Quantitative optical nanophysiology of Ca2+ signaling at inner hair cell active zones

Abstract

Ca2+ influx triggers the release of synaptic vesicles at the presynaptic active zone (AZ). A quantitative characterization of presynaptic Ca2+ 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 Ca2+ channels at AZs of mouse cochlear inner hair cells (IHCs). Establishing STED Ca2+ imaging, we analyze presynaptic Ca2+ signals at the nanometer scale and find confined elongated Ca2+ domains at normal IHC AZs, whereas Ca2+ domains are spatially spread out at the AZs of bassoon-deficient IHCs. Performing 2D-STED fluorescence lifetime analysis, we arrive at estimates of the Ca2+ concentrations at stimulated IHC AZs of on average 25 µM. We propose that IHCs form bassoon-dependent presynaptic Ca2+-channel clusters of similar density but scalable length, thereby varying the number of Ca2+ channels amongst individual AZs.

Electron Microscopic Reconstruction of Neural Circuitry in the Cochlea

“…In the mammalian cochlea, each IHC is contacted by 10-30 1 SGNs, whereby predominantly an AZ with a single ribbon drives firing in a single unbranched peripheral dendrite. These afferent connections exhibit great diversity in synaptic and 1 SGN dendritic morphology 13–20 , physiological properties 6, 13, 16, 17, 20–24 , and molecular 1 SGN profile 25–27 . What might appear as a peculiar biological variance at first glance, is becoming increasingly recognized as a fascinating parallel information processing mechanism by which the auditory system copes with encoding over a broad range of sound pressures downstream of cochlear micromechanics.…”

Distinct nanoscale calcium channel and synaptic vesicle topographies contribute to the diversity of synaptic function

“…In drosophila NMJ, super-resolution microscopy has demonstrated that VGCCs are arranged in a tight cluster (50 - 100 nm diameter) with vesicles being tethered around its perimeter (Liu et al, 2011). Recently at mouse inner hair cells super-resolution microscopy identified linear clusters of VGCCs (Neef et al, 2018; Wong et al, 2014) that in combination with VGCC cooperativity measurements and numerical simulations drive release from within ∼20 nm of the cluster perimeter (Pangrsic et al, 2015).…”

“…To date specific VGCCs-SV topographies have been proposed at Drosophila (VGCC clusters coupled to vesicles around its perimeter) and mammalian NMJ (rows of VGCC and SVs), and at hair cell synapses (rows of calcium channels coupled to SV around its perimeter; Dittrich et al, 2013; Luo et al, 2015; Neef et al, 2018; Pangrsic et al, 2015). However, the nanoscale organization of SVs and VGCCs underlying the functional diversity of CNS synapses is less well understood.…”

VGLUT3-p.A211V variant fuses stereocilia bundle and elongates synaptic ribbons in the human deafness DFNA25

“…In our experiments, the RRP is not affected in the IHCs from VGLUT3 A224V/A224V mice. In hair cells, the calcium channels are densely packed underneath the presynaptic element and the RRP correlates to the membraneproximal synaptic vesicles, i.e., the vesicles tether to the synaptic body and docked to the plasma membrane (Khimich et al, 2005;Schnee et al, 2005;Rutherford & Roberts, 2006;Meyer et al, 2009;Frank et al, 2010;Pangršič et al, 2010;Wong et al, 2014;Neef et al, 2018).…”