Developmental changes of the mitochondria in the murine anteroventral cochlear nucleus.

Mitochondria are key organelles to provide ATP for synaptic transmission. This study aims to unravel the structural adaptation of mitochondria to an increase in presynaptic energy demand and upon the functional impairment of the auditory system. We use the anteroventral cochlear nucleus (AVCN) of wild-type and congenital deaf mice before and after hearing onset as a model system for presynaptic states of lower and higher energy demands. We combine focused ion beam scanning electron microscopy and electron tomography to investigate mitochondrial morphology. We found a larger volume of synaptic boutons and mitochondria after hearing onset with a higher crista membrane density. In deaf animals lacking otoferlin, we observed a shallow increase of mitochondrial volumes toward adulthood in endbulbs, while in wild-type animals mitochondria further enlarged. We propose that in the AVCN, presynaptic mitochondria undergo major structural changes likely to serve higher energy demands upon the onset of hearing and further maturation.

RIM-Binding Protein 2 Organizes Ca2+ Channel Topography and Regulates Release Probability and Vesicle Replenishment at a Fast Central Synapse.

Rab-interacting molecule (RIM)-binding protein 2 (BP2) is a multidomain protein of the presynaptic active zone (AZ). By binding to RIM, bassoon (Bsn), and voltage-gated Ca2+ channels (CaV), it is considered to be a central organizer of the topography of CaV and release sites of synaptic vesicles (SVs) at the AZ. Here, we used RIM-BP2 knock-out (KO) mice and their wild-type (WT) littermates of either sex to investigate the role of RIM-BP2 at the endbulb of Held synapse of auditory nerve fibers (ANFs) with bushy cells (BCs) of the cochlear nucleus, a fast relay of the auditory pathway with high release probability. Disruption of RIM-BP2 lowered release probability altering short-term plasticity and reduced evoked EPSCs. Analysis of SV pool dynamics during high-frequency train stimulation indicated a reduction of SVs with high release probability but an overall normal size of the readily releasable SV pool (RRP). The Ca2+-dependent fast component of SV replenishment after RRP depletion was slowed. Ultrastructural analysis by superresolution light and electron microscopy revealed an impaired topography of presynaptic CaV and a reduction of docked and membrane-proximal SVs at the AZ. We conclude that RIM-BP2 organizes the topography of CaV, and promotes SV tethering and docking. This way RIM-BP2 is critical for establishing a high initial release probability as required to reliably signal sound onset information that we found to be degraded in BCs of RIM-BP2-deficient mice in vivoSIGNIFICANCE STATEMENT Rab-interacting molecule (RIM)-binding proteins (BPs) are key organizers of the active zone (AZ). Using a multidisciplinary approach to the calyceal endbulb of Held synapse that transmits auditory information at rates of up to hundreds of Hertz with submillisecond precision we demonstrate a requirement for RIM-BP2 for normal auditory signaling. Endbulb synapses lacking RIM-BP2 show a reduced release probability despite normal whole-terminal Ca2+ influx and abundance of the key priming protein Munc13-1, a reduced rate of SV replenishment, as well as an altered topography of voltage-gated (CaV)2.1 Ca2+ channels, and fewer docked and membrane proximal synaptic vesicles (SVs). This hampers transmission of sound onset information likely affecting downstream neural computations such as of sound localization.

Ultrastructural maturation of the endbulb of Held active zones comparing wild-type and otoferlin-deficient mice.

Endbulbs of Held are located in the anteroventral cochlear nucleus and present the first central synapses of the auditory pathway. During development, endbulbs mature functionally to enable rapid and powerful synaptic transmission with high temporal precision. This process is accompanied by morphological changes of endbulb terminals. Loss of the hair cell-specific protein otoferlin (Otof) abolishes neurotransmission in the cochlea and results in the smaller endbulb of Held terminals. Thus, peripheral hearing impairment likely also leads to alterations in the morphological synaptic vesicle (SV) pool size at individual endbulb of Held active zones (AZs). Here, we investigated endbulb AZs in pre-hearing, young, and adult wild-type and Otof -/- mice. During maturation, SV numbers at endbulb AZs increased in wild-type mice but were found to be reduced in Otof -/ - mice. The SV population at a distance of 0-15 nm was most strongly affected. Finally, overall SV diameters decreased in Otof -/- animals during maturation.