Diffusion of Ca²⁺ from Small Boutons en Passant into the Axon Shapes AP-Evoked Ca²⁺ Transients

Not only the amplitude but also the time course of a presynaptic Ca²⁺ transient determine multiple aspects of synaptic transmission. In small bouton-type synapses, the mechanisms underlying the Ca²⁺ decay kinetics have not been fully investigated. Here, factors that shape an action-potential-evoked Ca²⁺ transient were quantitatively studied in synaptic boutons of neocortical layer 5 pyramidal neurons. Ca²⁺ transients were measured with different concentrations of fluorescent Ca²⁺ indicators and analyzed based on a single-compartment model. We found a small endogenous Ca²⁺-binding ratio (7 ± 2) and a high activity of Ca²⁺ transporters (0.64 ± 0.03 ms^-1), both of which enable rapid clearance of Ca²⁺ from the boutons. However, contrary to predictions of the single-compartment model, the decay time course of the measured Ca²⁺ transients was biexponential and became prolonged during repetitive stimulation. Measurements of [Ca²⁺]_i along the adjoining axon, together with an experimentally constrained model, showed that the initial fast decay of the Ca²⁺ transients predominantly arose from the diffusion of Ca²⁺ from the boutons into the axon. Therefore, for small boutons en passant, factors like terminal volume, axon diameter, and the concentration of mobile Ca²⁺-binding molecules are critical determinants of Ca²⁺ dynamics and thus Ca²⁺-dependent processes, including short-term synaptic plasticity.