Mapping developmental maturation of inner hair cell ribbon synapses in the apical mouse cochlea

Abstract

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 membraneanchored 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. synaptogenesis | presynaptic development | ribbon synapse maturation | synaptic heterogeneity I n mammals, synaptic sound encoding occurs at the first auditory synapse between cochlear inner hair cells (IHCs) and postsynaptic neurites of afferent spiral ganglion neurons (SGNs). The highly specialized IHC presynaptic active zones (AZs) are characterized by the presence of proteinaceous electron-dense bodies, called "synaptic ribbons," which are primarily composed of the structural cytomatrix protein RIBEYE (1, 2). Ribbons provide presynaptic scaffolding, cluster and functionally regulate presynaptic Ca 2+ channels at the AZ membrane (3-5), and tether a halo of synaptic vesicles (SVs) (6). This latter feature is thought to enable rapid and indefatigable vesicular replenishment to the release site-even during periods of persistent stimulation (3,5,7,8).In mice, hearing onset occurs around postnatal day (P)12 (9) before which, IHC presynaptic AZs undergo a range of structural and functional refinements. For example, extrasynaptically localized Ca 2+ channels are progressively eliminated from the nonsynaptic basolateral plasma membrane and form-in concert with the corresponding postsynaptic glutamate receptor patchestightly confined clusters at mature presynaptic AZs (3, 10). Moreover, otoferlin-a large Ca 2+ -binding multi-C 2 domain protein (11, 12)-likely replaces synaptotagmins as the putative Ca 2+ sensor of IHC release during the first postnatal week (13). This finding reflects a key landmark of functional maturation of this unconventional high-throughput release machinery and is essentially required to faithfully orchestrate vesicular...

Electron Microscopic Reconstruction of Neural Circuitry in the Cochlea

“…3 b). As previously reported for the apex of the mouse cochlea 18 , AZs with multiple ribbons preferred the modiolar IHC side (20 out of 35) also in our mid-cochlear dataset.…”

“…Volume EM techniques have been recently employed to investigate ribbon synapse morphology in the apical cochlea of C57BL/6 mice at different developmental stages 18 . Our SBEM dataset now provides detailed structural insight into the mature mid-turn and, hence, most sound sensitive region of the cochlea 14, 23, 42 in the CBA mouse strain.…”

“…So far, the presence of multiple ribbons at a single AZ was considered the exception to the rule and, if encountered, to occur at modiolar AZs 12, 15, 18, 19 . Here, we found that about 15 % afferent contacts contain more than one ribbon in the middle turn of the mouse cochlea (Fig.…”

Pou4f1 Defines a Subgroup of Type I Spiral Ganglion Neurons and Is Necessary for Normal Inner Hair Cell Presynaptic Ca2+ Signaling

“…Interestingly, a similar observation was made in the third postnatal week of CBA mice (Liberman and Liberman, 2016), with a gradient for larger synaptic ribbons on the pillar side, in contrast to our previous report at the same ages (Ohn et al, 2016, C57BL/6J). Nonetheless, future studies, ideally involving electron microscopy, should follow up on this observation over several devel-opmental stages (Liberman and Liberman, 2016;Michanski et al, 2019) and compare with previous studies on mouse cochleae from different mouse strains (Liberman et al, 2011;Liberman and Liberman, 2016;Ohn et al, 2016).…”

A sensory cell diversifies its output by varying Ca2+ influx-release coupling among presynaptic active zones for wide range intensity coding

“…In the cat 6 , back-tracing experiments linked morphology to function and showed that low-SR SGNs have thinner radial fibers (peripheral neurites) with fewer mitochondria than high-SR. Low-SR SGNs preferentially innervate the modiolar (or neural) side of the IHC, where they face larger and more complex presynaptic AZs [6][7][8][9] . Larger and more complex AZs at the modiolar side of IHCs were also found in mouse [10][11][12] , guinea pig 13,14 , and gerbil 15 , suggesting some features of SGN morphology and synaptic connectivity may be shared across species. In the mouse, RNA sequencing of individual SGNs indicated three distinct subtypes [16][17][18] that were suggested to correspond to low, medium, and high-SR SGNs based on the spatial segregation of their IHC innervation.…”