Morphology of neuroglia in the hypothalamus of the opossum (Didelphis virginiana), armadillo (Dasypus novemcinctus mexicanus) and cat (Felis domestica)

The hypothalamus of the opossum (Didelphis virginiana), the armadillo (Dasypus novemcinctus mexicanus), and the cat (Felis domestica) was studied using Del Rio Hortega's silver carbonate technique, as modified by Scharenberg ('60). This technique demonstrates astrocytes, oligodendroglia, and neuronal perikarya, but does not impregnate microglia. The morphology of macroglia was observed in ten comparable nuclei in each of the three species. The subpial and subependymal areas were also examined. Astrocytes display more cell body angularity and have more processes in most hypothalamic regions of the cat when compared to similar regions of the opossum and armadillo. In the anterior hypothalamic nucleus, the ventromedial and the dorsomedial hypothalamic nuclei, and the medial mammillary nucleus of all three species, astrocytes send processes to neurons, but neuronal and astrocytic perikarya are usually not directly contiguous. However, oligodendrocytes in a perisomatic position on neurons are a consistent feature in these nuclei. A closer relationship appears to exist between astrocytes and neurons in the neurosecretory nuclei. In the supraoptic nucleus and paraventricular nucleus of all three species a basket-like structure, designated a ?pericellular envelope”? was observed surrounding neuronal perikarya. This structure is composed of astrocytic and oligodendroglial cell bodies and processes, and is most highly developed in the cat. A dense astrocytic plexus was observed in the suprachiasmatic nucleus of the cat, and in the comparable nuclei of the armadillo and opossum. The most prominent macroglial cell type of the lateral hypothalamic and lateral mammillary nuclei of all three species is the interfascicular oligodendrocyte. The posterior hypothalamic nucleus of each species has many perisomatic oligodendrocytes, and in the armadillo and cat astrocytes are closely related to the larger neurons. A subpial plexus, consisting of a palisade of small glial cells with many processes, is present in the hypothalamus of the three species. Ependymal cells have long projecting processes throughout the length of the third ventricle in the armadillo hypothalamus, but such processes are only apparent in the region of the infundibular nucleus and median eminence in the opossum and cat.     

Interactions between cerebellar Purkinje cells and their associated astrocytes

Some neurons, including cerebellar Purkinje cells, are completely ensheathed by astrocytes. When granule cell neurons and functional glia were eliminated from newborn mouse cerebellar cultures by initial exposure to a DNA synthesis inhibitor, Purkinje cells lacked glial sheaths and there was a tremendous sprouting of Purkinje cell recurrent axon collaterals, terminals of which hyperinnervated Purkinje cell somata, including persistent somatic spines, and formed heterotypical synapses with Purkinje cell dendritic spines, sites usually occupied by parallel fiber (granule cell axon) terminals. Purkinje cells in such preparations failed to develop complex spikes when recorded from intracellularly, and their membrane input resistances were low, making them less sensitive to inhibitory input. If granule cells and oligodendrocytes were eliminated, but astrocytes were not compromised, sprouting of recurrent axon collaterals occurred and their terminals projected to Purkinje cell dendritic spines, but the Purkinje cells had astrocytic sheaths, their somata were not hyperinnervated, the somatic spines had disappeared, complex spike discharges predominated, and membrane input resistance was like that of Purkinje cells in untreated control cultures. When cerebellar cultures without granule cells and glia were transplanted with granule cells and/or glia from another source, a series of changes occurred that included stripping of excess Purkinje cell axosomatic synapses by astrocytic processes, reduction of heterotypical axospinous synapses in the presence of astrocytes, disappearance of Purkinje cell somatic spines with astrocytic ensheathment, and proliferation of Purkinje cell dendritic spines after the introduction of astrocytes. Dendritic spine proliferation was followed by formation of homotypical axospinous synapses when granule cells were present or persistence as unattached spines in the absence of granule cells. The results of these studies indicate that astrocytes regulate the numbers of Purkinje cell axosomatic and axospinous synapses, induce Purkinje cell dendritic spine proliferation, and promote the structural and functional maturation of Purkinje cells.     

Isolation and Culture of Mouse Cortical Astrocytes

Astrocytes are an abundant cell type in the mammalian brain, yet much remains to be learned about their molecular and functional characteristics. In vitro astrocyte cell culture systems can be used to study the biological functions of these glial cells in detail. This video protocol shows how to obtain pure astrocytes by isolation and culture of mixed cortical cells of mouse pups. The method is based on the absence of viable neurons and the separation of astrocytes, oligodendrocytes and microglia, the three main glial cell populations of the central nervous system, in culture. Representative images during the first days of culture demonstrate the presence of a mixed cell population and indicate the timepoint, when astrocytes become confluent and should be separated from microglia and oligodendrocytes. Moreover, we demonstrate purity and astrocytic morphology of cultured astrocytes using immunocytochemical stainings for well established and newly described astrocyte markers. This culture system can be easily used to obtain pure mouse astrocytes and astrocyte-conditioned medium for studying various aspects of astrocyte biology.     

A fourth type of neuroglial cell in the adult central nervous system

Labeling central nervous tissue from mature animals with antibodies to NG2 chondroitin sulfate proteoglycan reveals the existence of large numbers of NG2 positive cells, at least some of which are oligodendroglial progenitors. It is generally agreed that these cells differ from the classically defined neuroglia, since they are antigenetically different from astrocytes, oligodendrocytes, or microglial cells. Although the NG2 positive cells have been well characterized in light microscopic preparations, examination of the labeled cells by electron microscopy have not led to general agreement about their morphological features. The basic reason for this is that it is difficult to obtain good preservation of the fine structure of NG2 labeled neurons. Since these NG2 positive cells are abundant in the central nervous system, it was decided to examine routinely prepared tissue from the brains of mature monkeys and rats by electron microscopy to determine if there is a neuroglial cell type whose presence has been overlooked. It soon became evident that there is a fourth type of neuroglial cell. These cells have pale, irregular shaped nuclei with a thin rim of heterochromatin beneath the nuclear envelope, and they have pale cytoplasm. Superficially they resemble astrocytes, which is the probable reason why the presence of this fourth type of neuroglial cell has been largely overlooked. However, the fourth type of neuroglial cell, here referred to as a ß neuroglial cell, has no intermediate filaments in its cytoplasm, the mitochondria are thinner than those of astrocytes, centrioles are frequently encountered in their cytoplasm, and when they are adjacent to capillaries they are always separated from the basal membrane by an astrocytic processes.     

Differential Localization of Glutathione-S-Transferase Yp and Yb Subunits in Oligodendrocytes and Astrocytes of Rat Brain

Glutathione-S-transferase Yb subunits were recently identified in rat brain and localized to astrocytes, ependymal cells lining the ventricles, subventricular zone cells, and tanycytes. Another isoform, Yp (pi family), was detected in rat brain by immunoblotting, and its mRNA was detected by Northern hybridizations. Double immunofluorescence localized Yb and Yp in different glial cells. The strongly Yp-positive cells were identified as oligodendrocytes by virtue of their arrangement in rows in white-matter tracts, colocalization in strongly carbonic anhydrase-positive cells, and association with myelinated tracts in the corpus striatum. Ependymal cells in the choroid plexus and ventricular lining were also strongly Yp positive, whereas Yb was not detected in the choroid plexus. The occurrence of Yp at low levels in astrocytes was indicated after immunostaining by a sensitive peroxidase-antiperoxidase method, which revealed weak staining of those cells in the molecular layer of the cortex. The data suggest that Yb and Yp subunits are primarily localized to astrocytes and oligodendrocytes, respectively, and that both are absent from neurons. The glutathione-S-transferase in oligodendrocytes may participate in the removal of toxins from the vicinity of the myelin sheath. The finding of glutathione-S-transferases in ependymal cells and astrocytes in the brain also suggests that this enzyme could be a first line of defense against toxic substances.     

The filum terminale of the frog spinal cord

Summary The filum terminale, or terminal portion of the spinal cord, was studied in normal adult frogs (Rana pipiens) by means of light and electron microscopy. Astroglial cells are the predominant elements in this region. The rostral portion of the filum terminale consists mainly of (1) a peripheral dense ring of myelinated and some unmyelinated nerve fibers, and processes of astrocytes terminating at the subpial space; (2) an intermediate zone, in which astrocytes are the main cellular elements in addition to a few degenerated neurons; and (3) a central region where the central canal is lined by dark and light ependymal cells. In the caudal portion of the filum terminale, the amount of neuropil is greatly reduced. This region is formed mainly by astrocytic glial cells and very few neuronal elements. The central canal in the caudal portion is located ventrally and contains a lining consisting almost exclusively of dark ependymal cells.     

Intermediate filaments reminiscent of immature cells expressed by goldfish (Carassius auratus) astrocytes and oligodendrocytes in vitro

The expression of intermediate filaments is developmentally regulated. In the mammalian embryo keratins are the first to appear, followed by vimentin, while the principal intermediate filament of the adult brain is glial fibrillary acidic protein. The intermediate filaments expressed by a cell thus reflect its state of differentiation. The differentiation state of cells, and especially of glial cells, in turn determines their ability to support axonal growth. In this study we used three new antibodies directed against three fish intermediate filaments (glial fibrillary acidic protein, keratin 8 and vimentin), in order to determine the identity and level of expression of intermediate filaments present in fish glial cells in culture. We found that fish astrocytes and oligodendrocytes are both able to express keratin 8 and vimentin. We further demonstrate that under proliferative conditions astrocytes express high keratin 8 levels and most oligodendrocytes also express keratin 8, whereas under nonproliferative conditions the astrocytes express only low keratin 8 levels and most oligodendrocytes do not express keratin 8 at all. These results suggest that the fish glial cells retain characteristics of immature cells. The findings are also discussed in relation to the fish glial lineage.     

Combination of in situ hybridization and immunocytochemistry to detect messenger RNAs in identified CNS neurons and glia in tissue culture

We have developed a technique in which immunofluorescence is combined with in situ hybridization using cDNA and RNA probes to assess the expression and distribution of messenger RNAs (mRNA) by neurons and neuroglia in tissue cultures of the rat dentate gyrus. The probes used in this study include a cDNA probe for ribosomal RNA (rRNA) and an RNA probe (cRNA) for glial fibrillary acidic protein (GEAP), an intermediate filament protein subunit expressed by astrocytes in the central nervous system. Both ubiquitous (tubulin) and cell type-specific (MAP-2 and GEAP) antibodies were used to identify neurons and neuroglia in culture. Using this procedure, the mRNA for rRNA was found in the cell bodies and large processes of MAP-2-positive neurons and throughout the cytoplasm of GEAP-positive flat astrocytes. In process-bearing astrocytes, GEAP mRNA is concentrated in the cell body, although some hybridization also occurred in astrocyte cell processes. With this combined in situ hybridization-immunofluorescence technique, the expression and distribution of an mRNA can be examined in different immunocytochemically identified cell types under identical culture and hybridization conditions. It is also possible to determine if there is a differential subcellular distribution of an mRNA in a single cell and if the distribution of the mRNA reflects the distribution of the protein itself. Finally, this technique can be utilized to verify the specificity of probes for cell type-specific mRNAs and to determine appropriate hybridization conditions to produce a specific signal.     

Immunocytochemical localization of S-100 protein in the brain of adult rat

Summary The cellular and subcellular distribution of the nervous system-specific S-100 protein has been investigated in the brain of adult rat at the ultrastructural level by the pre-embedding unlabelled antibody PAP method. The protein is found in both fibrous and protoplasmic astrocytes and in the ependymal cells. The neurons, the oligodendrocytes as well as the microglial cells are lacking S-100. The labelled cells show a reaction product diffusely distributed in the cytoplasmic matrix and on specialized membranes, namely plasma membranes, outer mitochondrial membranes and membranes of the endoplasmic reticulum and Golgi apparatus. The astrocytic filaments and the axonemes of the ependymal cilia exhibit a strong immunoreactivity. The reaction product is also present in the nucleoplasm of the astrocytes and ependymal cells but it is absent from the nucleolus and nuclear envelope. This immunocytochemical data on tissue with satisfactory ultrastructural preservation, provides new information on the localization of the S-100 protein, and could contribute to the understanding of the biological role of the protein.     

The fine structure of the area postrema of the sheep

The ultrastructural features of the area postrema (AP) were investigated in the suckling lamb, weaned lamb and adult sheep. No morphological differences were observed between lambs and sheep. Unciliated ependymal cells, linked by zonulae adherentes-type junctions and gap junctions, cover the AP ventricular surface. Clusters of pyriform neurons, glial cells, and axons are present in the parenchyma. The blood vessels are surrounded by wide perivascular spaces, which present an inner and outer basal lamina. The capillaries are of the fenestrated type. Perivascular glial cells rest on the outer basal lamina of the perivascular space and form a continuous ensheathment with their cell bodies or with flattened interdigitating processes. Along adjacent perivascular glial processes gap junctions are present. From our ultrastructural observations it appears that the overall cellular morphology of AP of the sheep does not differ substantially from that of monogastric mammals.     

Localization of Glycogen Synthase in Brain

Antisera against glycogen synthase from canine brain were prepared and used for investigation of the localization of the enzyme in the brain. Antisera cross-reacted only with the 88-kilodalton protein that is the subunit of brain glycogen synthase. Immunoreactivity of glycogen synthase was universally distributed in all regions of the brain, although hippocampus, cerebral cortex, caudatoputamen, and cerebellar cortex had relatively high immunoreactivity. Light microscopic examination revealed that the immunoreactivity was found in all cell types, such as neurons in several regions, astrocytes, ependymal cells surrounding the ventricle, oligodendrocytes, and epithelial cells of the choroid plexus in the ventricle. Immunoreactive intensity was more prominent in neurons than glial cells. Immunostaining may be a useful tool for investigation of the state of glycogen metabolism under normal and pathological conditions.     

A light and electron microscopic study of the iron transporter protein DMT-1 in the monkey cerebral neocortex and hippocampus

We have studied by immunocytochemistry, the distribution of DMT-1, a cellular iron transporter responsible for transport of metal irons from the plasma membrane to endosomes, in the normal monkey cerebral neocortex and hippocampus. Light to moderate DMT-1 staining was observed in glial cell bodies in the neocortex, the subcortical white matter, and the hippocampus. Despite light labeling of cell bodies, glial end feet around cortical and subcortical blood vessels were heavily labeled. In the neocortex, the glial cell bodies displayed the morphological features of protoplasmic astrocytes. Labeled glial cells in the subcortical white matter contained dense bundles of glial filaments and were identified as fibrous astrocytes. The observation that DMT-1 was present on astrocytic endfeet suggests that these cells are involved in uptake of iron from endothelial cells. It is possible that the iron could then be redistributed into the extracellular space in the brain parenchyma.     

Immunocytochemical localization of cell type-specific markers in reaggregating cell cultures of mouse cerebellum

Summary Reaggregate cultures were obtained from single-cell suspensions of fetal and early postnatal cerebellum, and fetal telencephalon and mesencephalon from C57BL/6J and NMRI mice and maintained in suspension under constant rotation as described previously (Seeds 1971). The percentage of dead cells in the aggregates as measured by the uptake of the fluorescent dye propidium iodide was always less than 5% of all cells. During the initial phase of reaggregation up to 20 h in vitro (hiv) several immunocytochemically defined cell types had a random distribution within the aggregate. Astrocytes were identified by indirect immunofluorescence by the use of the markers glial fibrillary acidic protein (GFAP), C1 and M1 antigens; neurons by NS-4 antigen and tetanus-toxin receptors; fibroblasts or fibroblast-like cells by fibronectin and laminin; and oligodendrocytes by myelin basic protein (MBP). Choleratoxin receptors and M 2 antigen served to distinguish the more mature from the less mature neurons. In reaggregates of early postnatal cerebellar cells neurons had started to redistribute after 40 hiv, forming an outer region containing more immature neurons and a core with more mature neurons. After 5 days in vitro (div) immature neurons were no longer detectable. From 3–8 div M1-and GFAP-positive astrocytic processes in the outer region showed a tendency for radial orientation. At later stages the processes appeared more randomly distributed and formed a dense glial network. Few oligodendrocytes and fibronectin-positive cells were present in the reaggregates. When reaggregates were prepared from 15 day-old embryonic cerebella, formation of radially oriented astrocytic processes and redistribution of neurons proceeded more slowly, but in a similar pattern as described for early postnatal cerebellum. GFAP was detectable at earlier ages than in situ. In reaggregates of 15 to 17 day old embryonic telencephalic anlage or midbrain, radially oriented astrocytic processes were not detectable. Similar to cerebellar reaggregates, accumulation of neurons in the inner region was observed.     

Glial cell lineage in vivo in the mouse cerebellum

Glial cells of the cerebellum originate from cells of the ventricular germinative layer, but their lineage has not been fully elucidated. For studying the glial cell lineage in vivo by retrovirus-mediated gene transfer, we introduced a marker retrovirus into the ventricular germinative layer of embryonic day 13 mice. In the resulting adult cerebella, virus-labeled glial cells were grouped in discrete clusters, and statistical analysis showed that these clusters represented clones in high probability. Of 71 of the virus-labeled glial clusters, 33 clusters were composed of astrocytes/Bergmann glia, 10 were composed of only white matter astrocytes, and 24 were composed of only oligodendrocytes. No glial clusters contained virus-labeled neurons. These results suggest that astrocytes/Bergmann glia, white matter astrocytes and oligodendrocytes immediately arise from separate glial precursors: these three glial lineages may diverge in the course of cerebellar development.     

Carbonic Anhydrase Immunostaining in Astrocytes in the Rat Cerebral Cortex

Carbonic anhydrase is known to occur in the choroid plexus, oligodendrocytes, and myelin, and to be virtually absent from neurons, in the mammalian CNS; however, there is significant controversy whether it is also present in astrocytes. When brain sections from adult rats were stained for simultaneous immunofluorescence of carbonic anhydrase and the astrocyte marker glutamine synthetase, both antigens were detected in the same glial cells in the cortical gray matter, whereas the oligodendrocytes and myelinated fibers in and adjacent to the white matter showed immunofluorescence only for carbonic anhydrase. Some glial cells in the gray matter also showed double immunofluorescence for carbonic anhydrase and glial fibrillary acidic protein. These results indicate that there is carbonic anhydrase in some astrocytes in the mammalian CNS.     

Transgenic overexpression of BMP4 increases astroglial and decreases oligodendroglial lineage commitment

Bone morphogenetic proteins (BMPs) promote astrocytic differentiation of cultured subventricular zone stem cells. To determine whether BMPs regulate the astrocytic lineage in vivo, transgenic mice were constructed that overexpress BMP4 under control of the neuron-specific enolase (NSE) promoter. Overexpression of BMP4 was first detectable by Western analysis on embryonic day 16 and persisted into the adult. The overexpression of BMP4 resulted in a remarkable 40% increase in the density of astrocytes in multiple brain regions accompanied by a decrease in the density of oligodendrocytes ranging between 11 and 26%, depending on the brain region and the developmental stage. No changes in neuron numbers or the pattern of myelination were detected, and there were no gross structural abnormalities. Similar phenotypes were observed in three independently derived transgenic lines. Coculture of transgenic neurons with neural progenitor cells significantly enhanced astrocytic lineage commitment by the progenitors; this effect was blocked by the BMP inhibitor Noggin, indicating that the stimulation of astrogliogenesis was due to BMP4 release by the transgenic neurons. These observations suggest that BMP4 directs progenitor cells in vivo to commit to the astrocytic rather than the oligodendroglial lineage. Further, differentiation of radial glial cells into astrocytes was accelerated, suggesting that radial glia were a source of at least some of the supernumerary astrocytes. Therefore, BMPs are likely important mediators of astrocyte development in vivo.     

Gap junctions between astrocytes during growth and differentiation in organ culture systems

Summary Fetal rat neocortex maintained in organ culture systems with the use of sponge foam matrices and millipore filter platforms undergoes growth and cytodifferentiation along classical neuronal and glial lines up to 36 days in vitro (DIV). Astrocytic differentiation is characterized by accumulation of 80–90 Å glial filaments in the cell bodies and processes of astrocytes. Gap or nexus junctions closely resembling those formed in mammalian brain in situ are identified by 15 DIV. By 36 DIV, interastrocytic gap junctions are numerous and frequently join extensive lengths of adjacent glial plasma membranes. The results suggest that these organ culture systems may provide a favorable environment for the study of cellular structure and function of coupled neuroglia.This work was supported by a research grant from the Veterans Administration. Skilled technical assistance was provided by Robin L. Isaacs and Marilyn Woodward     

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.     

Immunocytochemical and RT-PCR analysis of connexin36 in cultures of mammalian glial cells

The expression of connexin36 (Cx36) was studied in primary cultures of rat brain glial cells: mature astrocytes, ameboid and ramified microglia and immature oligodendrocytes (at middle period of myelinogenesis). The data from these cells were compared with those obtained from cultures of neocortical and hypothalamic neurons. mRNA encoding Cx36 was investigated by RT-PCR, the Cx36 protein by immunocytochemistry using a polyclonal antibody against Cx36 in cells characterized by antibodies specific for the single cell types. The Cx36 was found in oligodendrocytes, both ameboid and ramified microglial cells and in neurons. Astrocytes showed no detectable expression of the Cx36. The expression of Cx36 in oligodendrocytes and microglial cells suggests an involvement of the direct cell-cell communication channels formed by Cx36 in myelin formation and in brain development, damage and repair processes.     

Improvement in Double Staining With Fluoro-Jade C and Fluorescent Immunostaining: FJC Staining Is Not Specific to Degenerating Mature Neurons

Fluoro-Jade C (FJC) staining has been used to detect degenerating neurons in tissue sections. It is a simple and easy staining procedure and does not depend on the manner of cell death. In some experiments, double staining with FJC and fluorescent immunostaining (FI) is required to identify cell types. However, pretreatment for FJC staining contains some processes that are harsh to fluorophores, and the FI signal is greatly reduced. To overcome this issue, we improved the double staining protocol to acquire clear double-stained images by introducing the labeled streptavidin–biotin system. In addition, several studies indicate that FJC can label non-degenerating glial cells, including resting/reactive astrocytes and activated microglia. Moreover, our previous study indicated that degenerating mesenchymal cells were also labeled by FJC, but it is still unclear whether FJC can label degenerating glial cells. Acute encephalopathy model mice contained damaged astrocytes with clasmatodendrosis, and 6-aminonicotinamide-injected mice contained necrotic astrocytes and oligodendrocytes. Using our improved double staining protocol with FJC and FI, we detected FJC-labeled degenerating astrocytes and oligodendrocytes with pyknotic nuclei. These results indicate that FJC is not specific to degenerating neurons in some experimental conditions: