AMPA receptor protein expression and function in astrocytes cultured from hippocampus.

Glutamate receptors guide the proliferation, migration, and differentiation of glial cells. Here, we characterize AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid) and NMDA receptor protein expression and function and mRNA expression in hippocampal glial cultures. By immunocytochemistry, GluR2 (the subunit that limits the Ca(2+) permeability of AMPA receptors) exhibited prominent labeling in hippocampal glial cultures. Double-labeling of GluR2 with GFAP and with A2B5 revealed GluR2 subunit expression on type-1 and type-2 astrocyte lineage cells. GluR1 subunit expression was more prominent in type-1 than in type-2 astrocytes. To characterize functional properties of glutamate receptors expressed in cultured hippocampal astrocytes, we performed whole-cell patch clamp recording. Application of L-glutamate, AMPA, and kainate, but not NMDA, to small, rounded cells (morphologically identified as type-2 astrocytes) elicited inward currents which were blocked by the AMPA/kainate antagonist 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX). Cyclothiazide potentiated AMPA- and kainate-elicited currents, indicative of AMPA-preferring receptors. Current voltage analysis indicated that type-2 astrocyte AMPA receptors were electrically linear, indicative of GluR2-containing, Ca(2+)-impermeable AMPA receptors. By Northern blot analysis, GluR1 mRNA was highest in astrocyte cultures from cerebellum and hippocampus and moderate in astrocyte cultures from neocortex and striatum. GluR3 mRNA was detectable in astrocyte cultures from cerebellum and neocortex. GluR2 and NR1 mRNA expression were not detected in astrocytes cultured from any brain region examined. In situ hybridization studies showed wide expression of GluR1 mRNA in cultured astrocytes; GluR2 and GluR3 mRNAs were near background levels. Thus, cultured type-2 astrocytes express functional AMPA receptors in a cell-specific and region-specific manner, consistent with their role in neuronal-glial communication.     

Interferon-gamma induced IA antigen expression on cultured neuroglial cells and brain macrophages from rat spinal cord and cerebrum.

The inducibility of major histocompatibility complex class II (Ia) antigens on glial cells of the brain suggests that neuroglia have immunoregulatory functions within the central nervous system (CNS), i.e., recognition and presentation of antigens. The aim of the present study was to investigate rat recombinant-interferon-gamma (r-IFN-gamma) induced Ia antigen expression in rat cerebral cultures containing type-1 astrocytes and macrophages, and in rat spinal cord cultures enriched in type-2 astrocytes or oligodendrocytes. We compared induction of Ia antigen expression in glial cell cultures derived from Lewis rats, which are very susceptible to experimental allergic encephalomyelitis (EAE), with those from Wistar rats, which are but modestly EAE susceptible. After 5 days in culture we found in Wistar rat type-1 astrocyte-enriched cultures that Ia antigens were expressed by 19% of the astrocytes, whereas we found that in Lewis rat type-1 astrocyte cultures a considerably higher number of astrocytes expressed Ia antigens (53%). However, no significant difference were found in Ia antigen expression between type-2 astrocytes derived from Wistar rat spinal cord (49%) and Lewis rat type-2 astrocytes (56%). In contrast, in oligodendrocyte-enriched cell cultures derived from either Lewis or Wistar rats no Ia antigen expression was found. Interestingly, we found in type-1 astrocyte-enriched cerebral cultures a large number (approx. 46% of the cells) of brain macrophages (amoeboid microglia), all expressing Ia antigens after treatment with r-IFN-gamma.     

Transferrin gene expression and secretion by rat brain cells in vitro

We have previously shown by immunocytochemistry in rat primary glial cultures that transferrin (Tf) is an early developmental marker for oligodendrocytes. The present work addresses the issue of Tf gene expression and synthesis by neural cells in vitro. For this purpose, we used rat embryonic neuronal cultures and newborn glial cultures of astrocytes and oligodendrocytes. Cultured fibroblasts and C6 glioma cells were used as negative controls. We found that Tf mRNA is present in oligodendrocytes, astrocytes, and neurons. However, oligodendrocytes and astrocytes, but not neurons, were shown to synthesize and secrete Tf. Neither fibroblasts nor C6 glioma cells expressed detectable amounts of Tf mRNA. Tf mRNA levels in astrocyte cultures appeared to be under hormonal control since hydrocortisone markedly reduced message levels. These results show that both astrocytes and oligodendrocytes can synthesize and secrete Tf under cell culture conditions. However, epigenetic factors, such as hydrocortisone, may repress the expression of Tf in astrocytes in vivo.     

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.     

Transplanted cultured type-1 astrocytes can be used to reconstitute the glia limitans of the CNS: the structure which prevents Schwann cells from myelinating CNS axons

Transplantation of different glial cells into areas of demyelination made in the adult rat spinal cord allows insights into the cell-cell interaction necessary to reconstruct a glial environment around demyelinated axons. Such studies have shown that type-1 astrocytes are central to the exclusion of Schwann cells from areas of glia-free demyelination. However, for these cells to be established in a manner which prevents Schwann cell remyelination of CNS axons, cells of the O-2A lineage are also required. If cultures of isogeneic rat type-1 astrocytes and mouse O-2A cells are transplanted into lesions made in non-immunosuppressed animals. Schwann cell remyelination is limited and extensive oligodendrocyte remyelination is achieved. This paradigm creates a model of immune mediated demyelination in which the immune response is not primarily directed at oligodendrocyte specific epitopes.     

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.     

Tyrosine phosphorylated proteins decrease during differentiation of neuronal and glial cells.

Tyrosine kinases have been implicated in the development of the nervous system. To investigate their role, immunoblotting with phosphotyrosine antibodies was used to identify substrates of tyrosine kinases during glial and neuronal differentiation in the rat and mouse. Fourteen prominent phosphotyrosine-containing proteins were detected in oligodendrocyte-type 2 astrocyte (O2A) progenitor cells. When O2A cells differentiated into type 2 astrocytes, a phosphotyrosine-modified protein of 74 kilodaltons (kDa) decreased 15-fold in abundance, and phosphotyrosine-containing proteins of 36-40 kDa declined. When O2A cells differentiated into oligodendrocytes, a prominent 71-kDa phosphotyrosine-modified protein became undetectable. During retinoic acid-induced neuronal differentiation of the mouse embryonal carcinoma cell line P19S101A1 (P19), an 80-kDa phosphotyrosine-containing protein decreased from high levels in the undifferentiated state to undetectable levels after 96 h in aggregation. Retinoic acid treatment also caused a rapid decrease in levels of a 96-kDa phosphotyrosine-containing protein. Cell-cell contact occurring as a result of aggregation resulted in decreases in 130- and 90-kDa phosphotyrosine-containing proteins in both retinoic acid-induced and control cultures. Cultured rat central nervous system cerebral cortical neurons and peripheral nervous system dorsal root ganglion neurons exhibited prominent phosphotyrosine-modified proteins of 90 and 46 kDa the same sizes as those in P19 neurons. The phosphotyrosine-containing proteins involved in the retinoic acid-induced differentiation of P19 cells to neurons were different from those altered in the glial differentiation of O2A cells to astrocytes or oligodendrocytes, indicating that the tyrosine kinase substrates modified during nervous system differentiation may be cell-type-specific.     

Establishment of highly enriched type-2 astrocyte cultures and quantitative determination of intense glutamine synthetase activity in these cells.

We report procedures that allow one to develop and maintain cultures highly enriched in rat neopallial type-2 astrocytes. Even after four weeks such cultures consist of more than 90% type-2 astrocytes, approximately 5% O-2A progenitors and fewer than 2% type-1 astrocytes. Their survival for more than 5 days requires the addition of conditioned medium from type-1 astrocyte cultures. The type-2 astrocytes have an intense glutamine synthetase activity whose basal level is sevenfold higher than in type-1 astrocytes. The glutamine synthetase activities of both the type-2 and type-1 astrocytes are increased after exposure to cortisol. Thus, type-2 astrocytes express the two quint-essential astrocytic features: glial fibrillary acidic protein (previously reported by others) and glutamine synthetase.     

Brain-specific angiogenesis inhibitor-1 expression in astrocytes and neurons: implications for its dual function as an apoptotic engulfment receptor

Brain-specific angiogenesis inhibitor-1 (BAI1) is a transmembrane protein highly expressed in normal brain that has been ascribed two apparently distinct functions: inhibition of angiogenesis and recognition and engulfment of apoptotic cells by phagocytes. A previous localization study reported BAI1 expression only in neurons. Because a phagocytic function of BAI1 could be important for neuroglial antigen processing and presentation, we performed immunolocalization studies in adult mouse brain and cultured neural cells, using a pair of antibodies directed against N- and C-terminal epitopes. BAI1 immunoreactivity is enriched in gray matter structures and largely excluded from myelinated axon tracts. Neuronal BAI1 expression was readily detectable in the cerebellar molecular layer as well as in primary hippocampal cultures. In some brain regions, especially olfactory bulb glomeruli, BAI1 was expressed by GFAP-positive astrocytes. Cultured cortical astrocytes show small (∼0.4 μm²) BAI1 immunoreactive membrane puncta as well as prominent focal adhesion localization in a subset of cells. In mixed neuronal-glial cultures, BAI1-expressing astrocytes frequently contained engulfed apoptotic debris. Cultured astrocytes engulfed apoptotic targets, and BAI1 showed accumulation within the phagocytic cup. We hypothesize that glial BAI1 may subserve an engulfment function in adult brain regions such as olfactory bulb with ongoing apoptotic turnover, whereas neuronal-derived BAI1 may serve primarily as an anti-angiogenic factor in the mature neuropil.     

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.     

Identification of mouse type-2-like astrocytes: demonstration of glutamate and GABA transmitter activated responses.

We have identified mouse type-2-like astrocytes and examined some of their electrophysiological properties. Cultures were prepared from P4 mouse neopallia. We demonstrate that mouse type-2-like astrocytes can be identified using the following criteria: presence of glial fibrillary acidic protein (GFAP), presence of chondroitin sulfate polysaccharide, and presence of gamma-aminobutyric acid (GABA). A2B5-binding is not a sufficient criterion to identify O2A lineage cells in mouse neopallial glial cultures since the monoclonal antibody A2B5 binds not only to O2A lineage cells but also to a subpopulation of large, flat type-1-like astrocytes. Mouse type-2-like astrocytes have resting membrane potentials of -76.2 +/- 2.1 mV-i.e., similar to that of mouse type-1-like astrocytes. The input resistance of 44.2 +/- 0.5 M omega is an order of magnitude greater than that of type-1-like astrocytes suggesting the type-2-like astrocytes are not extensively electrically coupled either to each other or to type-1-like astrocytes. Glutamate application caused an 8.8 +/- 1.7 mV depolarization of type-2-like astrocytes. Application of glutamate to barium treated astrocytes caused a fast depolarization with a peak amplitude of 21.4 +/- 1.8 mV; the cells repolarized from this peak by about 10 mV and upon removal of glutamate returned to its pre-glutamate value. Application of GABA caused a transient depolarization of 14.0 +/- 1.7 mV. The presence of barium resulted in a steady-state GABA-induced depolarization of 10.3 +/- 2.0 mV. Neither SITS nor beta-alanine interfered with the amplitude of the glutamate and GABA responses.     

Mature astrocytes transform into transitional radial glia within adult mouse neocortex that supports directed migration of transplanted immature neurons

Neuronal migration is an essential step in normal mammalian neocortical development, and the expression of defined cellular and molecular signals within the developing cortical microenvironment is likely crucial to this process. Therapy via transplanted or manipulated endogenous precursors for diseases which involve neuronal loss may depend critically on whether newly incorporated cells can actively migrate to repopulate areas of neuronal loss within the adult brain. Previous studies demonstrated that embryonic neurons and multipotent precursors transplanted into the neocortex of adult mice undergoing targeted apoptosis of pyramidal neurons migrate long distances into neuron-deficient regions, undergo directed differentiation, accept afferent synaptic input, and make appropriate long-distance projections. The experiments presented here: (1) use time-lapse digital confocal imaging of neuronal migration in living slice cultures to assess cellular mechanisms utilized by immature neurons during such long distance migration, and (2) identify changes within the host cortical astroglial population that may contribute to this migration. Prelabeled embryonic day 17 mouse neocortical neurons were transplanted into adult mouse primary somatosensory cortex undergoing targeted apoptotic degeneration of callosal projection neurons. Four to 7 days following transplantation, living slice cultures containing the region of transplanted cells were prepared and observed. Sequential time-lapse images were recorded using a video-based digital confocal microscope. Transplanted cells displayed bipolar morphologies characteristic of migrating neuroblasts and moved in a saltatory manner with mean rates of up to 14 microm/h. To investigate whether a permissive glial phenotype may provide a potential substrate for this directed form of neuronal migration, slice cultures were immunostained with the RC2 monoclonal antibody, which identifies radial glia that act as a substrate for neuronal migration during corticogenesis. RC2 does not label mature stellate astrocytes, which express glial fibrillary acidic protein (GFAP). RC2 expression was observed in glial cells closely apposed to migrating donor neurons within the slice cultures. The timing and specificity of RC2 expression was examined immunocytochemically at various times following transplantation. RC2 immunostaining within regions of neuronal degeneration was transient, with peak staining between 3 and 7 days following transplantation. Strongly RC2-immunoreactive cells that did not express GFAP were found within these regions, but not in distant cortical regions or within control brains. RC2-positive cells were identified in recipient transgenic mice which express beta-galactosidase under a glial specific promoter. Coexpression of RC2 and beta-galactosidase identified these cells as host astroglia. These results demonstrate that adult cortical astrocytes retain the capacity to reexpress an earlier developmental phenotype that may partially underlie the observed active migration of transplanted neurons and neural precursors. Further understanding of these processes could allow directed migration of transplanted or endogenous precursors toward therapeutic cellular repopulation and complex circuit reconstruction in neocortex and other CNS regions.     

Primary culture of cortical neurons, type-1 astrocytes, and microglial cells from cynomolgus monkey (Macaca fascicularis) fetuses

We established selective primary cultures of neurons, astrocytes, and microglial cells from cryopreserved fetal cerebral cortex of cynomolgus monkeys (Macaca fascicularis). At 14 days in serum-containing medium, the cell cultures of the fetal cerebral cortex consisted primarily of neurons, astrocytes, and floating microglial cells. At 21 days, we observed a small number of myelin basic protein (MBP)-positive oligodendrocytes. The addition of cytosine arabinoside (a selective DNA synthesis inhibitor) at 2 days in culture eliminated proliferative glial cells, allowing adequate numbers of neurons to survive selectively. A chemically defined serum-free medium successfully supported neuronal survival at a level equivalent to that supported by the serum-containing medium. Brain-derived neurotrophic factor (BDNF) significantly affected the survival of primate neurons. Glutamate induced a significant degree of neuronal cell death against primate neurons and MK-801, a selective N-methyl-D-aspartate receptor (NMDAR) antagonist, blocked cell death, which suggests that primate cortical neurons have NMDAR and the glutamate-induced cell toxicity is mediated by NMDAR. In the serum-free medium, type-1 astrocytes responded to dibutyryl cyclic AMP and showed a process-bearing morphology. The growth of type-1 astrocytes in the serum-free medium was stimulated by epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and hydrocortisone, which are known growth factors in rat type-1 astrocytes. Cultured microglial cells expressed CD68, a monocyte marker. Macrophage-colony stimulating factor (M-CSF) stimulated microglial cell growth in the serum-free medium. These selective primary culture systems of primate cerebral cortical cells will be useful in issues involving species specificity in neuroscience.     

In vitro identification and functional characterization of glial precursor cells in human gliomas

Human gliomas including astrocytomas and oligodendrogliomas are defined as being composed of neoplastic astrocytes and oligodendrocytes respectively. Here, on the basis of in vitro functional assays, we show that gliomas contain a mixture of glial progenitor cells and their progeny. We have set up explant cultures from pilocytic astrocytomas, glioblastomas and oligodendrogliomas and studied antigens that characterize glial lineage, from the precursor cells (glial restricted precursors and oligodendrocyte-type2-astrocyte/oligodendrocyte precursor cells expressing the A2B5 ganglioside) to the differentiated cells (oligodendrocyte and type-1 and type-2 astrocytes). All tumoral explants contain A2B5+ cells and can generate migrating cells with distinctive functional properties according to glioma subtypes. In pilocytic astrocytomas, very few migrating cells are dividing and can differentiate in type-2 astrocytes or towards the oligodendrocyte lineage. In glioblastomas, most migrating cells are dividing, express A2B5 or glial fibrillary acid protein (GFAP) and can generate oligodendrocytes and type-1 and type-2 astrocytes in appropriate medium. Oligodendroglioma explants are made by actively dividing glial precursor cells expressing A2B5 or PSA-NCAM. Only few cells can migrate and differentiation towards oligodendrocyte lineage does not occur. Isolated A2B5+ cells from both glioblastomas and oligodendrogliomas showed similar genetic alterations as the whole tumour. Therefore, pilocytic astrocytomas contain slowly dividing oligodendrocyte-type2-astrocyte/oligodendrocyte precursor cells in keeping with their benign behaviour whereas both glioblastomas and oligodendrogliomas contain neoplastic glial restricted precursor cells. In oligodendrogliomas, these cells are trapped in undifferentiated and proliferating state. The precursor cells properties present in gliomas give new insight into their histogenesis and open up new avenues for research in the field of gliomagenesis.     

Cell-type-specific expression of protein tyrosine kinase-related receptor RYK in the central nervous system of the rat

The mammalian RYK is an orphan receptor that contains a catalytically inactive tyrosine-kinase-related domain. Its Drosophila homolog, Lio/Drl, is required for axon pathfinding in developing brain. Our previous study suggested that RYK mRNA is expressed in nestin-positive progenitor cells and neurons. In the present study, immunohistochemistry has been used to further localize RYK in the central nervous system of rats to identify the lineage of the RYK-expressing cells. In the embryonic forebrain, RYK colocalized with nestin in the ventricular zone and with MAP2 in the cortical plate, suggesting that RYK is expressed in neural progenitor cells and neurons. Localization of RYK in embryonic spinal cord also suggested its expression in both cell types. In primary cultures of rat cerebrum, RYK expression was observed in all neurons, as well as in a significant population of oligodendrocytes, O-2A progenitor cells, and type-2 astrocytes. However, no RYK expression was detected in type-1 astrocytes or microglia. Multipotent neural stem cell line MNS-70 was also analyzed for expression of RYK, and most of the cells were positive for both RYK and nestin in the undifferentiated stage. In the differentiated stage, expression of RYK was detected in the neurons, but not in type-1 astrocytes. In conclusion, RYK is expressed in nestin-positive progenitor cells and neurons, and in a certain population of oligodendrocytes, O-2A progenitor cells, and type-2 astrocytes in developing CNS. These findings show that expression of RYK in rat CNS is tightly regulated in a cell-type-specific manner.