Optogenetics and electron tomography for structure-function analysis of cochlear ribbon synapses.

Ribbon synapses of cochlear inner hair cells (IHCs) are specialized to indefatigably transmit sound information at high rates. To understand the underlying mechanisms, structure-function analysis of the active zone (AZ) of these synapses is essential. Previous electron microscopy studies of synaptic vesicle (SV) dynamics at the IHC AZ used potassium stimulation, which limited the temporal resolution to minutes. Here, we established optogenetic IHC stimulation followed by quick freezing within milliseconds and electron tomography to study the ultrastructure of functional synapse states with good temporal resolution in mice. We characterized optogenetic IHC stimulation by patch-clamp recordings from IHCs and postsynaptic boutons revealing robust IHC depolarization and neurotransmitter release. Ultrastructurally, the number of docked SVs increased upon short (17-25 ms) and long (48-76 ms) light stimulation paradigms. We did not observe enlarged SVs or other morphological correlates of homotypic fusion events. Our results indicate a rapid recruitment of SVs to the docked state upon stimulation and suggest that univesicular release prevails as the quantal mechanism of exocytosis at IHC ribbon synapses.

AP180 promotes release site clearance and clathrin-dependent vesicle reformation in mouse cochlear inner hair cells.

High-throughput neurotransmission at ribbon synapses of cochlear inner hair cells (IHCs) requires tight coupling of neurotransmitter release and balanced recycling of synaptic vesicles (SVs) as well as rapid restoration of release sites. Here, we examined the role of the adaptor protein AP180 (also known as SNAP91) for IHC synaptic transmission by comparing AP180-knockout (KO) and wild-type mice using high-pressure freezing and electron tomography, confocal microscopy, patch-clamp membrane capacitance measurements and systems physiology. AP180 was found predominantly at the synaptic pole of IHCs. AP180-deficient IHCs had severely reduced SV numbers, slowed endocytic membrane retrieval and accumulated endocytic intermediates near ribbon synapses, indicating that AP180 is required for clathrin-dependent endocytosis and SV reformation in IHCs. Moreover, AP180 deletion led to a high prevalence of SVs in a multi-tethered or docked state after stimulation, a reduced rate of SV replenishment and a hearing impairment. We conclude that, in addition to its role in clathrin recruitment, AP180 contributes to release site clearance in IHCs.This article has an associated First Person interview with the first author of the paper.

Intrinsic planar polarity mechanisms influence the position-dependent regulation of synapse properties in inner hair cells.

Encoding the wide range of audible sounds in the mammalian cochlea is collectively achieved by functionally diverse type I spiral ganglion neurons (SGNs) at each tonotopic position. The firing of each SGN is thought to be driven by an individual active zone (AZ) of a given inner hair cell (IHC). These AZs present distinct properties according to their position within the IHC, to some extent forming a gradient between the modiolar and the pillar IHC side. In this study, we investigated whether signaling involved in planar polarity at the apical surface can influence position-dependent AZ properties at the IHC base. Specifically, we tested the role of Gαi proteins and their binding partner LGN/Gpsm2 implicated in cytoskeleton polarization and hair cell (HC) orientation along the epithelial plane. Using high and superresolution immunofluorescence microscopy as well as patch-clamp combined with confocal Ca2+ imaging we analyzed IHCs in which Gαi signaling was blocked by Cre-induced expression of the pertussis toxin catalytic subunit (PTXa). PTXa-expressing IHCs exhibited larger CaV1.3 Ca2+-channel clusters and consequently greater Ca2+ influx at the whole-cell and single-synapse levels, which also showed a hyperpolarized shift of activation. Moreover, PTXa expression collapsed the modiolar-pillar gradients of ribbon size and maximal synaptic Ca2+ influx. Finally, genetic deletion of Gαi3 and LGN/Gpsm2 also disrupted the modiolar-pillar gradient of ribbon size. We propose a role for Gαi proteins and LGN in regulating the position-dependent AZ properties in IHCs and suggest that this signaling pathway contributes to setting up the diverse firing properties of SGNs.