Enrichment of differentiated, stellate astrocytes in cerebellar interneuron cultures as studied by GFAP immunofluorescence and autoradiographic uptake patterns with [ 3H] d-aspartate and [ 3H]GABA

Abstract

A study was undertaken to correlate the morphology expressed by astroglial cells in post-natal cerebellar, interneuron-enriched primary cultures, and the ability of these cells to accumulate putative neurotransmitter amino acids. Astroglial cell morphology, as studied by GFAP immunofluorescence staining showed considerable changes during the culture period considered (up to 12 days in vitro). While the total number of GFAP-positive cells decreased with time (cell multiplication was prevented by cytosine arabinoside), a progressive enrichment of stellate astrocytes (cells bearing multiple radially arranged processes) and a striking increase in size of these cells was noted. In 12 DIV cultures stellate astrocytes accounted for 70–80% of the astrocytes present, and could reach a diameter of over 300 μm. The 4l-glutamate analogue, [ 3H] d-aspartate, was avidly taken up by all the astrocytes, independently of their shape and stage of differentiation. Astroglial cell morphology as delineated by [ 3H] d-aspartate autoradiography was identical to that evidenced by GFAP staining. On the other hand, [ 3H]GABA was accumulated in substantial amounts only by the stellate astrocytes, that is by the cells showing greater morphological differentiation. Astrocytes of other shapes were only lightly labelled by [ 3H]GABA in 2 DIV and 5 DIV cultures, and even less at later stages. Even within the stellate astrocyte population, the extent of [ 3H]GABA labelling was very variable, from one cell to another. Autoradiographic examinations and the determination of the IC 50s for GABA uptake inhibitors consistently indicated that the GABA transport system present in stellate astrocytes did not have the features generally attributed to a glial transport system. In fact, β-alanine was a very weak inhibitor, while nipecotic acid and ACHC were strongly inhibitory; DABA inhibitory potency fell somewhere in between. [ 3H]GABA uptake into the inhibitory interneurons present in the cultures showed similar sensitivity to GABA transport inhibitors.