Radioautographic investigation of gliogenesis in the corpus callosum of young rats II. Origin of microglial cells

Microglial cells are absent from the corpus callosum of newborn rats. In the hope of finding out when and how microglial cells appear with age, ³H-thymidine was given intraperitoneally as single or three shortly spaced injections to 5-day-old rats weighing about 15 g; and these animals were sacrificed at various time intervals from 2 hours to 35 days later. Pieces of corpus callosum were taken near the superior lateral angle of the lateral ventricles; and semithin sections were radioautographed and stained with toluidine blue. The corpus callosum of 5-day-old rats is composed of loosely arranged unmyelinated fibers and scattered cells. Among these cells, microglia are rare; there are a few astrocytes, many immature glial cells, rare pericytes, and 6-7% of phagocytic “ameboid cells” consisting of a few monocytes and many macrophages. In the animals sacrificed two hours after ³H-thymidine administration, label is present only in immature cells and “ameboid cells.” As time elapses and the fibers of corpus callosum become myelinated, oligodendrocytes and, later, microglial cells appear. At the age of 12 days, microglial cells are present in substantial number; and by 19 days, the number doubles to reach a plateau. Many of the new microglial cells are labeled, e.g., 78.1% in 12-day-old animals (7 days after ³H-thymidine administration). The labeled microglial cells must have come from the transformation of cells that acquired label early, that is, from the immature cells or the “ameboid cells.” The height of the peaks of labeling – 59.8% at nine days for immature cells and 77.8% at 12 days for “ameboid cells” – points to the latter as precursors of the highly labeled microglial cells. Furthermore, the “ameboid cells” disappear as microglial cells appear and there are transitional elements between these two cell types. Cell counts suggest that about a third of the “ameboid cells” transform into microglial cells, while the others degenerate and die. Thus, the microglial cells which appear in the corpus callosum during the first three weeks of life result from transformation of the “ameboid cells” – a group of macrophages showing various stages of transition from monocytes. As for the occasional microglial cell appearing after the third week or in the adult, they presumably come directly from monocytes. In either case, monocytes would be the intial precursors.     

Brain mechanisms and feeding behavior in the pigeon (Columba livia). II. Analysis of feeding behavior deficits after lesions of quinto-frontal structures

Strong labeling of the cells in the subependymal layer was produced by stereotaxic injection of 5 μCi of ³H-thymidine into the left lateral ventricle of the brain of one and a quarter month old rats weighing about 100 gm. These animals were sacrificed by glutaraldehyde perfusion from two hours to 21 days later. Blocks of corpus callosum with adjacent subependymal and ependymal layers were excised from the injected and non-injected sides, and embedded in Epon; 0.5 μ thick sections were radioautographed and stained with toluidine blue. In the subependymal region, on both injected and non-injected sides, there was an immediate uptake of label by many cells followed by an increase and later a decrease in the percent cells labeled. In the corpus callosum while at first the percent labeling of glial cells was rather low, it did increase slowly with time and, after seven days, exceeded that in the subependymal region. These results were interpreted as indicating that cells arising in the subependymal layer had migrated into the corpus callosum. Up to four days after injection, most of the label in corpus callosum was present in immature-looking cells resembling the cells of the subependymal layer and referred to as free subependymal cells. With time, the percent labeling decreased in these cells while increasing in some of the glial cells. A labeling peak was observed for light oligodendrocytes at four to seven days and for dark oligodendrocytes at 21 days, whereas labeling of medium shade oligodendrocytes occurred at intermediate times. The succession of labeling peaks indicated a sequence of development from free subependymal cells through light and medium shade to dark oligodendrocytes. Few astrocytes carried label at any time; those which did seemed to have arisen from the transformation of labeled free subependymal cells. Microglia were unlabeled at two hours, but their percent labeling was high at 4–14 days. While the labeling of other glial cells reflected their physiological behavior, the labeling of microglia was a consequence of the trauma produced by the injection 0f tracer into the ventricle. In conclusion, cells coming from the subependymal layer appear to migrate into the corpus callosum where, in 100 gm rats, many of them transform into oligodendrocytes and a few into astrocytes.     

Proliferation and turnover of glial cells in the forebrain of young adult mice as studied by repeated injections of 3 H-thymidine over a prolonged period of time

The magnitude of glial cell renewal was studied on young adult mice using repeated intraperitoneal injections of ³H-thymidine every eight hours over a period of 30 days. Mean labeling indices one hour after the last injection were as follows: Glial cells of the subependymal layer of the lateral ventricle, 61.5%; oligodendrocytes (various sites), 24 to 36.2%; astrocytes (various sites), 14.3 to 30.8%, and satellites in the cerebral cortex, 32.7%. Since DNA synthesis time of the proliferating, immature glial cells is unknown and may be shorter than the time interval of eight hours chosen for repeated injections of ³H-thymidine, these results are interpreted as representing minimum values for turnover, during 30 days, of the various cell types in different areas of the forebrain. The significance of a marked proliferative activity of the glial cells as related to differentiation and possible migration of subependymal cells, is discussed.     

Correlation of glial proliferation with age in the mouse brain

Radioautographs of brain sections were prepared after injection of ³H-thymidine into mice aged 23, 100, 200 or 400 days. The presence of a small number of labeled cells in all animals indicates that neuroglia do proliferate even at an advanced age. Proliferation is most active in the corpus callosum and least so in the corpus striatum. Comparison of the counts of labeled and unlabeled nuclei suggests that glial cells are produced in numbers exceeding growth requirements and, accordingly, that they turn over, although slowly.     

A qualitative and quantitative study of the glial cells in normal and athymic mice

A qualitative and quantitative light and electron microscopic analysis of the glial cells in the supraventricular part of the corpus callosum of the neonatal and adult homozygous athymic nude (nu/nu) and normal BALB/c (+/+) mice was carried out to determine the possible contribution of nude gene mutation to glial cell development. Quantitative cell counts using toluidine blue stained serial callosal sections of 0.5 μm thickness showed that the overall glial cell population was significantly reduced in both neonatal and adult athymic mice. The number of glioblasts, astrocytes and microglia of 5-day-old athymic mouse was reduced by 10%, 27%, and 39%, respectively, when compared to the 5-day-old normal mouse. The frequency of necrotic cells in the neonatal athymic mouse increased by 70% when compared with the normal mouse. In the 13-week-old adult athymic mouse, the number of oligodendrocytes, astrocytes, and microglia decreased by 19%, 31%, and 33%, respectively, when compared to normal mouse. There was no significant difference in the area covered by the corpus callosum in 5-day-old and adult nude mice versus the normal ones of corresponding ages. Except for microglia and astrocytes, the ultrastructural features of the other glial cell types in both strains were comparable. Most of the microglial cells of the neonatal normal mouse were round and were selectively marked by Mac-1 monoclonal antibody at their plasma membrane. The immunoreactivity appeared to be more intense in the normal than the athymic mouse, suggesting a down regulation of CR3 receptors and reduced phagocytic activity of this cell type in the athymic mouse. It is proposed that the increased number of necrotic cells in the neonatal athymic mouse may be attributed to the delay in the removal of dead cells normally phagocytosed by microglia. The microglia in both strains of mouse showed comparable lectin staining intensity at the plasma membrane, indicating that their glycoprotein binding receptors to lectin remained unchanged. Some astrocytes in the adult athymic mice showed hypertrophy. The reduced number of glial cells may be the direct result of genetic mutation or consequential to the lack of certain trophic factors arising from the genetic mutation. Thus, the reduction of microglial cells in both neonatal and adult athymic mice may be due to the lack of thymic hormones which, together with lymphokines have been shown to affect the maturation of bone marrow derived cells including monocytes, the putative precursor cells of microglia. The reduction in the number of glioblasts and astrocytes may be attributed to the diminution of T lymphocytes or consequential to the reduction of microglia which are known to secrete interleukin-1 that would influence gliogenesis and produce specific growth factors for promoting astrocyte proliferation. Last, as interaction exists between astrocytes and oligodendrocytes, the products of astrocytes may affect the development of oligodendrocytes and vice vasa. The present findings point to a relation between glial cell development and immune network system. © 1995 Wiley-Liss, Inc.     

The fine structure of pulse labeled (3H-thymidine cells) in degenerating rat optic nerve

The ultrastructure of pulse labeled (³H-thymidine) cells in rat optic nerve undergoing Wallerian degeneration is described. The study was limited to the first ten days after enucleation since cell proliferation during this interval is greater than in normal optic nerve (Skoff and Vaughn, '71). Approximately one-third of the pulse labeled cells are astrocytes. The majority of the proliferating astrocytes are in a reactive state, having changed their normal fibrous appearance to one showing a paucity of filaments. Thirty percent of the pulsed cells can be classified as microglia. Only immature oligodendrocytes proliferate, and they account for less than 10% of the pulse labeled cells. About 30% of the labeled population are undifferentiated glial precursor cells. Electron microscopic autoradiographic data obtained from normal optic nerve and presented in this paper indicates that glial precursor cells which have divided shortly before enucleation continue to proliferate after it. The evidence suggests that recently formed glial precursor cells transform into phagocytes following enucleation. Less than 3% of the pulse labeled cells examined in this study are ultrastructurally similar to mononuclear leukocytes. The results of the present study together with previous studies of degenerating optic nerve indicate that most phagocytes in Wallerian degeneration are derived from proliferation of intrinsic glia rather than from an invasion of exogenous cells.     

Progenitor cells of the adult mouse subventricular zone proliferate, migrate and differentiate into oligodendrocytes after demyelination

Identifying a source of cells with the capacity to generate oligodendrocytes in the adult CNS would help in the development of strategies to promote remyelination. In the present study, we examined the ability of the precursor cells of the adult mouse subventricular zone (SVZ) to differentiate into remyelinating oligodendrocytes. After lysolecithin-induced demyelination of the corpus callosum, progenitors of the rostral SVZ (SVZa) and the rostral migratory pathway (RMS), expressing the embryonic polysialylated form of the neural cell adhesion molecule (PSA-NCAM), increased progressively with a maximal expansion occurring after 2 weeks. This observation correlated with an increase in the proliferation activity of the neural progenitors located in the SVZa and RMS. Moreover, polysialic acid (PSA)-NCAM-immunoreactive cells arizing from the SVZa were detected in the lesioned corpus callosum and within the lesion. Tracing of the constitutively cycling cells of the adult SVZ and RMS with 3H-thymidine labelling showed their migration toward the lesion and their differentiation into oligodendrocytes and astrocytes but not neurons. These data indicate that, in addition to the resident population of quiescent oligodendrocyte progenitors of the adult CNS, neural precursors from the adult SVZ constitute a source of oligodendrocytes for myelin repair.     

Glial expression of fibroblast growth factor-9 in rat central nervous system.

We examined the expression of fibroblast growth factor (FGF)-9 in the rat central nervous system (CNS) by immunohistochemistry and in situ hybridization studies. FGF-9 immunoreactivity was conspicuous in motor neurons of the spinal cord, Purkinje cells, and neurons in the hippocampus and cerebral cortex. In addition to the neuronal localization of FGF-9 immunoreactivity that we reported previously, the present double-label immunohistochemistry clearly demonstrated that the immunoreactivity was present in glial fibrillary acidic protein (GFAP)-positive astrocytes preferentially present in the white matter of spinal cord and brainstem of adult rats and in CNPase-positive oligodendrocytes that were arranged between the fasciculi of nerve fibers in cerebellar white matter and corpus callosum of both adult and young rats. There was a tendency for FGF-9 immunoreactivity in oligodendrocytes to be more pronounced in young rats than in adult rats. The variation of oligodendrocyte FGF-9 immunoreactivity in adult rats was also more pronounced than that in young rats. With in situ hybridization, FGF-9 mRNA was observed in astrocytes in the white matter of rat spinal cord and oligodendrocytes in the white matter of cerebellum and corpus callosum of adult and young rats. The expression of FGF-9 mRNA in glial cells was lower than in neurons, and not all glial cells expressed FGF-9. In the present study, we demonstrated that FGF-9 was expressed not only in neurons but also in glial cells in the CNS. FGF-9 was considered to have important functions in adult and developing CNS.     

Developmental changes in the membrane current pattern, K+ buffer capacity, and morphology of glial cells in the corpus callosum slice

Recent studies indicated that glial cells in tissue culture can express a variety of different voltage-gated channels, while little is known about the presence of such channels in glial cells in vivo. We used a mouse corpus callosum slice preparation, in which after postnatal day 5 (P5) more than 99% of all perikarya belong to glial cells (Sturrock, 1976), to study the current patterns of glial cells during their development in situ. We combined the patch-clamp technique with intracellular labeling using Lucifer yellow (LY) and subsequent ultrastructural characterization. In slices of mice from P6 to P8, we predominantly found cells expressing delayed-rectifier K+ currents. They were similar to those described for cultured glial precursor cells (Sontheimer et al., 1989). A-type K+ currents or Na+ currents were not or only rarely observed, in contrast to cultured glial precursors. LY labeling revealed that numerous thin processes extended radially from the perikaryon of these cells, and ultrastructural observations suggested that they resemble immature glial cells. In slices of older mice (P10-13), when myelination of the corpus callosum has already commenced, many cells were characterized by an almost linear current-voltage relationship. This current pattern was similar to cultured oligodendrocytes (Sontheimer et al., 1989). Most processes of LY-filled cells with such a current profile extended parallel to each other. Electron microscopy showed that these processes surround thick, unmyelinated axons. We suggest that cells with oligodendrocyte-type electrophysiology are promyelinating oligodendrocytes. In contrast to cultured oligodendrocytes, membrane currents of promyelinating oligodendrocytes in the slice decayed during the voltage command. This decay was due not to inactivation, but to a marked change in the potassium equilibrium potential within the voltage jump. This implies that, in the more mature corpus callosum, small membrane polarizations in a physiological range can lead to extensive changes in the K+ gradient across the glial membrane within a few milliseconds.     

Investigation of glial cells in semithin sections. II. Variation with age in the numbers of the various glial cell types in rat cortex and corpus callosum

Semithin Epon sections stained with toluidine blue were used to enumerate astrocytes, microglia, the three subtypes of oligodendrocytes, and cells referred to as free subependymal cells, in the corpus callosum and cerebral cortex of male Sherman rats of various ages. The period covered extended from a few days before weaning (3/4 month of age) until the time when growth became negligible (5 months of age). The total number of glial cells increases with age in both cortex and corpus callosum. However, the investigation of individual cell types reveals that the number of microglia remains fairly constant throughout the period under study. The number of astrocytes in corpus callosum increases up to the age of one month, but remains constant thereafter, while their number in the cortex is the same at all investigated times. In the case of oligodendrocytes, the three subtypes behave differently. About two-thirds of the oligodendrocytes in rats aged three-quarters of a month are of the light or medium shade types, but the number of these gradually decreases with age and becomes very low in five-month-old rats. In contrast, the dark cells which constitute about one-third of the oligodendrocytes in young rats make up nearly the whole of this group in adults. Finally, free subependymal cells are absent in cortex throughout the period under study, but are present in corpus callosum, where their number steadily declines with age. In conclusion, the numbers of astrocytes and microglia seem to remain constant in growing rats after the age of one month. Dark oligodendrocytes markedly increase in number with age, while the other types of oligodendrocytes and the free subependymal cells are reduced to negligible numbers by the age of five months.     

Histological localization of nervous-system antigens in the cerebellum by immunoperoxidase labeling

The indirect immunoperoxidase method was used to localize histologically on sagittal sections of mouse cerebellum antigenic determinants detected by the following antisera: anti-NS-2, anti-NS-3, anti-NS-4, rabbit anti-bovine corpus callosum, rabbit anti-mouse brain, rabbit anti-glial fibrillary acidic protein, and rabbit anti-neurofilament protein. Anti-α-bungarotoxin serum and normal rabbit serum were used as negative controls. The various sera showed similarities in staining pattern as well as differences. Anti-NS-2 antiserum labeled the somata of interneurons in the molecular layer, granule cell bodies, glial cells in the white matter, and along the surfaces of blood vessels. A similar pattern of staining is produced by the anti-NS-3 antiserum except that glial cells are less prominent in the white matter and the blood vessels are not visible at all. Anti-NS-4 antiserum does not label interneurons but does label glomeruli and, less intensely, granule cell bodies in the granular layer. Rabbit anti-mouse brain antiserum is similar to anti-NS-4 antiserum except that fiber tracts in the white matter are stained more intensely. Rabbit anti-bovine corpus callosum labels only white matter. Antisera to neurofilament and glial fibrillary acidic proteins label Bergmann glia and fibrous astrocytes.     

Localization of mu class glutathione-S-transferase in the forebrains of neonatal and young rats: implications for astrocyte development.

The Yb (Mu class) isoform of glutathione-S-transferase has recently been localized in ependymal cells, subependymal cells, and astrocytes in the forebrains of rats 3 weeks to adult in age. It was not known, however, at what age Mu might first be observed during postnatal development and whether the first cells in which it was found would be immature astrocytes or some less differentiated glial precursor cell, if the latter were present in vivo. Tissue sections from the forebrains of neonatal to 16 day old rats were immunostained with antibodies against Mu. In neonates Mu was observed in vimentin-positive cells and their processes adjacent to the lateral ventricles, and in the corpus striatum. The colocalization with vimentin suggested that these were subependymal cells and radial glia. In the corpus striatum the radial glia, while still vimentin-positive, rapidly lost Mu from their radial cell processes, whereas the cell-bodies remained Mu-positive. During the first postnatal week the Mu-positive, glial-fibrillary-acidic-protein (GFAP)-positive cell bodies of immature astrocytes appeared in the corpus striatum. The earliest Mu-positive cells in the immature white matter of the corpus callosum were vimentin-positive and had striking longitudinal processes that also were vimentin- and Mu-positive. Like the processes of radial glia, the longitudinal processes lost their Mu-immunoreactivity, only later and more gradually. Mu-positive, GFAP-positive cells appeared later in the corpus callosum than in the corpus striatum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Astrocytes, oligodendrocytes, and Schwann cells share a common antigenic determinant that cross-reacts with myelin basic protein: identification with monoclonal antibody

We have produced a monoclonal antibody against myelin basic protein that reacts with astrocytes, oligodendrocytes, and Schwann cells. This antibody was generated by fusion of mouse myeloma cells with spleen cells from BALB/c mice immunized with delipidated white matter from adult rat corpus callosum. The antibody was characterized via solid-phase radioimmunoassay, immunoblot of SDS-PAGE, and by indirect immunofluorescence staining of monolayer cultures containing oligodendrocytes, astrocytes, and Schwann cells. Myelin basic protein (MBP) was shown previously to be present only in myelin producing cells in CNS and PNS (oligodendroglia and Schwann cells) and not in astrocytes. The binding of this monoclonal antibody to all 3 cell types suggests that these cells share a common epitope. This epitope may be related to a common progenitor cell.     

Gliogenesis of astrocytes and oligodendrocytes in the neocortical grey and white matter of the adult rat: Electron microscopic analysis of light radioautographs

The identification of newly formed glial cells in the normal adult cerebral cortex is unresolved, since the identification of cells incorporating [H³] thymidine has not been demonstrated in the adult by electron microscopy. In the present study, this problem has been studied by combining the resolution of the electron microscope with radioautography of 1-μm sections. Four normal male rats were injected at 90 days of age with [H³] thymidine and allowed to survive for 30 days. Labeled cells were found in 1-μm sections of the visual cortex of these adult rats, and electron micrographs of selected cells from these same sections demonstrated clearly two types of cells labeled, astrocytes and oligodendrocytes, in both grey and white matter. The few cells that were tentatively identified as labeled microglia in the light microscope proved to resemble oligodendrocytes when examined in the electron microscope. In 1-μm sections of the cortical grey matter, heavily labeled astrocytes (13 or more silver grains over the nucleus) represent about 0.08% of the total astrocytic population, and heavily labeled oligodendrocytes also were about 0.08% of their population. In the cortical white matter, about 0.03% heavily labeled astrocytes were observed, compared to about 0.07% heavily labeled oligodendrocytes. For all neuroglial cells in both white and grey matter, the average percent heavily labeled cells was 0.066%, a value large enough to suggest a slow turnover of neuroglial cells during the lifespan of the rats.     

Defined astrocytic expression of human amyloid precursor protein in Tg2576 mouse brain

Transgenic Tg2576 mice expressing human amyloid precursor protein (hAPP) with the Swedish mutation are among the most frequently used animal models to study the amyloid pathology related to Alzheimer's disease (AD). The transgene expression in this model is considered to be neuron-specific. Using a novel hAPP-specific antibody in combination with cell type-specific markers for double immunofluorescent labelings and laser scanning microscopy, we here report that—in addition to neurons throughout the brain—astrocytes in the corpus callosum and to a lesser extent in neocortex express hAPP. This astrocytic hAPP expression is already detectable in young Tg2576 mice before the onset of amyloid pathology and still present in aged Tg2576 mice with robust amyloid pathology in neocortex, hippocampus, and corpus callosum. Surprisingly, hAPP immunoreactivity in cortex is restricted to resting astrocytes distant from amyloid plaques but absent from reactive astrocytes in close proximity to amyloid plaques. In contrast, neither microglial cells nor oligodendrocytes of young or aged Tg2576 mice display hAPP labeling. The astrocytic expression of hAPP is substantiated by the analyses of hAPP mRNA and protein expression in primary cultures derived from Tg2576 offspring. We conclude that astrocytes, in particular in corpus callosum, may contribute to amyloid pathology in Tg2576 mice and thus mimic this aspect of AD pathology.     

Developmental and post‐injury cortical gliogenesis: A Genetic fate‐mapping study with Nestin‐CreER mice

The primary sources of cortical gliogenesis, either during development or after adult brain injury, remain uncertain. We previously generated Nestin‐CreER mice to fate‐map the progeny of radial glial cells (RG), a source of astrocytes and oligodendrocytes in the nervous system. Here, we show that Nestin‐CreER mice label another population of glial progenitors, namely the perinatal subventricular zone (SVZ) glioblasts, if they are crossed with stop‐floxed EGFP mice and receive tamoxifen in late embryogenesis (E16–E18). Quantification showed E18 tamoxifen‐induction labeled more perinatal SVZ glioblasts than RG and transitional RG combined in the newborn brain (54% vs. 22%). Time‐lapse microscopy showed SVZ‐glioblasts underwent complex metamorphosis and often‐reciprocal transformation into transitional RG. Surprisingly, the E10‐dosed RG progenitors produced astrocytes, but no oligodendrocytes, whereas E18‐induction fate‐mapped both astrocytes and NG2+ oligodendrocyte precursors in the postnatal brain. These results suggest that cortical oligodendrocytes mostly derive from perinatal SVZ glioblast progenitors. Further, by combining genetic fate‐mapping and BrdU‐labeling, we showed that cortical astrocytes cease proliferation soon after birth (<P10) and only undergo nonproliferative gliosis (i.e., increased GFAP expression without cell‐division) after stab‐wound injury in adult brains. By contrast, 9.7% of cortical NG2+ progenitors remained mitotic at P29, and the ratio rose to 13.8% after stab‐wound injury. Together, these results suggest NG2+ progenitors, rather than GFAP+ astrocytes, are the primary source of proliferative gliosis after adult brain injury. © 2008 Wiley‐Liss, Inc.     

Sodium and Calcium Currents in Glial Cells of the Mouse Corpus Callosum Slice

We studied Na⁺ and Ca²⁺ currents in glial cells during the development of the corpus callosum in situ. Glioblasts and oligodendrocytes from frontal brain slices of postnatal day (P) 3 to P18 mice were identified based on morphological and ultrastructural features after characterization of the currents with the patch-clamp technique. Slices from P3-P8 mice contained predominantly glioblasts with immature morphological features. These cells showed Na⁺ and Ca²⁺ currents, but the population with these currents decreased between P3 and P8. Na⁺ currents were blocked in Na⁺-free bathing solution and in the presence of tetrodotoxin, Ca²⁺ currents were only observed when a high concentration of extracellular Ba²⁺ was present. The cells from the corpus callosum of P10 – P18 mice predominantly had morphological features of oligodendrocytes. In these cells, which in some cases were shown to form myelin, neither Na⁺ nor Ca²⁺ currents were detected. To compare these in situ results with those from the electrophysiologically and immunocytochemically well-characterized cultured glial cells, we determined the expression pattern of stage-specific antigens in the corpus callosum in situ. The first O4 antigen-positive glial precursors were observed at P1, the earliest stage examined. The oligodendrocytic antigens O7 and O10 appeared at P6 and P14, respectively, and prominent labelling with the corresponding markers was seen at P12 and P18, respectively. Despite the existence of numerous mature, O10-positive oligodendrocytes at P18, which expressed Ca²⁺ channels in vitro , we failed to detect Ca²⁺ currents in situ at this stage.     

Cell-type-specific markers for distinguishing and studying neurons and the major classes of glial cells in culture

We have used 4 cell-type-specific markers to identify individual glial and neuronal cells in dissociated cell cultures of neonatal rat sciatic nerve, dorsal root ganglia (DRG), optic nerve, cerebellum, corpus callosum, cerebral cortex and leptomeninges. Schwann cells were identified with antibodies against rat neural antigen-1 (Ran-1), neurons with tetanus toxin, astrocytes with antibody against the glial fibrillary acidic protein (GFAP) and oligodendrocytes with antibody against galactocerebroside. All of these ligands react with cell surface molecules except for anti-GFAP antibody which binds to intracellular glial filaments. Using two-fluorochrome immunofluorescence we have studied the distribution of various glycoproteins and glycolipids on these 4 major neural cell types in short-term cultures. We have found that (1) although Ran-1 is expressed on glial and neuronal tumours, it was not found on normal astrocytes, oligodendrocytes or neurons; (2) Thy-1 was present on fibroblasts and some neurons but not on the majority of leptomeningeal cells or on oligodendrocytes or astrocytes in short-term cultures (however, it was expressed on some astrocytes in longer term cultures); (3) the 'large external transformation sensitive' (LETS) protein could be detected on fibroblasts and leptomeningeal cells but not on neurons or glial cells; (4) GM1 was present on all neurons, most oligodendrocytes and approx. 50% of other cell types; sulfatide and GM3 were only detectable on oligodendrocytes, while globoside was only found on some neurons. In addition, we were able to identify putative microglial cells by the presence of cell surface receptors for IgG and by their phagocytic activity; they did not express and of the cell-type-specific defining markers.     

Stage‐specific requirement for cyclin D1 in glial progenitor cells of the cerebral cortex

Despite the vast abundance of glial progenitor cells in the mouse brain parenchyma, little is known about the molecular mechanisms driving their proliferation in the adult. Here we unravel a critical role of the G1 cell cycle regulator cyclin D1 in controlling cell division of glial cells in the cortical grey matter. We detect cyclin D1 expression in Olig2‐immunopositive (Olig2+) oligodendrocyte progenitor cells, as well as in Iba1+ microglia and S100β+ astrocytes in cortices of 3‐month‐old mice. Analysis of cyclin D1‐deficient mice reveals a cell and stage‐specific molecular control of cell cycle progression in the various glial lineages. While proliferation of fast dividing Olig2+ cells at early postnatal stages becomes gradually dependent on cyclin D1, this particular G1 regulator is strictly required for the slow divisions of Olig2+/NG2+ oligodendrocyte progenitors in the adult cerebral cortex. Further, we find that the population of mature oligodendrocytes is markedly reduced in the absence of cyclin D1, leading to a significant decrease in the number of myelinated axons in both the prefrontal cortex and the corpus callosum of 8‐month‐old mutant mice. In contrast, the pool of Iba1+ cells is diminished already at postnatal day 3 in the absence of cyclin D1, while the number of S100β+ astrocytes remains unchanged in the mutant. GLIA 2014;62:829–839     

Characteristics of fish glial cells in culture: possible implications as to their lineage.

Regeneration of injured central nervous system axons is largely dependent on the response of the associated nonneuronal glial cells to injury. Glial cells of the mammalian central nervous system, unlike those of fish, are apparently not conducive to axonal regeneration. While the lineage of rat glial cells is well characterized and its role in the support or inhibition of regenerative growth is beginning to be understood, little is known about fish glial cells. Accordingly, glial cells in cultures of adult goldfish brain and of newly hatched goldfish larvae were studied in an attempt to establish their lineage. The cells were identified by means of indirect immunofluorescence, using antibodies against fish astrocytes and oligodendrocytes. The cell count in the cultures increased from a small number of cells at 24 h after plating to a large number of both astrocytes and oligodendrocytes after 1 week in culture. Both of these cell types had originated from proliferating cells, as shown by their uptake of tritiated thymidine and by the inhibition of cell proliferation by 5-fluoro-2'-deoxyuridine. Both astrocytes, i.e., glial fibrillary acidic protein-positive cells, and oligodendrocytes, i.e., 6D2-positive cells, were positively labeled also by A2B5 antibodies, which are known to label progenitors of type-2 astrocytes and oligodendrocytes in the rat optic nerve. The results suggest that A2B5 positive progenitor cells in the goldfish central nervous system, as in the rat optic nerve, might be a common progenitor of astrocytes and oligodendrocytes.