Morphological Changes within the Rat Lateral Ventricle after the Administration of Proteasome Inhibitors

The broad variety of substances that inhibit the action of the ubiquitin-proteasome system (UPS)—known as proteasome inhibitors—have been used extensively in previous studies, and they are currently frequently proposed as a novel form of cancer treatment and as a protective factor in intracerebral hemorrhage treatment. The experimental data on the safest route of proteasome inhibitor administration, their associated side effects, and the possible ways of minimizing these effects have recently become a very important topic. The aim of our present study was to determine the effects of administering of MG-132, lactacystin and epoxomicin, compounds belonging to three different classes of proteasome inhibitors, on the ependymal walls of the lateral ventricle. Observations were made 2 and 8 weeks after the intraventricular administration of the studied substances dissolved in dimethyl sulfoxide (DMSO) into the lateral ventricle of adult Wistar rats. Qualitative and quantitative analysis of brain sections stained with histochemical and inmmunofluorescence techniques showed that the administration of proteasome inhibitors caused a partial occlusion of the injected ventricle in all of the studied animals. The occlusion was due to ependymal cells damage and subsequent ependymal discontinuity, which caused direct contact between the striatum and the lateral nuclei of the septum, mononuclear cell infiltration and the formation of a glial scar between these structures (with the activation of astroglia, microglia and oligodendroglia). Morphologically, the ubiquitin-positive aggregates corresponded to aggresomes, indicating impaired activity of the UPS and the accumulation and aggregation of ubiquitinated proteins that coincided with the occurrence of glial scars. The most significant changes were observed in the wall covering the striatum in animals that were administered epoxomicin, and milder changes were observed in animals administered lactacystin and MG-132. Interestingly, DMSO administration also caused damage to some of the ependymal cells, but the aggresome-like structures were not formed. Our results indicate that all of the studied classes of proteasome inhibitors are detrimental to ependymal cells to some extent, and may cause severe changes in the ventricular system. The safety implications of their usage in therapeutic strategies to attenuate intracerebral hemorrhagic injury and in brain cancer treatment will require further studies.     

Glial fibrillary acidic protein-like immunoreactive ependymal elements in the third ventricle of the rat. A study at different stages of development.

Using the peroxidase-antiperoxidase method a study was made of the cells immunoreactive to glial fibrillary acidic protein (GFAP) anti-serum in the ependyma of the third ventricle of the rat at different stages of growth. Most of the ependymal cells of the third ventricle were seen to be unreactive to this protein; however, it was sometimes possible to observe some GFAP-immunoreactive ependymocytes and occasionally other immunoreactive cellular types, such as tanycytes and supraependymal cells. Despite this, the most frequent localization of the elements immunoreactive to the protein adopted the shape of an immunoreactive subependymal band situated parallel to the ventricular wall. As the weights of the animals increased an increase in the elements immunoreactive to this protein could be observed in all the zones considered, there being no differences between the male and female animals.     

Localization of metallothionein in the brain of rat and mouse.

Metallothionein (MT) is a low molecular mass protein inducible by heavy metals such as cadmium (Cd), zinc, and copper, and having high affinity for these metals. In the present study, we investigated the immunohistological localization of MT in the brains of rats and mice. In adult rat brain, almost no MT immunostaining was observed, whereas in adult mouse brain strong MT immunostaining was found in the ependymal cells, some glial cells, arachnoid, and pia mater. No immunostaining was detected in neurons and endothelial cells. In younger rats (1-3 weeks old), strong MT immunostaining was observed in ependymal cells, choroid plexus epithelium, arachnoid, and pia mater. The overall MT concentration in adult mouse brain appeared higher than that of the brains of young and adult rats. When adult rats were administered Cd, MT was induced not only in some glial cells, ependymal cells, arachnoid, and pia mater but also in endothelial cells. Although Cd treatment resulted in an increase in the MT immunostaining in the specific cells described above, the MT induction was not great enough to significantly affect the overall MT level in the brain. The present result suggest a possible link of MT with cell growth of choroid plexus epithelium and ependymal cells, as well as a detoxifying role of MT in the blood-brain barrier and the cerebrospinal fluid-brain barrier.     

Scanning electron microscopic observations of the ependymal cell and the subependymal layer of the third brain ventricle in the rat

This investigation was undertaken to clarify the three dimensional ultrastructure of the subependymal layer in relation with the ependymal cell layer in rat brain using the scanning electron microscope (SEM). The subependymal layer existing below the ependyma of the third ventricle in the brain of mature albino rats was examined with S E M. The hypothalamus freshly excised after median sagittal section was treated by collagenase with or without trypsin for a short while to remove the ependymal cells at the ventricular wall. After the enzymatic pretreatment of the specimen, many ependymal cells were removed and the subependymal layer was partially exposed. Most of the ciliated ependymal cells remaining at the ventricular wall extended long, single basal processes which then penetrated into the subependymal layer. The subependymal layer was composed of a delicate framework of thin processes of glial cells, ependymal cells and, in addition nerve cells. Scattered among the neuropil just beneath the ependymal cell layer, there were relatively small, globular subependymal cells. Occasionally, there were large bundles of unmyelinated nerve fibres in the subependymal layer. The individual nerve fibres distinctly showed many axonal varicosities within the fibres. Intermingled with the nerve fibres, glial processes of various forms were present. The structure of the ependymal cells and the subependymal layer was compared with the findings already reported in the studies using light and transmission electron microscope.     

Radial astrocytes and ependymocytes in the spinal cord of the adult toad (Bufo bufo L.)

Summary Immunohistochemical and ultrastructural techniques have been used to demonstrate glial fibrillary acidic protein (GFAP) immuno-positive cells in the adult toad spinal cord. Two types of GFAP-immunoreactive cells were observed: ependymocytes and radial astrocytes. GFAP-positive ependymocytes were scarce and contained the immunoreactive product in their processes. They showed intermediate filaments in the basal pole and in their processes when studied with the electron microscope. These immuno-positive ependymocytes represent the tanycytic form of ependymal cells because their processes ended at the subpial zone. The radial astrocytes showed a more intensive immunoreactive product in somata and processes when they were located far away from the ependymal layer. Cell bodies and processes were also associated with blood vessels, but most of the processes ended at the subpial zone forming a continuous subpial glia limitans. The GFAP-positive processes, which form this subpial glia limitans in the toad spinal cord, belong to both tanycytic ependymocytes and radial astrocytes, whose somata are located in the grey matter. These findings lead us to suggest that both types of GFAP-immunopositive cells might be the functional equivalents of mammalian astrocytes.     

Morphological and neurochemical diversity of neuronal nitric oxide synthase-positive amacrine cells in the turtle retina

By gently scraping off the surface of the lateral ventricles of adult bovine brains, we obtained sheets containing the ependymal layer and some attached sub-ependymal cells. Explants were cultured in serum-free medium or in two media enriched with 20% fetal calf serum or 20% adult bovine cerebrospinal fluid, and processed for different time intervals from 4 h to 60 days. For characterization of the ependymal cells we used antisera against S-100 protein, vimentin and glial fibrillary acidic protein (GFAP). For comparison, the ependyma of adult bovines and of fetuses from days 60 to 120 post coitum was studied in situ. The adult ependyma consisted of a ciliated, cuboid cell monolayer with short basal processes; it displayed S-100 immunoreactivity but only scarce deposits of vimentin and no GFAP. The fetal ependyma had the appearance of a pseudostratified epithelium with elongated nuclei and basal processes containing S-100 and vimentin from day 80 post coitum and GFAP from day 100 post coitum. In explants, no differences were seen between the three culture media; the ependyma became pseudostratified, developed basal processes and showed increasing amounts of S-100 and vimentin first, and subsequently also GFAP. These changes were concomitant with the onset of mitotic activity in the subependymal layer leading to the production of numerous cells. The morphological and immunocytochemical features of ependymal cells in cultured explants resembled those of fetal ependyma. Our results indicate that the culture of ependymal explants from adult bovine lateral ventricles is an useful model system for morphological and functional studies of the ependyma and for the analysis of cell proliferation in the subependymal layer.     

Recognition of Bergmann glial and ependymal cells in the mouse nervous system by monoclonal antibody

A monoclonal antibody designated anti-Cl was obtained from a hybridoma clone isolated from a fusion of NS1 myeloma with spleen cells from BALB/c mice injected with homogenate of white matter from bovine corpus callosum. In the adult mouse neuroectoderm, C1 antigen is detectable by indirect immunohistology in the processes of Bergmann glial cells (also called Golgi epithelial cells) in the cerebellum and of Muller cells in the retina, whereas other astrocytes that express glial fibrillary acidic protein in these brain areas are negative for C1. In addition, C1 antigen is expressed in most, if not all, ependymal cells and in large blood vessels, but not capillaries. In the developing, early postnatal cerebellum, C1 antigen is not confined to Bergmann glial and ependymal cells but is additionally present in astrocytes of presumptive white matter and Purkinje cell layer. In the embryonic neuroectoderm, C1 antigen is already expressed at day 10, the earliest stage tested so far. The antigen is distinguished in radially oriented structures in telencephalon, pons, pituitary anlage, and retina. Ventricular cells are not labeled by C1 antibody at this stage. C1 antigen is not detectable in astrocytes of adult or nearly adult cerebella from the neurological mutant mice staggerer, reeler, and weaver, but is present in ependymal cells and large blood vessels. C1 antigen is expressed not only in the intact animal but also in cultured cerebellar astrocytes and fibroblastlike cells. It is localized intracellularly.     

Differential immunocytochemical staining for glial fibrillary acidic (GFA) protein,S-100 protein and glutamine synthetase in the rat subcommissural organ,nonspecialized ventricular ependyma and adjacent neuropil

Summary Antibodies raised against glial fibrillary acidic protein (GFA), S-100 protein (S100) and glutamine synthetase (GS) are currently used as glial markers. The distribution of GFA, S100 and GS in the ependyma of the rat subcommissural organ (SCO), as well as in the adjacent nonspecialized ventricular ependyma and neuropil of the periaqueductal grey matter, was studied by use of the immunocytochemical peroxidase-antiperoxidase technique. In the neuropil, GFA, S100 and GS were found in glial elements, i.e., in fibrous (GFA, S100) and protoplasmic astrocytes (S100, GS). The presence of S100 in the majority of the ventricular ependymal cells and tanycytes, and the presence of GFA in a limited number of ventricular ependymal cells and tanycytes confirm the glial nature of these cells. The absence of S100, GFA and GS from the ependymocytes of the SCO, which are considered to be modified ependymal cells, suggests either a non-astrocytic lineage of these cells or an extreme specialization of the SCO-cells as glycoprotein-synthesizing and secreting elements, a process that may have led to the disappearance of the glial markers.     

Experimental protein malnutrition in squirrel monkeys: reaction of the nissl substance in the motor neurons of the spinal cord

Summary Experimental protein malnutrition was induced in juvenile squirrel monkeys by feeding them synthetic diets which contained 2% protein for a period of 15 weeks. Along with their controls who were fed 25% protein food and laboratory monkey chow, groups of monkeys were sacrificed at nine, 11, 13, and 15 weeks of feeding schedule. The spinal cords were stained with chromalum-gallocyanin, PAS, Trichrome Masson's method and Alcian Blue. The neurons and the surrounding glial cells showed significant cytoohemical changes in different groups of the malnourished animals. The motor neurons of the spinal cord showed a varying but dramatic reaction to protein deficiency, which reflects a profound physiological distortion. These observations have been discussed in relation to probable functional derangement under the influence of protein deficiency.     

Rat brain glial cells in culture: Effects of brain extracts on the development of oligodendroglia-like cells

Cells dissociated from brains of newborn rats and grown on plastic surfaces develop into a glial culture, composed of at least three morphologically different cell types. The predominating cell type consists of astroglial cells, which form a monolayer. The second cell type, rarely observed, consists of ependymal cells. The third type consists of small cells scattered upon the astroglial layer. After 3 weeks very few of these small cells remain and the glial culture develops into a more homogenous appearance, mainly composed of astroglial cells. The effects of various brain extracts on the development of the small cell type was investigated. The treatment by either rat or chick brain extracts caused an increase in the number of these cells, which were seen to form clusters. Brain extracts from older animals have a stronger effect than brain extracts from younger animals. These data suggest that factors contained in the brain during and after the myelination period influence the development of this cell type in dissociated cultures. The small cells were tentatively identified as oligodendroglial cells by ultrastructural and histochemical criteria. They did not contain acetylcholinesterase (AChE) and did not bind tetanus toxin. Furthermore, they did not contain glial fibrillary acidic (GFA) protein. But carbonic anhydrase II (CAII) was found in them at light and electron-microscopical level. CAII was found to be localized essentially on the plasmic membrane and on the endoplasmic reticulum of these cultured oligodendroglial cells.     

Comparative marker analysis of the ependymocytes of the subcommissural organ in four different mammalian species

Summary The subcommissural organ (SCO), classified as one of the circumventricular organs, is composed mainly of modified ependymal cells, attributable to a glial lineage. Nevertheless, in the rat, these cells do not possess glial markers such as glial fibrillary acidic protein (GFAP), protein S100, or the enzyme glutamine synthetase (GS). They receive a synaptic 5-HT input and show pharmacological properties for uptake of GABA resembling the uptake mechanism of neurons. In this study, we examine the phenotype of several mammalian SCO (cat, mouse, rabbit) and compare them with the corresponding features of the rat SCO. In all these species, the SCO ependymocytes possess vimentin as an intermediate filament, but never express GFAP or neurofilament proteins. They do not contain GS as do glial cells involved in GABA metabolism, and when they contain protein S100 (rabbit, mouse), its rate is low in comparison to classical glial or ependymal cells. Thus, these ependymocytes display characteristics that differentiate them from other types of glial cells (astrocytes, epithelial ependymocytes and tanycytes). Striking interspecies differences in the capacity of SCO-ependymocytes for uptake of GABA might be related to their innervation and suggest a species-dependent plasticity in their function.     

Ultramicroscopic organization of the vascular plexus in the rabbit cerebral lateral ventricles and its changes under the effect of pyrogens

By means of electron microscopical methods organization of various components of the vascular plexus in the rabbit cerebral lateral ventricles--ependymal and supraependymal cells, as well as capillaries, making the base of the hemato-encephalic and hemato-liquor barriers of the plexus have been studied. Injection of bacterial pyrogen (pyrogenal) to the animals is accompanied with an increasing permeability in the barriers of the vascular plexus for certain blood cells (lymphocytes, monocytes), their activation and transformation into plasma cells and macrophages. Under pyrogenal effect in the ventricle cavities activation of the supraependymal cells and intensification of their interaction with the underlying ependyma take place. In the ependymal layer local dilatation of intercellular spaces and intensification of exocytic processes are observed. The changes revealed demonstrate an active reaction of some elements of the vascular plexus to fever, dependent on injection of the bacterial pyrogens into the organism.     

Up-regulation of neural stem cell markers suggests the occurrence of dedifferentiation in regenerating spinal cord

Following tail amputation in urodele amphibians, an ependymal tube, that resembles a developing neural tube, forms from ependymal cells that migrate from the cord stump and elongates by cell proliferation. Expression of the keratin pair 8 and 18 has been observed in the developing urodele nervous system and is maintained in the ependymal cells of the mature cord. We show here that expression of these keratins is not unique to urodeles, but is also observed in the radial glia of the human spinal cord, suggesting that these proteins might play a role both in neural development and regeneration. Analysis of their expression in the regenerating spinal cord following tail amputation shows that their expression, as well as that of glial fibrillary acidic protein (GFAP), is maintained in the ependymal tube during regeneration, though differences in their levels of expression are observed along the anteroposterior axis and appear to be related to the progression of morphogenesis. In addition, we show that following tail amputation the ependymal tube expresses the neural stem cell markers nestin and vimentin, which are undetectable in normal urodele spinal cord. This up-regulation of neural stem cell markers shows that the ependymal cells undergo a phenotypic change. Whereas maintenance of keratin and GFAP expression in the adult ependyma may reflect a higher plasticity of these cells in adult urodeles than in other vertebrates, re-expression of markers of early neural development suggests the occurrence of a dedifferentiation process in the spinal cord in response to injury.Edited by J. Campos-Ortega     

Glial fibrillary acidic protein and vimentin immunoreactivity of astroglial cells in the central nervous system of the African lungfish, Protopterus annectens (Dipnoi: Lepidosirenidae)

The distribution of glial intermediate filament molecular markers, glial fibrillary acidic protein (GFAP), and vimentin, in the brain and spinal cord of the African lungfish, Protopterus annectens, was examined by light microscopy immunoperoxidase cytochemistry. Glial fibrillary acidic protein immunoreactivity is clear and is evident in a radial glial system. It consists of fibers of different lengths and thicknesses that are arranged in a regular radial pattern throughout the central nervous system (CNS). They emerge from generally immunopositive radial ependymoglia (tanycytes), lining the ventricular surface, and are directed from the ventricular wall to the meningeal surface. These fibers give rise to endfeet that are apposed to the subpial surface and to blood vessel walls forming the glia limitans externa and the perivascular glial layer, respectively. GFAP-immunopositive star-shaped astrocytes were not found in P. annectens CNS. In the gray matter of the spinal cord, cell bodies of immunopositive radial glia are displaced from the ependymal layer. Vimentin-immunopositive structures are represented by thin fibers mostly localized in the peripheral zones of the brain and the spinal cord. While a few stained fibers appear in the gray matter, the ependymal layer shows no antivimentin immunostaining. In P. annectens the immunocytochemical response of the astroglial intermediate filaments is typical of a mature astroglia cell lineage, since they primarily express GFAP immunoreactivity. This immunocytochemical study shows that the glial pattern of the African lungfish resembles that found in tetrapods such as urodeles and reptiles. The glial pattern of lungfishes is comparable to that of urodeles and reptiles but is not as complex as that of teleosts, birds, and mammals.     

Expression of vimentin and glial fibrillary acidic protein in human developing spinal cord

Summary Expression of intermediate filament proteins was studied in human developing spinal cord using immunoperoxidase and double-label immunofluorescence methods with monoclonal antibodies to vimentin and glial fibrillary acidic protein (GFAP). Vimentin was found in the processes of radial glial cells in 6-week embryos, while GFAP appeared in vimentin-positive astroglial cells at 8–10 weeks. GFAP and vimentin were present in approximately equal amounts in differentiating astrocytes in 23-week spinal cord. In 30-week fetuses, astrocytes reacted strongly for GFAP, while both the reaction intensity and the number of vimentin-positive cells fluctuated predominantly in the grey matter. No clear-cut transition from vimentin to GFAP was noticed during the development of astrocytes. The majority of ependymal cells in 23-week fetuses contained vimentin but only a few of them reacted for GFAP. The expression of vimentin continued during the whole development of the ependymal layer, in contrast to the reactivity for GFAP which disappeared between the 30th week and term.     

The ventricular surface of the area postrema and the adjacent area subpostrema in the rabbit brain]

The ependymal surface of the area postrema (rabitt) was examined by scanning and transmission electron microscopy. The flattened ependymal cells show few microvilli. Towards the central canal, the ependymal cells change gradually to a columnar shape; the number of microvilli increases concomitantly. The area postrema ependymal cell surface mostly bears a single cilia. In contrast, a region immediately adjacent to the area postrema, which has been named area subpostrema (Gwyn and Wolstencroft 1968), shows cilia arranged in bunches. These cilia are regularly covered with colloid -- like droplets. A period-acid-bisulfit-aldehydthionine method (Specht 1970) permits to identify these droplets with glyproteids.it has been suggested that the droplets might derive from the area subpostrema ependymal cells. Above the ependymal surface of the area postrema, a great number of fine unmyelinated neuronal processes and thicker processes are observed. Some of them show bulb-like endings. These terminals contain small vesicles, dense cored vesicles (400...800 A), and mitochondria which are mostly characterized by a single central prismatic tubule. The plasmalemma of some bulbs is in a synaptic contact with the apical plasmalemma of the ependyma, while other bulbs see to end freely in the ventricle. Some neuronal processes penetrate between ependymal cells of the area postrema into the ventricular lumen.     

Immunocytochemical demonstration of vimentin in astrocytes and ependymal cells of developing and adult mouse nervous system

The occurrence of vimentin, a specific intermediate filament protein, has been studied by immunoflourescence microscopy in tissue of adult and embryonic brain as well as in cell cultures from nervous tissue. By double imminofluorescence labeling, the distribution of vimentin has been compared with that of subunit proteins of other types of intermediate filaments (glial fibrillary acidic [GFA] protein, neurofilament protein, prekeratin) and other cell-type specific markers (fibronectin, tetanus toxin receptor, 04 antigen). In adult brain tissue, vimentin is found not only in fibroblasts and cells of larger blood vessels but also in ependymal cells and astrocytes. In embryonic brain tissue, vimentin is detectable as early as embryonic day 11, the earliest stage tested, and is located in radial fibers spanning the neural tube, in ventricular cells, and in blood vessels. At all stages tested, oligodendrocytes and neurons do not express detectable amounts of vimentin. In primary cultures of early postnatal mouse cerebellum, a coincident location of vimentin and GFA protein is seen in astrocytes, and both types of filament proteins are included in the perinuclear aggregates formed upon exposure of the cells to colcemid. In cerebellar cell cultures of embryonic-day-13 mice, vimentin is seen in various cell types of epithelioid or fibroblastlike morphology but is absent from cells expressing tetanus toxin receptors. Among these embryonic, vimentin-positive cells, a certain cell type reacting neither with tetanus toxin nor with antibodies to fibronectin or GFA protein has been tentatively identified as precursor to more mature astrocytes. The results show that, in the neuroectoderm, vimentin is a specific marker for astrocytes and ependymal cells. It is expressed in the mouse in astrocytes and glial precursors well before the onset of GFA protein expression and might therefore serve as an early marker of glial differentiation. Our results show that vimentin and GFA protein coexist in one cell type not only in primary cultures in vitro but also in the intact tissue in situ.     

A steroid isolated from the water mold Achlya heterosexualis induces neurogenesis in vitro and in vivo

Using 22R-hydroxycholesterol as a sub-structure to screen natural compound databases, we identified a naturally occurring steroid (sc-7) with a 16-acetoxy-22R-hydroxycholesterol moiety, in which the hydroxyl groups in positions 3 and 22 are esterified by an acetoxy group and in which the carbon in position 26 carries a functional diacetylamino. sc-7 is an analog of the sex steroids dehydro-oogoniol and antheridiol, can be isolated from the water mold Achlya heterosexualis, and promoted neurogenesis in vitro and in vivo. Mouse embryonic teratocarcinoma P19 cells exposed to sc-7 for 2days followed by a 5-day wash-out differentiated into cholinergic neurons that expressed specific neuronal markers and displayed axonal formation. Axons continued growing up to 28days after treatment. In vivo, infusion of sc-7 for 2weeks into the left ventricle of the rat brain followed by a 3-week wash-out induced bromodeoxyuridine uptake by cells of the ependymal layer and subventricular zone that co-localized with doublecortin and glial fibrillary acidic protein immunostaining, demonstrating induction of proliferation and differentiation of neuronal progenitors. Migrating neuroblasts were also observed in the corpus callosum. Thus, under these experimental conditions, adult ependymal cells resumed proliferation and differentiation. Taken together, these results suggest that sc-7 is an interesting molecule for stimulating in situ neurogenesis from resident neuronal progenitors as part of neuron replacement therapy. sc-7 did not bind to nuclear steroid receptors and was not metabolized as a steroid, supporting our hypothesis that the neurogenic effect of sc-7 is not likely due to a steroid-like effect.     

A new protein (J1-31 antigen, 30 kD) is expressed by astrocytes,Müller glia and ependyma

Monoclonal antibody (MAb) J1-31 raised using human brain homogenate as immunogen in mice can be used as a cell type marker for certain types of CNS macroglia, namely astrocytes, Müller cells and tanycytes as well as ciliated ependymal cells. Except for the ciliated ependymal cells, these types of macroglia express glial fibrillary acidic protein (GFAP). J1-31 antigen is an intracellular protein which has a MW of 30 kD under reducing conditions for gel electrophoresis (Singhet al., 1986). This protein is distinct from GFAP (MW 50 kD) and vimentin (MW 55 kD), the two core proteins of 10 nm IFs known to be expressed in the above types ofmacroglia. This conclusion is based on several criteria including temporal differences in the onset of expression of GFAP and J1-31 antigen during development of the rat cerebellum. Also, there is no detectable (by immunofluorescence microscopy) expression of J1-31 antigen in the prenatal CNS or outside the CNS where vimentin has been reported to be abundant. The most direct evidence that J 1-31 antigen and GFAP are distinct proteins comes from studies on the mature ciliated ependymal cells which do not express GFAP and yet show intense immunostaining for J1-31 antigen.     

Fetal hypothalamic transplants in the third ventricle of the adult rat brain

Summary Blocks of anterior hypothalamus were transplanted from 19 day-old fetuses of Wistar/Lewis rats into the third ventricle of adult male Brattleboro rats. Physiological changes in graft recipients and in sham-operated animals were monitored daily. Twenty days after surgery, the graft recipients and shamoperated animals were killed and their brains examined by correlative scanning and transmission electron microscopy. Host animals that exhibited both decreased polydipsia and increased urine concentration were found to have viable grafts within the third ventricle. The observed physiological changes suggested that synthesis and release of vasopressin occurred in the transplanted neurons. Grafts were well vascularized by vessels arising from the host hypothalamus. Neurons, with perikarya ranging from 8 to 30 m in diameter, glial cells, and neurites were located throughout the transplants. A neurohemal contact zone, similar to that normally seen in the median eminence, could not be demonstrated in the grafts. The absence of complete glial and ependymal barriers indicates a relatively close association between cells in the transplants and the cerebrospinal fluid. A large increase in supraependymal neurons and their processes, including an eruption of neurons through the floor of the third ventricle in one animal, was observed in graft recipients but not in shamoperated animals.Supported by NIH Grants NS 15109 and NS 13717