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Journal of Neuroscience, Vol 7, 2721-2731, Copyright © 1987 by Society for Neuroscience
Plasticity of developing cerebellar cells in vitro studied with antibodies against the NG2 antigen
JM Levine and WB Stallcup
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.
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