title: Calcium microdomains at presynaptic active zones of vertebrate hair cells unmasked by stochastic deconvolution.
abstract: Signal transduction by auditory and vestibular hair cells involves an impressive ensemble of finely tuned control mechanisms, strictly dependent on the local intracellular Ca(2+) concentration ([Ca(2+)](i)). The study of Ca(2+) dynamics in hair cells typically combines Ca(2+)-sensitive fluorescent indicators (dyes), patch clamp and optical microscopy to produce images of the patterns of fluorescence of a Ca(2+) indicator following various stimulation protocols. Here we describe a novel method that combines electrophysiological recordings, fluorescence imaging and numerical simulations to effectively deconvolve Ca(2+) 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 Ca(2+) entry at individual presynaptic active zones followed by diffusion, buffering, extrusion and release of Ca(2+). Our results indicate that changes of the hair cell [Ca(2+)](i) during synaptic transmission are primarily controlled by the Ca(2+) endogenous buffers both at short (<1mu) and at long (tens of microns) distances from the active zones. We provide quantitative estimates of concentration and kinetics of the hair cell endogenous Ca(2+) buffers and Ca(2+)-ATPases. We finally show that experimental fluorescence data collected during Ca(2+) influx are not interpreted correctly if the [Ca(2+)](i) is estimated by assuming that Ca(2+) 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 Ca(2+) dynamics in diverse biological systems.
authors.affiliation: Foundation for Advanced Biomedical Research, Venetian Institute of Molecular Medicine, via G. Orus 2, 35129 Padova, Italy.
authors.forename: Mario
authors.initials: M
authors.lastname: Bortolozzi
authors.forename: Andrea
authors.initials: A
authors.lastname: Lelli
authors.forename: Fabio
authors.initials: F
authors.lastname: Mammano
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels; D020013:Calcium Signaling; D002614:Chelating Agents / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004058:Diffusion; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005453:Fluorescence; D006198:Hair Cells, Auditory / Q000502:physiology*; D018069:Hair Cells, Vestibular / Q000502:physiology*; D021962:Membrane Microdomains; D018408:Patch-Clamp Techniques; D017729:Presynaptic Terminals / Q000502:physiology*; D011892:Rana catesbeiana; D009435:Synaptic Transmission
chemical_list: D015220:Calcium Channels; D002614:Chelating Agents; D004533:Egtazic Acid; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium