Quantitation and simulation of single action potential-evoked Ca2+ signals in CA1 pyramidal neuron presynaptic terminals

Abstract

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 calbindin28K 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.