Quantitation and Simulation of Single Action Potential-Evoked Ca(2+) Signals in CA1 Pyramidal Neuron Presynaptic Terminals

Presynaptic Ca(2+) evokes exocytosis, endocytosis, and synaptic plasticity. However, Ca(2+) 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(2+) using Ca(2+) dyes as buffers, and constructed models of Ca(2+) dispersal. Action potentials evoked Ca(2+) 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(2+) buffering capacity, action potential-evoked free [Ca(2+)](i), and total Ca(2+) amounts entering terminals were determined using Ca(2+) dyes as buffers. These data constrained Monte Carlo (MCell) simulations of Ca(2+) entry, buffering, and removal. Simulations of experimentally-determined Ca(2+) fluxes, buffered by simulated calbindin(28K) well fit data, and were consistent with clustered Ca(2+) entry followed within 4 ms by diffusion throughout the varicosity. Repetitive stimulation caused free varicosity Ca(2+) to sum. However, simulated in nanometer domains, its removal by pumps and buffering was negligible, while local diffusion dominated. Thus, Ca(2+) within tens of nanometers of entry, did not accumulate. A model of synaptotagmin1 (syt1)-Ca(2+) binding indicates that even with 10 µM free varicosity evoked Ca(2+), syt1 must be within tens of nanometers of channels to ensure occupation of all its Ca(2+)-binding sites. Repetitive stimulation, evoking short-term synaptic enhancement, does not modify probabilities of Ca(2+) fully occupying syt1’s C2 domains, suggesting that enhancement is not mediated by Ca(2+)-syt1 interactions. We conclude that at spatiotemporal scales of fusion machines, Ca(2+) necessary for their activation is diffusion dominated.