Calcium microdomains at presynaptic active zones of vertebrate hair cells unmasked by stochastic deconvolution

SummarySignal transduction by auditory and vestibular hair cells involves an impressive ensemble of finely tuned control mechanisms, strictly dependent on the local intracellular Ca2+ concentration ([Ca2+]i). The study of Ca2+ dynamics in hair cells typically combines Ca2+-sensitive fluorescent indicators (dyes), patch clamp and optical microscopy to produce images of the patterns of fluorescence of a Ca2+ indicator following various stimulation protocols. Here we describe a novel method that combines electrophysiological recordings, fluorescence imaging and numerical simulations to effectively deconvolve Ca2+ signals within cytoplasmic microdomains that would otherwise remain inaccessible to direct observation. The method relies on the comparison of experimental data with virtual signals derived from a Monte Carlo reaction–diffusion model based on a realistic reconstruction of the relevant cell boundaries in three dimensions. The model comprises Ca2+ entry at individual presynaptic active zones followed by diffusion, buffering, extrusion and release of Ca2+. Our results indicate that changes of the hair cell [Ca2+]i during synaptic transmission are primarily controlled by the Ca2+ endogenous buffers both at short (<1 μ) and at long (tens of microns) distances from the active zones. We provide quantitative estimates of concentration and kinetics of the hair cell endogenous Ca2+ buffers and Ca2+-ATPases. We finally show that experimental fluorescence data collected during Ca2+ influx are not interpreted correctly if the [Ca2+]i is estimated by assuming that Ca2+ equilibrates instantly with its reactants. In our opinion, this approach is of potentially general interest as it can be easily adapted to the study of Ca2+ dynamics in diverse biological systems.