Kapoor, R.; Kim, H.; Garlick, E.; Lima, M. A. d. R. B. F.; Ruhwedel, T.; Moebius, W.; Wolf, F.; Moser, T.

For us to hear, the cochlea encodes sounds into neural signals at synapses of inner hair cells (IHCs) and the auditory nerve with remarkable fidelity. To achieve the high rates of temporally precise synaptic transmission over long periods of time, IHCs employ sophisticated ribbon-type active zones (AZ). In order for us to understand synaptic sound encoding, we need to decipher the underpinning molecular topography of these synapse which had remained challenging due to technological limitations. Here we applied 3-dimensional minimal flux optical nanoscopy to mouse IHC synapses to chart the position of key pre- and postsynaptic proteins with single digit nanometre resolution of imaging. We demonstrate that nanoclusters of channels and interacting proteins govern the topography of AZs and postsynaptic densities (PSDs). We count synaptic proteins, their nanoclusters and determine their spatial organization feeding into computational modelling 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.