The functional and structural border of the neurohemal region of the median eminence

Summary In stressed rats the tanycytes of the ventrolateral wall of the third ventricle exhibit by light microscopic immunohistochemistry a positive staining for neurohormones which is distinctly limited to the distal perivascular end of the tanycyte process. Since by electron microscopic immuncytochemistry the tanycyte cytoplasm does not show any reaction product, the light microscopic reaction most likely results from a labeling of the intercellular space in the direct vicinity of the subendothelial cleft. Whether this subendothelial space is permeable to neurohormones was tested by injection of HRP¹. In the region of the arcuate nucleus 30 min after intravenous application, the marker is affixed to the membranes of the perivascular tanycyte processes in the subendothelial cleft of capillaries possessing non-fenestrated endothelia. Occasionally, HRP penetrates for a short distance between the tanycytes. Then the labeling of the intercellular cleft ends abruptly. Here, several parallel ridges of tight junctions between the perivascular distal tanycyte processes are found by the freezeetching technique. Since HRP cannot reach the subendothelial clefts of this region by passing through capillary walls due to the presence of a blood-brain barrier, it is suggested that the marker penetrates from the median eminence this far via the subendothelial extracellular space. It is prevented from spreading further by the tight junctions of the perivascular tanycyte endings. The same way may be taken by the neurohormones. Hence, a border area exists adjacent to the dorsolateral aspect of the neurohemal region of the median eminence where the tanycytes isolate the neuropil from the cerebrospinal fluid not only by their apical tight junctions, but also by basal tight junctions from the subendothelial cleft. This communicates with the perivascular space of the portal vessels.Supported by the Deutsche Forschungsgemeinschaft (Grant Nr. Kr. 569/2) and Stiftung VolkswagenwerkDedicated to Professor Dr. R. Ortmann on the occasion of his 65th birthday.The skilful technical assistance of Miss K. Bielenberg, Mrs. A. Hinz and Mrs. H. Prien is thankfully acknowledged     

Ependymoneuronal specializations between LHRH fibers and cells of the cerebroventricular system

Summary Light-and electron-microscopic immunocytochemistry (LM-ICC and EM-ICC) were used to visualize luteinizing hormone-releasing hormone (LHRH) in fibres associated with ventricular ependyma and tanycytes of the median eminence. LM-ICC suggests that LHRH fibers appear to enter the third ventricle. However, with EM-ICC, LHRH fibers are in fact found within ependymal canaliculi formed by adjacent ependymal cells. The canaliculi contain other myelinated and unmyelinated axons in addition to immunoreactive LHRH fibers. Thin slips of ependymal and tanycyte processes project into the canaliculi and enclose axons to varying degrees. At the median eminence many LHRH fibers bend sharply downwards from their ventricular course and travel with tanycytic processes towards their common destination — the perivascular space of the hypophysial-portal vascular system. Here, EM-ICC reveals that LHRH fibers closely contact basal processes of tanycytes. Lateral processes from tanycytes form glioplasmic sheaths which surround some individual LHRH fibers. A few LHRH terminals contact the perivascular space directly but more often are separated from the perivascular space by intervening glia. It is hypothesized that: (1) glia of this region responds to the physiological state of the animal and may determine the degree of LHRH secretion by varying the extent of glial investment of LHRH terminals; and (2) may play a role during development by providing direction and support for LHRH fibers similar to that described for radial and other glial cells.     

Changes in pericytic expression of NG2 and PDGFRB and vascular permeability in the sensory circumventricular organs of adult mouse by osmotic stimulation

The blood–brain barrier (BBB) is a barrier that prevents free access of blood‐derived substances to the brain through the tight junctions and maintains a specialized brain environment. Circumventricular organs (CVOs) lack the typical BBB. The fenestrated vasculature of the sensory CVOs, including the organum vasculosum of the lamina terminalis (OVLT), subfornical organ (SFO) and area postrema (AP), allows parenchyma cells to sense a variety of blood‐derived information, including osmotic ones. In the present study, we utilized immunohistochemistry to examine changes in the expression of NG2 and platelet‐derived growth factor receptor beta (PDGFRB) in the OVLT, SFO and AP of adult mice during chronic osmotic stimulation. The expression of NG2 and PDGFRB was remarkably prominent in pericytes, although these angiogenesis‐associated proteins are highly expressed at pericytes of developing immature vasculature. The chronic salt loading prominently increased the expression of NG2 in the OVLT and SFO and that of PDGFRB in the OVLT, SFO and AP. The vascular permeability of low‐molecular‐mass tracer fluorescein isothiocyanate was increased significantly by chronic salt loading in the OVLT and SFO but not AP. In conclusion, the present study demonstrates changes in pericyte expression of NG2 and PDGFRB and vascular permeability in the sensory CVOs by chronic osmotic stimulation, indicating active participation of the vascular system in osmotic homeostasis. Copyright © 2013 John Wiley & Sons, Ltd.     

Tanycyte absorption affected by the hypothalamic deafferentation in Japanese quail,Coturnix coturnix japonica

In Japanese quail, Coturnix coturnix japonica the tanycytes of the median eminence absorbed peroxidase injected into the third ventricle. The number of tanycytes showing peroxidase reaction was greater in the posterior median eminence than in the anterior median eminence. Following hypothalamic deafferentation, the tanycyte absorption was augmented both in the posterior and anterior median eminence. These findings suggest that axons of some neurons, which have inhibitory action on the tanycyte absorption, were transected by deafferentation resulting in augmentation of tanycyte absorption. A considerable number of ependymal cells lining the upper portion of the third ventricle and those of the pars nervosa also absorbed peroxidase. In birds with a deafferented hypothalamus, photostimulated ovarian growth was completely inhibited.     

Ultrastructural demonstration of neurohaemal contacts in the internal zone of the median eminence of the Mongolian gerbil (Meriones unguiculatus): correlation with synaptophysin immunohistochemistry

Summary Electron microscopy of the median eminence (ME) of the Mongolan gerbil (Meriones unguiculatus) revealed that, unlike most other mammalian species, abundant neurohaemal contacts were present not only in the external zone (EZ), but also in the internal zone (IZ) up to the subependymal layer. In the IZ, nerve terminals with dense core vesicles and/or small clear vesicles abutted on the outer basal lamina of the perivascular space of portal capillaries, alternating with tanycyte processes. In addition to these neurohaemal contacts, several layers of vesicle-filled varicosities surrounded the portal vasculature. An analysis of serial thin sections showed that the latter varicosities could also reach the perivascular basal lamina or contact it through small extensions in other planes of section. Apparently at least some of the nerve terminals making neurohaemal contacts were en passant in nature. A correlative investigation of synaptophysin (a major integral membrane protein of small synaptic vesicles) immunoreactivity at the light microscopical level demonstrated a conspicuously dense immunostaining around portal capillaries in both EZ and IZ of the proximal and distal ME (neural stalk). Since this perivascular accumulation of immunoreactivity coincides precisely with the ultrastructural accumulation of vesicle-filled axons which establish numerous neurohaemal contacts, it is concluded that this pattern of synaptophysin immunostaining indicates sites of neurohaemal contacts at the light microscopical level. During postnatal development, the perivascular concentration of synaptophysin immunoreactivity in the IZ appeared concomitantly with the early postnatal expansion of long portal capillary loops into the IZ. By direct electron-microscopical demonstration and indirect immunohistochemical evidence, the present study of the gerbil ME reveals that the whole extent of portal capillaries up to the subependymal layer constitutes an area for numerous neurohaemal contacts. Hence, the common view that neurohaemal contacts are restricted to the EZ of the mammalian ME is not generally valid.     

Immunohistochemical localization of gonadotropin-releasing hormone (GnRH) in the fetal and early postnatal mouse brain.

The objectives were to (a) determine the age in development when GnRH is first detectable in the brain and (b) observe the distribution of GnRH throughout the fetal and early postnatal period. GnRH was localized immunohistochemically in fetal (15, 16, 17 and 19 days of gestation) and early postnatal (1- and 7-day-old) mice with the peroxidase-antiperoxidase (PAP) method of Sternberger. In the organum vasculosum of the lamina terminalis (OVLT) and in the median eminence of the fetus, GnRH was first detected at 17 days of gestation. In the OVLT, GnRH was found ventral to the preoptic recess of the third ventricle near the ventral surface of the brain. In addition, GnRH was located adjacent to the superficial portal capillaries near the surface of the median eminence. At 19 days of gestation, the distribution of GnRH was similar to that observed at 17 days and there was a marked increase in amount. In the newborn mouse, GnRH was undetectable in the OVLT and its content in the median eminence was decreased as compared to that observed in the fetus. By the seventh postnatal day, a considerable accumulation of GnRH had occurred in the OVLT and median eminence. In the OVLT, it was associated with capillaries ventral to the preoptic recess, and its distribution in the median eminence was similar to that in the adult mouse. In both the OVLT and median eminence of the fetal and early postnatal mouse GnRH appeared to be stored in axons and axon endings, but was not detectable in nerve cell bodies or ependymal cells. These observations suggest that the potential for neuroendocrine control of gonadotropin secretion exists in the fetal mouse early as 17 days of gestation.     

Mitotic Figures in the Median Eminence of the Hypothalamus

The median eminence of the hypothalamus is part of the avenue by which neurosecreted hormones from the hypothalamic nuclei reach the pars nervosa (neural lobe) of the pituitary and eventually the bloodstream. Lithium treatment and osmotic stress increases the transport of neurosecretory hormones to the pituitary in the adult rat. Specialized astrocytes termed pituicytes in the pars nervosa of the pituitary participate in the secretory process and also develop considerable mitotic activity. The present work reveals similar mitotic figures in cells within the median eminence following 3 days of lithium treatment. The location and appearance of these mitoses add to the evidence that pituicytes are present in the median eminence. Moreover, mitoses occur within the ependymal (tanycyte) layer of the median eminence. Thus, the present results suggest that the tanycyte layer may contain pituicytes, indicating that the hypothalamus possesses specialized cells for modulating neurosecretion in response to osmotic challenges.     

Ultrastructural demonstration of neurohaemal contacts in the internal zone of the median eminence of the Mongolian gerbil (Meriones unguiculatus): correlation with synaptophysin immunohistochemistry

Electron microscopy of the median eminence (ME) of the Mongolian gerbil (Meriones unguiculatus) revealed that, unlike most other mammalian species, abundant neurohaemal contacts were present not only in the external zone (EZ), but also in the internal zone (IZ) up to the subependymal layer. In the IZ, nerve terminals with dense core vesicles and/or small clear vesicles abutted on the outer basal lamina of the perivascular space of portal capillaries, alternating with tanycyte processes. In addition to these neurohaemal contacts, several layers of vesicle-filled varicosities surrounded the portal vasculature. An analysis of serial thin sections showed that the latter varicosities could also reach the perivascular basal lamina or contact it through small extensions in other planes of section. Apparently at least some of the nerve terminals making neurohaemal contacts were en passant in nature. A correlative investigation of synaptophysin (a major integral membrane protein of small synaptic vesicles) immunoreactivity at the light microscopical level demonstrated a conspicuously dense immunostaining around portal capillaries in both EZ and IZ of the proximal and distal ME (neural stalk). Since this perivascular accumulation of immunoreactivity coincides precisely with the ultrastructural accumulation of vesicle-filled axons which establish numerous neurohaemal contacts, it is concluded that this pattern of synaptophysin immunostaining indicates sites of neurohaemal contacts at the light microscopical level. During postnatal development, the perivascular concentration of synaptophysin immunoreactivity in the IZ appeared concomitantly with the early postnatal expansion of long portal capillary loops into the IZ.(ABSTRACT TRUNCATED AT 250 WORDS)     

Permeability of the csf-brain barrier in the median eminence of rats in the perinatal period

The ultrastructure and permeability of the CSF-brain barrier in the median eminence were studied during the perinatal period in rats with electron microscopy, electron-microscopic radioautography and electron-dense markers. It was shown that the ependymal cells forming the ventral portion of the 3rd ventricle (infundibular recess) are joined by the specialized junctions. The specialized junctions are similar in ultrastructure in different parts of infundibular recess all through the perinatal period. They consist of the tight junctions which are sometimes in series with the gap junctions and adhesion zones. An electron-dense marker, La3+, injected into the cerebral ventricles, penetrates through the foetal ependymal linins both via the ependymal cells and intercellularly in all parts of the infundibular recess. In neonatal rats La3+ penetrates in the same way in the rostral part of infundibular recess only, while in the caudal part its permeability is markedly limited, apparently by the tight junctions formed de novo around the cells. 3H-dopamine, introduced into the ventricles, crosses over the ependymal linins apparently in the same way as La3+. Then the labeled dopamine is transported into the pericapillary space of the primary portal plexus and, finally, into the capillary lumen. Thus, the results obtained point to the permeability of the CSF-brain barrier in the foetal median eminence for the markers including neurohormones. In the postnatal period the permeability of the ependyma becomes limited due to the formation of the tight junctions surrounding the ependymal cells.     

Immunocytochemical distribution of LHRH neurons and processes in the rat: Hypothalamic and extrahypothalamic locations

Summary The distribution of luteinizing hormone-releasing hormone (LHRH)-immunoreactive perikarya and processes was examined, in the untreated rat, with the unlabeled antibody enzyme method of immunocytochemistry on thick 50 m vibratome sections. LHRH neurons were primarily observed in the preoptico-anterior hypothalamic and septal areas. Projections from these cell bodies to the median eminence form three distinct pathways, one laterally along the course of the optic tracts, one medially through the periventricular stratum of the third ventricle, and one through the tractus infundibularis. In addition, some of these cell bodies project to the organum vasculosum of the lamina terminalis (OVLT) and the subfornical organ (SFO). LHRH immunoreactive neurons were also noted in the anterior olfactory regions; they project along the medial olfactory tract to the olfactory bulb.     

Direct evidence for the co-expression of URP and GnRH in a sub-population of rat hypothalamic neurones: anatomical and functional correlation

Urotensin-II-related peptide (URP) is an eight amino-acid neuropeptide recently isolated from rat brain and considered as the endogenous ligand for the GPR14 receptor. Using single and double immunohistochemical labelling, in situ hybridization and ultrastructural immunocytochemistry, we explored the cellular and subcellular localization of URP in the male rat brain. URP peptide was detected in numerous varicose fibres of the median eminence (ME) and organum vasculosum laminae terminalis (OVLT) as well as in neuronal cell bodies of the medial septal nucleus and diagonal band of Broca where corresponding mRNA were also detected. Combining in situ hybridization with immunohistochemistry, we showed that cell bodies of the rat anterior hypothalamus contained both URP mRNA and GnRH peptide. In addition, double ultrastructural immunodetection of URP and GnRH peptides clearly revealed, in the median eminence, the co-localization of both peptides in the same neuronal processes in the vicinity of fenestrated portal vessels. This remarkable cellular and subcellular distribution led us to test the effect of URP on the GnRH-induced gonadotrophins release in the anterior pituitary, and to discuss its putative role at the level of the median eminence.     

Fine structure of ependymal cells in the median eminence of the frog and mouse revealed by freeze-etching

Summary Freeze-etched preparations of the ventricular surfaces of ependymal cells clearly reveal the presence of pinocytotic vesicles opening into the third ventricle and large vacuoles formed by broad cell projections. The density of the vesicular openings is approximately 20 per m². The ependymal cells in the median eminence of the frog are adjoined by tight junctions comprised of five to eight interconnected junctional strands, whereas near the median eminence in the mouse only one to two such strands form the tight junction of the ependymal cells. Gap junctions between the adjacent ependymal cells are detected near the median eminence in the mouse but not in the frog.This study was supported in part by a grant from the Japanese Ministry of Education (No. 067670)     

Immunocytochemical localization of TRH and autoradiographic determination of 3H-TRH-binding sites in the arcuate nucleus-median eminence of the rat

Immunocytochemical and autoradiographic localization of thyrotropin-releasing hormone (TRH)- and 3H-TRH-binding sites was studied in the arcuate nucleus-median eminence region of the rat. TRH-like immunoreactivity was found in dense granular vesicles (90-140 nm in diameter) in TRH-like immunoreactive nerve fibers and terminals. In the median eminence, the immunoreactive terminals were observed to be in direct contact with the perivascular basal lamina of the portal vessel and to form synaptoid contacts with tanycytes. In the arcuate nucleus, the immunoreactive terminals were often found to form axosomatic and axo-axonic, and/or axo-dendritic synapses. The uptake of tritiated TRH into the nerve fibers and terminals of the median eminence was also observed by autoradiography and the distribution and localization of silver grains in them were analyzed quantitatively by circle analysis. Thirty minutes after intraventricular infusion of 3H-TRH, radioactive labeling occurred in type-2 and 3-nerve fibers and terminals containing dense granular vesicles in the median eminence. It is therefore suggested that the neurons labeled after 3H-TRH infusion possess certain functions as physiological recognition sites or receptors for TRH.     

Organization and development of the perivascular space system in the neurohypophysis of the laboratory mouse

Summary The organization of the system of perivascular space around the capillaries in the neurohypophysis was studied in the adult and developing laboratory mouse by the use of histological silver impregnation and electron microscopical techniques.In the median eminence short and long extensions, arising mainly from the shallow space around capillary loops of the primary plexus of the portal system, formed radiations into the adjacent neural tissue of the external zone. The tissue of the neural lobe was separable into non-vascular regions dominated by undilated portions of neurosecretory nerve fibres and pituicytes, and neurovascular regions with perivascular space extensions forming an extensive system of connections between neighbouring capillaries.In the median eminence, the system of extensions of the perivascular space was estimated to increase the neurovascular contact surface area by at least 50%, implying an increased efficiency of the organ without a notable increase of its volume. The possibility that the ramifications of the perivascular space imply an enhanced uptake rate into the bloodstream and a subsequent increased concentration of the neurohormones in the portal blood, was discussed.During development of the median eminence, differentiation of perivascular space extensions of the adult type started in the juvenile of about three weeks of age, when shallow capillary loops had been formed. In the neural lobe, perivascular space ramifications were already present when the internal capillaries were formed and were fairly frequent in ten-day young. At the age of three to four weeks the organization of the system was similar to that of the adult animal.     

Effects of forebrain circumventricular organ ablation on drinking or salt appetite after sodium depletion or hypernatremia

In many previous studies, one or the other forebrain circumventricular organ, the subfornical organ (SFO) or organum vasculosum laminae terminalis (OVLT), was lesioned to test whether it was critical for the behavioral or physiological responses to sodium depletion and hypernatremia. These studies conflict in their conclusions. The present study was designed to create discrete lesions of both the SFO and OVLT in the same animals and to compare these with rats having a lesion of only the SFO or OVLT. Both the OVLT-lesioned group and the combined SFO + OVLT-lesioned group drank significantly more water and saline on a daily basis than Controls or SFO-lesioned rats. In both sodium depletion and hypertonic saline testing, rats with SFO lesions displayed transient deficits in salt appetite or thirst responses, whereas the rats with single OVLT lesions did not. In the sodium depletion test, but not in the hypernatremia test, rats with lesions of both the SFO and OVLT exhibited the largest deficit. The data support the hypothesis that a combined lesion eliminates redundancy and is more effective than a single lesion in sodium depletion tests. The interpretation of the OVLT lesion-only data may have been complicated by a tendency to drink more fluid on a daily basis, because some of those animals drank copious water in addition to saline even very early during the salt appetite test.     

Estrogen receptor-alpha expression in osmosensitive elements of the lamina terminalis: regulation by hypertonicity

The subfornical organ (SFO), median preoptic nucleus (MnPO), and organum vasculosum lamina terminalis (OVLT), which are associated with the lamina terminalis, are important in the control of body fluid balance. Neurons in these regions express estrogen receptor (ER)-alpha, but whether the ER-alpha neurons are activated by hypertonicity and whether hypertonicity regulates ER-alpha expression are not known. Using fluorescent, double-label immunocytochemistry, we examined the expression of ER-alpha-immunoreactivity (ir) and Fos-ir in control and water-deprived male rats. In control animals, numerous ER-alpha-positive neurons were expressed in the periphery of the SFO, in both the dorsal and ventral MnPO, and in the dorsal cap of the OVLT. Fos-positive neurons were sparse in euhydrated rats but were numerous in the SFO, MnPO, and the dorsal cap of the OVLT after 48-h water deprivation. Most ER-alpha-ir neurons in these areas were positive for Fos, indicating a significant degree of colocalization. To examine the effect of dehydration on ER-alpha expression, animals with and without lesions surrounding the anterior and ventral portion of the 3rd ventricle (AV3V) were water deprived for 48 h. Water deprivation resulted in a moderate increase in ER-alpha-ir in the SFO of sham-lesioned rats (P = 0.03) and a dramatic elevation in AV3V-lesioned animals (P < 0.05). This was probably induced by the significant increase in plasma osmolality in both dehydrated groups (P < 0.001) rather than a decrease in blood volume, because hematocrit was significantly increased only in the dehydrated sham-lesioned animals. Thus these studies implicate the osmosensitive regions of the lamina terminalis as possible targets for sex steroid effects on body fluid homeostasis.     

The microvascular pattern and perivascular linings of the area postrema

Summary The microvasculature and perivascular linings of the area postrema (A.P.) were studied electron microscopically with the ultrathin section and freeze-etching techniques. Special attention was given to the intercellular contacts of the different cellular entities. Two types of microvascular segments were identified. The endothelium of these vascular segments reveals fenestrations and a high pinocytotic activity. There are no significant differences in the frequency and distribution of the endothelial openings between both types of capillaries. The endothelium of the blood vessels, however, is joined by different types of tight junctions. Focal tight junctions occur between pericytes and the endothelium, and between leptomeningeal cellular elements in the perivascular space. The cell membrane of the perivascular glia shows intramembrane particles which are either distributed at random or organized in the form of membrane-associated orthogonal particle complexes (MOPC, Dermietzel, 1974). The significance of these findings is discussed with respect to the modified blood-brain barrier mechanism in the A.P.Supported by a grant of the Deutsche Forschungsgemeinschaft (SFB 114 Bionach) to R. DermietzelThe authors are indebted to Mrs. D. Schünke and Mr. R. Eichner for technical assistance and Mr. A. Stapper for preparing the diagram     

Fine structure of the median eminence and the pars nervosa of the bullfrog,Rana catesbeiana

Summary The hypothalamic neurosecretory system of the bullfrog, Rana catesbeiana, was studied with light- and electron microscopy. The median eminence is roughly divided into two portions. The upper portion mostly consists of ependymal cells, glial cells and preoptico-hypophysial nerve tract, whereas in the lower portion, neurosecretory axons, glial cells, processes of glial and ependymal cells, and fine blood vessels of the hypothalamic portal vein are located. A part of the neurosecretory axons of the preoptico-hypophysial tract proceeds to the lower portion of the median eminence. These axons are arranged perpendicularly to the capillaries of the hypothalamic portal vein. The glial cells are densely located in the area of the median eminence where neurosecretory material is abundant. The neurosecretory material in the neurosecretory cells, their axons, the median eminence and the pars nervosa of the bullfrog shows a positive reaction to PAS treatment.The neurohemal area of the median eminence is occupied by many neurosecretory and non-neurosecretory axons, containing neurosecretory granules and/or synaptic vesicles. The axonal portions with the synaptic vesicles which are considered to be the nerve endings abut on the capillaries of the portal system. The size of synaptic vesicles in the axon terminals containing few neurosecretory granules is larger than those in the endings with many neurosecretory granules. Infrequently glial and ependymal processes are interposed between the nerve endings and the capillary wall.In the hilar region of the infundibulum, synapses are frequently observed between the thin fibers with or without neurosecretory granules and dendrites of non-neurosecretory neurons. The probable functions of these synapses are briefly discussed on the basis of our findings. Both in the hilar region of the infundibulum and in the pars nervosa, electron-dense neurosecretory granules of two different sizes were observed. The median eminence contains only one type of granules.The fine structure of the pars nervosa shows similar structures to those of the median eminence. Both in the median eminence and the pars nervosa, the fenestrated endothelium of the capillaries was frequently observed. The thick perivascular connective tissue space containing fibroblasts and collagen fibrils was observed both in the median eminence and the pars nervosa. Vesicles in the cytoplasm of the endothelial cells which appear to take a part in the transendothelial transport were observed.This investigation was supported in part by United States Public Health Service Research Grant, No. A-3678, to Hideshi Kobayashi from the National Institute of Arthritis and Metabolic Diseases and partly by a grant for Fundamental Scientific Research from the Ministry of Education of Japan. The authors wish to express their thanks to Prof. K. Takewaki for his kind encouragement.     

Antibodies obtained by xenotransplantation of organ-cultured median eminence specifically recognize hypothalamic tanycytes

Tanycytes are specialized ependymal cells lining the infundibular recess of the third ventricle of the cerebrum. Early and recent investigations involve tanycytes in the mechanism of gonadotropin-releasing hormone (GnRH) release to the portal blood. The present investigation was performed to obtain a specific immunological marker of tanycytes and to identify the compound(s) responsible for this labeling. After 30 days of organ culture, explants of bovine median eminence formed spherical structures mostly constituted by tanycytes. These tanycyte spheres were xenotransplanted to rats, and the antibodies raised by the host animals against the transplanted living tanycytes were used for immunochemical studies of the bovine and rat median eminence. This antiserum immunoreacted with two compounds of 60 kDa and 85 kDa present in extracts of bovine and rat median eminence. The individual immunoblotting analysis of rat medial basal hypothalami showed a decrease in the amount of the 85-kDa compound in castrated rats as compared to control rats processed at oestrus and dioestrus. The antiserum, labeled as anti-P85, when used for immunostaining of sections throughout the rat central nervous system, immunoreacted specifically with the hypothalamic tanycytes. Within tanycytes, P-85 immunoreactivity was exclusively present in the basal processes. It is suggested that the 85-kDa and 60-kDa compounds correspond to two novel proteins selectively expressed by tanycytes. The possibility that they are secretory proteins involved in GnRH release is discussed. Anti-P85 appears to be the first specific marker of hypothalamic tanycytes.     

Access and distribution of exogenous substances in the intercellular clefts of the rat adenohypophysis

In model experiments with the use of horseradish peroxidase (HRP), two pathways of transport of substances to the adenohypophysis were studied, as well as the distribution of the tracer in the latter organ. The first pathway allows the tracer to penetrate from the intercellular milieu of the median eminence below the meningeal sheath covering the adenohypophysis to the surface of the pituitary gland. The second pathway transports the tracer via the capillaries of the hypophysial portal circulation to the interior of the glandular parenchyma. These results show (i) that the meningeal sheath establishes a barrier between the hemal milieu of the pituitary and the hemal milieu of the general circulation, and (ii) that the tracer reaching the adenohypophysis via both routes is found in the intercellular clefts of the glandular parenchyma only to a limited extent. By means of conventional electron microscopy, intercellular contacts between hormone-producing adenohypophysial cells are observed resembling focal tight junctions. Between the membranes of entwined processes of stellate cells, only small maculae adhaerentes are found. Freeze-etch studies on unfixed adenohypophyses reveal zonulae occludentes between the durafacing layers of the meningeal sheath and focal maculae occludentes between parenchymal cells. Additional tissue-culture experiments with adenohypophysial cells directly exposed to HRP reveal a gradual cessation of the labeling process in the intercellular clefts in accord with the observations from the in-vivo experiments, as well as intercellular focal tight junctions between individual hormone-producing cells.