Charting the nanotopography of inner hair cell synapses using MINFLUX nanoscopy

The cochlea encodes sounds into neural signals at the synapses of inner hair cells (IHCs) and spiral ganglion neurons (SGNs) with remarkable fidelity. To achieve high rates of precise synaptic transmission over long periods, IHCs use ribbon-type active zones (AZs). To understand synaptic sound encoding, we need to decipher the underlying molecular topography of these synapses, which has remained challenging because of technological limitations. Here, we applied three-dimensional minimal flux optical nanoscopy to mouse IHC-SGN synapses to chart the positions of key pre- and postsynaptic proteins with single-digit nanometer resolution. We demonstrate that nanoclusters of ion channels and their interacting proteins govern the topography of AZs and postsynaptic densities (PSDs). We count synaptic proteins and their nanoclusters and determine their spatial organization, feeding into computational modeling of AZ function. In conclusion, this study reveals a nanocluster-based molecular AZ and PSD topography, likely serving as functional modules in synaptic sound encoding.