We studied the role of the synaptic ribbon for sound encoding at the synapses between inner hair cells (IHCs) and spiral ganglion neurons (SGNs) in mice lacking RIBEYE (RBEKO/KO). Electron and immunofluorescence microscopy revealed a lack of synaptic ribbons and an assembly of several small active zones (AZs) at each synaptic contact. Spontaneous and sound-evoked firing rates of SGNs and their compound action potential were reduced, indicating impaired transmission at ribbonless IHC-SGN synapses. The temporal precision of sound encoding was impaired and the recovery of SGN-firing from adaptation indicated slowed synaptic vesicle (SV) replenishment. Activation of Ca2+-channels was shifted to more depolarized potentials and exocytosis was reduced for weak depolarizations. Presynaptic Ca2+-signals showed a broader spread, compatible with the altered Ca2+-channel clustering observed by super-resolution immunofluorescence microscopy. We postulate that RIBEYE disruption is partially compensated by multi-AZ organization. The remaining synaptic deficit indicates ribbon function in SV-replenishment and Ca2+-channel regulation.

Ants rely heavily on olfaction for communication and orientation. Here we provide the first detailed structure-function analyses within an ant's central olfactory system asking whether in the carpenter ant, Camponotus floridanus, the olfactory pathway exhibits adaptations to processing many pheromonal and general odors. Using fluorescent tracing, confocal microscopy, and 3D-analyses we demonstrate that the antennal lobe (AL) contains up to approximately 460 olfactory glomeruli organized in seven distinct clusters innervated via seven antennal sensory tracts. The AL is divided into two hemispheres regarding innervation of glomeruli by either projection neurons (PNs) with axons leaving via the medial (m) or lateral (l) antennocerebral tract (ACT). M- and l-ACT PNs differ in their target areas in the mushroom-body calyx and lateral horn. Three additional ACTs project to the lateral protocerebrum only. We analyzed odor processing in AL glomeruli by retrograde loading of PNs with Fura-2 dextran and fluorimetric calcium imaging. Odor responses were reproducible and comparable across individuals. Calcium responses to pheromonal and nonpheromonal odors were very sensitive (10(-11) dilution) and patterns were partly overlapping, indicating that processing of both odor classes is not spatially segregated within the AL. Response patterns to the main trail-pheromone component nerolic acid remained stable over a wide range of intensities (7-8 log units), while response durations increased indicating that odor quality is maintained by a stable pattern and intensity is mainly encoded in response durations. The structure-function analyses contribute new insights into important aspects of odor processing in a highly advanced insect olfactory system.

The mechanisms underlying the large amplitudes and heterogeneity of excitatory postsynaptic currents (EPSCs) at inner hair cell (IHC) ribbon synapses are unknown. Based on electrophysiology, electron and superresolution light microscopy, and modeling, we propose that uniquantal exocytosis shaped by a dynamic fusion pore is a candidate neurotransmitter release mechanism in IHCs. Modeling indicated that the extended postsynaptic AMPA receptor clusters enable large uniquantal EPSCs. Recorded multiphasic EPSCs were triggered by similar glutamate amounts as monophasic ones and were consistent with progressive vesicle emptying during pore flickering. The fraction of multiphasic EPSCs decreased in absence of Ca(2+) influx and upon application of the Ca(2+) channel modulator BayK8644. Our experiments and modeling did not support the two most popular multiquantal release interpretations of EPSC heterogeneity: (1) Ca(2+)-synchronized exocytosis of multiple vesicles and (2) compound exocytosis fueled by vesicle-to-vesicle fusion. We propose that IHC synapses efficiently use uniquantal glutamate release for achieving high information transmission rates.

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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 2l (AP-2l) is required for release site replenishment and hearing. We show that hair cell-specific disruption of AP-2l 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 endosomelike vacuoles, fewer clathrin-coated endocytic intermediates, and vesicle depletion of the membrane-distal synaptic ribbon in AP-2l-deficient IHCs, indicating a further role of AP-2l 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.

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ABSTRACT

Ca2+ influx triggers the release of synaptic vesicles at the presynaptic active zone (AZ). A quantitative characterization of presynaptic Ca2+ signaling is critical for understanding synaptic transmission. However, this has remained challenging to establish at the required resolution. Here, we employ confocal and stimulated emission depletion (STED) microscopy to quantify the number (20–330) and arrangement (mostly linear 70 nm × 100–600 nm clusters) of Ca2+ channels at AZs of mouse cochlear inner hair cells (IHCs). Establishing STED Ca2+ imaging, we analyze presynaptic Ca2+ signals at the nanometer scale and find confined elongated Ca2+ domains at normal IHC AZs, whereas Ca2+ domains are spatially spread out at the AZs of bassoon-deficient IHCs. Performing 2D-STED fluorescence lifetime analysis, we arrive at estimates of the Ca2+ concentrations at stimulated IHC AZs of on average 25 µM. We propose that IHCs form bassoon-dependent presynaptic Ca2+-channel clusters of similar density but scalable length, thereby varying the number of Ca2+ channels amongst individual AZs.