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    • "One possible interpretation of the experimentally observed mixed exponential/gamma distribution is the parallel action of only four release sites per IHC AZ, each modeled with only two states (Peterson et al, 2014). However, IHC AZs likely feature 10 or more release sites (Frank et al, 2010; Pangr si c et al, 2010), each comprising more than 2 functional states (Andor-Ardo et al, 2010). Therefore , we reason that the gamma process may relate to clearance at each of the 10–15 release sites. "
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    ABSTRACT: Active zones (AZs) of inner hair cells (IHCs) indefatigably release hundreds of vesicles per second, requiring each release site to reload vesicles at tens per second. Here, we report that the endocytic adaptor protein 2μ (AP-2μ) is required for release site replenishment and hearing. We show that hair cell-specific disruption of AP-2μ slows IHC exocytosis immediately after fusion of the readily releasable pool of vesicles, despite normal abundance of membrane-proximal vesicles and intact endocytic membrane retrieval. Sound-driven postsynaptic spiking was reduced in a use-dependent manner, and the altered interspike interval statistics suggested a slowed reloading of release sites. Sustained strong stimulation led to accumulation of endosome-like vacuoles, fewer clathrin-coated endocytic intermediates, and vesicle depletion of the membrane-distal synaptic ribbon in AP-2μ-deficient IHCs, indicating a further role of AP-2μ in clathrin-dependent vesicle reformation on a timescale of many seconds. Finally, we show that AP-2 sorts its IHC-cargo otoferlin. We propose that binding of AP-2 to otoferlin facilitates replenishment of release sites, for example, via speeding AZ clearance of exocytosed material, in addition to a role of AP-2 in synaptic vesicle reformation.
    No preview · Article · Oct 2015 · The EMBO Journal
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    • "Bassoon was also shown to regulate RRP size, Ca 2+ current amplitude, and occupancy of release sites at the ribbon synapse of the inner hair cell (Frank et al., 2010). These latter findings may be limited to ribbon synapses, since they seem to correlate with the detachment of the ribbon from the AZ (Frank et al., 2010; Jing et al., 2013). In our study, we could not detect any changes in RRP size or calcium current amplitude , but we noticed a slight, non-significant reduction in the replenishment rate during high-frequency trains. "
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    ABSTRACT: Mover, a member of the exquisitely small group of vertebrate-specific presynaptic proteins, has been discovered as an interaction partner of the scaffolding protein Bassoon, yet its function has not been elucidated. We used adeno-associated virus (AAV)-mediated shRNA expression to knock down Mover in the calyx of Held in vivo. Although spontaneous synaptic transmission remained unaffected, we found a strong increase of the evoked EPSC amplitude. The size of the readily releasable pool was unaltered, but short-term depression was accelerated and enhanced, consistent with an increase in release probability after Mover knockdown. This increase in release probability was not caused by alterations in Ca(2+) influx but rather by a higher Ca(2+) sensitivity of the release machinery, as demonstrated by presynaptic Ca(2+) uncaging. We therefore conclude that Mover expression in certain subsets of synapses negatively regulates synaptic release probability, constituting a novel mechanism to tune synaptic transmission. Copyright © 2015 Elsevier Inc. All rights reserved.
    Full-text · Article · Jul 2015 · Neuron
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    • " with 60 nm lateral resolution ( Hell and Wichmann , 1994 ; Nägerl et al . , 2008 ; Liu et al . , 2011 ; Urban et al . , 2011 ) . STED microscopy was instrumental in detecting a disorganization of Ca 2+ channel punctae in inner ear hair cells lacking Bassoon , demonstrating its role in organizing the precise localization of Ca 2+ channels at AZs ( Frank et al . , 2010 ) . Detection efficiency in immuno - EM is limited by many factors , including embedding material , epitope preservation , lack of specific antibodies and difficulty achieving adequate tissue penetration . Correlative light and EM microscopy ( CLEM ) has begun to address these issues by combining the advantages of EM with those of fluor"
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    ABSTRACT: Synapses are the fundamental functional units of neural circuits, and their dysregulation has been implicated in diverse neurological disorders. At presynaptic terminals, neurotransmitter-filled synaptic vesicles are released in response to calcium influx through voltage-gated calcium channels activated by the arrival of an action potential. Decades of electrophysiological, biochemical, and genetic studies have contributed to a growing understanding of presynaptic biology. Imaging studies are yielding new insights into how synapses are organized to carry out their critical functions. The development of techniques for rapid immobilization and preservation of neuronal tissues for electron microscopy has led to a new renaissance in ultrastructural imaging that is rapidly advancing our understanding of synapse structure and function.
    Full-text · Article · May 2015 · Frontiers in Cellular Neuroscience
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