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.     

Astrocytes cultured from mature brain derive from glial precursor cells

We have previously shown that enriched preparations of oligodendrocytes from either mature bovine brain or 30-d-old rat brain, when cultured in serum-free medium, yield mixed cultures of oligodendrocytes and astrocytes even though no GFAP+ cells were present after 24 hr in culture (Norton et al., 1986, 1988). To test the possibility that the astrocytes in these cultures arose from glial precursor cells, we followed the expression of ganglioside GD3, galactosylceramide (GC), glial fibrillary acidic protein (GFAP), and vimentin in the cultures. GD3 has already been shown to be a marker of immature neuroectodermal cells, which in the postnatal brain are glial progenitor cells (Goldman et al., 1984, 1986). The cultures from both species contained at 1 DIV only two populations of cells; 90-95% GC+/GD3- oligodendrocytes and 4-10% GD3+/GC- small, round cells. With time, the oligodendrocytes remained GD3-/GFAP-/vimentin-. The kinetics of antigen expression of the GD3+ cells could best be interpreted by the following sequence: (sequence; see text) We interpret these results to show that the astrocytes arose from a small population of GD3+ glial precursor cells present in the brain that were co-isolated with oligodendroglia. No evidence was obtained that these GD3+ cells could also differentiate into oligodendrocytes.     

Secretion of nerve growth factor in cultures of glial cells and neurons derived from different regions of the mouse brain

The regional ability of central neurons and glial cells to produce nerve growth factor (NGF) was studied in vitro. NGF secretion was compared in cultures of perinatal astrocytes or embryonic neurons that were derived from various mouse brain structures. No regional differences were detected among cultures of post-natal day 2 glial cells of hippocampal, cortical, striatal, or mesencephalic origin. In all cases, levels of NGF released by the cells were very similar. They were closely correlated to the growth rate as shown by the fact that exponentially growing cells produced relatively more factor than did confluent cells, a finding in agreement with previous observations. Unlike growth-phase cells, primary astrocytes immediately plated at high cell density did not secrete any assayable factor before the 7th day of culture. Levels of NGF found during the following days remained low. In contrast, striking differences were observed among cultures of embryonic neurons. NGF was found in relatively large amounts in cultures of embryonic day 17 or 19 striatal neurons, whereas media conditioned by neurons from the mesencephalon, cortex, or septum contained much less factor. Amounts of NGF assayed in cultures of hippocampal neurons varied with the time of sampling of this brain structure. Levels of factor were significantly higher in media conditioned by embryonic day 19 neurons than in media of embryonic day 17 neurons. However, amounts of NGF found in supernatants of hippocampal neurons remained smaller than those present in cultures of striatal nerve cells. Altogether, the results suggest that, in addition to astrocytes, central neurons may also synthesize and secrete NGF in vitro and that this phenomenum is dependent on both the origin and the developmental stage of the neuronal population.     

Cellular distribution and regulation by cAMP of the GABA transporter (GAT-1) mRNA

The high-affinity GABA transporter in neurons and glial cells is the primary means of inactivating synaptic GABA. In the present study, a rat GABA transporter (GAT-1)-specific probe was used to quantitate GAT-1 mRNA in cultured neurons and glial cells from rat brain. GAT-1 mRNA is expressed in neurons but not in pure cultures of astrocytes. Incubation of neurons with forskolin led to concentration- and time-dependent decreases in GAT-1 mRNA. This effect could be also achieved by chronic exposure of neurons to 8-Br-cAMP and dib-cAMP but not with 1,9-dideoxyforskolin. This effect on the levels of GAT-1 mRNA correlates with a decrease in the Na⁺-dependent GABA transport activity in neurons. Treatment with agents that increase cellular levels of cAMP did not affect GABA transport or GAT-1 mRNA expression in glial cells.     

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.     

Effects of interleukin-1β and tumor necrosis factor-α on the expression of glial fibrillary acidic protein and transferrin in cultured astrocytes

Recent evidence suggests that interleukin (IL)-1 and tumor necrosis factor (TNF) may play a role in astrogliosis following injury to the CNS. The short-term biochemical effects of these immune-related cytokines were determined on cultured rat polygonal and process-bearing astrocytes. Both IL-1 and TNF stimulated the rate of thymidine incorporation in polygonal astrocytes up to 137% and 215%, respectively, over the level observed in untreated controls. By contrast, thymidine incorporation was relatively unaffected by these cytokines in process-bearing astrocytes. The cytokines did not significantly affect the level of glial fibrillary acidic protein (GFAP) within polygonal astrocytes, even though they appeared to downregulate the expression of GFAP mRNA by as much as 62%. Both cytokines increased the intracellular expression of transferrin (Tf) within some polygonal astrocytes. In untreated control cultures, fewer than 2% of polygonal astrocytes were immunoreactive for Tf. By contrast, approximately 30% of polygonal astrocytes treated with IL-1 or TNF-α became strongly immunoreactive for Tf. Neither IL-2 nor a number of other known growth factors appeared to alter the level of immunoreactive Tf in these cells. Process-bearing astrocytes were negative for Tf, regardless of the treatment used. Northern blot analysis demonstrated that the level of Tf mRNA in cultures of polygonal astrocytes increased 148% above the level observed in untreated controls following treatment with either IL-1 or TNF, whereas no change was observed following treatment with IL-2. These results suggest that increased levels of particular cytokines known to be present in injured CNS can produce pronounced biochemical alterations within a subtype of cultured astrocytes. © 1993 Wiley-Liss, Inc.     

MeCP2 Differentially Regulate the Myelin MBP and PLP Protein Expression in Oligodendrocytes and C6 Glioma

MeCP2 (methyl-CpG binding protein 2), an epigenetic regulator, has been shown to regulate the function of neurons and glial cells. Our previous study has demonstrated that MeCP2 repress the myelin gene expression in rat oligodendrocytes but whether MeCP2 bind to myelin gene MBP and PLP is not yet known. Besides oligodendrocytes, C6 glioma also expresses myelin genes and could be used as a model system to study myelin gene expression. In the present study, we determined that MeCP2 directly bind to MBP, PLP, and BDNF promoter in oligodendrocytes. Further, it was found that MeCP2 differentially regulates the myelin gene expression in oligodendrocytes and C6 glioma. In contrast to oligodendrocytes, MeCP2 does not bind to promoter region of MBP and PLP in C6 glioma suggest that MeCP2 differentially regulates the gene expression in different cell types.     

Evidence for neuronal regulation of oligodendrocyte development: Cellular localization of platelet-derived growth factor α receptor and A-chain mRNA during cerebral cortex development in the rat

Oligodendrocyte responses in vitro to platelet-derived growth factor (PDGF) include proliferation, survival, migration, and changes in cell morphology and molecular expression. Studies of mixed glial cultures established that astrocytes secrete PDGF; thus astrocytes are considered to be key regulators of oligodendrocyte development in vitro. We previously demonstrated PDGF α receptor mRNA expression by oligodendrocyte progenitors and preoligodendrocytes during postnatal development of rat cerebral cortex. In the present study, we have mapped the spatial and temporal expression of PDGF A-chain ligand mRNA and α receptor mRNA to determine if the cell-cell interactions that form the basis for PDGF regulation of oligodendrocyte development in vitro are also present in vivo. By in situ hybridization (ISH) we demonstrate that at embryonic day 17 (E17) cells expressing receptor mRNA (PDGFRα+) are initially in the subventricular zone, at a distance from cells expressing ligand mRNA (PDGF+) in the cortical plate. By E20 PDGFRα+ cells are found throughout the corpus callosum and cortical gray matter. PDGF+ cells are restricted to the cortical plate prenatally and only appeared in the corpus callosum postnatally. Combined immunocytochemistry and ISH demonstrated the PDGF+ cells colocalized with neurofilament, but not with GFAP. These data establish that PDGF is expressed by neurons during PDGFRα+ oligodendrocyte progenitor migration from the subventricular zone to the corpus callosum and gray matter. Furthermore, neurons continue to express PDGF during the generation and differentiation of appropriate numbers of oligodendrocytes needed to myelinate axons as the nervous system matures. © 1996 Wiley-Liss, Inc.     

Possible involvement of 5alpha-reduced neurosteroids in adrenergic and serotonergic stimulation of GFAP gene expression in rat C6 glioma cells

Influence of adrenergic and serotonergic stimulation on glial fibrillary acidic protein (GFAP) gene expression in rat C6 glioma cells was first examined as an in vitro model experiment for investigating the neuronal regulation of glial cell differentiation. Stimulation of these cells with isoproterenol and serotonin elevated GFAP mRNA levels followed by an increase in its protein contents, thus suggesting that both adrenergic and serotonergic stimulation might induce the differentiation of the glioma cells. In addition, progesterone and its 5alpha-reduced metabolite dihydroprogesterone also elevated GFAP mRNA levels in rat C6 glioma cells, consistent with their stimulatory actions on GFAP gene expression observed in rat astrocytes. Further studies showed that the elevation of GFAP mRNA levels induced by isoproterenol and serotonin as well as progesterone was abolished by pretreatment of the glioma cells with finasteride, an inhibitor of 5alpha-reduced steroid production. Moreover, the stimulatory actions of isoproterenol and serotonin on GFAP gene expression were inhibited by pretreatment with a GABA(A) receptor antagonist bicuculline and a progesterone receptor antagonist RU486. These findings suggest that both adrenergic and serotonergic stimulation may indirectly activate GFAP gene expression probably through the production of 5alpha-reduced steroid metabolites in rat C6 glioma cells, proposing the possibility that 5alpha-reduced neurosteroids may play a potential role in the neuronal regulation of glial cell differentiation.     

Expression of myelin proteolipid and basic protein mRNAs in cultured cells

Studies were undertaken to investigate the regulation of myelin-specific mRNA expression in cultured cells. Three experimental systems were investigated: primary oligodendrocytes grown as enriched cell populations, primary oligodendrocytes grown in the presence of chick spinal cord neurons, and C6 cells. cDNA probes specific for the myelin proteolipid mRNA and the myelin basic protein mRNA were used to quantitate proteolipid and myelin basic protein mRNA levels in cells under different experimental conditions. C6 cells expressed less than 0.2% of the proteolipid mRNA that was expressed in primary oligodendrocytes. Primary oligodendrocytes expressed the myelin-specific mRNAs for at least 104 days in culture, and the level of these mRNAs in cultures was elevated fourfold by coculturing rat oligodendrocytes with chick spinal cord neurons.     

Trk A, B, and C are commonly expressed in human astrocytes and astrocytic gliomas but not by human oligodendrocytes and oligodendroglioma

Neurotrophins regulate the proliferation and differentiation of neurons in the central nervous system via a family of specialized receptors, including TrkA, TrkB, and TrkC. As little is known about their expression or potential role in human glial tissues and glial tumors, we undertook an immunohistochemical analysis of human glia, glioma tissues and cell cultures of glial tumors to characterize the expression of Trk family members (full-length TrkA, TrkB, the truncated form of TrkB, and TrkC). In normal human brain Trk A, B, and C immunoreactivity was found in neurons and some weak staining was also seen astrocytes. No Trk expression was seen on oligodendrocytes. Strong reactivity was seen in reactive astrocytes in a glial scar. In a total of 34 glioma tissue specimens, which included 16 astrocytic tumors (4 low-grade astrocytomas and 12 glioblastomas multiforme) and 15 oligodendrogliomas (8 low-grade and 7 anaplastic) as well as 3 oligoastrocytomas (WHO grade II), TrkA, B, and C immunoreactivity was observed exclusively in specimens from astrocytic gliomas (16/16), but not in any of the oligodendrocytic gliomas (0/15). In the oligoastrocytomas, staining was restricted to the astrocytic component. In the astrocytoma and oligodendroglioma specimens, Trk A, B, and C immunoreactivity was also seen in the surrounding reactive astrocytes. Trk expression was independent of age, sex or histological grade of the investigated tumors. In six primary cell cultures, one derived from human astrocytes and five established from malignant astrocytomas, only TrkA immunoreactivity could be detected, while TrkB (both full-length and truncated isoforms) and TrkC were absent. The TrkA expression in primary cell cultures decreased with continuous cell passaging, and no Trk could be detected in established cell lines derived from glioblastoma. In conclusion, our data suggest that in human glial tissues Trk A, B, and C may be expressed in a lineage-restricted manner, thereby distinguishing between astrocytes and oligodendrocytes in a marker-like fashion. Trk expression, like GFAP expression appears to be increased in activated (reactive)/ neoplastic astrocytes. Received: 15 January 1998 / Revised, accepted: 27 March 1998     

Expression pattern of galectin-3 in neural tumor cell lines

Galectin-3 is a member of the galectin family of beta-galactoside-specific animal lectins. Here we show that galectin-3 is constitutively expressed in 15 out of 16 glioma cell lines tested, but not by normal or reactive astrocytes, oligodendrocytes, glial O-2A progenitor cells and the oligodendrocyte precursor cell line Oli-neu. Galectin-3 is also expressed by one oligodendroglioma cell line, but not by primitive neuroectodermal tumor and 4 neuroblastoma cell lines tested so far. In all galectin-3 expressing cell lines, the lectin is predominantly, if not exclusively, localized intracellularly and carries an active carbohydrate recognition domain (shown for C6 rat glioma cells). Moreover, in contrast to primary astrocytes, glioma cells do not or only weakly adhere to substratum-bound galectin-3, probably reflecting an unusual glycosylation pattern. Our findings indicate that the expression of galectin-3 selectively correlates with glial cell transformation in the central nervous system and could thus serve as a marker for glial tumor cell lines and glial tumors.     

Influence of cell density and thyroid hormone on glial cell development in primary cultures of embryonic rat cerebral hemisphere

The influence of cell density and thyroid hormone (TH) on the development of astrocytes and oligodendrocytes was investigated in primary cultures prepared from rat cerebral hemisphere on embryonic day (E)18. At the beginning of the culture, most of the cells were microtubule-associated protein 2 (MAP2)-positive neurons, whereas O1-positive oligodendrocytes and glial fibrillary acidic protein (GFAP)-positive astrocytes were rarely observed. After the cells were maintained in serum-free defined medium, astrocytes developed at high cell density but rarely at a low one. When leukemia inhibitory factor (LIF) was supplemented in low-density cultures, the levels of GFAP expression markedly increased to almost the same extent as in high-density culture without TH. This suggests that, in low-density cultures, astrocyte progenitors could not differentiate because of insufficient astrocyte-inducing factors. Interestingly, the addition of TH increased GFAP expression levels only at high density. The number of oligodendrocytes increased with TH addition at both cell densities, although the effects were more remarkable at high density. These results suggest that cell density and TH are pivotal factors in the development of both astrocytes and oligodendrocytes. It is also suggested that the effects of TH on glial cell development could be accelerated via cell-cell communications.     

Immunocytochemical Localization of Glia-Derived Nexin,Laminin and Fibronectin on the Surface or Extracellular Matrix of C6 Rat Glioma Cells,Astrocytes and Fibroblasts

The expression and cellular distribution of glia-derived nexin (GDN), laminin and fibronectin on C6 rat glioma cells, rat brain astrocytes and rat fibroblasts were investigated by immunoblotting and immunocytochemistry. Western blot analysis of C6 cell homogenates confirmed the specificities of the antibodies. Immunocytochemical staining of C6 cells, astrocytes and fibroblasts showed that laminin, fibronectin and GDN were abundant on the surface of glioma cells and astrocytes whereas on fibroblasts fibronectin was abundant though only traces of GDN and laminin could be detected. The light microscopy data were confirmed by ultrastructural studies showing that each antigen was present on the surface of the C6 rat glioma cells as numerous spots with slightly different distribution patterns for each of the antigens. In fibroblast cultures, the antigens were also localized in the extracellular matrix in the vicinity of the cells. Migrating fibroblasts but not migrating glioma cells or astrocytes deposit the matrix-proteins onto the substratum leaving behind a track of GDN, laminin and fibronectin. When the cells were treated with heparin prior to antibody incubation, the GDN immunoreactivity completely disappeared, whereas the distribution and abundance of laminin and fibronectin was not affected. Our data show that GDN binds, possibly by a heparin-like molecule, to the outer surface of cells or to the extracellular matrix and may protect cells and matrix proteins against proteolytic degradation.     

Neuronal and non-neuronal catechol-O-methyltransferase in primary cultures of rat brain cells

Previous biochemical and histochemical studies have suggested that catechol-O-methyltransferase (COMT) is a predominantly glial enzyme in the brain. The aim of this work was to study its localization and molecular forms in primary cultures, where cell types can be easily distinguished with specific markers. COMT immunoreactivity was studied in primary astrocytic cultures from newborn rat cerebral cortex, and in neuronal cultures from rat brain from 18-day-old rat embryos using antisera against rat recombinant COMT made in guinea pig. Double-staining studies with specific cell markers to distinguish astrocytes, neurons and oligodendrocytes were performed. COMT immunoreactivity colocalized with a specific oligodendrocyte marker galactocerebroside in cells displaying oligodendrocyte morphology, flat cells displaying type-1 astrocyte morphology and glial fibrillary acidic protein, in branched cells displaying type-2 astrocyte morphology and in cell bodies of neurons, the processes of which displayed neurofilament immunoreactivity. Western blots detected both soluble 24 kDa and membrane-bound 28-kDa COMT proteins in neuronal and astrocyte cultures. The results suggest that COMT is synthesized by cultured astrocytes, oligodendrocytes and neurons.