Neurotransmitter release is modulated by multiple regulatory mechanisms that control several stages of synaptic vesicle (SV) exocytosis. At the final stage, SV fusion with the presynaptic membrane requires calcium influx through voltage-gated calcium channels, and regulatory mechanisms that alter the surface expression or conductance of calcium channels have large effects on neurotransmitter release. To determine how these mechanisms contribute to synapse-specific modulations of neurotransmitter release and synaptic strength, we require a means to monitor presynaptic calcium transients at individual synapses. Genetically encoded calcium indicators (GECIs), engineered proteins that change their fluorescence emission properties upon calcium binding, generally lack the sensitivity to measure such transients in response to isolated stimuli. Therefore, we modified the GECI, GCaMP3, by altering its sensitivity for calcium. Our results suggest the modified GCaMP-based presynaptically targeted GECIs are excellent tools to quantify presynaptic calcium transients at individual synapses in response to isolated action potentials.

Presynaptic terminals of neurons are optimized for neurotransmitter release, which is tightly controlled by presynaptic calcium. Here, we evaluate the role of calcium influx through voltage-gated calcium channels (VGCCs) in regulating the initial vesicular release probability (p) and the number of vesicles available for release by action potentials (effective RRP) at the calyx of Held synapse in mice. Two established methods of estimating effective RRP size and p reveal that both are calcium dependent. Reducing calcium influx by blocking R-type (VGCCs) or P/Q-type VGCCs also reduces EPSC amplitude via p and effective RRP size. Furthermore, activation of gamma-aminobutryic acid class B (GABAB) receptors, which reduces presynaptic calcium by regulating VGCCs without other significant effects on release, also reduces the effective RRP size and p. These findings suggest that the calcium dependence of RRP size may influence the manner in which certain neuromodulators affect neurotransmitter release.

Short-term synaptic plasticity results from use-dependent activity, lasts on the timescale of milliseconds to minutes, and is thought to underlie working memory and neuronal information processing. Here, we focus on two forms of short-term plasticity: 1) post-tetanic potentiation (PTP), which is induced by high-frequency stimulation, and 2) presynaptic ionotropic receptor-activated synaptic enhancement, which can be produced by the activation of presynaptic glycine receptors. Potentiation of evoked and spontaneous responses is thought to arise from elevations in presynaptic residual Ca2+, which activates one or more molecular targets to increase neurotransmitter release. However, the Ca2+ sensor protein has not yet been identified. The overall goal of this work is to elucidate the Ca2+-dependent mechanisms of short-term plasticity.

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