Subpopulations of rat cerebellar astrocytes in primary culture: Morphology, cell surface antigens and [ 3H]GABA transport

Abstract

Glial fibrillary acidic protein (GFAP)-positive astrocytes in preconfluent cultures derived from postnatal rat cerebellum have been previously shown to display two distinct morphologies, one stellate and the other irregularly epithelioid. The immunofluorescence studies described here showed that these cells also possess unique surface characteristics. In cultures derived from 8-day-old animals stellate cells bound the monoclonal antibody A 2B 5 whereas the epithelioid cells bound another monoclonal antibody against rat neural antigen-2 (RAN 2). Some stellate cells derived from 2-day-old animals also bound tetanus toxin. The A 2B 5 labelling of the stellate cells made it possible to follow their fate in vitro. In confirmation of previous time-lapse studies, they underwent a shape transformation as confluence was approached, ultimately attaining a form resembling that of the epithelioid cells. Autoradiographic transport studies using two tritiated γ-aminobutyric acid (GABA) analogues cis-1,3-aminocyclohexane carboxylic acid (ACHC) and β-alanine revealed further differences between the two types of astrocytes. Whereas [ 3H]ACHC was taken up solely by the stellate cells [ 3H]β-alanine was transported by both cell types. In other experiments in which various inhibitors of [ 3H]GABA transport were used ACHC virtually eliminated uptake into the stellate astrocyte, but had little effect on the epithelioid ones. The ‘neuron-like’ [ 3H]GABA transport process in the stellate astrocytes was confirmed in experiments comparing the effect of another compound which has been proposed as an astrocyte-selective GABA transport inhibitor, 4,5,6,7-tetrahydroisoxazolo-(4,5-C)pyridin-3-ol (THPO). No discrimination was found in its effect on the uptake of [ 3H]GABA into either neurons or stellate astrocytes. Further autoradiographic studies following the uptake of [ 3H]GABA by postnatal cerebellar slices showed that astrocytes in all layers of the cerebellar cortex and white matter transported [ 3H]GABA in contrast to the situation in culture where the amino acid is taken up predominantly by the stellate astrocytes. The possibility is discussed that the stellate astrocytes represent a population of cerebellar fibrous astrocytes whereas the identity of the epithelioid astrocytes is less certain.