and saturation studies, performed by means of in vitro binding, revealed presence of a single class high affinity binding sites. The & values, melatonin, and 2-iodomelatonin Kj values were in the low picomolar range. Coincubation with GTP@ inhibited 2-[ '25J]iodomelatonin binding, demonstrating that these putative receptors are linked to a G protein in their signaltransduction pathway. The hypothalamus was devoid of specific binding. In conclusion, the results suggest that in these species, the hypophysis may be a principal target for the melatonin action on the reproductive system.
The permeability of fenestrated capillaries in the mouse pineal gland to proteins and peptides wa... more The permeability of fenestrated capillaries in the mouse pineal gland to proteins and peptides was demonstrated by means of ultrastructural tracers. Horseradish peroxidase (HRP) and microperoxidase (MP) were injected intravenously and allowed to circulate for approximately 30 s, 1 min, 5 rain, 1 or 2 h. The tissue was then fixed by vascular perfusion or by immersion with aldehydes. In all experiments a pronounced extravasation of HRP and MP occurred. Transendothelial vesicular transport seemed to have occurred across the fenestrated capillaries. The most pronounced tracer labeling of vesicles was found after 1 min of MP-or HRP-circulation. The vesicles were uncoated and more than 70 ~ of the HRP-and MP-containing vesicles exhibited diameters between 50 and 110nm. Furthermore, three other transcapillary pathways taken by the tracers are suggested: 1) via intercellular junctions, 2) through fenestrae and 3) via channels formed by fusion of vesicles with the luminal and abluminal cell membranes. Based on these results, it is assumed that the capillaries in the mouse pineal gland are also permeable to peptides synthesized and secreted by the pineal gland.
An immunohistochemical investigation of the mink pineal gland was performed by use of antibodies ... more An immunohistochemical investigation of the mink pineal gland was performed by use of antibodies raised in rabbits against neuropeptide Y (NPY) and Cys-NPY(32-36)-amide recognizing neuropeptide Y with an amidation at position 36 (NPYamide). NPY-immunoreactive nerve fibers were located predominantly in the rostral part of the pineal gland and in the pineal stalk. Immunoreactive nerve fibers were found throughout the pineal gland, but the number of fibers in the caudal part of the gland was low. The fibers were present both in the perivascular spaces and between the pinealocytes. Many NPY-immunoreactive fibers were also located in the posterior and habenular commissures; some of these fibers were connected with the fibers in the rostral part of the mink pineal gland, indicating that at least some of the NPY-immunoreactive nerve fibers are of central origin. The nerve fibers immunoreactive to amidated NPY were distributed in a similar manner. However, the number of fibers immunoreactive to NPYamide was lower than the number of fibers immunoreactive to NPY itself. After removal of the superior cervical ganglia bilaterally 22 days or 12 months before sacrifice, NPY-immunoreactive nerve fibers remained in the gland. This immunohistochemical study of the mink pineal gland therefore shows that the NPY/NPYamide-immunoreactive nerve fibers innervating the pineal gland in this species are a component of the central innervation or originate from extracerebral parasympathetic ganglia.
The perivascular space of the rat pineal gland is known to contain phagocytic cells that are immu... more The perivascular space of the rat pineal gland is known to contain phagocytic cells that are immunoreactive for leukocyte antigens, and thus they appear to belong to the macrophage/microglial cell line. These cells also contain MHC class II proteins. We investigated this cell type in the pineal gland of mice. Actively phagocytosing cells with a prominent lysosomal system were found in the pericapillary spaces of the mouse pineal gland following intravenous injection of horseradish peroxidase. The cells also exhibited strong acid phosphatase activity. Perivascular cells were immunopositive for MHC class II protein and for CD68, a marker of monocytes/ phagocytes. This study verifies that perivascular phagocytes with antigen-presenting properties are present in the mouse pineal gland.
The vertebrate retina contains a circadian clock participating in adaptations to day and night vi... more The vertebrate retina contains a circadian clock participating in adaptations to day and night vision. This peripheral clock is independent of the master clock in the suprachiasmatic nucleus (SCN). The retinal clock is located in several cell types, including the photoreceptors. To investigate the role of the circadian clock of the photoreceptor cells in regulation of retinal protein rhythms, we analysed diurnal protein expression in the photoreceptor-deficient cone-rod homeobox knockout mouse (Crx(-/-) ) and the 129/Sv mouse. 2D gels were made from retinal homogenates of 129/Sv and Crx(-/-) mice killed at midday and midnight. Stained gels were analysed by use of PDQuest 2D gel analysis software. After trypsin digestion of differential expressed spots, the proteins were identified by LC-MS/MS using a nano-liquid chromatograph connected to a Q-TOF Premier mass spectrometer. These data were used to search the SWISS-PROT database. Both the retinae of the control and the Crx(-/-) mice e...
Journal of the Medical Association of Thailand Chotmaihet Thangphaet, Jul 1, 2005
The human pineal gland secretes melatonin in a circadian rhythm manner. The rhythm of melatonin s... more The human pineal gland secretes melatonin in a circadian rhythm manner. The rhythm of melatonin synthesis is primarily controlled by the noradrenergic sympathetic system originating from the superior cervical ganglion. Several neurotranmitters/neuropeptides have been reported to influence the production of melatonin in the pineal glands of many mammalian species. Both opioid peptide, a pain suppressing peptide and substance P, a pain inducing peptide were also reported to be present in the pineal gland of several kinds of mammals. However, few studies have been demonstrated in humans. Therefore, in the present study, the immunohistochemical investigation was performed in the human pineal gland by using antisera raised against leu-enkephalin, met-enkephalin and beta-endorphin to demonstrate an opioidergic system; and antisera raised against substance P, neurokinin A, and neurokinin B to study a tachykinin system. A high amount of leu-and met-enkephalin immunoreactivities were observed in intrapineal neuronal-like cells while very few were presented in nerve fibers. This result suggests a local regulatory function or paracrine opioidergic control in human pineal. Substance P-and neurokinin A-immunoreactivities, but not neurokinin B were observed in the human pineal gland. They are located mostly in nerve fibers but a few in neuronal-like cells. The tachykininergic control of human pineal is mainly from the nerve fibers which have their perikaryal origin outside the gland. Some of the nerve fibers might originate from neurons in the brain and/or from a peripheral ganglion.
The retinohypothalamic tract (RHT) relays photic information from the eyes to the suprachiasmatic... more The retinohypothalamic tract (RHT) relays photic information from the eyes to the suprachiasmatic nucleus (SCN). Activation of this pathway plays a role in adjusting circadian timing to the light/dark environment. Two transmitters, glutamate and pituitary adenylate cyclase activating polypeptide (PACAP) having phase shifting capacity during the night and day, respectively, are located in the RHT. Using double staining immunohistochemistry at the light and electron microscopic level, we showed that PACAP was co-stored with glutamate in a subset of retinal ganglion cells and in nerve terminals in the retino-recipient area of the SCN. These findings provide an anatomical basis for the recent demonstration of the interaction between these two transmitters on the SCN phase response at night.
The parenchymal cells of the mammalian pineal gland are the hormone-producing pinealocytes and th... more The parenchymal cells of the mammalian pineal gland are the hormone-producing pinealocytes and the interstitial cells. In addition, perivascular phagocytes are present. The phagocytes share antigenic properties with microglial and antigen-presenting cells. In certain species, the pineal gland also contains neurons and/or neuron-like peptidergic cells. The peptidergic cells might influence the pinealocyte by a paracrine secretion of the peptide. Nerve fibers innervating the mammalian pineal gland originate from perikarya located in the sympathetic superior cervical ganglion and the parasympathetic sphenopalatine and otic ganglia. The sympathetic nerve fibers contain norepinephrine and neuropeptide Y as neurotransmitters. The parasympathetic nerve fibers contain vasoactive intestinal peptide and peptide histidine isoleucine. Recently, neurons in the trigeminal ganglion, containing substance P, calcitonin gene-related peptide, and pituitary adenylate cyclaseactivating peptide, have been shown to project to the mammalian pineal gland. Finally, nerve fibers originating from perikarya located in the brain containing, for example, GABA, orexin, serotonin, histamine, oxytocin, and vasopressin innervate the pineal gland directly via the pineal stalk. Biochemical studies have demonstrated numerous receptors on the pinealocyte cell membrane, which are able to bind the neurotransmitters located in the pinealopetal nerve fibers. These findings indicate that the mammalian pinealocyte can be influenced by a plethora of neurotransmitters.
Serial frontal and sagittal sections through the organum vasculosum laminae terminalis (OVLT) of ... more Serial frontal and sagittal sections through the organum vasculosum laminae terminalis (OVLT) of the male Mongolian gerbil (Meriones unguiculatus) were incubated with an antibody against substance P and stained by the indirect peroxidase anti-peroxidase technique. A high density of nerve fibres with large-sized terminals exhibiting substance P-immunoreactivity was observed in the OVLT in close relation to the blood vessels both in the internal and the external zone of the organ. In addition, a few fibres penetrated into the ependyma covering the organ. These results indicate that substance P is released into the bloodstream from the OVLT and in addition might influence the ependymal cells bordering the organ towards the third ventricle.
Succinate dehydrogenase activity was investigated histochemically in the rat pineal gland. The in... more Succinate dehydrogenase activity was investigated histochemically in the rat pineal gland. The influence of fixation on the activity pattern, the possible diffusion of enzyme, the nothing dehydrogenase reaction, and the substantivity of the tetrazolium salts and formazans were investigated in control experiments. In rats maintained on a 17/7 h light/dark schedule a distinct circadian rhythm of the succinate dehydrogenase was demonstrated in the pineal gland. Activity was lowest during the day and highest during the night. The dorsocaudal part of the gland showed the highest activity and within the same part of the gland the activity varied between individual pinealocytes. A relative lack of endogenous coenzyme Q, as well as a circadian rhythm of this coenzyme, highly influenced the activity of succinate dehydrogenase. It is concluded that succinate dehydrogenase activity in the pineal gland of the rat is regulated by changing the concentration of the active enzyme itself as well as the level of the endogenous coenzyme Q. Whether this is caused by a circadian rhythm in the synthesis or in the catabolism of the enzyme and the coenzyme was not revealed by the present study .
Otx2 and Crx are vertebrate orthologs of the orthodenticle family of homeobox genes, which are in... more Otx2 and Crx are vertebrate orthologs of the orthodenticle family of homeobox genes, which are involved in retinal development. In this study, the temporal expression patterns of Otx2 and Crx in the rat retina during embryonic and postnatal stages of development were analyzed in detail. This confirmed the presence of Otx2 mRNA in both the embryonic retinal pigment epithelium and the developing neural retina. During development, the expression of Otx2 persists in the pigment epithelium, whereas Otx2 expression of the neural retina becomes progressively restricted to the outer nuclear layer and the outer part of the inner nuclear layer. Immunohistochemistry revealed that Otx2 protein is also present in cell bodies of the ganglion cell layer, which does not contain the Otx2 transcript, suggesting that Otx2 protein is synthesized in cell bodies of the bipolar neurons and then transported to and taken up by cells in the ganglion cell layer. Crx is also highly expressed in the outer nuclear layer starting at E17 and postnatally in the inner nuclear layer. The onset of expression of Crx lags behind that of Otx2 consistent with evidence that Otx2 activates Crx transcription. These expression patterns are consistent with evidence that Otx2 and Crx function during retinal development and extend the period of probable functionality to the adult. In this regard, these results provide an enhanced and expanded temporal and spatial framework for understanding the multiple roles of Otx2 and Crx in the developing and mature mammalian retina.
An immunohistochemical investigation of the mink pineal gland was performed by use of antibodies ... more An immunohistochemical investigation of the mink pineal gland was performed by use of antibodies raised in rabbits against neuropeptide Y (NPY) and Cys-NPY(32-36)-amide recognizing neuropeptide Y with an amidation at position 36 (NPYamide). NPY-immunoreactive nerve fibers were located predominantly in the rostral part of the pineal gland and in the pineal stalk. Immunoreactive nerve fibers were found throughout the pineal gland, but the number of fibers in the caudal part of the gland was low. The fibers were present both in the perivascular spaces and between the pinealocytes. Many NPY-immunoreactive fibers were also located in the posterior and habenular commissures; some of these fibers were connected with the fibers in the rostral part of the mink pineal gland, indicating that at least some of the NPY-immunoreactive nerve fibers are of central origin. The nerve fibers immunoreactive to amidated NPY were distributed in a similar manner. However, the number of fibers immunoreactive to NPYamide was lower than the number of fibers immunoreactive to NPY itself. After removal of the superior cervical ganglia bilaterally 22 days or 12 months before sacrifice, NPY-immunoreactive nerve fibers remained in the gland. This immunohistochemical study of the mink pineal gland therefore shows that the NPY/NPYamide-immunoreactive nerve fibers innervating the pineal gland in this species are a component of the central innervation or originate from extracerebral parasympathetic ganglia.
Light and electron microscope autoradiography indicated that 3H-labelled inulin was taken up by n... more Light and electron microscope autoradiography indicated that 3H-labelled inulin was taken up by neurons and glia cells of rat brain cortex in vitro. The mechanism, by which inulin passed the cell membranes, was studied by comparing the transport of inulin (molecular weight 5000) with the transport of dextran (molecular weight 75000). The half-time for the cellular in-and efflux for the two molecules was the same although their diffusion coefficients differed by a factor of 4-5. The transport mechanism was therefore interpreted as bulk transport, and vesicular transport is suggested. Efflux of inulin from brain cortex exposed to inulin in vivo indicated that cellular uptake of inulin also occured in vivo.
The ultrastructure of the pineal gland of 18 human fetuses (crown-rump lengths 30-178 mm) was inv... more The ultrastructure of the pineal gland of 18 human fetuses (crown-rump lengths 30-178 mm) was investigated. The pineal gland exhibits a pyramidal shape and consists of an anterior and posterior lobe. Only one parenehymal cell type, the pinealocyte, was observed. Few neuroblasts were seen between the pinealoeytes and in the extended perivascular space. The pinealocytes possess all the organelles necessary for hormone synthesis. No specific secretory granule could be observed. The organ is abundantly vascularized and richly innervated. The morphology of the capillaries indicates the existence of a blood-brain barrier. The ultrastructure of the human fetal pineal gland suggests that the gland has a secretory function in early intrauterine life.
The rat pineal gland has been extensively used in studies of melatonin synthesis. However, the ce... more The rat pineal gland has been extensively used in studies of melatonin synthesis. However, the cellular localization of melatonin synthesis in this species has not been investigated. Here we focus on the localization of melatonin synthesis using immunohistochemical methods to detect the last enzyme in melatonin synthesis, acetylserotonin O-methyltransferase (ASMT), and in situ hybridization techniques to study transcripts encoding ASMT and two other enzymes in melatonin synthesis, tryptophan hydroxylase 1 (TPH1) and aralkylamine N-acetyltransferase (AANAT). In sections of the rat pineal gland, marked cell-to-cell differences were found in ASMT immunostaining intensity and in the abundance of Tph1, Aanat and Asmt transcripts. ASMT immunoreactivity was localized to the cytoplasm in pinealocytes in the parenchyma of the superficial pineal gland, and immunopositive pinealocytes were also detected in the pineal stalk and in the deep pineal gland. ASMT was found to inconsistently colocali...
Silver-impregnated series of cat and monkey (Cereopithecus aethiops) brains were studied in an at... more Silver-impregnated series of cat and monkey (Cereopithecus aethiops) brains were studied in an attempt to demonstrate the existence of nervous connections between the brain and the pineal gland via the pineal stalk (central pineal connections). The presence of such connections between both the pineal gland and the habenular area, and between the pineal gland and the posterior commissure was verified in this study. A well defined median nerve tract between the pineal gland and the posterior commissure is described.
With some exceptions, in most of the mammals the pituitary pars tuberalis and the hypothalamic su... more With some exceptions, in most of the mammals the pituitary pars tuberalis and the hypothalamic suprachiasmatic nuclei are reportedly the main targets for the pineal hormone melatonin. However, it is not known if the conspicuous diversity in the distribution pattern of melatonin binding sites in these areas depicts differences in reproductive behavior observed in the seasonally breeding species in the temperate zones. We explored the distribution and the characteristics of melatonin binding sites in the hypothalamus and pituitary of three species (bovine, horse, and donkey) different in terms of seasonal reproductive competence. The topographical localization, investigated by in vitro autoradiography, revealed 2-[1251]iodomelatonin binding sites only in the pituitary gland in all three species, primarily in the pars tuberalis (PT), but also in the pars distalis (PD) and pars intermedia (PI). Kinetic, inhibition, and saturation studies, performed by means of in vitro binding, revealed presence of a single class high affinity binding sites. The & values, melatonin, and 2-iodomelatonin Kj values were in the low picomolar range. Coincubation with GTP@ inhibited 2-[ '25J]iodomelatonin binding, demonstrating that these putative receptors are linked to a G protein in their signaltransduction pathway. The hypothalamus was devoid of specific binding. In conclusion, the results suggest that in these species, the hypophysis may be a principal target for the melatonin action on the reproductive system.
The permeability of fenestrated capillaries in the mouse pineal gland to proteins and peptides wa... more The permeability of fenestrated capillaries in the mouse pineal gland to proteins and peptides was demonstrated by means of ultrastructural tracers. Horseradish peroxidase (HRP) and microperoxidase (MP) were injected intravenously and allowed to circulate for approximately 30 s, 1 min, 5 rain, 1 or 2 h. The tissue was then fixed by vascular perfusion or by immersion with aldehydes. In all experiments a pronounced extravasation of HRP and MP occurred. Transendothelial vesicular transport seemed to have occurred across the fenestrated capillaries. The most pronounced tracer labeling of vesicles was found after 1 min of MP-or HRP-circulation. The vesicles were uncoated and more than 70 ~ of the HRP-and MP-containing vesicles exhibited diameters between 50 and 110nm. Furthermore, three other transcapillary pathways taken by the tracers are suggested: 1) via intercellular junctions, 2) through fenestrae and 3) via channels formed by fusion of vesicles with the luminal and abluminal cell membranes. Based on these results, it is assumed that the capillaries in the mouse pineal gland are also permeable to peptides synthesized and secreted by the pineal gland.
An immunohistochemical investigation of the mink pineal gland was performed by use of antibodies ... more An immunohistochemical investigation of the mink pineal gland was performed by use of antibodies raised in rabbits against neuropeptide Y (NPY) and Cys-NPY(32-36)-amide recognizing neuropeptide Y with an amidation at position 36 (NPYamide). NPY-immunoreactive nerve fibers were located predominantly in the rostral part of the pineal gland and in the pineal stalk. Immunoreactive nerve fibers were found throughout the pineal gland, but the number of fibers in the caudal part of the gland was low. The fibers were present both in the perivascular spaces and between the pinealocytes. Many NPY-immunoreactive fibers were also located in the posterior and habenular commissures; some of these fibers were connected with the fibers in the rostral part of the mink pineal gland, indicating that at least some of the NPY-immunoreactive nerve fibers are of central origin. The nerve fibers immunoreactive to amidated NPY were distributed in a similar manner. However, the number of fibers immunoreactive to NPYamide was lower than the number of fibers immunoreactive to NPY itself. After removal of the superior cervical ganglia bilaterally 22 days or 12 months before sacrifice, NPY-immunoreactive nerve fibers remained in the gland. This immunohistochemical study of the mink pineal gland therefore shows that the NPY/NPYamide-immunoreactive nerve fibers innervating the pineal gland in this species are a component of the central innervation or originate from extracerebral parasympathetic ganglia.
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Papers by Morten Møller