Characterization of GABAergic neurons in cerebellar primary cultures and selective neurotoxic effects of a serum fraction

Abstract

The morphological and functional differentiation of GABAergic interneurons present in cerebellar primary cultures has been examined by means of [3H]gamma-aminobutyric acid (GABA) autoradiography and [3H]GABA depolarization-evoked release. At 2 days in vitro these neurons showed scarce accumulation of radioactivity and no Ca2+- dependent K+-evoked or veratridine-induced release of [3H]GABA. At 5 days in vitro GABAergic interneurons appeared more intensely labeled and had grown out long and often branched neuritic processes; a large Ca2+-dependent release of [3H] GABA could be evoked by high K+. At later stages the progressive increase in labeling and branching of the neuritic processes was paralleled by a further increase in the amount and Ca2+ dependence of [3H]GABA release; a tetrodotoxin-sensitive, veratridine-stimulated release was also demonstrated. The [3H]GABA- accumulating stellate astrocytes present in the culture were not responsible for the observed release of the amino acid. GABAergic neurons were also identified by indirect immunofluorescence, using antibodies to the specific marker glutamic acid decarboxylase. Total renewal of the culture medium at 7 days in vitro caused a drastic (90%) reduction in the number of GABAergic neurons and a concomitant decrease in the amount of [3H]GABA uptake and release in the cultures. The disappearance of GABAergic neurons was caused by a low molecular weight (Mr less than 1000) fraction of the serum used to supplement the basal culture medium. This serum component did not significantly influence the survival of the major neuronal population of the culture (the granule cells) and appeared to be selectively toxic for GABAergic neurons only after they had reached a quite advanced degree of morphological and functional differentiation in vitro. The toxic activity was no longer present in neuronal or glial conditioned media.