Abstract

Calcium regulation of neurotransmitter release is essential for maintenance of synaptic transmission. However, the temporal and spatial organization of Ca²⁺ dynamics that regulate synaptic vesicle (SV) release efficacy in sympathetic neurons is poorly understood. Here, we investigate the N-type Ca²⁺ channel–mediated kinetic structure of Ca²⁺ regulation of cholinergic transmission of sympathetic neurons. We measured the effect of Ca²⁺ chelation with fast 1,2-bis(2-aminophenoxy) ethane-tetraacetic acid (BAPTA) and slow ethyleneglycol-tetraacetic acid (EGTA) buffers on exocytosis, synaptic depression, and recovery of the readily releasable vesicle pool (RRP), after both single action potential (AP) and repetitive APs. Surprisingly, postsynaptic potentials peaking at ∼12 milliseconds after the AP was inhibited by both rapid and slow Ca²⁺ buffers suggests that, in addition to the well known fast Ca²⁺ signals at the active zone (AZ), slow Ca²⁺ signals at the peak of Ca²⁺ entry also contribute to paired-pulse or repetitive AP responses. Following a single AP, discrete Ca²⁺ transient increase regulated synaptic depression in rapid (<30-millisecond) and slow (<120-millisecond) phases. In contrast, following prolonged AP trains, synaptic depression was reduced by a slow Ca²⁺ signal regulation lasting >200 milliseconds. Finally, after an AP burst, recovery of the RRP was mediated by an AP-dependent rapid Ca²⁺ signal, and the expansion of releasable SV number by an AP firing activity–dependent slow Ca²⁺ signal. These data indicate that local Ca²⁺ signals operating near Ca²⁺ sources in the AZ are organized into discrete fast and slow temporal phases that remodel exocytosis and short-term plasticity to ensure long-term stability in acetylcholine release efficacy.