Properties of synchronous and asynchronous release during pulse train depression in cultured hippocampal neurons

Neurotransmitter release displays at least two kinetically distinct compone nts in response to a single action potential. The majority of release occur s synchronously with action-potential-triggered Ca2+ influx; however, delay ed release-also called asynchronous release-persists for tens of millisecon ds following the peak Ca2+ transient. In response to trains of action poten tials, synchronous release eventually declines, whereas asynchronous releas e often progressively increases, an effect that is primarily attributed to the buildup of intracellular Ca2+ during repetitive stimulation. The precis e relationship between synchronous and asynchronous release remains unclear at central synapses. To gain better insight into the mechanisms that regul ate neurotransmitter release, we systematically characterized the mio compo nents of release during repetitive stimulation at excitatory autaptic hippo campal synapses formed in culture. Manipulations that increase the Ca2+ inf lux triggered by an action potential-elevation of extracellular Ca2+ or bat h application of tetraethylammonium (TEA)-accelerated the progressive decre ase in synchronous release (peak excitatory postsynaptic current amplitude) and concomitantly increased asynchronous release. When intracellular Ca2was buffered by extracellular application of EGTA-AM, initial depression of synchronous release was equal to or greater than control; however, it quic kly reached a plateau without further depression. In contrast, asynchronous release was largely abolished in EGTA-AM. The total charge transfer follow ing each pulse-accounting for both synchronous and asynchronous release-rea ched a steady-state level that was similar between control and EGTA-AM. A p ortion of the decreased synchronous release in control conditions therefore was matched by a higher level of asynchronous release. We also examined th e relative changes in synchronous and asynchronous release during repetitiv e stimulation under conditions that highly favor asynchronous release by su bstituting extracellular Ca2+ with Sr2+. Initially, asynchronous release wa s twofold greater in Sr2+. By the end of the train, the difference was simi lar to 50%; consequently, the total release per pulse during the plateau ph ase was slightly larger in Sr2+ compared with Ca2+. We thus conclude that w hile asynchronous release-like synchronous release-is limited by vesicle av ailability, it may be able to access a slightly larger subset of the readil y releasable pool. Our results are consistent with the view that during rep etitive stimulation, the elevation of asynchronous release depletes the ves icles immediately available for release, resulting in depression of synchro nous release. This implies that both forms of release share a small pool of immediately releasable vesicles, which is being constantly depleted and re filled during repetitive stimulation.