Two Photon Imaging of Calcium Signalling at the Mouse Inner Hair Cell Ribbon Synapse

Abstract

The information sent from each cochlear inner hair cell (IHC) to the afferent nerve is determined by 10-20 ribbon synapses, structures specialised for rapid release of vesicles upon cell depolarization. To study the IHC calcium domains during transmitter release in mature wild-type mice, we have imaged and simultaneously measured currents in IHCs through an apical opening in the isolated temporal bone. Cells were recorded on the stage of an upright 2PCLSM at room temperature, superfused with medium containing 2mM Ca2+. IHCs could be visualised either with oblique optics or by using 830nm trans-illumination through bone structures. Using whole-cell tight seal recording, with Cs+ containing pipettes to reduce large outward currents, the I-V curve of the IHCs exhibit a Ca current with peak magnitude of approx 80pA near −20mV. To observe the distribution of Ca2+ entry in the vicinity of the ribbon sites, cells we pipette-loaded IHCs with either high or low affinity Ca2+ dyes (200uM OGB1 or OGB5N respectively) and imaged the basal IHC pole up to maximal rates of 70 frames/s during 20 ms or 100ms depolarizing steps to 0mV. At the fastest rates, the images derived from within single cells showed an initial punctuate rise of Ca2+ at the presumed synaptic sites with a larger increase at the neural side, a possible correlate of differing afferent thresholds known to characterise auditory nerve fibres. The sites were correlated with fluorescent hotspot distribution identified by IHC FM-dye uptake. The distribution of sites, the localisation of signal maxima close to (<3um) the plasma-membrane and recovery time constant (∼100ms) of Ca2+ influx also suggests that intrinsic Ca2+ buffering near the ribbon synapse was not significantly perturbed. Supported by EuroHear, the Physiological Society (SC), and College de France (JBM).