Selective release of glutamate from cerebellar granule cells differentiating in culture

Abstract

The aim of the present study was to assess whether endogenous and newly synthesized glutamate can be released from differentiating cultured cerebellar granule cells in a way compatible with a neurotransmitter role. Granule cells from 8-day-old rat cerebella were grown in basal Eagle's medium with 10% fetal calf serum for 2-12 days in vitro (DIV), then washed with Krebs-Ringer medium, and labeled for 45 min with tracer amounts of radioactive glutamine. Subsequently, the release of endogenous glutamate and of newly formed radioactive glutamate was measured in basal conditions and upon depolarization with elevated K + concentration or veratridine. At 2 DIV, the release of endogenous and newly synthesized glutamate evoked by high K + concentration was small and Ca 2+ independent, but it progressively and steadily increased (up to 8- to 10-fold) and became Ca 2+ dependent (up to 80-85%) at later stages (4, 8, and 12 DIV). Veratridine was almost ineffective with cells at 2 DIV but greatly increased glutamate release (endogenous and neosynthesized) at 8 DIV, and its action was totally antagonized by tetrodotoxin. The level and synthesis of glutamate remained fairly constant in cells from 2 to 12 DIV. γ-Aminobutyric acid synthesis from radioactive glutamine was about 3% of that of glutamate, and γ-aminobutyric acid release (endogenous and neosynthesized) was not measurable. Aspartate synthesis was about 10% of that of glutamate, and the high K + concentration-evoked release of this amino acid was modest and scarcely affected by Ca 2+ . Neither high K + concentration nor veratridine was able to induce glutamate release from confluent cerebellar astrocyte cultures at 14 DIV, although the level and synthesis of the amino acid were comparable to those in granule cells. In conclusion, the data show that a stimulus-coupled release of endogenous and neosynthesized glutamate is progressively expressed by cerebellar granule cells differentiating in culture, and this strongly supports the concept that glutamate is the neurotransmitter of these cells.