Confocal Imaging of Calcium Signal and Exocytosis at Individual Hair Cell Synapses

Hearing over a wide range of sound intensities is thought to require complementary coding by functionally diverse spiral ganglion neurons (SGNs), each changing its firing rate only over a limited sub-range. The means for establishing SGN diversity are not well understood, but likely involves differences among their inputs − the presynaptic active zones (AZs) of inner hair cells (IHCs). In this study, I investigated the Ca2+ signal in IHCs, which were previously demonstrated to be heterogeneous among AZs in mature IHCs. In particular, I explored the changes of synaptic Ca2+ signal during postnatal development of the mouse cochlea, and probed the functional consequence thereof. Moreover, I attempted to establish analogous measurement of exocytosis using the pH-sensitive fluorescent protein pHluorin. The results showed that depolarization-evoked Ca2+ signal in IHC became progressively confined to AZs during development, mostly mediated by a loss of extrasynaptic channels revealed by immunohistochemistry of CaV1.3 channels. Parallel to the confinement of Ca2+ channel, the IHC switches from a Ca2+ microdomain control of exocytosis to a tighter Ca2+ nanodomain control, without a change in intrinsic Ca2+ dependence of vesicle fusion. At the level of individual AZs, Ca2+ action potentials in prehearing IHCs drove robust Ca2+ transients. On the other hand, receptor-potential-like stimuli were able to drive synaptic Ca2+ signals in mature IHCs up to 1 kHz, even given experimental limitations. An increase in heterogeneity of synaptic Ca2+ influx among the AZs of IHCs was observed, mediated by the strengthening of Ca2+ signaling at a subset of AZs through a gain of Ca2+ channels. The emergence of SGN with high spontaneous rate around the onset of hearing suggests that AZ with stronger Ca2+ influx drive these postsynaptic SGNs which are more sensitive to sound. However, other mechanisms including differences in voltage-dependence of Ca2+ channel, number of postsynaptic glutamate receptors and efferent modulation cannot be excluded. Depolarization of mature IHCs expressing vglut1-pHluorin revealed localized exocytic activity at the AZs, similar to the confined Ca2+ signal. However, the signal-to-noise ratio is yet to be improved for quantitative measurement of exocytosis. The establishment of a single AZ measurement of vesicle release will help to confirm whether the heterogeneity of synaptic Ca2+ signal indeed lead to difference in output.