Endogenous Amino Acid Release from Cultured Cerebellar Neuronal Cells: Effect of Tetanus Toxin on Glutamate Release

Abstract

Endogenous amino acid release was measured in developing cerebellar neuronal cells in primary culture. In the presence of 25 mM K⁺ added to the culture medium, cerebellar cells survived more than 3 weeks and showed a high level of differentiation. These cultures are highly enriched in neurons, and electron-microscopic observation of these cells after 12 days in vitro (DIV) confirmed the presence of a very large proportion of cells with the morphological characteristics of granule cells, making synapses containing many synaptic vesicles. Synaptogenesis was also confirmed by immunostaining the cells with antisera against synapsin I and synaptophysin, two proteins associated with synaptic vesicles. From these cultures, endogenous glutamate release stimulated by 56 m M K⁺ was already detected after only a few days in culture, the maximal release value (1,579% increase over basal release) being reached after 10 DIV. In addition to that of glutamate, the release of aspartate, asparagine, alanine, and, particularly, γ-aminobutyric acid (GABA) was stimulated by 56 mM K⁺ after 14 DIV, but to a lesser extent. No increase in serine, glutamine, taurine, or tyrosine release was observed during K⁺ depolarization. The effect of K⁺ on amino acid release was strictly Ca²⁺-dependent. Stimulation of the cells with veratridine resulted in a qualitatively similar effect on endogenous amino acid release. In the absence of Ca²⁺, 30% of the veratridine effect persisted. The Ca²⁺-dependent release was quantitatively similar after stimulation by veratridine and K⁺. Treatment of cerebellar cells with tetanus toxin (5 μ/ml) for 24 h resulted in a total inhibition of the Ca²⁺-dependent component of the glutamate release evoked by K⁺ or veratridine. It is concluded that glutamate is the main amino acid neurotransmitter of cerebellar cells developed in primary culture under the present conditions and that glutamate is probably mainly released through the exocytosis of synaptic vesicles.