Abstract

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 vivo.SIGNIFICANCE 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.

Highlights

  • Active zones (AZs) are specialized regions at the presynaptic terminals where neurotransmitter release occurs

  • Deletion of Rab-interacting molecule (RIM)-BP2 impairs synchronous transmitter release at the endbulb of held synapse To determine the functional role of RIM-BP2, we studied synaptic transmission at the endbulb of Held synapse in acute parasagittal slices of the brainstem of constitutive RIM-BP2 KO mice (RIM-BP2 KO; Grauel et al, 2016) recording spontaneous EPSCs

  • We probed the role of RIM-BP2, thought to serve as molecular linker between CaV and release sites, and alternatively in synaptic vesicle (SV) priming via Munc13, in synaptic transmission at the endbulb of Held synapse

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Summary

Introduction

Active zones (AZs) are specialized regions at the presynaptic terminals where neurotransmitter release occurs. The function and abundance of CaV (for recent review, see Pangrsic et al, 2018; Dolphin and Lee, 2020) is positively regulated by auxiliary subunits and multidomain proteins of the AZ such as Rab-interacting molecule (RIM)-binding protein (BP), RIM, piccolo, bassoon (Bsn), CAST, and ELKS. Several of these proteins promote the clustering of Ca21 channels at the AZ and/or their interaction with the SV release sites (Gundelfinger and Fejtova, 2012; Südhof, 2012; Moser et al., 2020).

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