Plasticity of developing cerebellar cells in vitro studied with antibodies against the NG2 antigen

Abstract

The NG2 antigen, a chondroitin-sulfate proteoglycan, is a cell surface marker for a class of smooth protoplasmic astrocytes found throughout the brain and at high frequency in the cerebellar molecular layer (Levine and Card, 1987). To study the development of the cerebellar astrocytes at the level of the single cell, we have analyzed the distribution of the NG2 antigen by indirect immunofluorescence in dissociated cell cultures prepared from postnatal cerebella and compared that distribution to the distribution of several other cell surface and intracellular antigens that identify specific cell types in cultures of nervous tissue. When cerebellar cells from 5 d rat pups were grown in a medium containing 10% fetal calf serum, the NG2-labeled cells, which constituted 0.1-1.0% of the total glial cells present, contained glial fibrillary acidic protein (GFAP)-immunoreactive filaments and bound monoclonal antibody A2B5, a surface marker for neurons and some astrocytes. Approximately 30% of the NG2-labeled cells were also labeled with tetanus toxin, an additional surface marker for neurons and immature astrocytes. Less than 2% of the cells were labeled with antibodies against galactocerebroside or with monoclonal antibody O1, both of which are surface markers for oligodendrocytes. About half the NG2-labeled cells exhibited high-affinity uptake of 3H-GABA, and this uptake was partially inhibited by both beta-alanine and DABA. Thus, the NG2 antigen is a cell surface marker for a subpopulation of the type II or fibrous astrocytes present in the cultures. When the cerebellar cells were grown in a chemically defined, serum-free medium, the NG2-labeled cells had a stellate morphology and between 50-60% of the cells bound tetanus toxin. Although almost all the cells bound antibody A2B5, less than 5% of the cells expressed either of the oligodendrocyte surface markers or GFAP immunoreactivity. As was the case with cells grown in serum-containing medium, 60% of the NG2-labeled cells had high-affinity uptake of 3H-GABA. However, this uptake was inhibited by DABA but not by beta-alanine. This phenotype may be the in vitro analog of the NG2-labeled, filament-lacking, smooth protoplasmic astrocytes identified in the intact adult cerebellum. The expression of these 2 phenotypes could be reversed by switching the tissue culture medium within 5 d of plating the cells. These results demonstrate that the in vitro environment can influence the phenotypic properties expressed by developing cerebellar astrocytes and suggest that smooth protoplasmic astrocytes may be developmentally related to glial cells of the O-2A lineage.