Immunohistochemical co-localization of glycogen phosphorylase with the astroglial markers glial fibrillary acidic protein and S-100 protein in rat brain sections.

Immunofluorescence double-labelling and immunoenzyme double-staining methods were used to examine the location of glycogen phosphorylase brain isozyme with the astrocyte markers glial fibrillary acidic protein (GFAP) and S-100 protein in formaldehyde-fixed, paraffin-embedded slices from adult rat brain. Astrocytes in the cerebellum and the hippocampus, which express GFAP or S-100 protein immunoreactivity, show glycogen phosphorylase immunoreactivity. Regional intensity and intracellular distribution of the three antigens vary characteristically. In ependymal cells, glycogen phosphorylase immunoreactivity is co-localized with S-100 protein immunoreactivity, but not with GFAP immunoreactivity. These findings confirm that glycogen phosphorylase in the rat brain is exclusively localized in astrocytes and ependymal cells. All astrocytes, as far as they express GFAP or S-100 protein, do contain glycogen phosphorylase.     

Immunohistochemical co-localization of glycogen phosphorylase with the astroglial markers glial fibrillary acidic protein and S-100 protein in rat brain sections

Summary Immunofluorescence double-labelling and immunoenzyme double-staining methods were used to examine the location of glycogen phosphorylase brain isozyme with the astrocyte markers glial fibrillary acidic protein (GFAP) and S-100 protein in formaldehydefixed, paraffin-embedded slices from adult rat brain. Astrocytes in the cerebellum and the hippocampus, which express GFAP or S-100 protein immunoreactivity, show glycogen phosphorylase immunoreactivity. Regional intensity and intracellular distribution of the three antigens vary characteristically. In ependymal cells, glycogen phosphorylase immunoreactivity is co-localized with S-100 protein immunoreactivity, but not with GFAP immunoreactivity. These findings confirm that glycogen phosphorylase in the rat brain is exclusively localized in astrocytes and ependymal cells. All astrocytes, as far as they express GFAP or S-100 protein, do contain glycogen phosphorylase.     

Immunocytochemical localization of S-100 beta beta protein in olfactory and supporting cells of lamb olfactory epithelium

By immunocytochemistry, we have identified two novel cell types, olfactory and supporting cells of lamb olfactory epithelium, expressing S-100 beta beta protein. S-100 immune reaction product was observed on ciliary and plasma membranes, on axonemes and in the cytoplasm adjacent to plasma membranes and to basal bodies of olfactory vesicles. A brief treatment of olfactory mucosae with Triton X-100 before fixation is necessary for detection of S-100 beta beta protein within olfactory vesicles. In the absence of such a treatment, the immune reaction product is restricted to ciliary and plasma membranes. On the other hand, irrespective of pre-treatment of olfactory mucosae, S-100 beta immune reaction product in supporting cells is restricted to microvillar and plasma membranes. The anti-S-100 beta antiserum used in these studies does not bind to basal cells of the olfactory epithelium or to cells of the olfactory glands, whereas it binds to Schwann cells of the olfactory nerve. An anti-S-100 alpha antiserum does not bind to cellular elements of the olfactory mucosa, Schwann cells, or axons of the olfactory nerve. The present data provide, for the first time, evidence for the presence of S-100 beta beta protein in mammalian neurons (olfactory cells).     

Differential localization of "brain-specific" S-100 and its subunits in rat salivary glands

In the rat, the S-100 antigens in the submandibular gland were found to be immunochemically identical with those in the brain (glial cells) when compared using crossed immunoelectrophoresis. Specific antibodies against the S-100a non-beta and against the S-100 beta subunit were prepared from antibodies against crude S-100 protein and from S-100 components (S-100a and b) by affinity chromatography. In the rat salivary glands a differential distribution of subunit immunoreactivity was clearly evidenced using indirect immunofluorescence. Certain intercalated duct cells of the submandibular gland as well as Schwann cells contained the S-100 beta subunit immunoreactivity exclusively, while other duct cells in parotid, submandibular, and sublingual glands contained S-100a non-beta subunit immunoreactivity. Both subunits were present in astrocytes and ependymal cells. The immunocytochemical localization of alpha and beta subunits is a promising technique for the classification of various types of S-100-containing cells.     

Formation of S-100 containing cells of the hypothalamus and adenohypophysis in normal ontogenesis and in protein deficiency

By means of the indirect immunohistochemical method distribution of S-100 containing cells has been studied in sections of the mediobasal hypothalamus (astrocytes) and adenohypophysis (follicular-stellate cells) in newborn, 10- and 21-day-old rats under normal development and under protein insufficiency. For this the animals are given the diet containing 6% of protein (control--25% of protein). S-100 containing cells are revealed in the hypothalamus and adenohypophysis in 10- and 21-day-old animals. In the brain of the newborn rats S-100 immunoreactive cells are not revealed. At the ultrastructural level the diaminobenzidine (DAB) reaction products in the immunoreactive cells are revealed diffusely along the whole cytoplasm of the cells, in nuclei the DAB reaction products are absent. Part of S-100 containing cells is essentially lowered, comparing with the control. In the rat adenohypophysis part of S-100 containing cells from the 10th up to the 21st day also decreases. Unlike the hypothalamus, however, content of cells, immunopositive to S-100 exceeds the analogous index in the control rats of the corresponding age groups.     

Detection of S-100b protein in Triton cytoskeletons: an immunocytochemical study on cultured Schwann cells

We investigated the subcellular distribution of S-100b protein in primary cultures of Schwann cells. The subcellular localization of the protein in cells fixed and then permeabilized is similar, if not identical, to that seen in Schwann cells in peripheral nerves, i.e., S-100b protein is found in the cytoplasm and associated with membranes and filamentous structures. In cells either fixed in the presence of Triton X-100 or exposed to Triton X-100 for a short time before fixation (Triton cytoskeletons), the immune reaction product is considerably less intense, and the protein is associated with filaments running parallel to the long axis of the cell as well as in a submembranous position. Including CaCl2 in the buffer during fixation in the presence of Triton X-100 does not result in any increase in the intensity of the immune reaction product in Triton cytoskeletons, suggesting that, within the limits of the technique employed, no binding of additional S-100b protein to the Triton X-100-resistant material can be induced. On the other hand, including EGTA results in a substantial decrease in the intensity of the immune reaction product in Triton cytoskeletons. Altogether, these findings suggest that a remarkable fraction of S-100b protein in cultured Schwann cells is associated with elements of the cytoskeleton and that Ca2+ exerts some regulatory role in the association of S-100b protein with the cytoskeleton.     

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.     

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.     

Immunocytochemical localization of S-100b protein in degenerating and regenerating rat sciatic nerves

We studied the cellular and subcellular distribution of S-100b protein in normal, crushed, and transected rat sciatic nerves by an immunocytochemical procedure. In uninjured nerves, S-100b protein was restricted to the cytoplasm and membranes of Schwann cells, with no reaction product present in the nucleus or in axons. Similar images were seen from the first to the thirtieth day after the crush in activated Schwann cells during the degeneration period, i.e., up to the seventh post-lesion day, and in normal Schwann cells reappearing during the regeneration period, i.e., after the seventh post-lesion day, in the zone of the crush and proximal and distal to it. By the technique employed, there seemed to be no differences in the intensity of the immune reaction product in normal and activated Schwann cells. Also, similar images were seen in the proximal stump of transected nerves. Only a slight S-100b protein immune reaction product could be observed in the rare activated Schwann cells present in the distal stump around the seventh post-lesion day, the majority of cell types being represented by fibroblasts and elongated cells at this stage and thereafter. By immunochemical assays, similar results as those presented here have been reported and interpreted as indicative of the presence of S-100 protein in axons or, alternatively, of axonal control over expression of S-100 protein in Schwann cells. Our immunocytochemical data clearly show that the strong reduction in the S-100 protein content of the distal stump of transected nerves is owing to the paucity of Schwann cells and to the decrease in the S-100 protein content of these cells, rather than to degeneration of axons.     

Immunocytochemical localization of annexin V (CaBP33), a Ca(2+)-dependent phospholipid- and membrane-binding protein, in the rat nervous system and skeletal muscles and in the porcine heart.

We investigated the ultrastructural localization of annexin V a Ca(2+)-dependent phospholipid- and membrane-binding protein in the nervous system, heart, and skeletal muscles. The results indicate that in the cerebellum the protein is restricted to glial cells, where it is found diffusely in the cytoplasm as well as associated with plasma membranes. Bergmann glial cell bodies and processes and astrocytes in the cerebellar cortex and oligodendrocytes in the cerebellar white matter displayed an intense immune reaction product. In sciatic nerves, the protein was exclusively found in Schwann cells with a subcellular localization similar to that seen in glial cells in the cerebellum. Pituicytes in the neurohypophysis were intensely immunostained, whereas axons were not. In the heart, annexin V was restricted to the sarcolemma, transverse tubules, and intercalated discs. In skeletal muscles the protein was localized to the sarcolemma and transverse tubules. No evidence for the presence of the protein in the sarcoplasm or in association with mitochondria, the sarcoplasmic reticulum, or contractile elements was obtained. The observation that plasma membranes in cells expressing annexin V have the protein associated with them is in agreement with previous data on Ca(2+)-dependent binding of the protein to brain and heart membranes, and on existence of both EGTA- and Triton X-100-extractable and resistant fractions of annexin V in these membranes. The present data support the hypothesis that annexin V might be involved in membrane trafficking and suggest a role for this protein in the regulation of cytoplasmic activities in glial cells.     

Choroid plexus ependymal cells enhance neurite outgrowth from dorsal root ganglion neurons in vitro

The epithelial cells of the choroid plexus are a continuation of the ventricular ependymal cells and are regarded as modified ependymal cells. The present study was carried out to determine the influence of choroid plexus ependymal cells (CPECs) on axonal growth in vitro. Choroid plexuses were dissected from the fourth ventricle of postnatal day-1–10 mice, mechanically dissociated, and plated in fibronectin-coated culture dishes. CPECs had spread into monolayers with few endothelial cells in 3-week cultures. Some macrophages were scattered on the monolayer of CPECs. Dorsal root ganglia (DRG) were excised from mouse fetuses of 14-day gestation, dissociated with trypsin and cocultured on the CPEC monolayers. For comparison, dissociated DRG neurons were cocultured on astrocyte monolayers or cultured on laminin-coated plates. After 4.5 h culturing, the cultures were fixed and immunohistochemically double-stained for neurites and CPECs using antibodies against β-tubulin III and S-100 β, respectively. It was demonstrated that neurons extended many long neurites with elaborate branching on the surface of S-100-stained CPECs. In contrast, DRG neurons cultured on the astrocytes and on the laminin-coated plates had much shorter primary neurites with fewer branches than those cultured on the CPECs. The total length of neurites including primary neurites and their branches, of a single DRG neuron was 285 ± 14, 395 ± 15 and 565 ± 12 μm on the laminin-coated plates, on astrocytes and on CPECs, respectively. Scanning electron microscopy revealed extension of neurites with well-developed growth cones on the ependymal cells. These results suggest that CPECs have a great capacity to promote neurite outgrowth from DRG neurons in vitro.     

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.     

Light- and electron-microscopic investigation of the rat subcommissural organ grafted under the kidney capsule,with particular reference to immunocytochemistry and lectin histochemistry

Summary There is increasing evidence that, in the rat, a serotonin-mediated neural input may have an inhibitory influence on the secretory activity of the subcommissural organ (SCO). In the present investigation the rat SCO was studied 7, 30 and 90 days after transplantation under the kidney capsule, an area devoid of local serotonin-containing nerves. The grafted tissue was examined by use of immunocytochemistry employing a series of primary antisera, lectin histochemistry and transmission electron microscopy. The grafted SCO survived transplantation and contained, in addition to secretory ependymal and hypendymal SCO-cells, also elements immunoreactive with antisera against glial fibrillary acidic protein or S-100 protein. In transplants, SCO-cells produced a material displaying the characteristic immunocytochemical and lectin-binding properties of SCO-cells observed under in-situ conditions. The ependymal cells lined 1–3 small cavities, which contained secretory material. A fully developed structural equivalent of Reissner's fiber was, however, never found. The immunocytochemical and ultrastructural study of the grafted SCO showed an absence of nerve fibers within the graft and suggested a state of enhanced secretory activity. A network of protruding basal lamina structures connected the secretory cells to the newly formed capillaries revascularizing the SCO. One week after transplantation, long-spacing collagen started to appear in expanded areas of such laminar networks and also in the perivascular space. It is suggested (i) that the formation of long-spacing forms of collagen is triggered by factors provided by the SCO-secretory cells, and (ii) that secretory material of the ependymal and hypendymal cells may reach the reticular extensions of the basal lamina. In contrast to the SCO in situ, the grafted SCO-cells showed a positive immunoreaction for neuron-specific enolase. They became surrounded by a S-100-immunoreactive glial sheath that separated them from other transplanted cell types and the adjacent kidney tissue of the host.Supported by Grant I/63 476 from the Stiftung Volkswagenwerk, Federal Republic of Germany, Grants 187 and 0890/88 from Fondo Nacional de Desarrollo Cientifico y Tecnológico, Chile, and Grant S-85-39 from the Directión de Investigaciones, Universidad Austral de Chile. The authors wish to acknowledge the valuable help of Ms. Elizabeth Santibañez and Mr. Genaro Alvial (Valdivia) and Ms. Inge Lyncker (Giessen)     

Immunocytochemical localization of S-100 protein in astrocytes and Müller cells in the rabbit retina

Summary The localization of S-100 protein was studied in histological sections of retinae from adult rabbits. By use of double-immunolabeling techniques it was shown that most but not all radially oriented vimentin-positive Müller cells were co-labeled by an antiserum to S-100 protein. Glial fibrillary acidic protein-positive astrocytes, which in the rabbit retina are restricted to the medullary rays formed by myelinated optic nerve fibers, consistently showed S-100 protein immunoreactivity. The present report shows that, with respect to S-100 protein staining, Müller cells represent a heterogeneous population of glial elements.     

Primary astroglial cultures

Conclusion Primary cultures from neonatal rat brain consist mainly of astroglial cells, immunohistochemically identified by GFAp and S-100. As other cells than astrocytes may survice in the culture, specific markers for the expected cells were used. Cells with phagocytic properties, endothelial-like cells, oligoblasts, ependymal cells and mesenchymal cells were identified. No neurons have so far been detected.The astroglial cells have a high-affinity uptake for glutamate, aspartate GABA, taurine and hypotaurine, while there is probably a non-saturable uptake of norepinephrine, dopamine and 5-HT. The enzymes MAO, COMT, GABA-T and GS have been demonstrated. It thus seems that astrocytes take part in the inactivation of neurotransmitters, although amino acids and monoamines are taken up with different mechanisms.The presence of receptors for different neurotransmitters and neuromodulators has been demonstrated on astrocytes.Astroglial-enriched cultures from various brain regions have shown that the cells express specialized functional properties concerning neurotransmitter uptake, metabolizing enzymes and receptor density.Astroglial cell differentiation in culture is shortly reviewed and one possibility to affect this maturation by co-cultivation with neuronal containing cultures is point out.     

Intermolecular effects in the polymerization of hemoglobin S

Monolayer cultures of astrocytes from newborn rat brain hemispheres have been analysed for the glial-specific protein S-100, during their growth cycle. In primary cultures S-100 protein level increases with a pattern close to that observed with rat brain hemispheres $\text{in vivo}$. This finding suggests that some biochemical maturation of the astrocytes occurs $\text{in vitro}$. In secondary cultures the level of S-100 protein decreases and then increases at the end of the proliferation phase. This modulation, similar to that observed in a clonal culture of tumor cells from rat brain (C6) provides a model to study the relationship between gene expression and the phase of growth of the cells and will allow parallel investigations in normal and tumor cells.     

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.     

Folliculostellate cells in pituitary pars distalis of male viscacha: immunohistochemical,morphometric and ultrastructural study

Folliculostellate cells (FSC) have been reported in pituitary of several mammalian species. FSC morphology and secreted substances have been instrumental to the understanding of their function. The purpose of this work was to perform an immunohistochemical, morphometric and ultrastructural study of the pituitary pars distalis FSC in adult male viscacha and to analyze their relation with hormone secreting cells. Immunohistochemistry and image analysis were carried out in different sectors of the gland, from the middle (sector 1) to the glandular periphery (sector 5). Transmission electron microscopy with lanthanum as electrodense tracer was used. FSC formed follicles with PAS-positive colloid inside. They expressed S-100 protein mainly in both nucleus and cytoplasm. FSC were stellate-like in shape and exhibited short cytoplasmic processes that contacted with blood vessels and endocrine cells. In addition, some follicular colloids were immunostained with anti-S-100 protein. A few FSC were immunostained with anti-glial fibrillary acidic protein (GFAP) and anti-vimentin. The morphometric parameters analyzed (percentages of S-100-positive total, cellular and colloidal areas) increased from sector 1 to sector 3 and then decreased to sector 5. Hormone secreting cells, mainly lactotrophs, gonadotrophs and corticotrophs were associated with FSC and follicles. The ultrastructural study demonstrated that FSC developed junctional complexes and desmosomes between their lateral membranes. Lanthanum freely penetrated the spaces between granulated cells and FSC, but did not penetrate into the follicular lumen. In conclusion: 1) the differential expression of S-100 protein, GFAP and vimentin may indicate different physiological stages of FSC; 2) the expression of these proteins suggests a neuroectodermic origin of these cells; 3) FSC spatial distribution, association with endocrine cells, and the generation of an intercellular communication network suggest that FSC are involved in the pituitary pars distalis paracrine regulation of the viscacha.Key words: Lagostomus, pituitary, folliculostellate cells, immunohistochemistry, distribution, ultrastructure.     

S-100 antigen in satellite cells of the adrenal medulla and the superior cervical ganglion of the rat

Summary Measurable amounts of the nervous-system specific S-100 protein were detected by microcomplement fixation assay both in the superior cervical ganglion and in the adrenal medulla of adult rats, though at a significantly higher concentration in the ganglion. By the unlabeled antibody PAP method, the antigen was localized at: he ultrastructural level in the Schwann cells and in the satellite cells of the ganglion, but not in neurons. Similarly, the protein was found in the Schwann cells of the adrenal medulla, but not in the chromaffin cells. Moreover, the S-100 immunolabeling allowed detection of a class of satellite cells closely enveloping the chromaffin cells. In the labeled cells of both organs the reaction product was diffusely distributed in the cytoplasmic matrix as well as in the nucleoplasm.The presence of the S-100 antigen in the satellite cells of the sympathetic ganglion and in satellite cells of the adrenal medulla suggests a possible homology for the two cell types, and one could hypothesize the presence in peptide hormone-secreting endocrine organs of glia-like cells exhibiting functional relationships with the secretory cells comparable to those of the glial cells with the neurons.     

S-100 protein immunoreactivity in mammalian testis and epididymis

The present study deals with immunohistochemical localization of S-100 protein in mouse, bank vole and pine vole testis and epididymis. S-100 protein immunoreactivity was observed in the endothelia of capillaries and lymphatic sinusoids of pine vole testis. A reaction to S-100 protein of the same intensity as that noted in the endothelia of testicular capillaries was found in myoid cells of pine vole and bank vole seminiferous tubules. Moreover, a positive reaction to S-100 protein was observed in bank vole and mouse Leydig cells. In the epididymis, a weaker reaction to S-100 occurred in smooth muscles of pine vole and mouse epididymal duct. Despite difficult interpretation of physiological role of S-100 protein we suggest that it may be a part of the blood-testis barrier. It may also participate in the processes of transcytosis and contractility; its cellular expression is regulated by local factors. However, location of S-100 is not specific to the representatives of the same order.