A biochemical approach to study sub-second endogenous release of diverse neurotransmitters from central nerve terminals

Exocytosis in central nerve terminals is rapidly triggered by the influx of calcium through high voltage sensitive Ca2+-channels. Mainly due to their small size, studies in which neurotransmitter release from these terminals was determined at the sub-second time-scale are still rather limited. Here we describe the use of a pneumatic rapid mixing device, allowing application of short (≥50 ms) K+-depolarizing pulses to purified nerve terminals, synaptosomes, to trigger endogenous release of different transmitter types. A consistent, Ca2+-dependent exocytotic release of the amino acid transmitters, glutamate and GABA, from synaptosomes purified from rat and mouse brain was observed after 100 ms depolarization. For determination of amino acid release after longer depolarizations (>100 ms), transporter blockers had to be added to prevent clearance of the vesicularly released transmitters. Ca2+-dependent release of the neuropeptide cholecystokinin occured only after 250 ms depolarization. In addition, the time-courses of amino acid and cholecystokinin release were clearly different. The fast Ca2+-dependent release of all transmitters was selectively and strongly inhibited by the P/Q-type Ca2+-channel blocker ω-Agatoxin IVA. In conclusion, this approach allows direct measurement of Ca2+-dependent release of diverse endogenous neurotransmitters from central nerve terminals upon depolarization pulses at a physiologically relevant, sub-second, time scale.