Quantitation and Simulation of Single Action Potential-Evoked Ca Signals in CA1 Pyramidal Neuron Presynaptic Terminals.

Presynaptic Ca evokes exocytosis, endocytosis, and synaptic plasticity. However, Ca flux and interactions at presynaptic molecular targets are difficult to quantify because fluorescence imaging has limited resolution. In rats of either sex, we measured single varicosity presynaptic Ca using Ca dyes as buffers, and constructed models of Ca dispersal. Action potentials evoked Ca transients with little variation when measured with low-affinity dye (peak amplitude 789 ± 39 nM, within 2 ms of stimulation; decay times, 119 ± 10 ms). Endogenous Ca buffering capacity, action potential-evoked free [Ca], and total Ca amounts entering terminals were determined using Ca dyes as buffers. These data constrained Monte Carlo (MCell) simulations of Ca entry, buffering, and removal. Simulations of experimentally-determined Ca fluxes, buffered by simulated calbindin well fit data, and were consistent with clustered Ca entry followed within 4 ms by diffusion throughout the varicosity. Repetitive stimulation caused free varicosity Ca to sum. However, simulated in nanometer domains, its removal by pumps and buffering was negligible, while local diffusion dominated. Thus, Ca within tens of nanometers of entry, did not accumulate. A model of synaptotagmin1 (syt1)-Ca binding indicates that even with 10 µM free varicosity evoked Ca, syt1 must be within tens of nanometers of channels to ensure occupation of all its Ca-binding sites. Repetitive stimulation, evoking short-term synaptic enhancement, does not modify probabilities of Ca fully occupying syt1's C2 domains, suggesting that enhancement is not mediated by Ca-syt1 interactions. We conclude that at spatiotemporal scales of fusion machines, Ca necessary for their activation is diffusion dominated.