Calcium-sensing receptors and parathyroid hormone-related protein in the caudal neurosecretory system of the flounder (Platichthys flesus)

The caudal neurosecretory system of the flounder (Platichthys flesus) has been examined by immunocytochemistry and in situ hybridization for the expression of parathyroid hormone-related protein (PTHrP) and calcium-sensing receptors (CaSR). The N-terminus nucleotide and deduced amino acid sequences of flounder PTHrP were determined and used to prepare oligonucleotide probes and homologous antiserum. The Dahlgren cells of the posterior spinal cord and their axons contained PTHrP protein which was also detected around the capillaries of the urophysis. PTHrP gene expression was abundant in the Dahlgren perikarya and axons in the spinal cord, but it was absent from nerve endings in the urophysis. Calcium-sensing receptor protein was present in the Dahlgren perikarya and axons, also with abundant gene expression, but there was neither protein nor mRNA in the urophysis. There were no apparent differences between freshwater- and seawater-adapted fish in either CaSR or PTHrP expression in the caudal neurosecretory system. These observations suggest that Dahlgren cells produce PTHrP which may be released from axons abutting capillaries in the urophysis. However, the sensing of ionic calcium appears to be confined to the perikarya of the Dahlgren cells in the spinal cord neuropil, suggesting that they are responsive to calcium in the central nervous system rather than the general circulation.     

Immunocytochemical, Golgi and electron microscopic characterization of putative dendrites in the ventral glial lamina of the rat supraoptic nucleus

Processes of magnocellular neurosecretory cells in the rat supraoptic nucleus which project along the pial surface in the ventral glial lamina were investigated using immunocytochemistry, Golgi stains and electron microscopy. Immunocytochemical studies revealed that although both oxytocin- and vasopressin-containing processes were evident in the ventral glial lamina, vasopressin-containing processes predominated. Ventral processes were thicker and of a different morphology than dorsal axon-like processes which joined the hypothalamo-neurohypophysial tract and exhibited large varicosities along their length or at their apparent termination. Golgi stains revealed that classically defined dendrites of supraoptic neurons projected primarily ventrally and often invaded the ventral glial lamina. No axons were traced to the lamina. Ultrastructurally, processes within the ventral glial lamina characterized as dendrites could be stained immunocytochemically for neurophysin and were post-synaptic to a variety of presynaptic elements. The results suggest that many dendrites from magnocellular neurosecretory cells in the supraoptic nucleus project to the ventral glial lamina and form a restricted, receptive plexus. The previously demonstrated coexistence of catecholamine-containing varicosities and other axon types with these processes in the lamina indicates an important role for supraoptic dendrites in integrating a wide variety of information relevant to neurohypophysial hormone release.     

Structural plasticity in the hypothalamic supraoptic nucleus at lactation affects oxytocin-, but not vasopressin-secreting neurones

Magnocellular neurones in the supraoptic nucleus of the hypothalamus are usually separated by neuropil and glial elements. In lactating animals, however, the surface membranes of many neurosecretory somata and dendrites are frequently in direct apposition, without any glial interposition. A significant number of such neurones are also bridged by the same presynaptic terminal ("double synapses"). As the supraoptic nucleus is composed of two types of neurosecretory cell, secreting either oxytocin or vasopressin, we carried out comparative quantitative analyses on identified supraoptic neurones of virgin and lactating rats to determine which neurones were affected by the structural changes and to what extent. The neurones were identified in: (i) normal and Brattleboro homozygote rats by electron microscopic immunocytochemistry (pre- and post-embedding procedures) using antisera raised against oxytocin, vasopressin and oxytocin-related neurophysin I, and (ii) in homozygous Brattleboro rats by their neuronal content of approximately 170 nm neurosecretory granules. We report here that, in virgin animals under normal conditions, a small proportion of both types of neurone show neuronal appositions. At lactation, neuronal appositions are far more numerous and extensive, as are "double synapses". These changes affect exclusively the oxytocinergic neurones. The increased appositions cannot result solely from glial retraction because the hypertrophied oxytocin cells have a greater absolute, though smaller proportional, coverage by glial processes than cells in the control animals. From the present observations, and those obtained in chronically dehydrated animals (see accompanying article), it is clear that the plastic changes in the supraoptic nucleus are closely related to the activity of its oxytocinergic neurones. During lactation, these structural modifications may serve to facilitate and maintain the characteristic synchronized electrical activity of these neurones at milk ejection. On a few occasions, we also found appositions between one oxytocinergic and one vasopressinergic neurone, which may account for the rare cases of electrophysiological interactions between the two types of cell.     

Microglia in the neurohypophysis associate with and endocytose terminal portions of neurosecretory neurons

The rat neurohypophysis contains a population of microglial cells, the majority of which occupy a pericapillary position in the resting gland. The microglia are immunocytochemically identifiable by the presence of macrophage-associated antigens and resemble microglia of the CNS. Morphometry at light and electron microscopic levels reveals that such cells constitute approximately 19% of the intrinsic cell population, excluding the endothelial cells. Two other populations of neurohypophysial glial cells, parenchymatous pituicytes and fibrous pituicytes, do not express macrophage-associated antigens. The microglia have long processes which surround and, in some cases, engulf apparently viable portions of the magnocellular neurosecretory nerve terminals. A sequence of stages of selective endocytosis and degradation of the engulfed nerve terminals can be visualized within pericapillary microglia. Some phagosomes and secondary lysosomes contain morphologically intact neurosecretory granules; others contain partially destroyed neurosecretory granules or amorphous material all of which are identifiable as originating from the magnocellular neurosecretory terminals by their immunoreactivity for oxytocin- or vasopressin-neurophysin. This finding indicates a novel role for the microglial cells in remodelling terminal aborizations of neurosecretory neurons and in processing or degrading hormones and peptides they contain. Because of their close and selective associations with other cellular elements of the neurohypophysis, any substances produced by microglia also have the potential to influence hormone secretion, pituicyte proliferation and neurohypophysial vasculature.     

Ependymal cells of the filum terminale in fish (poecilia sphenops) adapted to freshwater and saltwater: Electron microscopic study

The ependymal lining of the central canal of the filum terminale and spinal cord in the vicinity of the caudal neurosecretory system in P. sphenops was examined in this study. Two general cell types based on shape and location were observed in the ependymal lining: cuboidal ependyma located in dorsal aspects of the filum terminal and columnar to pseudostratified ependymal cells found in ventrolateral and ventral aspects of the filum terminale. Comparison of the ependymal lining was made in animals adapted to saltwater and freshwater. In animals adapted to saltwater there was an increase in the basal infoldings of the cell membrane of the dorsal cuboidal ependyma. Infolding of the basal cell membrane is a phenomenon shared by cells known to participate in transport of electrolytes. Since a possible functional relationship between the ependyma of the third ventricle and median eminence has been shown, in future studies on the osmoregulatory function of the caudal neurosecretory system the ependymal lining of the central canal in this region should be considered.     

Morphological plasticity that occurs in the neurohypophysis following activation of the magnocellular neurosecretory system can be mimicked in vitro by beta-adrenergic stimulation

The osmotic stress to rats of replacing drinking water with 2% NaCl for four days (salt loading) led to dramatic ultrastructural morphological changes in the neural lobe of the pituitary, including a decrease in the ratio of glial to neurosecretory terminal coverage of the pericapillary basal lamina. The decrease in the proportion of the basal lamina covered by pituicytes, the specialized astrocytic glial cells of the neural lobe, was due to a decrease in the number of pituicyte processes reaching the pericapillary spaces. The concomitant increase in the proportion of neuronal coverage was due to the combination of an increase in the length of individual nerve terminals and a change in the number of terminals. Similar structural changes to those seen in vivo were produced by incubation of the isolated neural lobe with the beta-adrenergic agonist isoprenaline. A decrease in the ratio of glial to neuronal coverage of the basal lamina was achieved within 1 h and could be blocked by inclusion of the beta-adrenergic antagonist propranolol. It is proposed that the morphological plasticity is explained by the active movement of pituicyte cytoplasm.     

Maturation of the goldfish preoptic area: Ultrastructural correlates of position and maturation of neurosecretory cells

A gradation of small rostral to larger caudal neurosecretory cells is found in the preoptic area of fish. Regional variations in ultrastructural features were assessed in this region of fish ranging widely in size (1.7–283.1 g), some of which had received intraperitoneal injections of horseradish peroxidase in order to label neurons projecting beyond the blood-brain barrier (i.e. neurosecretory cells). In small (≤6.6. g) fish, neurosecretory cells of the caudal preoptic area were 10–15 μm in diameter and contained dense core vesicles. More rostral smaller cells had few or no dense core vesicles, but were labeled following intraperitoneal horseradish peroxidase injections. Cells in both areas were progressively larger, with larger nuclei and more granular reticulum in larger fish. In large fish, neurons that contained dense core vesicles ranged from 10 μm in diameter rostrally to 70 μm in diameter caudally. Some intermediate and large cells were extensively vacuolated and contained extremely convoluted and possibly multiple nuclei. Degenerating material was seen in apparently normal large fish. A few neurosecretory cells were extensively surrounded by perineuronal electron-dense glial cells. Most neurosecretory cells were involved in extensive soma-somatic apposition, which was especially pronounced in fish which were removed from cold water in late fall. Gap junctions were also clearly present on the somata of preoptic neurosecretory cells of these fall fish.These data imply that increased capacity for hormone secretion in large fish may be accomplished in part by neuronal hypertrophy. Maturational and seasonal variation in structure suggest that physiological characteristics are also variable in this nucleus.     

不同禁水时间老龄大鼠下丘脑大细胞分泌神经元的免疫电镜观察

电镜观察了不同禁水时间老龄大鼠下丘脑视上核和室旁核分泌神经元，即加压素和催产素神经元及胶质细胞的超微结构变化，结果显示，禁水6与12h后，上述两核团中的分泌神经元胞体增大，胞质中粗面内质网（RER）排列紧密且规则，高尔基（Golgi)器的未成熟分泌颗粒及神经分泌颗粒增多，轴突内神经分泌颗粒少见，胶质细胞成份减少，突起回缩；相邻两神经元胞膜直接接触，质膜并列现象及突触增多，而在禁水24h后，神经元胞体内的神经分泌颗粒有减少，轴突中的神经分泌颗粒却增多并聚集成膨大区域，以上结果提示老龄大鼠视上核和室旁核的分泌神经元在禁水时其合成激素的功能是活跃的，而且催产素神经元的结构变化与加压素神经元结构变化是相似的，胶质细胞的结构变化为神经元质膜并列及突触的形式提供了有利条件。     

Neuronal/glial plasticity in the supraoptic dendritic zone: Dendritic bundling and double synapse formation at parturition

The magnocellular neurosecretory cells of the supraoptic nucleus increase production and release of oxytocin and/or vasopressin under such conditions as parturition, lactation and dehydration. These stimuli have been shown to result in increased direct apposition of neuronal membranes and the formation of double synapses (one presynaptic terminal contacting two postsynaptic elements) within the supraoptic nucleus at the level of the cell bodies. These morphological changes are due to the retraction of the thin glial processes which are normally interposed between adjacent neurons. The present study was undertaken to ascertain whether, and to what extent, neuronal/glial plasticity occurs in the dendritic zone (i.e. the ventral glial laminar area) of the supraoptic nucleus. The instances of two or more dendrites with membrane in direct apposition (dendritic bundles), the number of dendrites per bundle, the amount of dendritic membrane in direct apposition and the percentage of dendrites contacted by double synapses were quantified at the ultrastructural level in virgin female, prepartum (21 days of gestation), postpartum (day of parturition) and lactating rats. All parameters measured varied significantly with the hormone demand states created by pregnancy and lactation, apparently due to glial retraction. Moreover, in the 2–24 h period between pre- and postpartum there was a significant increase in the number of dendrites per bundle, dendritic membrane in direct apposition and the percentage of dendrites contacted by double synapses. This time course corresponds to the known increased release of oxytocin and vasopressin at parturition.These findings constitute the first demonstration that dendritic bundles and double synapses occur in the ventral glial lamina/dendritic zone of the supraoptic nucleus and vary under the physiological conditions of pregnancy, parturition and lactation.     

The lack of a structured blood-brain barrier in the onychophoran Peripatus acacioi

Summary Onychophorans are ‘living fossils’ frequently purported to have evolved from the same ancestor as the arthropods and annelids. In the CNS ofPeripatus acacioi, beneath an outer acellular neural lamella, glial cells ensheath the cerebral ganglion and the nerve cords. These glial cells are, however, attenuated and rather few in number and, although they interdigitate with one another, they seem to lack intercellular junctions. Exogenous tracers penetrate between them and into the underlying neuropile, suggesting that there is no structural blood-brain barrier. Throughout the nervous tissue, extracellular spaces occur which contain banded collagen fibrils embedded in a matrix material. Thin glial cell processes, characterized by dense filaments, surround these regions and frequently form hemi-desmosomes with the extracellular matrix. The peripheral nerve cell bodies have a range of diameters; some have the characteristics of neurosecretory neurons. Granules in such neurons are produced by the Golgi saccules and associated fenestrated membranes which also possess many coated vesicles. Comparable granules are also found in axonal tracts, but no distinct peripheral neurohaemal areas have been found. Lysosomes are common in the nerve cell bodies and are frequently in the form of multivesicular bodies or large phagocytic vacuoles. Beneath the outer nerve cells lie many tracheae, arranged as a ring around the central neuropile which consists of glial processes, extracellular matrix, axons and nerve terminals. These nerve terminals occur throughout the central neuropile and are characterized by dense pyramidal presynaptic specializations and postsynaptic subsurface cisternae. The nervous system ofPeripatus is relatively simple in its organization, in the lack of glial intercellular junctions and in the ready accessibility of substances from the external milieu.     

A system of cells in the unstimulated rat brain characterized by preferential accumulation of [3H]deoxyglucose

An autoradiographic technique using cryostat sections is described which permits visualization of a system of small, probably glial cells accumulating 2-[3H]deoxyglucose (2-[3H]DG). These cells are localized mainly in periventricular regions and in certain parts of white matter. Deoxyglucose uptake of these cells is not influenced by various experimental conditions. An attempt to demonstrate by this technique activation of hypothalamic neurosecretory centers following water withdrawal for 3 days was unsuccessful.     

Characterization of a new neurosecretory cell type containing gastrin-cholecystokinin-like peptide in the locust pars intercerebralis: ultrastructural and immunocytochemical studies, in situ and in vitro

A new neurosecretory cell type of the locust pars intercerebralis, immunolabelled with an antiserum against a vertebrate peptide related to gastrin-cholecystokinin (CCK-8(s)), was characterized both in situ and in primary cell cultures. Semithin sections of pars intercerebralis were first immunostained in order to identify neurosecretory cells containing CCK-like material and then examined by electron microscopy. The neurosecretory cells containing CCK-like material were paraldehyde fuchsin negative and were unequivocally identified in ultrathin sections adjacent to immunostained semithin sections. They exhibited neurosecretory vesicles of variable electron density, ranging in diameter from 150 to 250 nm. Immunogold labelled ultrathin sections adjacent to unlabelled ultrathin sections allowed for the unambiguous localization of CCK-like immunoreactive material over the neurosecretory vesicles of the cells containing CCK-like material. Immunoreactivity towards CCK-8(s)-like peptide could also be detected in pars intercerebralis neurosecretory neurons grown in vitro. The CCK-like positive neurons showed a multipolar morphology with fine processes radiating from the cell body. The positive cells had the same ultrastructural characteristics as the in situ CCK-like neurons. The pattern of neurite outgrowth on reactive CCK-like neurosecretory cells in vitro and the neuroanatomical pathway of the CCK-like immunoreactive neurosecretory cells in situ could be correlated. On the basis of their number, size and localization in the locust pars intercerebralis, it is possible that the CCK-like neurosecretory cells correspond to neurosecretory cell type C, which has not, to date, been identified at the ultrastructural level.     

Ultrastructural studies on the ontogenesis of the caudal neurosecretory system in the roach, Leuciscus rutilus

Summary The ontogenesis of the urophysial system (caudal neurosecretory system) in the roach (Leuciscus rutilus) was ultrastructurally analyzed. In newly hatched spawns the urophysial components are differentiated in the form of neurosecretory perikarya, axons and terminals and display the characteristics of a functional system although the neurohemal area is not developed. It is assumed that the system is active simultaneously with the preopticohypophysial system during ontogenesis. On the contrary, organogenesis of the urophysis is late, and only at the 14 mm stage do the neurosecretory axons first penetrate the meninx to participate in the formation of the organ.Assumed aminergic cell types associated with the urophysial system are differentiated at the time of hatching, thus indicating a functional relationship. A secretory ependyma releasing its granules into the central canal is described.Supported by grants from the Swedish Natural Science Research Council (No 2502-1-7).     

Morphological adaptability at neurosecretory axonal endings on the neurovascular contact zone of the rat neurohypophysis

To compare the effects of a variety of acute and chronic stimuli that bring about or terminate hormone release the ultrastructure of nerve terminal contact at the basal lamina of the neurohypophysial neurovascular contact zone was examined quantitatively in young adult rats of the following treatment groups: untreated virgin females, untreated male rats, prepartum (day 21 of gestation), postpartum (on the day of parturition), lactating (14 days of suckling), mothers 10 days after their pups were weaned, 48 h water-deprived males, males given 2% saline solution (dehydrated) for 10 days, males given 2% saline as described then given tap water to rehydrate for 2 or 5 weeks. Morphometric analysis of electron micrographs revealed that all stimuli leading to increased hormone release were accompanied by both increased occupation of the basal lamina by nerve terminals as well as decreased enclosure of neurosecretory processes by pituicyte cytoplasm. Neural occupation of the basal lamina remained significantly elevated 10 days post-weaning and at 2 weeks (but not 5 weeks) of rehydration following 10 days of dehydration. Pituicyte enclosure of neurosecretory axons had returned to control values in the postweaning and 5 week (but not 2 week) rehydrated animals. The mean length of individual nerve terminal contact with the basal lamina was found to increase under some, but not all, conditions associated with increased hormone release (i.e. parturition, acute and chronic dehydration, but not during lactation) and to decrease below control values in prepartum females and after 5 weeks of rehydration. During lactation, in contrast, the greater neural coverage of the basal lamina was accompanied by an increased number of terminals per unit length of basal lamina without an increase in the average contact length of the individual terminal.These data provide further evidence that neurohypophysial nerve terminal contact at the basal lamina and the relationship of pituicytes to neurosecretory processes can change under physiological conditions in a manner that is probably functionally related to controlling the contact area for hormone release.     

State of the hypothalamic-pituitary neurosecretory system in rabbits with anaphylactic shock

The hypothalamic-pituitary neurosecretory system of rabbits with anaphylactic shock was investigated by morphometric and histochemical methods. The volumes of the perikarya were increased and the dimensions of the nuclei and nucleoli of the neurosecretory cells were reduced, and the content of neurosecretory substance throughout the neurosecretory system was increased. In rabbits dying from shock the volumes of the nuclei and nucleoli were reduced by a lesser degree, the dimensions of the perikarya were changed, and the content of neurosecretory substance in the posterior lobe of the pituitary was reduced. In animals surviving shock the synthesis of neurohormones by the neurosecretory cells was thus sharply stimulated, but liberation of neurohormones from the posterior lobe was inhibited. In animals dying from shock hormone formation in the neurosecretory cells was stimulated to a lesser degree, but the processes of liberation of neurohormones from the posterior lobe of the pituitary were probably intensified.     

Evidence for dynamic interactions between pituicytes and neurosecretory axons in the rat

Several ultrastructural features of pituicytes as well as their relationship with neurosecretory axons were measured in neural lobes from rats either: non-deprived; water-deprived for 4, 12, or 24 h; or rehydrated for 12 or 24 h after an initial 24 h dehydration. A significant increase in pituicyte lipid bodies occurs with as little as 4 h of water deprivation. This is a time when several dehydration-related changes are known to occur in the somata of those neurosecretory cells sending axons to the neural lobe. Thus, increases in pituicyte lipid bodies may be related to the initiation of hormone release. In the neural lobes of water replete, non-deprived rats, pituicytes were found to contain neurosecretory axons completely enclosed by their cytoplasm. The number of these per unit area of cytoplasm decreased by 64% with 24 h of water deprivation and returned to normal levels with 24 h of rehydration. Since few of the enclosed axons were found to be degenerating, enclosure is unlikely to represent the early stages of phagocytosis.These findings suggest that during periods of little hormone secretion the neurosecretory axons are enclosed by the glial cells, but are released under conditions of increased hormone demand. This may provide the morphological framework for pituicyte involvement in inhibition and facilitation of hormone release from the neurohypophysis.     

Osmotic stimulation causes structural plasticity of neurone-glia relationships of the oxytocin but not vasopressin secreting neurones in the hypothalamic supraoptic nucleus

A comparative quantitative analysis was carried out on identified supraoptic neurones of male and female Wistar and Long Evans rats under normal conditions and after chronic osmotic stimulation, and in homozygous Brattleboro rats suffering from diabetes insipidus. The neurones were identified by immunocytochemical or morphological means. Osmotic stimulation resulted in significant increases in the number and extent of direct neuronal appositions and in the number of presynaptic terminals contacting two neurosecretory cells simultaneously ("double" synapses). In the supraoptic nuclei of both sexes these increases were restricted to the oxytocin secreting neurones. In Brattleboro homozygous rats treated with vasopressin, the proportion of oxytocinergic neurones in apposition was not modified, but the number of appositions per soma profile decreased as did the incidence of "double" synapses. In nuclei of osmotically stimulated rats, increase in cell volume affected both types of neurosecretory cell and was accompanied by an increase of the absolute extent of glial coverage. However, the extent of glial coverage of the oxytocinergic neurones did not match the hypertrophy of the cells, resulting in a decrease in their relative glial coverage, compared to normal hydrated animals. The increased neuronal appositions, therefore, cannot result simply from a retraction of glial processes. The structural reorganization of the oxytocinergic system observed during chronic osmotic stimulation was as extensive as that observed at lactation. Moreover, the changes were as extensive in Wistar as in Brattleboro lactating rats, although the latter have an added osmotic stimulus. This implies that lactation and osmotic stimulation do not produce additive effects.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ependymal changes in experimental hydrocephalus

A morphologic investigation of ependyma over gray matter (caudate nucleus) and over periventricular white matter (tapetum) of the rabbit lateral ventricle was performed four months after the induction of experimental hydrocephalus. Ependymal cells over the caudate nucleus are not modified by hydrocephalus. They remain cuboidal and heavily ciliated. Numerous microvilli cover the cell surface. The extracellular space of the neuropil is not expanded. Ependymal cells over the periventricular white matter are markedly modified. The characteristic response of these ependymal cells is to enlarge and to form lacunae in their apical cytoplasm. Their apical, horizontal cytoplasmic processes elongate as adjacent ependymal cells separate. The ex-tracellular space of the neuropil is expanded. It is proposed that the changes seen in ependymal cells over periventricular white matter are a response to enlargement of the ventricular surface permitted by the orientation of neuronal and glial fibers parallel to the ventricular surface. With expansion of the ventricular surface, overlapping apical processes become elongated and modified, containing a terminal web. With further enlargement, sliding of an overlapping apical process of one cell uncovers the apical process of its neighboring cell. By this mechanism, the ventricular surface area of any ependymal cell whose surface has been partially covered by its neighbor is increased. With further progression, this compensation fails and the neuropil is exposed to the ventricular cavity. Over caudate nucleus, expansion of ventricular surface is hindered by the disposition of fascie adherentes along intercellular clefts oriented perpendicular to the ventricular surface. Lateral sliding of horizontal apical processes does not occur as such processes are not found in ependyma over the caudate nucleus. The differential response of the ventricular surface in these two areas characteristically seen in hydrocephalus is; determined by regional differences in the morphology of their ependymal cells and underlying neuropil.     

Paraganglioma of the Cauda Equina: An Ultrastructural and Immunohistochemical Study of Two Cases

The ultrastructural and immunohistochemical features of 2 paragangliomas arising in the cauda equina are described. In both cases the tumor cells were arranged in small nests or cords and contained characteristic neurosecretory granules, lamellar stacks of rough endoplasmic reticulum (RER), and some well-developed Golgi apparatuses in their cytoplasm. The cells varied in electron density; the darker cells, occasionally resembling sustentacular cells, were probably dehydrated light cells because they contained a few neurosecretory granules. Sustentacular cells were difficult to identify by electron microscopy, but irregularly distributed S-100 protein and glial fibrillary acidic protein (GFAP) were found in these cells by immunostaining. Many tumor cells contained abundant neurofilaments. Curiously, a few cytokeratin-positive cells were found in 1 case. On microscopic examination, a small area of ganglioneuroma was found associated with the paraganglioma in 1 case. Ganglionic differentiation was concluded to be frequent in paragangliomas of the cauda equina region as in duodenal paragangliomas.     

Light- and electron-microscopic immunocytochemistry of glutamic acid decarboxylase (GAD) in the basal hypothalamus: Morphological evidence for neuroendocrine γ -aminobutyrate (GABA)

GABAergic cells and axon terminals were localized in the basal hypothalamus of different species (rat, mouse and cat), by means of an immunocytochemical approach using a specific and well-characterized antiserum to the GABA biosynthetic enzyme, glutamate decarboxylase. Lightmicroscopic visualization was performed with an indirect immunofluorescence method and electron-microscopic observations were made on material with pre-embedding staining and use of the peroxidase-antiperoxidase procedure.At the light-microscopic level, a dense immunofluorescent plexus was observed over both the medial and lateral parts of the external layer of the median eminence. The labelling extended from the rostral part of the median eminence up to the pituitary stalk. Over the subependymal and internal layers only a few immunoreactive dots were visible, except around the blood vessels where they appeared more concentrated. Immunoreactive varicosities could be found following the outlines of the capillary loops and lining tanycyte processes, especially in the median eminance midportion.At the electron-microscopic level, the immunolabelling was exclusively found over neuronal profiles in the median eminence. The latter represented a small fraction of the total number of varicosities visible on the same section. Labelled profiles typically contained numerous small clear synaptic vesicles and only a few or no dense-core vesicles. In the subependymal and internal layers, rare labelled endings were found close to ependymal cells or among transversally cut fibers, respectively. In the palisadic zone, elongated positive boutons were visible intermingled with bundles of unlabelled axons and glial or ependymal processes. In the neurohemal contact zone, immunoreactive endings were observed among unlabelled neurosecretory endings in close vicinity to fenestrated capillary perivascular space.Small moderately intense immunofluorescent varicosities were observed all over the hypothalamus. The density of the glutamate decarboxylase-positive network was higher than in most diencephalic regions. Intraventricular or topical injection of colchicine allowed the visualization of small lightly immunoreactive cells in the diffusion area of colchicine. In the arcuate nucleus labelled axonal endings containing small pleomorphic synaptic vesicles and sometimes a few dense-core vesicles were observed at the electron-microscopic level. Typical synaptic junctions were commonly found between positive endings and unlabelled perikarya, or more frequently, unlabelled dendrites.These findings show that glutamate decarboxylase-containing endings are localized in several strategic sites for potential GABAergic neuroendocrine regulations. The GABAergic endings found among neurosecretory endings in the neurohemal contact zone may provide the morphological support for the release of γ -aminobutyrate into the portal blood flow as an hypothalamic hypophysiotropic hormone. Alternatively, neurosecretory cells might be under GABAergic control expressed either at their terminal level within the median eminence or the cell body level within the parvicellular hypothalamic nuclei.