Localization of glyoxylic acid-induced histofluorescence in surgically isolated medial basal hypothalamus of the rat

Summary Examination of glyoxylic acid-induced catecholamine histofluorescence in the hypothalamic median eminence of adult male rats revealed a linear pattern of fine varicosities coursing through the ependymal and fibrous zones, suggestive of juxtaposition to tanycytes. In order to determine the origin of these terminals, adult rats were subjected to complete isolation of the medial basal hypothalamus, using a small Halasz-Pupp knife. As rapidly as 24h after this deafferentation degenerative axon profiles were observed dorsal, as well as anterior and lateral, to the knife track. Occasionally at three days postoperatively, and routinely by seven days after surgery, fine-sized new fibres were seen passing through the knife wound. The linear profiles of varicosities observed in the normal median eminence remained traceable in the experimental preparations; the site of origin for these terminals therefore appears to be neurons of the arcuate (A12) and rostral periventricular (A14) regions. The results also indicate that fibres innervating the isolated area are capable of morphologically demonstrable new growth. The observations bear functional implications in assessing endocrine regulation following MBH isolation of the type used in this study.This study was supported by USPHS Postdoctoral Fellowship 5-F₂2-HD00630 (CT) and USPHS Grant NS-11642 (JRS). The authors wish to express their appreciation to Yvonne Cheung and Patricia Walker for technical assistance     

The primate median eminence II

Summary The ultrastructure of the normal median eminence of the male rhesus monkey (Macaca mulatta) is described using high-voltage electron microscopy. Surface specializations of ependymal cells lining the infundibular recess included cilia, apical extrusions, and microvilli. Supraependymal cells were predominantly macrophage-like, but examples of lymphocytic types were also seen. Tanycytes had long, branching, basal processes filled with numerous microtubules, some lipid droplets, and granules. The zona interna was composed of large unmyelinated neurosecretory fibers. A few myelinated fibers were also seen, but their character as neurosecretory fibers could not be established. The zona externa was composed of densely-packed profiles of neurosecretory fibers of small diameter, was well-vascularized and contained the terminations of tanycytes. Perivascular glial cells, vesiculated elements, pituicytes, and cellular elements common to connective tissue were observed. The intricate relationships between both the cellular and fibrous elements of the median eminence can be appreciated with the capability of high-voltage electron microscopy to discern ultrastructure in sections 10 times thicker than those used for low-voltage electron microscopy. The median eminence of this primate species has an ultrastructural organization similar to that described for most other species.Supported by USPHS Program Project Grant NS-11642 and USPHS HD-08867. The authors appreciate the excellent technical advice and assistance of Mr. George Wray in operation of the HVEMCareer Development Awardee K04-GM-70001     

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.     

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.     

Fine structure of neurons of the arcuate nucleus and median eminence of the hypothalamus of the golden hamster following immobilization

Summary The fine structure of arcuate neurons of the arcuate nucleus, the ependymal tanycytes and the contact zone of the median eminence was examined following immobilization, an acute stress which significantly activated the hypothalamo-pituitary-adrenal (HPA) axis. Arcuate neurons of immobilized adult male hamsters displayed morphological indications of heightened activity; the number of lysosomes and dense core vesicles (80–120 nm) was increased. A markedly greater number of dense core vesicles was present in axon terminals of the contact zone of the mid-central median eminence and the ventral proximal stalk.Tanycytes of the median eminence exhibited an augmented number of electron dense bodies in both perikarya and end processes. These results indicate that the arcuate neurons, the axons of the contact zone, and the ependymal tanycytes of the hamster medial basal hypothalamus (MBH) may be involved in the response to immobilization.This work was supported by Program Project Grant #NS-11642     

D1 receptor mechanisms in the median eminence and their inhibitory regulation of LHRH release

D1 receptor mechanisms in the median eminence have been studied by means of immunocytochemistry using antisera against dopamine and cyclic AMP-regulated phosphoprotein-32 (DARPP-32) and tyrosine hydroxylase (TH) and by autoradiography using the iodinated analogue of the D1 receptor antagonist SCH-23390. The co-distribution of DARPP-32 and TH immunoreactivity (IR) and of DARPP-32 and luteinizing hormone releasing hormone (LHRH) IR was analysed in the median eminence by means of computer-assisted morphometry and microdensitometry. Functional analysis involved studies on the role of D1 receptors in the regulation of LH serum levels in rats treated with nicotine in the absence and presence of the D1 receptor antagonist. LH serum levels were measured by means of radioimmunoassay procedures.The results on the co-distribution of TH and DARPP-32 IR in the median eminence which were obtained both by analysis of adjacent sections and by two-colour immunocytochemistry on the same section, demonstrated a high degree of overlap of TH and DARPP-32 IR nerve terminals and tanycytes within the medial and lateral palisade zone. Furthermore, studies on LHRH and DARPP-32 IR nerve terminals and tanycytes in the median eminence with the same methodologies demonstrated preferential overlaps within the lateral palisade zone. The overlap area was about 50% of the LHRH or DARPP-32 immunoreactive area in this region. Density maps were also obtained on the distribution of LHRH and DARPP-32 immunoreactive profiles at various rostrocaudal levels. Correlation studies demonstrated a significant and positive co-distribution of LHRH and DARPP-32 immunoreactive terminals and tanycytes within the lateral palisade zone and the subependymal layer (when all DARPP-32 positive squares were considered) of the median eminence. Instead within the medial palisade zone a significant negative correlation coefficient was found, when all the LHRH positive squares were considered.In the receptor autoradiographical analysis a weak-to-moderate labelling was obtained of the part outside the mediobasal hypothalamus using the D1 receptor radioligand [¹²⁵I]SCH-23982, while hardly any labelling was found within the median eminence and the arcuate nucleus.SCH-23390 was found to counteract, in a dose-related way, the inhibitory effects of intermittent nicotine treatment on serum LH levels. The D2 receptor antagonist raclopride in a dose of 1 mg/kg did not counteract the inhibitory effects of nicotine on serum LH levels.The present immunocytochemical, autoradiographic and functional studies suggest the existence of a D1 receptor in the median eminence which can be blocked by the D1 receptor antagonist SCH-23390 in behaviourally relevant doses and which is masked under basal conditions in the male rat. It is proposed that one type of median eminence D1 receptor is located on the axon terminals, not linked to DARPP-32, and which may make possible a rapid regulation of hypothalamic hormone release, e.g. LHRH release from the nerve terminals in the lateral palisade zone as indicated in the present morphological and functional experiments. The other type of median eminence D1 receptor may be located on the tanycytes and linked to DARPP-32. It is suggested that this D1 receptor is responsible for a long-term regulation of hypothalamic hormone release inter alia LHRH release from the terminal and preterminal parts of the LHRH axons in the lateral palisade zone and subependymal layer, respectively.     

Immunogold electron microscopic localization of glial fibrillary acidic protein (GFAP) in neurohypophyseal pituicytes and tanycytes of the Mongolian gerbil (Meriones unguiculatus)

The post-embedding immuno gold staining (IGS) technique was used for the ultrastructural localization of glial fibrillary acidic protein (GFAP) in pituicytes and tanycytes of the neurohypophysis. IGS was applied to LR White embedded neurohypophyseal tissue of the Mongolian gerbil (Meriones unguiculatus), a species which contains abundant GFAP-positive pituicytes and tanycytes. GFAP-immunoreactivity could be demonstrated on pituicytic intermediate filaments (IF's) in situ. Thus, it was shown that pituicytes contain GFAP in its filamentous form, what had been a matter of speculation. At the ultrastructural level, gerbil tanycytes and tanycyte-like cells in the external zone of the median eminence were characterized by a great amount of densely packed IF's, which were labeled by both GFAP- or vimentin-antibodies. Sequential immunostaining of serial semithin sections with GFAP- and vimentin-antibodies revealed an invariable coexpression of the two IF proteins in somata and processes of these median eminence cells.     

Immunogold electron microscopic localization of glial fibrillary acidic protein (GFAP) in neurohypophyseal pituicytes and tanycytes of the Mongolian gerbil (Meriones unguiculatus)

Summary The post-embedding immuno gold staining (IGS) technique was used for the ultrastructural localization of glial fibrillary acidic protein (GFAP) in pituicytes and tanycytes of the neurohypophysis. IGS was applied to LR White embedded neurohypophyseal tissue of the Mongolian gerbil (Meriones unguiculatus), a species which contains abundant GFAP-positive pituicytes and tanycytes. GFAP-immunoreactivity could be demonstrated on pituicytic intermediate filaments (IF's) in situ. Thus, it was shown that pituicytes contain GFAP in its filamentous form, what had been a matter of speculation. At the ultrastructural level, gerbil tanycytes and tanycyte-like cells in the external zone of the median eminence were characterized by a great amount of densely packed IF's, which were labeled by both GFAP- or vimentin-antibodies. Sequential immunostaining of serial semithin sections with GFAP- and vimentin-antibodies revealed an invariable coexpression of the two IF proteins in somata and processes of these median eminence cells.     

Ultrastructural study of the uptake of peroxidase by the rat median eminence

An active role of the ependymal cells (tanycytes) of the median eminence in the transport of hypothalamic hormones has been recently suggested. In order to investigate the fate of material present in the cerebrospinal fluid, a protein tracer, horse-radish peroxidase (HRP) was injected into the left lateral ventricle of rats. Two minutes after the injection, HRP had largely diffused between tanycytes and hypendymal cells. As soon as 5 min after the injection, HRP had completely penetrated all the layers of the median eminence. A few labelled vesicles and lysosomes were occasionally seen in ependymal and glial cells. At longer time intervals (20 min, 1 and 4 hrs), a reaction was observed in the lumen of fenestrated capillaries of the pituitary portal plexus. In many nerve endings of the external zone, vesicles and lysosomes were seen to contain HRP. An interesting observation was the localization of HRP between nerve endings and cells in both the pars nervosa and the pars intermedia of the pituitary gland. No reaction was recorded in the anterior pituitary and the kidney. Seventeen hours after the injection, the extracellular space was free of reaction but a few positive intracellular structure were still found. These results clearly indicate that some material from the third ventricle can rapidly diffuse between cells and axons of the median eminence to reach the fenestrated capillaries of the pituitary portal plexus and the posterior pituitary without involving an active transport by tanycytes.     

Effects on gonadal function by lesioning tanycytes in the median eminence of the rat and Japanese quail

Summary The ependymal linings of the median eminence were destroyed by electric cautery or intraventricular injection of picric acid in the rat and Japanese quail. In these animals the ventricular lumen near the median eminence disappeared due to adhesion of lesioned walls on both sides of the third ventricle. Electric lesions of the ependymal layer containing tanycytes did not induce appreciable disturbance in the estrous cycles. Rats in which tanycytes were lesioned by picric acid displayed 4-day estrous cycles after prolonged diestrus (10–22 days). After destruction of tanycytes in the quail, a photostimulated gonadal growth was observed. It is concluded that the tanycyte transport of the ventricular fluid to capillaries of the portal vessels appears unnecessary for maintenance of adenohypophysial gonadotrophic activities.Supported by a grant from the Ministry of Education of Japan and by a grant from the Ford Foundation. The authors wish to express their gratitude to Dr. B.K. Follett for providing chicken LH (IRC2) and to the late Mr. T. Asai for assay of serum LH     

Immunohistochemical research on the insulin-sensitive elements of the hypothalamic median eminence

The indirect peroxidase-labeled antibody method was used to localize insulin receptors in the median eminence of rat hypothalamus. For experiments the rabbit antisera against synthetic peptides correspond to the deduced amino acid sequences of alpha- and beta-subunits of human placenta insulin receptor used as the first sera. The antisera were produced by prof. N. Yanaihara. Results have shown that after intraventricular injection of 1 U insulin the electron dense particles of DAB reaction were distributed on the surface membrane of tanycytes and axon terminals in the median eminence of hypothalamus. The functional properties of revealed receptors corresponded to IGF-1 receptors. Results support the hypothesis of central regulatory action of insulin in brain by its interaction with central IGF-1 receptors on the membrane of neuroependymal elements in the median eminence of hypothalamus.     

Elevated expression of glucose transporter-1 in hypothalamic ependymal cells not involved in the formation of the brain-cerebrospinal fluid barrier

Glucose transporters play an essential role in the acquisition of glucose by the brain. Elevated expression of glucose transporter-1 has been detected in endothelial cells of the blood-brain barrier and in choroid plexus cells of the blood-cerebrospinal fluid barrier. On the other hand, there is a paucity of information on the expression of glucose transporters in the ependymal cells that line the walls of the cerebral ventricles. The tanycytes are specialized ependymal cells localized in circumventricular organs such as the median eminence that can be segregated into at least three types, alpha, beta1 and beta2. The beta2 tanycytes form tight junctions and participate in the formation of the cerebrospinal fluid-median eminence barrier. Using immunocytochemistry and in situ hybridization, we analyzed the expression of hexose transporters in rat and mouse hypothalamic tanycytes. In both species, immunocytochemical analysis revealed elevated expression of glucose transporter-1 in alpha and beta1 tanycytes. Intense anti-glucose transporter-1 staining was observed in cell processes located throughout the arcuate nucleus, in the end-feet reaching the lateral sulcus of the infundibular region, and in cell processes contacting the hypothalamic capillaries. On the other hand, there was very low expression of glucose transporter-1 in beta2 tanycytes involved in barrier function. In contrast with the results of the cytochemical analysis, in situ hybridization revealed that tanycytes alpha, beta1, and beta2 express similar levels of glucose transporter-1 mRNA. Further analysis using anti-glial fibrillary acidic protein antibodies to identify areas rich in astrocytes revealed that astrocytes were absent from areas containing alpha and beta1 tanycytes, but were abundant in regions containing the barrier-forming beta2 tanycytes. Overall, our data reveal a lack of correlation between participation in barrier function and expression of glucose transporter-1 in hypothalamic tanycytes. Given the virtual absence of astrocytes in areas rich in alpha and beta1 tanycytes, we speculate whether the tanycytes might have astrocyte-like functions and participate in the metabolic coupling between glia and neurons in the hypothalamic area.     

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.     

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.     

Identification of separate vasopressin-neurophysin II and oxytocin-neurophysin I containing nerve fibres in the external region of the bovine median eminence

Summary Immuno-enzyme histochemical investigations showed the presence, in the external region of the bovine median eminence, of accumulations of vasopressin-neurophysin II-and oxytocin-neurophysin I-complexes. These two hormone-neurophysin complexes are located in separate fine varicose nerve fibres. The results strongly plead against an important role of tanycytes in the transport of vasopressin, oxytocin and neurophysins from the cerebrospinal fluid to the hypophysial portal blood-vessels.This work was supported by a grant from the Belgian Nationaal Fonds voor Geneeskundig Wetenschappelijk Onderzoek.     

Hypothalamic ependymal-glial cells express the glucose transporter GLUT2, a protein involved in glucose sensing

The GLUT2 glucose transporter and the K-ATP-sensitive potassium channels have been implicated as an integral part of the glucose-sensing mechanism in the pancreatic islet beta cells. The expression of GLUT2 and K-ATP channels in the hypothalamic region suggest that they are also involved in a sensing mechanism in this area. The hypothalamic glial cells, known as tanycytes alpha and beta, are specialized ependymal cells that bridge the cerebrospinal fluid and the portal blood of the median eminence. We used immunocytochemistry, in situ hybridization and transport analyses to demonstrate the glucose transporters expressed in tanycytes. Confocal microscopy using specific antibodies against GLUT1 and GLUT2 indicated that both transporters are expressed in alpha and beta tanycytes. In addition, primary cultures of mouse hypothalamic tanycytes were found to express both GLUT1 and GLUT2 transporters. Transport studies, including 2-deoxy-glucose and fructose uptake in the presence or absence of inhibitors, indicated that these transporters are functional in cultured tanycytes. Finally, our analyses indicated that tanycytes express the K-ATP channel subunit Kir6.1 in vitro. As the expression of GLUT2 and K-ATP channel is linked to glucose-sensing mechanisms in pancreatic beta cells, we postulate that tanycytes may be responsible, at least in part, for a mechanism that allows the hypothalamus to detect changes in glucose concentrations.     

Golgi-like immunostaining of pituicytes and tanycytes positive for glial fibrillary acidic protein in the neurohypophysis of the Mongolian gerbil (Meriones unguiculatus)

Glial cells that contain the glial fibrillary acidic protein (GFAP; the major protein constituent of glial filaments) were stained immunohistochemically in thick frozen sections of the neurohypophysis of the Mongolian gerbil (Meriones unguiculatus). The resulting Golgi-like images provided informations on cytological features and distributional patterns of tanycytes and pituicytes. In the proximal median eminence, numerous bundled processes of tanycytes were revealed. They emerged from the ependymal and sub-ependymal layer and mostly reached the brain surface. Several tanycytic processes extended into the anatomical neural stalk. In the whole neural lobe, a dense network of GFAP-immunoreactive pituicyte processes was visualized. Stained pituicytes were highly asymmetric and exhibited a great morphological variability. Immunopositive fibers which were encountered in the intermediate lobe might be derived from pituicytes. Electron-microscopically further evidence was obtained that GFAP-positive pituicytes correspond to filament-rich fibrous pituicytes at the ultrastructural level.     

Golgi-like immunostaining of pituicytes and tanycytes positive for glial fibrillary acidic protein in the neurohypophysis of the Mongolian gerbil (Meriones unguiculatus)

Summary Glial cells that contain the glial fibrillary acidic protein (GFAP; the major protein constituent of glial filaments) were stained immunohistochemically in thick frozen sections of the neurohypophysis of the Mongolian gerbil (Meriones unguiculatus). The resulting Golgi-like images provided informations on cytological features and distributional patterns of tanycytes and pituicytes. In the proximal median eminence, numerous bundled processes of tanycytes were revealed. They emerged from the ependymal and subependymal layer and mostly reached the brain surface. Several tanycytic processes extended into the anatomical neural stalk. In the whole neural lobe, a dense network of GFAP-immunoreactive pituicyte processes was visualized. Stained pituicytes were highly asymmetric and exhibited a great morphological variability. Immunopositive fibers which were encountered in the intermediate lobe might be derived from pituicytes. Electron-microscopically further evidence was obtained that GFAP-positive pituicytes correspond to filament-rich fibrous pituicytes at the ultrastructural level.     

Ultrastructural changes in the median eminence of the rat following deafferentation of the basal hypothalamus

Summary This report concerns a light and electron microscopic investigation of the median eminence and dorsal infundibular stem of the rat following surgical isolation (deafferentation). Using a modification of the Halász technique, the basal hypothalamus, including the arcuate nucleus and median eminence were surgically isolated from surrounding structures. Special attention was directed to the contact (external) zone of the median eminence and rostral infundibulum where tuberohypophyseal axons as well as ependymal cell processes abut upon the abluminal basement membrane of the portal perivascular space. The results of this study to date suggest that 9, 20, and 40 days following surgical isolation, there is a distinct increase in the population of tuberohypophyseal dense core vesicles. It is suggested that deafferentation abolishes inhibitory and excitatory input that serves to modify the cellular dynamics of tuberohypophyseal neurosecretory elements. Comments are also made on the presence of cistern-like structures in the lateral median eminence; the presence of vesicle-like inclusions in terminal ependymal processes is discussed in relationship to the role that ependyma may play in linking the third ventricle with the adenohypophysis.This research was supported by USPHS Grants NB 08171 and AM 10002. The authors are indebted to the excellent technical assistance provided by G. Krobisch Dudley. Further, the authors wish to express their thanks to Dr. Adolph Weindl for his valuable advice and criticism, and to Matilde Holzwarth for her helpful assistance.     

Microglia and reactive “M” cells of degenerating central nervous system: Does similar morphology and function imply a common origin?

Summary Penetration of the median eminence and ventrolateral walls of the III ventricle by intraventricularly injected horseradish peroxidase was studied in rats. Experimental times varied between 10 to 70 min (short-term experiments) and 12 hrs to 30 days (long-term experiments).In short-term experiments, the median eminence was found to be completely stained whereas the lateral walls of the III ventricle were penetrated only up to 1 mm in depth. Spreading of the tracer takes place predominantly through the extracellular space and cellular uptake and transport do not seem to play a role during the first 70 min following the injection.In long-term experiments, the tanycytes exhibit a variety of intracellular inclusions marked by HRP precipitate. Tanycytic perikarya contain dense bodies and lipofuscin-like aggregates. Lipoprotein granules are thought to arise from these and are interpreted as lysosomal residual bodies. In tanycytic processes and perivascular endfeet, accumulations of HRP-containing tubules and polymorphous granules are encountered suggesting a transport towards the blood vessels of the portal plexus or of the arcuate and ventromedial areas, respectively. Sporadic tanycytes which are completely and evenly stained from the ventricular surface to the end of their processes were observed in the arcuate and ventromedial area. Whether this appearance can be taken as a sign of normal cell function seems doubtful. Some possible routes of transport through the median eminence—extracellular and transcellular—are summarized in a schematic drawing, taking into account the present findings and those published elsewhere in the literature.We gratefully acknowledge an Anglo-German Collaboration Grant of the Wellcome Trust enabling stimulating discussions with other workers in the field.