Quantitation and Simulation of Single Action Potential-Evoked Ca²⁺ Signals in CA1 Pyramidal Neuron Presynaptic...

Presynaptic Ca²⁺ evokes exocytosis, endocytosis, and synaptic plasticity. However, Ca²⁺ 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 Ca²⁺ using Ca²⁺ dyes as buffers, and constructed models of Ca²⁺ dispersal. Action potentials evoked Ca²⁺ 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 Ca²⁺ buffering capacity, action potential-evoked free [Ca²⁺]_i, and total Ca²⁺ amounts entering terminals were determined using Ca²⁺ dyes as buffers. These data constrained Monte Carlo (MCell) simulations of Ca²⁺ entry, buffering, and removal. Simulations of experimentally-determined Ca²⁺ fluxes, buffered by simulated calbindin_28K well fit data, and were consistent with clustered Ca²⁺ entry followed within 4 ms by diffusion throughout the varicosity. Repetitive stimulation caused free varicosity Ca²⁺ to sum. However, simulated in nanometer domains, its removal by pumps and buffering was negligible, while local diffusion dominated. Thus, Ca²⁺ within tens of nanometers of entry, did not accumulate. A model of synaptotagmin1 (syt1)-Ca²⁺ binding indicates that even with 10 µM free varicosity evoked Ca²⁺, syt1 must be within tens of nanometers of channels to ensure occupation of all its Ca²⁺-binding sites. Repetitive stimulation, evoking short-term synaptic enhancement, does not modify probabilities of Ca²⁺ fully occupying syt1's C2 domains, suggesting that enhancement is not mediated by Ca²⁺-syt1 interactions. We conclude that at spatiotemporal scales of fusion machines, Ca²⁺ necessary for their activation is diffusion dominated.