Combining Cadherin Expression with Molecular Markers Discriminates Invasiveness in Growth Hormone and Prolactin Pituitary Adenomas.

Although growth hormone (GH)- and prolactin (PRL)-secreting pituitary adenomas are considered benign, in many patients, tumour growth and/or invasion constitute a particular challenge. In other tumours, progression relies in part on dysfunction of intercellular adhesion mediated by the large family of cadherins. In the present study, we have explored the contribution of cadherins in GH and PRL adenoma pathogenesis, and evaluated whether this class of adherence molecules was related to tumour invasiveness. We have first established, by quantitative polymerase chain reaction and immunohistochemistry, the expression profile of classical cadherins in the normal human pituitary gland. We show that the cadherin repertoire is restricted and cell-type specific. Somatotrophs and lactotrophs express mainly E-cadherin and cadherin 18, whereas N-cadherin is present in the other endocrine cell types. This repertoire undergoes major differential modification in GH and PRL tumours: E-cadherin is significantly reduced in invasive GH adenomas, and this loss is associated with a cytoplasmic relocalisation of cadherin 18 and catenins. In invasive prolactinomas, E-cadherin distribution is altered and is accompanied by a mislocalisation of cadherin 18, ß-catenin and p120 catenin. Strikingly, de novo expression of N-cadherin is present in a subset of adenomas and cells exhibit a mesenchymal phenotype exclusively in invasive tumours. Binary tree analysis, performed by combining the cadherin repertoire with the expression of a subset of known molecular markers, shows that cadherin/catenin complexes play a significant role in discrimination of tumour invasion.

Rat microglial cells secrete predominantly the precursor of interleukin-1beta in response to lipopolysaccharide.

Little is known on the forms of interleukin-1beta (IL-1beta) that are produced by microglial cells in the nervous system. Mixed glial cell cultures of rats produced IL-1beta in response to lipopolysaccharide (LPS). Using Western blot, pro-IL-1beta was found to be localized both intracellularly and in the supernatant, whereas mature IL-1beta was found only in the supernatant but in lower quantities than pro-IL-1beta. Immunocytochemistry confirmed that microglial cells are the exclusive source of IL-1beta. Blockade of the IL-1beta-converting enzyme (ICE) by Tyr-Val-Ala-Asp-aldehyde (YVAD-CHO) decreased the levels of mature IL-1beta but had no effect on pro-IL-1beta. Release of pro-IL-1beta was not associated with cell death nor with the extracellular release of ICE. Using gelatin zymography, glial cells were found to express constitutive matrix metalloproteinases (MMP) in the form of MMP-2. Exposure to LPS induced MMP-9 expression in a time-dependent manner similar to the pro-IL-1beta expression profile. MMP activation and inhibition experiments indicated a possible role of MMPs in the cleavage of pro-IL-1beta but not in the generation of mature IL-1beta. Microglial cells share with macrophages the ability to release large amounts of pro-IL-1beta of which the extracellular role remains to be determined.

Inactivation of the glial fibrillary acidic protein gene, but not that of vimentin, improves neuronal survival and neurite growth by modifying adhesion molecule expression.

Intermediate filaments (IFs) are a major component of the cytoskeleton in astrocytes. Their role is far from being completely understood. Immature astrocytes play a major role in neuronal migration and neuritogenesis, and their IFs are mainly composed of vimentin. In mature differentiated astrocytes, vimentin is replaced by the IF protein glial fibrillary acidic protein (GFAP). In response to injury of the CNS in the adult, astrocytes become reactive, upregulate the expression of GFAP, and reexpress vimentin. These modifications contribute to the formation of a glial scar that is obstructive to axonal regeneration. Nevertheless, astrocytes in vitro are considered to be the ideal substratum for the growth of embryonic CNS axons. In the present study, we have examined the potential role of these two major IF proteins in both neuronal survival and neurite growth. For this purpose, we cocultured wild-type neurons on astrocytes from three types of knock-out (KO) mice for GFAP or/and vimentin in a neuron-astrocyte coculture model. We show that the double KO astrocytes present many features of immaturity and greatly improve survival and neurite growth of cocultured neurons by increasing cell-cell contact and secreting diffusible factors. Moreover, our data suggest that the absence of vimentin is not a key element in the permissivity of the mutant astrocytes. Finally, we show that only the absence of GFAP is associated with an increased expression of some extracellular matrix and adhesion molecules. To conclude, our results suggest that GFAP expression is able to modulate key biochemical properties of astrocytes that are implicated in their permissivity.

Membrane lipid rafts are necessary for the maintenance of the (alpha)7 nicotinic acetylcholine receptor in somatic spines of ciliary neurons.

Calcium-permeable neurotransmitter receptors are concentrated into structurally and biochemically isolated cellular compartments to localize calcium-mediated events during neurotransmission. The cytoplasmic membrane contains lipid microdomains called lipid rafts, which can gather into microscopically visible clusters, and thus the association of a particular protein with lipid rafts can result in its redistribution on the cell surface. The present study asks whether lipid rafts participate in the formation and maintenance of the calcium-permeable alpha7-subunit nicotinic acetylcholine receptor (alpha7nAChR) clusters found in somatic spines of ciliary neurons. Lipid rafts and alpha7nAChR become progressively colocalized within somatic spines during synaptogenesis. To determine whether these rafts are required for the maintenance of alpha7nAChR aggregates, cholesterol was extracted from dissociated ciliary neurons by treatment with methyl-beta-cyclodextrin. This treatment caused the dispersion of lipid rafts and the redistribution of alpha7nAChR into small clusters over the cell surface, suggesting that the integrity of lipid rafts is required to maintain the receptor clustering. However, lipid raft dispersion also caused the depolymerization of the F-actin cytoskeleton, which can also tether the receptor at specific sites. To assess whether interaction between rafts and alpha7nAChR is independent of F-actin filaments, the lipid raft patches were stabilized with a combination of the cholera toxin B subunit (CTX), which specifically binds to the raft component ganglioside GM1, and an antibody against CTX. The stabilized rafts were then treated with latrunculin-A to depolymerize F-actin. Under these conditions, large patches of CTX persisted and were colocalized with alpha7nAChR, indicating that the aggregates of receptors can be maintained independently of the underlying F-actin cytoskeleton. Moreover, it was found that the alpha7nAChR is resistant to detergent extraction at 4 degrees C and floats with the caveolin-containing lipid-rich fraction during density gradient centrifugation, properties that are consistent with a direct association between the receptor and the membrane microdomains.

Tanycytes transplanted into the adult rat spinal cord support the regeneration of lesioned axons.

During past years a number of therapeutic strategies have been developed in order to stimulate axonal regeneration after traumatic injuries of the spinal cord. Recently, encouraging data have been obtained by grafting specific glial cells such as Schwann cells or olfactory ensheathing glial cells, known to support the regeneration of peripheral or central axons, respectively. In a recent series of studies, we have shown that tanycytes, a particular glial cell type present in the mediobasal hypothalamus, were able to support the regeneration of a variety of axons innervating this region. The aim of the present study was to determine whether tanycytes could also support the regeneration of lesioned spinal axons. Cultured hypothalamic tanycytes and cortical astrocytes were prelabeled with Fast blue (FB) and grafted into the thoracic spinal cord of adult rats. Three weeks after the transplantation, the animals were fixed and spinal cord sections treated for multiple fluorescence detection of the FB-labeled transplanted cells on the one hand and of various glial and neuronal markers on the other hand. We show here that in all the spinal cords examined, transplanted tanycytes or astrocytes formed large spherical clusters of about 0.5 mm in diameter, located in the mediolateral spinal cord layer. The immunodetection of glial markers showed that transplanted astrocytes exhibited intense immunostaining for both glial fibrillary acidic protein (GFAP) and vimentin (VIM), whereas transplanted tanycytes were intensely immunostained for VIM, but GFAP negative. The immunodetection of axonal markers showed that contrasting with astrocyte transplants, tanycyte transplants were invaded by numerous axonal fibers. These data indicate that tanycyte transplants may represent a useful therapeutic tool for the reparation of the lesioned spinal axons.

Tangential migration of young neurons arising from the subventricular zone of adult rats is impaired by surgical lesions passing through their natural migratory pathway.

In the brain of adult rodents, young neurons arising from the subventricular zone (SVZ) of the lateral ventricle migrate tangentially along the rostral migratory stream (RMS) toward the olfactory bulb. The aim of this study was to determine whether surgical lesions placed through the RMS could affect the rostral migration of these newly formed neurons. Confocal and electron microscopy were used to characterize their anatomical organization within the intact and lesioned forebrains. As soon as 7 days and up to 45 days after placing a surgical lesion through the proximal portions of the RMS, numerous cells immunostained for polysialylated neural cell adhesion molecule (PSA-NCAM) were detected both (1) throughout the lesional cavity extending from the cortex to the anterior commissura, and (2) within the tissue located caudal to the lesion. In both regions, these PSA-NCAM-immunostained cells were labeled for neuronal markers but were negative for glial fibrillary acidic protein (GFAP). After administration of the proliferation marker bromodeoxyuridine (BrdU), nuclear labeling was associated with cells immunostained for PSA-NCAM but GFAP-negative, that accumulated within the lesional cavity and in the tissue caudal to the lesion. For the longest postlesional delays, a number of the PSA-NCAM-immunostained neurons located in various portions of the lesional cavity exhibited intense immunostaining for gamma-aminobutyric acid, whereas only a few of them exhibited faint immunostaining for tyrosine hydroxylase. These data indicate that surgical lesions placed through the RMS of adult rats impede the migration toward the olfactory bulb of the neuroblasts arising from the SVZ, inducing their accumulation and their partial differentiation in forebrain regions caudal to the lesion.

Tanycytes present in the adult rat mediobasal hypothalamus support the regeneration of monoaminergic axons.

We have recently shown that tanycytes present in the median eminence (ME) constitute a preferential support for the regeneration of lesioned neurohypophysial oxytocinergic and vasopressinergic axons. However, although tanycytes are particularly abundant in the ME, they are also present along the third ventricle wall. This study was thus undertaken to determine whether tanycytes present in the mediobasal hypothalamus overlying the ME were also able to support the regeneration of the numerous monoaminergic axons innervating this region. Using confocal laser scanning microscopy combined with double or triple fluorescence immunostaining, we have compared the relationships occurring between glial cells and lesioned catecholaminergic and serotonergic axons at the levels of surgical cuts placed in the dorsomedial hypothalamus devoid of tanycytes or in the ventromedial hypothalamus containing numerous tanycyte processes. In dorsal lesions, catecholaminergic and serotonergic transected fibers were found to abut onto the scar formed along the surgical cut and composed of closely inderdigitating astrocyte processes strongly immunoreactive for both glial fibrillary acidic protein (GFAP) and vimentin (VIM). In ventral lesions, the lesional scar was composed of GFAP-immunoreactive (IR) and VIM-IR astrocyte processes and of VIM-IR but GFAP-negative processes that were identified as tanycytic processes. In all the ventral lesions examined, numerous catecholaminergic and serotonergic fibers were found to regenerate into the surgical cut in association with the VIM-IR, GFAP-negative tanycyte processes. On the other hand, such regenerating fibers were never found in scar portions containing only GFAP-IR astrocytic structures. These data indicate that, like in the ME, tanycytes present in the mediobasal hypothalamus of adult rat provide a substrate that favors the regeneration of lesioned axons.

PSA-NCAM and B-50/GAP-43 are coexpressed by specific neuronal systems of the adult rat mediobasal hypothalamus that exhibit remarkable capacities for morphological plasticity.

The present study was designed to determine whether the mediobasal hypothalamus of adult rats contains neurons that continue to coexpress the highly polysialylated neural cell adhesion molecule (PSA-NCAM) and B-50/GAP-43, two proteins coexpressed by virtually all of the neurons of the fetal and neonatal rat central nervous system. Confocal laser scanning microscopy combined with double- or triple-fluorescence immunostaining was used to identify the hypothalamic neurons that express high levels of both PSA-NCAM and B-50/GAP-43 and to study the possible modifications of their morphological organization following a surgical lesion through the mediobasal hypothalamus. In intact animals, PSA-NCAM and B-50/GAP-43 were found to be colocalized within numerous fibers projecting throughout the external layer of the median eminence that were immunoreactive for either gamma-aminobutyric acid (GABA) or tyrosine hydroxylase (TH). Three to 30 days after a lesion through this region, numerous regenerating axonal sprouts, triple-immunostained for PSA-NCAM, B-50/GAP-43, and either GABA or TH, were detected along the ventricular surface of, and throughout the perivascular layer of, the median eminence. Surprisingly, high levels of PSA-NCAM and B-50/GAP-43 were also associated with numerous supraependymal neurons that exhibited long ramified processes and were immunoreactive for GABA but TH-negative. The use of the proliferation marker, 3H-thymidine, further indicated that the emergence of such supraependymal neurons after median eminence lesion was not related to the proliferation of preexisting quiescent cells. These data indicate that the mediobasal hypothalamus of the adult rat contains two neuronal systems, in which the continued coexpression of PSA-NCAM and B-50/GAP-43 is related to remarkable capacities for postlesional, morphological plasticity.

Mab22C11 antibody to amyloid precursor protein recognizes a protein associated with specific astroglial cells of the rat central nervous system characterized by their capacity to support axonal outgrowth.

Amyloid precursor protein (APP) is a transmembrane glycoprotein which is believed to promote neural cell adhesion, neural survival, and neuritogenesis. The present study was undertaken to determine whether APP could be detected within different types of astroglial cells present in the central nervous system (CNS) of neonatal or adult rats. The localization of this protein within glial cells was studied by using a monoclonal antibody (Mab22C11) that recognizes all APP isoforms and in addition cross-reacts with APP-like proteins. In the brain of neonatal rats, Mab22C11 immunostaining was associated with numerous elongated radial glia-like structures. In the intact brain and spinal cord of adult rats, Mab22C11 immunostaining was associated with (i) numerous neuron-like structures and (ii) glial structures immunostained for glial fibrillary acidic protein (GFAP) and/or vimentin, including tanycytes mostly located in the mediobasal hypothalamus, fibrous astrocytes located in the white matter and ependymocytes bordering the ventricles. On the other hand, all the GFAP-immunostained astrocytes located in the grey matter were Mab22C11 negative. In the lesioned brain and spinal cord of adult rats, Mab22C11 immunostaining was associated with intensely GFAP-immunostained reactive astrocytes located close to a surgical lesion, but not with those induced by Wallerian degeneration that appear at a distance from a lesion. Electron microscopic observations further indicated that in all these labeled astroglial cells, Mab22C11 immunostaining was mainly localized to the limiting plasma membrane and the membrane of intracytoplasmic cisternae and vesicles. These data indicate that Mab22C11 antibody induces strong immunostaining of specific astroglial cells of the neonatal and adult rat CNS that support axonal outgrowth, therefore suggesting that an APP-like protein associated with these cells participates in their axonal outgrowth promoting properties.

[Neuronal regeneration and the glial barrier]. / Respousse axonale et obstacle glial.

The dogma of abortive axonal regrowth set by Cajal (1914) is now broken since the demonstration by Aguayo (1982) that severed axons can regrow in an appropriate environment. Over the last decade, the impediments to such a regrowth in the central nervous system of higher vertebrates have been identified, or, at least, some of them. On the one hand, the inhibitory molecules synthesized and secreted by oligodendrocytes have been counteracted by appropriate antibodies (Schnell & Schwab, 1990), which have permitted some regrowth of severed cortico-spinal axons in the rat spinal cord. On the other hand, the reduction by a pharmacological treatment of hypertrophy and hyperplasia of astrocytes has permitted some regrowth of monoaminergic axons in an hemisected cord (Gimenez y Ribotta et al. 1995). Finally, the identification of a subcategory of astrocytes, the tanycytes of the basal hypothalamus, as a permissive substrate for axonal regeneration opens a new avenue for future research.

Apoptosis occurs independently of the release of interleukin-1 beta in the anterior pituitary of end-lactating rats.

The mechanism of Interleukin-1 beta (IL-1 beta) release remains unknown. Because of the absence of typical peptide signal on the precursor, IL-1 beta is not secreted through the classical pathway. The aim of this study was to determine whether IL-1 beta is released during apoptosis, as has been reported for activated macrophages. We chose anterior pituitary cells of end-lactating rats because of their capacity to produce IL-1 beta spontaneously and because this organ undergoes cellular degeneration. The combination of two techniques, reverse haemolytic plaque assay (RHPA) and terminal transferase dUTP nick end labelling (TUNEL), allowed us to observe simultaneously the release of IL-1 beta and apoptosis. Our results show that in these conditions apoptosis is not the mechanism of IL-1 beta release.

Aged median eminence glial cell cultures promote survival and neurite outgrowth of cocultured neurons.

We have recently shown that tanycytes, a particular type of glial cell that has morphological and biochemical similarities with radial glial cells, constitute a preferential support for the regeneration of lesioned neurohypophysial axons. The present study was designed to explore the possible neurotrophic role of tanycytes in vitro. Glial cells derived from the median eminence or from the cerebral cortex of 10-day-old rats were cultured for 4-7 weeks. At these times the majority of the cells identified in the median eminence cultures exhibited immunostaining patterns of tanycytes, as detected in the mediobasal hypothalamus of 10-day-old and adult rats, i.e., they were immunoreactive to vimentin (VIM), to DARPP-32 (a dopamine- and adenosine 3':5'-monophosphate-regulated phosphoprotein), and to a lesser extent to glial fibrillary acidic protein (GFAP) antibodies. On the other hand, the majority of cells in cortex cultures showed immunostaining patterns of astrocytes, i.e., they were intensely immunoreactive to GFAP and VIM antibodies but negative to DARPP-32. Cells obtained from the dissociation of 3-day-old rat mesencephalon, cortex, and hypothalamus were cocultured on these glial monolayers, and the number of surviving neurons and their neurite length were quantified after 8 days. Our data showed that, when compared with astrocytes, tanycytes greatly improved both survival (six-to ten-fold higher) and neurite outgrowth (two- to five-fold longer) of cocultured neurons whatever their origin. Experiments performed by coculturing neurons on millicell inserts placed above the glial monolayers showed that diffusible factors from median eminence glial cells slightly increased survival (1.7-fold higher) of cocultured neurons but had no significant effect on neurite outgrowth. These observations indicate: 1) that aged tanycytes have a capacity to support survival and neurite outgrowth for a variety of postnatal neurons; and 2) that this neurotrophic effect is exerted mainly by means of specific molecules bound to the tanycytic plasmalemma limiting membrane and/or to the extracellular matrix.

Survival and regeneration of neurons of the supraoptic nucleus following surgical transection of neurohypophysial axons depend on the existence of collateral projections of these neurons to the dorsolateral hypothalamus.

The aim of the present study was to compare the postlesional responses of vasopressin-producing (VP) and oxytocin-producing (OT) neurons of the supraoptic nucleus (SON) to transection of neurohypophysial axons. At different times after sectioning the median eminence of adult rats, immunocytochemical staining of both types of neuronal cell bodies and axons indicated that: (1) the number of OT neurons detected within the SON was only slightly decreased as compared with controls (-20%), whereas the number of VP neurons was severely decreased (-60%); and (2) the large majority of axonal sprouts that regenerated into the external layer of the median eminence were OT neurohypophysial axons. The injection of a retrograde tracer into various areas surrounding the SON further showed that numerous SON neurons could be retrogradely labeled when the injection was centered in the lateral hypothalamus dorsal to the SON. The immunocytochemical identification of these retrogradely labeled neurons demonstrated that most of them were OT neurons. When animals were subjected to median eminence transection and to a unilateral surgical cut placed in the lateral hypothalamus above the SON, the survival of both OT and VP neurons was dramatically reduced in the SON ipsilateral to the hypothalamic lesion, as compared to the contralateral SON. Taken together, these data indicate that OT (and to a lesser extent VP) neurons of the SON display collateral projections towards the lateral hypothalamus that protect them from retrograde degeneration following the lesion of their neurohypophysial projections.

Immunocytochemical characterization of a new marker of fibrous and reactive astrocytes.

A specific monoclonal antiserum (Mab 6.17) inducing a strong immunostaining of the neuromuscular junction has been used to detect the possible occurrence of the corresponding antigen throughout the intact or lesioned central nervous system of adult rats. In intact animals, 6.17-immunolabeling was essentially detected in astrocyte-like structures located in white matter fasciculi of the brain, such as the optic tract, corpus callosum, fornix, and in the white matter of the spinal cord. The astroglial nature of such 6.17-immunolabeled profiles was verified by performing double or triple immunofluorescent labeling with Mab 6.17 and with specific antisera against astrocytic markers, such as S100 protein, glial fibrillary acidic protein and vimentin. In the white matter, all the structures reactive to Mab 6.17 were also reactive to antibodies against S100 protein, glial fibrillary acidic protein and vimentin. On the other hand, astrocytes of the grey matter that were immunoreactive to S100 and glial fibrillary acidic protein but negative to vimentin, were devoid of 6.17-immunoreactivity. After lesions including stab wound through the diencephalon or transection of the spinal cord, a marked increase of 6.17-immunostaining was noted in the regions surrounding the lesions. In these regions, 6.17-immunolabeling was associated with S100-, GFAP- and vimentin-positive astrocytes constituting the glial scar. The ultrastructural localization of 6.17-immunoreactivity indicated that, similar to glial fibrillary acidic protein and vimentin, the recognized antigen was mainly associated with gliofilaments. These observations indicate that, in the central nervous system of adult rats, Mab 6.17 recognizes a molecule associated with gliofilaments, which is essentially associated to reactive astrocytes expressing high levels of vimentin.

Immunocytochemical study of phenotypic plasticity of cultured dorsal root ganglion neurons during development.

Rat dorsal root ganglia (DRG) were cultured from different stages of development ranging from embryonic day-14 to adult. The expression of eight neurotransmitter phenotypes was examined with immunocytochemical detection and the percentages of each phenotype were calculated with reference to the whole neuronal population defined by the expression of neuron-specific enolase (NSE). The expression of peptides, calcitonin gene-related peptide (CGRP), substance P (SP), cholecystokinin (CCK) and neuropeptide Y (NPY) was always present whatever the age at onset of the cultures. Although the percentage of CGRP remained stable, that of the other peptides declined progressively. Their in-vitro expression did not differ markedly from that found in vivo. Another group of neurotransmitters, including 5-hydroxytryptamine (5-HT), thyrotropin-releasing hormone (TRH) and gamma-aminobutyric acid (GABA) was never expressed in situ in DRG neurons. In culture, they were expressed in a high percentage of neurons, especially for 5-HT and TRH, and they showed a similar evolution, with a decrease at early postnatal ages followed by a further increase. This profile suggests that the expression of these transmitters is strongly environment-dependent and may be repressed in situ. Finally, somatostatin (SOM) was found only in cultures prepared from adult tissues, whereas it was present in situ from the embryo onwards. The expression of this peptide would thus require a stabilization by a long exposure to environmental factors. We can conclude that the great diversity of phenotypic expression found in DRG neurons in situ is the result of a wide variety of influences occurring at different stages of development in a large potential repertory present in these neurons.

Non-dopaminergic catecholaminergic neurons of mesencephalic and medullary nuclei contain different levels of dopamine.

The present study addresses the question whether metabolic dopamine can be immunocytochemically detected within non-dopaminergic catecholaminergic axonal fibers. For this purpose, confocal microscopy was used to analyze sections treated for the double fluorescence immunostaining of dopamine and either noradrenaline or phenylethanolamine-N- methyltransferase (the enzyme in adrenergic neurons that converts noradrenaline into adrenaline). Our data demonstrate that throughout the brain and spinal cord, the majority of the axonal fibers that reacted with the anti-phenylethanolamine-N-methyltransferase antibodies also exhibited faint to intense dopamine immunoreactivity. Similarly noradrenaline and dopamine immunoreactivities were frequently colocalized within axonal fibers innervating brain and spinal cord regions that receive a dense innervation from medullary noradrenergic neurons. On the contrary, dopamine was rarely detected within noradrenaline-immunoreactive fibers in those regions where the noradrenergic innervation essentially arises from noradrenergic neurons of the locus coeruleus. A similar differential dopamine immunostaining was observed in the corresponding neuronal perikarya of the medulla oblongata and the locus coeruleus. These data indicate that two types of non-dopaminergic catecholaminergic neurons can be distinguished according to their content in dopamine: (i) the noradrenergic and adrenergic neurons located in the medulla oblongata, whose cell bodies and axons contain high concentrations of metabolic dopamine and (ii) the noradrenergic neurons located in the mesencephalon, which contain low levels of metabolic dopamine.

Transected axons of adult hypothalamo-neurohypophysial neurons regenerate along tanycytic processes.

In the intact hypothalamo-neurohypophysial system, oxytocinergic or vasopressinergic neurons project their axons throughout the internal layer of the median eminence towards the blood vessels of the hypophysial neural lobe. When transected at the level of the median eminence, these axons undergo massive sprouting towards the external layer of the organ and the underlying perivascular region containing hypophysial portal vessels. The present study was designed to explore the possible roles of median eminence glial cells in such a reorganization of transected neurohypophysial axons. The relationships between regenerating axons and glial cells were studied by laser scanning confocal microscopy and electron microscopy on vibratome sections immunostained with specific antibodies against neurohypophysial peptides and/or against glial markers. All along the intact median eminence, two main types of glial cells were identified: (1) tanycytes immunoreactive to vimentin and slightly immunoreactive to glial fibrillary acidic protein, and (2) classical astrocytes immunoreactive to glial fibrillary acidic protein but vimentin-negative. In the intact median eminence, neurohypophysial axons were associated with astrocytic processes located in the internal layer. After a lesion of the hypophysial stalk, peptidergic regenerating axonal sprouts were found to project massively towards the external layer and to penetrate the underlying perivascular region in close association with tanycytic-like processes immunoreactive to both vimentin and to glial fibrillary acidic protein. In contrast, regenerating sprouts were absent from those regions of the lesioned median eminence containing astrocytic processes immunoreactive to glial fibrillary acidic protein but vimentin-negative. When fixed lesioned median eminences were treated by placing crystals of the lipophilic dye DiI on their ventricular surface, regenerating axons were found to be closely associated with DiI-labelled tanycytic-like end feet terminating in the external layer, and with connected thin processes projecting through the external vascular region. These data indicate that in the median eminence of the adult rat, lesioned neurohypophysial axons regenerate in close association with tanycytic processes.