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In the mammalian cochlea, specialized ribbon-type synapses between sensory inner hair cells (IHCs) and postsynaptic spiral ganglion neurons ensure the temporal precision and indefatigability of synaptic sound encoding. These high-through-put synapses are presynaptically characterized by an electron-dense projection-the synaptic ribbon-which provides structural scaffolding and tethers a large pool of synaptic vesicles. While advances have been made in recent years in deciphering the molecular anatomy and function of these specialized active zones, the developmental assembly of this presynaptic interaction hub remains largely elusive. In this review, we discuss the dynamic nature of IHC (pre-) synaptogenesis and highlight molecular key players as well as the transport pathways underlying this process. Since developmental assembly appears to be a highly dynamic process, we further ask if this structural plasticity might be maintained into adulthood, how this may influence the functional properties of a given IHC synapse and how such plasticity could be regulated on the molecular level. To do so, we take a closer look at other ribbon-bearing systems, such as retinal photoreceptors and pinealocytes and aim to infer conserved mechanisms that may mediate these phenomena.

Ribbon synapses of cochlear inner hair cells (IHCs) undergo molecular assembly and extensive functional and structural maturation before hearing onset. Here, we characterized the nanostructure of IHC synapses from late prenatal mouse embryo stages (embryonic days 14-18) into adulthood [postnatal day (P)48] using electron microscopy and tomography as well as optical nanoscopy of apical turn organs of Corti. We find that synaptic ribbon precursors arrive at presynaptic active zones (AZs) after afferent contacts have been established. These ribbon precursors contain the proteins RIBEYE and piccolino, tether synaptic vesicles and their delivery likely involves active, microtubule-based transport pathways. Synaptic contacts undergo a maturational transformation from multiple small to one single, large AZ. This maturation is characterized by the fusion of ribbon precursors with membrane-anchored ribbons that also appear to fuse with each other. Such fusion events are most frequently encountered around P12 and hence, coincide with hearing onset in mice. Thus, these events likely underlie the morphological and functional maturation of the AZ. Moreover, the postsynaptic densities appear to undergo a similar refinement alongside presynaptic maturation. Blockwise addition of ribbon material by fusion as found during AZ maturation might represent a general mechanism for modulating ribbon size., Competing Interests: The authors declare no conflict of interest.

Ribbon synapses of cochlear inner hair cells (IHCs) operate with high rates of neurotransmission; yet, the molecular regulation of synaptic vesicle (SV) recycling at these synapses remains poorly understood. Here, we studied the role of endophilins-A1-3, endocytic adaptors with curvature-sensing and curvature-generating properties, in mouse IHCs. Single-cell RT-PCR indicated the expression of endophilins-A1-3 in IHCs, and immunoblotting confirmed the presence of endophilin-A1 and endophilin-A2 in the cochlea. Patch-clamp recordings from endophilin-A-deficient IHCs revealed a reduction of Ca 2+ influx and exocytosis, which we attribute to a decreased abundance of presynaptic Ca 2+ channels and impaired SV replenishment. Slow endocytic membrane retrieval, thought to reflect clathrin-mediated endocytosis, was impaired. Otoferlin, essential for IHC exocytosis, co-immunoprecipitated with purified endophilin-A1 protein, suggestive of a molecular interaction that might aid exocytosis-endocytosis coupling. Electron microscopy revealed lower SV numbers, but an increased occurrence of coated structures and endosome-like vacuoles at IHC active zones. In summary, endophilins regulate Ca 2+ influx and promote SV recycling in IHCs, likely via coupling exocytosis to endocytosis, and contributing to membrane retrieval and SV reformation., (© 2019 The Authors.)

At the presynaptic active zone (AZ), the related cytomatrix proteins CAST and ELKS organize the presynaptic release machinery. While CAST is known to regulate AZ size and neurotransmitter release, the role of ELKS and the integral system of CAST/ELKS together is poorly understood. Here, we show that CAST and ELKS have both redundant and unique roles in coordinating synaptic development, function, and maintenance of retinal photoreceptor ribbon synapses. A CAST/ELKS double knockout (dKO) mouse showed high levels of ectopic synapses and reduced responses to visual stimulation. Ectopic formation was not observed in ELKS conditional KO but progressively increased with age in CAST KO mice with higher rates in the dKO. Presynaptic calcium influx was strongly reduced in rod photoreceptors of CAST KO and dKO mice. Three-dimensional scanning EM reconstructions showed structural abnormalities in rod triads of CAST KO and dKO. Remarkably, AAV-mediated acute ELKS deletion after synapse maturation induced neurodegeneration and loss of ribbon synapses. These results suggest that CAST and ELKS work in concert to promote retinal synapse formation, transmission, and maintenance., (© 2018 Hagiwara et al.)

The timely and reliable processing of auditory and vestibular information within the inner ear requires highly sophisticated sensory transduction pathways. On a cellular level, these demands are met by hair cells, which respond to sound waves - or alterations in body positioning - by releasing glutamate-filled synaptic vesicles (SVs) from their presynaptic active zones with unprecedented speed and exquisite temporal fidelity, thereby initiating the auditory and vestibular pathways. In order to achieve this, hair cells have developed anatomical and molecular specializations, such as the characteristic and name-giving 'synaptic ribbons' - presynaptically anchored dense bodies that tether SVs prior to release - as well as other unique or unconventional synaptic proteins. The tightly orchestrated interplay between these molecular components enables not only ultrafast exocytosis, but similarly rapid and efficient compensatory endocytosis. So far, the knowledge of how endocytosis operates at hair cell ribbon synapses is limited. In this Review, we summarize recent advances in our understanding of the SV cycle and molecular anatomy of hair cell ribbon synapses, with a focus on cochlear inner hair cells., (© 2018 Federation of European Biochemical Societies.)

Synaptic vesicle glycoprotein (SV)2A is a transmembrane protein found in secretory vesicles and is critical for Ca(2+) -dependent exocytosis in central neurons, although its mechanism of action remains uncertain. Previous studies have proposed, variously, a role of SV2 in the maintenance and formation of the readily releasable pool (RRP) or in the regulation of Ca(2+) responsiveness of primed vesicles. Such previous studies have typically used genetic approaches to ablate SV2 levels; here, we used a strategy involving small interference RNA (siRNA) injection to knockdown solely presynaptic SV2A levels in rat superior cervical ganglion (SCG) neuron synapses. Moreover, we investigated the effects of SV2A knockdown on voltage-dependent Ca(2+) channel (VDCC) function in SCG neurons. Thus, we extended the studies of SV2A mechanisms by investigating the effects on vesicular transmitter release and VDCC function in peripheral sympathetic neurons. We first demonstrated an siRNA-mediated SV2A knockdown. We showed that this SV2A knockdown markedly affected presynaptic function, causing an attenuated RRP size, increased paired-pulse depression and delayed RRP recovery after stimulus-dependent depletion. We further demonstrated that the SV2A-siRNA-mediated effects on vesicular release were accompanied by a reduction in VDCC current density in isolated SCG neurons. Together, our data showed that SV2A is required for correct transmitter release at sympathetic neurons. Mechanistically, we demonstrated that presynaptic SV2A: (i) acted to direct normal synaptic transmission by maintaining RRP size, (ii) had a facilitatory role in recovery from synaptic depression, and that (iii) SV2A deficits were associated with aberrant Ca(2+) current density, which may contribute to the secretory phenotype in sympathetic peripheral neurons., (© 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

The afferent synapses between inner hair cells (IHC) and spiral ganglion neurons are specialized to faithfully encode sound with sub-millisecond precision over prolonged periods of time. Here, we studied the role of Rab3 interacting molecule-binding proteins (RIM-BP) 1 and 2 – multidomain proteins of the active zone known to directly interact with RIMs, Bassoon and Ca V 1.3 – in IHC presynaptic function and hearing. Recordings of auditory brainstem responses and otoacoustic emissions revealed that genetic disruption of RIM-BPs 1 and 2 in mice (RIM-BP1/2–/–) causes a synaptopathic hearing impairment exceeding that found in mice lacking RIM-BP2 (RIM-BP2–/–). Patch-clamp recordings from RIM-BP1/2–/– IHCs indicated a subtle impairment of exocytosis from the readily releasable pool of synaptic vesicles that had not been observed in RIM-BP2–/– IHCs. In contrast, the reduction of Ca2+-influx and sustained exocytosis was similar to that in RIMBP2–/– IHCs. We conclude that both RIM-BPs are required for normal sound encoding at the IHC synapse, whereby RIM-BP2 seems to take the leading role. [ABSTRACT FROM AUTHOR]