Quantitative optical nanophysiology of Ca signaling at inner hair cell active zones.

Ca influx triggers the release of synaptic vesicles at the presynaptic active zone (AZ). A quantitative characterization of presynaptic Ca signaling is critical for understanding synaptic transmission. However, this has remained challenging to establish at the required resolution. Here, we employ confocal and stimulated emission depletion (STED) microscopy to quantify the number (20-330) and arrangement (mostly linear 70 nm × 100-600 nm clusters) of Ca channels at AZs of mouse cochlear inner hair cells (IHCs). Establishing STED Ca imaging, we analyze presynaptic Ca signals at the nanometer scale and find confined elongated Ca domains at normal IHC AZs, whereas Ca domains are spatially spread out at the AZs of bassoon-deficient IHCs. Performing 2D-STED fluorescence lifetime analysis, we arrive at estimates of the Ca concentrations at stimulated IHC AZs of on average 25 µM. We propose that IHCs form bassoon-dependent presynaptic Ca-channel clusters of similar density but scalable length, thereby varying the number of Ca channels amongst individual AZs.