Synaptic vesicles (SVs) fuse at a specialized membrane domain called the active zone (AZ), covered by a conserved cytomatrix. How exactly cytomatrix components intersect with SV release remains insufficiently understood. We showed previously that loss of the Drosophila melanogaster ELKS family protein Bruchpilot (BRP) eliminates the cytomatrix (T bar) and declusters Ca²⁺ channels. In this paper, we explored additional functions of the cytomatrix, starting with the biochemical identification of two BRP isoforms. Both isoforms alternated in a circular array and were important for proper T-bar formation. Basal transmission was decreased in isoform-specific mutants, which we attributed to a reduction in the size of the readily releasable pool (RRP) of SVs. We also found a corresponding reduction in the number of SVs docked close to the remaining cytomatrix. We propose that the macromolecular architecture created by the alternating pattern of the BRP isoforms determines the number of Ca²⁺ channel-coupled SV release slots available per AZ and thereby sets the size of the RRP.

We studied the role of the synaptic ribbon for sound encoding at the synapses between inner hair cells (IHCs) and spiral ganglion neurons (SGNs) in mice lacking RIBEYE (RBE^KO/KO). Electron and immunofluorescence microscopy revealed a lack of synaptic ribbons and an assembly of several small active zones (AZs) at each synaptic contact. Spontaneous and sound-evoked firing rates of SGNs and their compound action potential were reduced, indicating impaired transmission at ribbonless IHC-SGN synapses. The temporal precision of sound encoding was impaired and the recovery of SGN-firing from adaptation indicated slowed synaptic vesicle (SV) replenishment. Activation of Ca²⁺-channels was shifted to more depolarized potentials and exocytosis was reduced for weak depolarizations. Presynaptic Ca²⁺-signals showed a broader spread, compatible with the altered Ca²⁺-channel clustering observed by super-resolution immunofluorescence microscopy. We postulate that RIBEYE disruption is partially compensated by multi-AZ organization. The remaining synaptic deficit indicates ribbon function in SV-replenishment and Ca²⁺-channel regulation.

Significance: Hearing relies on temporally precise and indefatigable transmission of sensory information mediated by inner hair cell (IHC) ribbon synapses. Synaptic ribbons are electron-dense structures that tether numerous vesicles at the active zone (AZ) and drive neural encoding of acoustical signals by neurotransmitter release. The mechanisms of ribbon formation, AZ targeting and subsequent functional maturation in murine IHCs remain elusive. By combining various high-resolution imaging techniques, we analyzed multiple developmental stages and provide a new hypothesis for the assembly of auditory ribbon synapses from the apical cochlear turn. Our findings suggest that fusion of presynaptic ribbons contributes to the morphological AZ development that is essential for the functional maturation of afferent synaptic transmission within the cochlea.

In the mammalian cochlea, sound is encoded at synapses between inner hair cells (IHCs) and type I spiral ganglion neurons (SGNs). Each SGN receives input from a single IHC ribbon-type active zone (AZ) and yet SGNs indefatigably spike up to hundreds of Hz to encode acoustic stimuli with submillisecond precision. Accumulating evidence indicates a highly specialized molecular composition and structure of the presynapse, adapted to suit these high functional demands. However, we are only beginning to understand key features such as stimulus–secretion coupling, exocytosis mechanisms, exo–endocytosis coupling, modes of endocytosis and vesicle reformation, as well as replenishment of the readily releasable pool. Relating structure and function has become an important avenue in addressing these points and has been applied to normal and genetically manipulated hair cell synapses. Here, we review some of the exciting new insights gained from recent studies of the molecular anatomy and physiology of IHC ribbon synapses.