Autoradiographic localization and depolarization-induced release of acidic amino acids in differentiating cerebellar granule cell cultures

Abstract

Granule cells from 8-day-old rat cerebella were grown in basal Eagle's medium with 10% fetal calf serum, for 2,5,8 or 12 days in vitro (DIV), in conditions giving a purity > 90%. The results obtained can be summarized as follows: (1) Light microscopic autoradiography showed that cultured granule cells and their processes can accumulate the glutamate analog [ 3H] d-aspartate once they have reached an advanced degree of morphological differentiation (8 and 12 DIV), but, even then, only a limited number of cells was heavily labeled. In contrast, astrocytes were heavily labeled at all stages. (2) Calcium-dependent, high [K +]-induced release, or tetrodotoxin-sensitive, veratridine-induced release of [ 3H] d-aspartate from granule cell-enriched cultures was detectable only in cultures of 8 or 12 DIV. (3) When subject to 3 consecutive depolarizations, cultured granule cells maintained their ability to release [ 3H] d-aspartate and endogenous glutamate almost unchanged. (4)_Newly synthesized [ 3H]glutamate was autoradiographically localized in both neurons and astrocytes (the latter, however, were not preferentially labeled as with [ 3H d-aspartate), but was specifically release from neuronal structures (perikarya and processes) by depolarizing stimuli.