Quantitative analysis of acetylcholine release in depolarized hippocampal slices

Time course of the hippocampal slice acetylcholine content and the rate of acetylcholine release were studied during high K(+)-induced depolarization for 4 to 60 min. At the end of the potassium exposure, both the acetylcholine remaining in the tissue and appearing in the incubation medium were quantitatively determined by gas chromatography using a nitrogen-sensitive detector. During prolonged K(+) incubation, the acetylcholine content of the slices decreased by 60%, reaching a steady state after 16 min. The increase in the acetycholine concentration of the depolarizing medium showed a biphasic pattern, with rate constants of 1.40 and 0.69 nmol/min/g in the early (0-16 min) and late (16-60 min) phase, respectively. K(+)-evoked acetylcholine release was Cal(+)-dependent, but addition of choline did not alter tissue levels of acetylcholine or the pattern of K(+)-evoked acetylcholine release. The rate of acetylcholine release was markedly decreased by inhibition of choline uptake with hemicholinium-3 or by addition of 4-(1-naphthylvinyl)pyridine which inhibits both ACh producing enzyme, choline acetyltransferase and choline uptake mechanism. These data confirm the essential role during depolarization of extracellular choline transport into the cholinergic terminals utilizing choline released by the slices during the incubation. It is concluded that drugs which can influence the processes of choline uptake and acetylcholine sythesis can alter the rate of acetylcholine release measured under similar conditions.