Pure astrocyte cultures derived from cells isolated from mature brain

Enriched preparations of oligodendrocytes, isolated either from adult bovine brain or from 30-day-old rat brain, eventually yield cultures in MEM-15% calf serum that contain, in addition to oligodendrocytes, proliferating astrocytes and variable numbers of fibroblast-like cells. If these cultures are switched to a serum-free defined medium during the 1st week, mixed cultures containing only oligodendrocytes and astrocytes are obtained. Bovine cultures can be replated and purified by selective adhesion to yield cultures that are greater than 99% astrocytes; similar procedures were not successful with rat cultures. Cytoskeletal preparations of the purified astrocyte cultures from mature bovine brain contain both vimentin and glial fibrillary acidic protein (GFAP), but vimentin is by far the major intermediate filament protein. Thus, the intermediate filament composition of these astrocytes is similar to that of astrocytes in primary cultures obtained from neonatal rat brain. Immunofluorescent studies of these cultures at 24 hr in vitro show that there are no GFAP+ cells in cultures of either species; the bovine cultures contain greater than 95% GC+ cells; and the rat cultures contain 90% GC+ cells. After a few days in vitro flat cells appear that are vimentin+/GFAP-/GC-. In serum-free medium these cells eventually become vimentin+/GFAP+. We propose that the astrocytes that grow in these cultures arise from a population of glial precursor cells, which are present even in adult brain and are isolated together with oligodendroglia, and that they do not derive from contaminating mature astrocytes. Thus, the astrocytes in our cultures may have the same origin as astrocytes grown in culture from dissociated neonatal brain.     

Infection of rat brain primary cell cultures with an avirulent A7 strain of Semliki Forest virus

The ability of A7 Semliki Forest Virus (SFV) to infect primary brain cell cultures has been examined using cultures prepared from 1-2-day neonatal rat cerebral hemispheres. These cultures, characterised immunocytochemically using cell-specified markers, contain mainly GFAP+ protoplasmic astrocytes and smaller multiprocessed A2B5+ cells, probably fibrous astrocytes. 10% of the cells are GC+ oligodendrocytes and some neurones are also present. These cultures support virus growth and a cytopathic effect was observed. Using double labelling techniques with the cell-specific markers and anti-SFV antibody A7 has been shown to readily infect cells which carry either the A2B5+ antigen or galactocerebroside marker. Protoplasmic astrocytes (GFAP+/A2B5-) are not readily infected under the conditions used. The protein labelling studies using [35S]methionine show that host cell protein synthesis is not completely shut off and continues in the astrocyte protein region. These results suggest that cells derived from a common A2B5+, GFAP-, GC- progenitor glial cell, i.e. GC+ oligodendrocytes and A2B5+/GFAP+ fibrous astrocytes, are more readily infected than other brain cell types including the protoplasmic astrocytes.     

Embryonic divergence of oligodendrocyte and astrocyte lineages in developing rat cerebrum

Oligodendrocyte and astrocyte lineages were traced in rat forebrain sections using single- and double-label immunoperoxidase and indirect immunofluorescent techniques. Antibodies were directed against antigenic markers, the expressions of which overlapped in time: GD3 ganglioside in immature neuroectodermal cells; vimentin in radial glia; glial fibrillary acidic protein (GFAP) in astrocytes; and carbonic anhydrase (CA) and galactocerebroside (GC) in oligodendrocytes. A histochemical stain for iron was also used as a marker of oligodendrocytes. Small cells of the subventricular zone (SVZ) were stained with anti-GD3 but not with the other antibodies. By 16 d of gestation (E16), the SVZ generated large, round cells and thick, process-bearing cells that were GD3+/CA+/iron+. These cells then appeared in the cingulum and, with time, increased in numbers and extended thick processes as they filled the subcortical white matter. These cells eventually lost their reactivity to anti-GD3 but became GC+/CA+ with processes extending to myelin sheaths. At E15 radial glia were stained with the anti-vimentin antibody but were negative for GFAP. At birth, only the vimentin+ radial glia midline between the 2 ventricles were GFAP+, but with time more vimentin+ cells became GFAP+. By 7 d of postnatal age all the vimentin+ cells were GFAP+ and had converged predominately on the cingulum. With time these cells condensed and took on characteristic shapes of astrocytes. The embryonic separation of the oligodendrocyte and the astrocyte lineage is supported by four pieces of evidence: (1) GD3+ cells were double labeled with anti-CA, and then went on to become GC+; (2) vimentin+ and GFAP+ cells were not also GD3+; (3) ultrastructural localization of anti-GD3 was confined to cells with characteristics consistent with developing oligodendrocytes; and (4) the shapes of GD3+, CA+, GC+, or iron+ cells did not resemble those of the vimentin+ or GFAP+ cells.     

Differentiation of astrocytes and oligodendrocytes from germinal matrix cells in primary culture

Primary cultures derived from neonatal rat forebrain grow almost entirely as glial cultures, with a large astrocytic preponderance and smaller numbers of oligodendrocytic cells. Although both astrocytic and oligodendrocytic characteristics are acquired in vitro, the origins of both types of glia in primary cultures have not been determined. We tested the hypothesis that glia differentiate in vitro from immature neuroectodermal cells by following the fate of germinal zone cells in primary cultures. A monoclonal antibody that binds GD3 ganglioside was used as a marker for cells of the subventricular zone (SVZ), since antibody binding in newborn rat forebrain could be detected by immunofluorescence only in the SVZ of newborn rats (Goldman et al., 1984). We followed the expression of glial fibrillary acidic protein (GFAP), an astrocytic marker; galactocerebroside (GC), an oligodendrocytic marker; and GD3 during the first several weeks of culture. Both GFAP and GC expression were first detected in cells that bound the GD3 antibody. Astrocytes developed during the first week in vitro; eventually, they lost the ability to bind the GD3 antibody and most became GD3-/GFAP+ cells. In high-density cultures, a population of small cells that resided on top of the astrocytic monolayer retained GD3 expression. GC-antibody binding was first observed in these cells of the upper layer, although it was not readily apparent until the second week of culture. Few GC+ cells were seen in cultures grown at low density, however.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunocytochemical characterisation of cell cultures grown from dissociated 1-2-day post-natal rat cerebral tissue. A developmental study

A range of cell-specific markers have been employed with immunocytochemical methods to characterise and quantitate the cell types present in mixed brain cell cultures derived from dissociated 1-2-day post-natal rat cerebral hemispheres and grown in the presence of FCS. Protoplasmic astrocytes (GFAP+, A2B5-) were the major cell type to develop in culture, a confluent monolayer forming in 5-8 days. A population of smaller round cells of oligodendrocyte-like morphology appeared on this astrocyte layer. Greater than 70% of these smaller cells were GC- and thus were not oligodendrocytes. The GC- cells were A2B5+ and, in early cultures, may therefore be progenitor glial cells. Examination of GFAP and A2B5 co-expression by these smaller cells was difficult due to the dense underlying GFAP+ astrocyte layer. In less dense areas of older cultures these smaller cells with processes were GFAP+ and A2B5+: these are Type 2, fibrous astrocytes. GC+ oligodendrocytes, comprising 5-10% of the total identified cell population, were initially distributed over the astrocyte monolayer; in older cultures (after about 8 days) GC+ cells were observed in clumps over places where NF+ cells were identifiable. Such GC+ cells mostly became MBP+. Neurones accounted for about 6% of the identifiable cells in early cultures but a lower percentage in older cultures. Minor populations of ependymal cells and macrophages were present; cells displaying fibronectin, fibroblasts, were rarely identified. Use of horse serum in place of FCS gave lower yields of GC+ cells in cultures, slowed down astrocyte development, and resulted in the formation of trunks of GFAP+ cells throughout cultures. Other sera gave lower numbers of GC+ cells.     

Comparative biochemical, morphological, and immunocytochemical studies between C-6 glial cells of early and late passages and advanced passages of glial cells derived from aged mouse cerebral hemispheres.

We have used C6 glial cells (2B clone), early and late passage, as well as advanced passages (8-17) of glial cells derived from aged (18-month-old) mouse cerebral hemispheres (MACH), as model systems for studying glial properties. In this study passages 20-24 were considered "early" and passages 73-90 were considered "late." Activities of glutamine synthetase (GS) and cyclic nucleotide phosphohydrolase (CNP) were used as biochemical markers for astrocytes and oligodendrocytes, respectively. Glial phenotypes were identified immunocytochemically using double staining for glial fibrillary acidic protein (GFAP) and A2B5 antigen (type 1 and type 2 astrocytes) or galactocerebroside (GalC) and A2B5 antigen (oligodendrocytes); cells positive for A2B5 and negative for both GFAP and GalC were considered to be precursor cells. Cultures were grown either in DMEM supplemented with 10% fetal bovine serum or in serum-free chemically defined medium (CDM) supplemented with insulin and transferrin. We report that early-passage C6 glial cells continue to be bipotential cells and when grown in the absence of serum express high GS and CNP activities correlating with the high number of GFAP- and GalC-positive cells, respectively. Late-passage cells continued to be committed to the type 2 astrocytic phenotype regardless of media composition (+/- serum). MACH cultures consist of protoplasmic type 1 astrocytes, differentiated type 2 astrocytes, and oligodendrocytes as well as glial progenitor cells. When these cultures were grown in CDM+transferrin, both GS and CNP activities increased, suggesting that transferrin has provided the signal for progenitor cells present in these cultures derived from aged brain to differentiate into type 2 astrocytes and oligodendrocytes.     

Developmental appearance, antigenic profile, and proliferation of glial cells of the human embryonic spinal cord: an immunocytochemical study using dissociated cultured cells.

We have investigated the time of appearance of the earliest differentiating glial cell types of human spinal cord using a panel of antigenic markers to identify them in cultures from 6- to 9-week-old human embryos. Immunolabeling performed at 14 h in vitro with the O4 mAb, an early oligodendrocyte marker, showed the presence of oligodendrocytes during the 7th week of age. At 8 weeks only a few of the O4+ cells expressed galactocerebroside (GalC), a marker of more differentiated oligodendrocytes. All the O4+ and GalC+ cells were vimentin+ and some of the GalC+ cells were A2B5+, GD3+ and SSEA-1+. During the first week in vitro many of the O4+ cells exhibiting a more immature, bi- or tri-polar morphology incorporated [3H]thymidine into their nuclei. Cells expressing the astrocyte-specific marker GFAP could be first observed at 8 weeks; almost all of these GFAP+ cells, which should correspond to radial glia on the basis of the current literature, were vimentin+, A2B5+, GD3+, and SSEA-1+. At 2 days in vitro incorporation of [3H]thymidine could be shown in a small fraction of these cells. The finding that radial glia and oligodendrocytes expressed similar antigenic features and the additional observation that a small, but consistent fraction of the cells were simultaneously labeled by O4 and anti-GFAP antibodies support the hypothesis that, in the human spinal cord, radial glial cells can give rise to both oligodendrocytes and astrocytes; in this respect, radial glial cells may be similar to the A2B5+, GD3+, vimentin+ bipotential glial progenitors previously identified in cultures from developing rat CNS, which also express A2B5, GD3, and vimentin.     

Bipotential glial precursor cells of the optic nerve express the NG2 proteoglycan

We have studied the expression of the NG2 chondroitin sulfate proteoglycan on bipotential glial precursor cells in cultures of postnatal rat optic nerve. Purified populations of these precursor cells were prepared by panning dissociated optic nerve cells on dishes coated with monoclonal A2B5 antibody. Using immunofluorescence double staining, we found that NG2 was present on almost 95% of the purified A2B5+ precursor cells. The NG2 core protein from optic nerve cells was identified by immune precipitation and PAGE and was found to be identical to the 300,000 Da NG2 core protein from a clonal rat cell line B49. Over a culture period of 5 d in medium containing 10% fetal calf serum, more than 80% of the NG2+ precursor cells acquired the glial fibrillary acidic protein (GFAP), an astrocyte-specific marker. Under these conditions, fewer than 10% of the NG2+ cells expressed galactocerebroside (GC), an oligodendrocyte-specific marker. These GFAP+GC- type II astrocytes continued to express the NG2 antigen for up to 10 d in culture. During a 5 d culture period in hormonally supplemented, serum-free medium, fewer than 15% of the NG2+ cells expressed GFAP, while up to 40% expressed GC. The NG2 antigen continued to be expressed for only a short period of time by these GFAP-GC+ oligodendrocytes, so that mature oligodendrocytes in the cultures became NG2-. These results support our previous suggestion that the NG2 antigen is found on a class of neural cells that can differentiate along more than one pathway.     

A distinct type of GD3+, flat astrocyte in rat CNS cultures.

We have identified what is apparently a distinct type of astrocyte in primary cultures from several regions of the neonatal rat CNS. These cells express GD3 ganglioside for long periods in vitro, and are GFAP+, but do not express the oligodendrocyte antigens O4 or galactocerebroside (GC). The majority, but not all, are A2B5+. The cells grow in a flat, highly spread morphology with many thin cytoplasmic processes. Gene transfer with a replication-deficient retrovirus combined with immunostaining for astro- and oligodendroglial markers (antibodies to GFAP, GD3 ganglioside, GC, and the A2B5 and O4 antibodies) demonstrated that in the neonatal rat CNS cultures these cells are clonally separate from oligodendrocytes and from the majority of (GD3-) astrocytes. The clonal analysis suggests a distinct progenitor cell and a distinct developmental sequence for these astrocytes.     

Expression of plasmolipin in oligodendrocytes.

Plasmolipin is a plasma membrane proteolipid which has recently been described as a component of myelin (Cochary et al.: Journal of Neurochemistry 55:602-610, 1990). The present study reports the expression and localization of plasmolipin in primary glial cultures and secondary oligodendrocyte cultures. Double-label immunofluorescence showed that plasmolipin was expressed by galactocerebroside (GC)-positive oligodendrocytes, but was absent from astrocytes, characterized by their positive staining for glial fibrillary acidic protein (GFAP). At 1 week in culture plasmolipin staining was relatively weak in the cell body of some of the GC-positive cells. During the following 3 weeks in culture plasmolipin staining of oligodendrocytes gradually increased and was present in the cell body, its plasma membrane, and all the processes. However, the plasmolipin antibodies did not stain regions of the flat membrane sheets. Western blot analysis of homogenates from primary glial cultures showed that plasmolipin levels gradually increased during the first 5 weeks in culture. We conclude that the presence of plasmolipin in myelin is a result of its expression by oligodendrocytes.     

Injuring neurons induces neuronal differentiation in a population of hippocampal precursor cells in culture

A novel population of hippocampal precursor cells (HPCs) that can be induced to differentiate into astrocytes and oligodendrocytes can be derived from hippocampal cultures grown in serum-free media. The HPCs are PDGF-responsive, do not proliferate with bFGF, and grow as sheets of cells rather than gathering into neurospheres. The HPCs share many markers (A2B5, GD3, poly-sialylated neuronal common adhesion molecule (PSA-NCAM), and NG2) with oligodendrocyte precursor cells (OPCs). The HPCs do not express markers for mature neurons, astrocytes, or oligodendrocytes. Like OPCs, the HPCs differentiate into glial fibrillary acidic protein (GFAP)+ astrocytes and GalC+ oligodendrocytes with the addition of bone morphogenetic protein-4 (BMP-4) and triiodothyronine (T3), respectively. They do not differentiate into neurons with the addition or withdrawal of basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor (BDNF), or retinoic acid (RA). These HPCs can be stimulated to differentiate into neuron-like cells by the induction of neuronal injury or cell death in nearby cultured neurons or by conditioned medium from injured neuronal cultures. Under these conditions, HPCs grow larger, develop more extensive dendritic processes, become microtubule-associated protein-2-immunoreactive, express large voltage-dependent sodium currents, and form synaptic connections. The conversion of endogenous pluripotent precursor cells into neurons in response to local brain injury may be an important component of central nervous system homeostasis.     

Differentiation of oligodendrocytes cultured from developing rat brain is enhanced by exogenous GM3 ganglioside

Cultures consisting primarily of O-2A progenitor cells and immature oligodendrocytes with a few microglia and astrocytes were obtained by shaking primary cultures from neonatal rat brain after 12--14 days in vitro. Addition of 50 μg/ml exogenous Neu-NAcα2-3Galβ1-1′ ceramide (GM3 ganglioside) to the cultures resulted in an increase in the number and thickness of cell processes that stained intensely for sulfatide and galactocerebroside (galC) in comparison to control cultures without added GM3. The treated cultures also contained fewer astrocytes than control cultures as revealed by immunostaining for glial fibrillary acidic protein (GFAP). Cells that immunostained for both GFAP and sulfatide/galC were very rare in control cultures but were frequently seen in the GM3-treated cultures, suggesting that these may represent cells changing their direction of differentiation away from type II astrocytes toward oligodendrocytes under the influence of GM3. These effects on the developing rat oligodendrocytes were specific for GM3 ganglioside and were not produced by adding GM1, GM2, GD3, or GD1a to the cultures. Lactosyl ceramide and neuraminyl lactose were also ineffective. When control cultures were initially plated on polylysine and incubated with [¹⁴C]galactose, GD3 was the principal labeled ganglioside. However, as the control cells differentiated over time in culture without the addition of exogenous GM3 and produced increasing amounts of myelin-related components, the incorporation of [¹⁴C]galactose into endogenous GM3 increased to become the predominant labeled ganglioside by 6 days after plating. Metabolic labeling of the GM3-treated oligodendrocytes with [¹⁴C]galactose revealed increased incorporation into galC and sulfatide in comparison to control cultures, but a decreased labeling of endogenous GM3. Similarly, incorporation of an amino acid precursor into the myelin-associated glycoprotein (MAG) was increased by GM3 treatment, but incorporation into myelin basic protein (MBP) was not affected. Although the overall effect of added GM3 was to decrease the phosphorylation of most proteins in the oligodendrocytes, including MBP, GM3 enhanced the phosphorylation of MAG. These findings indicate that GM3 ganglioside has an important role in the differentiation of cells of the O-2A lineage toward myelin production, since differentiation is associated with increased metabolic labeling of endogenous GM3 in control cultures and is enhanced by the addition of exogenous GM3. © 1994 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Cells positive for the O4 surface antigen isolated by cell sorting are able to differentiate into astrocytes or oligodendrocytes

Cells expressing the surface antigen O4 were isolated as pure populations from cultures of murine brain or cerebellum using fluorescence activated cell sorting. When these O4-positive cells were further cultured in the presence of fetal calf serum (FCS) many cells expressed both O4 and the astrocyte marker glial fibrillary acidic protein (GFAP) after 4 days of culture. Cells not exposed to FCS expressed O4, but never GFAP. GFAP-positive cells in the presence of fetal calf serum very rarely expressed the myelin associated glycoprotein (MAG) or O1, both of which are expressed on more differentiated oligodendrocytes, and never expressed O10 that is characteristic of even more mature oligodendrocytes. These results show that glial cells expressing O4, but not MAG, O1, O10 or GFAP are bipotential precursor cells able to differentiate into astrocytes or oligodendrocytes depending on the culture conditions and suggest that bipotentiality of glial precursor cells is retained up to a later developmental stage than that of the O2A progenitor cell.     

Transient expression of glial fibrillary acidic protein in developing oligodendrocytes in vitro

Expression of galactocerebroside (GC) and glial fibrillary acidic protein (GFAP) was studied in oligodendrocyte-enriched cultures of newborn mouse brains. In the cultures, GC was detectable as early as 1 day in vitro (1-DIV). Using double immunofluorescence labeling, some GC-positive cells were also stained homogeneously by the anti-GFAP serum. The intensity of GFAP staining increased until 9-DIV when the GC-positive cells revealed the typical morphology of oligodendrocytes, and the GFAP staining faded thereafter. The GFAP staining pattern of the GC-positive cells was not changed upon exposure to demecolcine, even though the GC-negative, GFAP-positive astrocytes showed perinuclear aggregation of GFAP. No intermediate filaments were observed in the oligodendrocytes by electron microscopy. The results suggest that the oligodendrocytes may have soluble GFAP in a certain period of early development.     

Ganglioside GD3 in radial glia and astrocytes in situ in brains of young and adult mice

Ganglioside GD3 occurs in immature cells in the neuroectoderm. However, with regard to particular cellular locations of GD3, rat brain has received more attention than mouse brain. In brains from neonatal mice the most intense GD3 immunostaining appears to occur in structures that differ from those that immunostain the most intensely in brains from neonatal rats (Cammer and Zhang: J Histochem Cytochem 44: 143–149, 1996). In the present study epifluorescence and confocal microscopy were used for the purpose of identifying the types of GD3-immunopositive structures in brains of neonatal, 2-week-old, and adult mice. Vibratome sections from mouse brains were double immunostained for GD3 and respective markers for macrophages, microglia, and cells belonging to the oligodendrocyte lineage. Surprisingly, none of those marker antigens immunostained intensely in the same respective structures as GD3. The GD3-positive structures, however, did resemble protoplasmic astrocytes and radial glia, some with GD3-positive end-feet at the glia limitans; however, we did not rule out the possibility that there might be some GD3 on the surfaces of prooligodendroblasts. The scarcity of glial fibrillary acidic protein (GFAP)-positive cells in brains of neonatal mice made it impractical to look for GD3+/GFAP+ structures that might belong to the astrocyte lineage. The Mu subunit of glutathione-S-transferase (Mu) was shown to label radial glia and the few GFAP-positive cells in brains of neonatal mice. Subsequently, confocal microscopy showed Mu and GD3 to be colocalized in radial glia and protoplasmic astrocytes in the neonate. In brains from mice ≥2 weeks of age GD3 immunostaining was demonstrated in GFAP-positive astrocytes, including reactive astrocytes. Much of the GD3 appeared to occur at the tips of astrocyte processes. It is suggested that GD3 in radial glia and astrocytes may function as a ligand enabling recognition of those structures by neurons or as a precursor of more complex gangliosides in neurons. © 1996 Wiley-Liss, Inc.     

Gliogenesis in rat spinal cord: evidence for origin of astrocytes and oligodendrocytes from radial precursors

We have examined glial cell lineages during rat spinal cord development by using a variety of antibodies that react with immature and mature glia. Radial glia in embryonic cord bound 1) A2B5, an antibody that reacts with a glial precursor cell population in optic nerve; 2) AbR24, which is directed against GD3 ganglioside and binds to immature neuroectodermal cells and to developing oligodendrocytes in forebrain and cerebellum; and 3) an antibody to the intermediate filament, vimentin. With time, two different populations emerged, both of which seemed to be derivatives of radial cells. One cell type expressed the astrocyte intermediate filament, GFAP, in addition to vimentin. GFAP-containing cells eventually took on the forms of astrocytes in gray and white matter. The other type expressed carbonic anhydrase, an enzyme characteristic of oligodendrocytes and enriched in myelin. Carbonic anhydrase-positive cells eventually developed into small cells with oligodendrocyte morphology. Our observations suggest a common lineage for astrocytes and oligodendrocytes from radial cells during spinal cord gliogenesis.     

Characterization of glial subpopulations in cultures of the ovine central nervous system

The cellular composition and in vitro development of glial cultures derived from the rat CNS has been well studied. However, less information is available on similar cultures from other species, particularly higher mammals. To study ovine glial development in vitro, cultures from 50-day fetal to adult animals were characterized with various immunocytochemical markers, which are frequently used to define neural cell subsets in rat cultures. As in rats, both A2B5+ and A2B5- astrocytes can be identified in ovine cultures. However, ovine A2B5+ and A2B5- could not be reliably differentiated by their morphology, which was more influenced by whether the cells were in serum-free or serum-containing media than by their A2B5-positive or -negative status. In addition, ovine A2B5+ astrocytes were present in cultures from early fetal brain before the development of identifiable oligodendrocytes, unlike rat type II astrocytes, which develop only after the appearance of oligodendrocytes. An A2B5+ cell, morphologically similar to the rat 02-A cell, can be found in cultures from fetal ovine cerebrum or cerebellum. A2B5+/glial fibrillary acidic protein (GFAP)- cells in cultures from 100- to 115-day ovine cerebellum appeared to differentiate into A2B5+ astrocytes in serum-containing media. However, in serum-free media, although the A2B5+ cells assumed a more "oligodendroglial-like" morphology, they did not express galactocerebroside or myelin basic protein, suggesting that these cells may not be bipotential as is the rat 02-A cell. Oligodendroglial differentiation was not induced by treatment with dibutyryl cyclic AMP or insulin-like growth factor I. Many cells in cultures from a variety of fetal ages did not label with any of the immunocytochemical markers used, suggesting the need for more cell-type-specific markers to identify neural cell subsets in higher mammals.     

Mesencephalic type I astrocytes mediate the survival of substantia nigra dopaminergic neurons in culture

We previously demonstrated that substantia nigra (SN) support cells selectively increase SN dopamine (DA) neuron survival in dissociated primary culture. Increased survival was elicited specifically by nigral support cells; glia from other brain regions exerted lesser effects. We now report that Type I astrocytes, the principal component of SN support cell monolayers, mediate the enhanced DA cell survival. Initially, the predominant glial subtypes in SN support cell cultures were identified. Postnatal day 1 rat SN was dissociated and cells were grown to confluence (7-9 days in vitro; DIV). Monolayers were immunostained with antibodies against glial fibrillary acidic protein (GFAP; an astrocyte-specific marker), myelin basic protein (MBP; an oligodendrocyte marker), or A2B5 (recognizes 0-2A progenitors and Type II astrocytes). The number of GFAP+ cells far exceeded MBP+ and A2B5+ cells, suggesting that astrocytes constituted the predominant subpopulation. Further, direct comparison of GFAP+ (Type I and Type II astrocytes) and A2B5+ (Type II astrocytes) cells indicated that the vast majority were Type I astrocytes. Greater than 98% of cells reacted with glial antibodies. To definitively characterize the cellular subtype that augments survival of DA neurons, glial subcultures were established. At 2 DIV, enriched populations of Type I or Type II astrocytes, or oligodendrocytes, were tested for the ability to elicit DA neuron survival. Embryonic day 16 rat SN dissociates were added and DA cell number was assessed with antibody against tyrosine hydroxylase (TH), the DA biosynthetic enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of gangliosides on glial and neuronal cells in normal and pathological adult human brain

Few studies have assessed the glycolipid phenotype of glial cells in the human central nervous system (CNS) in situ. We investigated by immunohistochemistry the expression and cellular distribution of a panel of gangliosides (GM1, GM2, acetyl-GM3, GD1a, GD1b, GD2, GD3, GT1b, GQ1b and the A2B5 antibody) in adult, human normal and pathological brain, namely multiple sclerosis (MS) and other neurological diseases (OND). In normal conditions, we found diffuse expression in the white matter of most gangliosides tested, with the exception of acetyl-GM3, GT1b and GQ1b. By double immunofluorescence with phenotypic markers, GM1 and GD1b were preferentially expressed on GFAP+ astrocytes, GD1a on NG2+ oligodendrocyte precursors, A2B5 immunostained both populations, while GD2 was selectively present on mature oligodendrocytes. In the gray matter, only GM1, GD2 and A2B5 were present on neuronal cells. Interestingly, those gangliosides present on astrocytes in normal conditions were preferentially expressed on NG2+ cells in chronic MS lesions and in OND. Selective expression of GT1b upon astrocytes and NG2+ cells was instead observed in MS lesions, but not in OND. The definition of the glycolipid phenotype of CNS glial cells may be useful to identify distinct biological glial subsets and provide insights on the potential autoantigenic role of gangliosides in CNS autoimmune diseases.