Optically Probing Heterogeneous Synaptic Activities In Hair Cells

At the first auditory synapse, the ribbon synapse of inner hair cell, neurotransmitter release is triggered by Ca2+ influx through clusters of Cav1.3 channels. Previous work in our lab has shown marked heterogeneity in the size of Ca2+-channel cluster (Meyer et al., 2009), as well as the amplitude and voltage dependence of microdomain Ca2+ (Frank et al. 2009) at active zones within single inner hair cells. Such heterogeneity of presynaptic Ca2+ signaling may contribute to the divergent sound coding properties of the postsynaptic spiral ganglion neurons that connect to the same hair cell. While potentially relevant for explaining large dynamic range sound encoding, its underlying mechanisms and the link to transmitter release are not well understood. Here we aimed at estimating the number of functional Ca2+ channels at the single synapses by means of an optical fluctuation analysis approach. The fluctuation analysis approach is compared to whole-cell Ca2+ current measurements in which single-synapse resolution is achieved by local pharmacological manipulation. In order to more systematically address synaptic heterogeneity we combined hair cell patch-clamp with spinning-disk confocal microscopy for simultaneous fast confocal Ca2+ imaging at multiple active zones. This method also allows us to analyze microdomain properties as a function of location in 3D-reconstructed hair cells. In addition, measurement of exocytosis at individual synapse is essential in linking Ca2+ signal amplitude to transmitter release. We are currently working on using optical reporters to monitor vesicle fusion at confocal resolution. Preliminary work demonstrates localized changes in VGLUT1-pHluorin fluorescence in response to voltage-gated Ca2+ influx.