The synaptic ribbon is critical for sound encoding at high rates and with temporal precision.

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.

Hair cells use active zones with different voltage dependence of Ca2+ influx to decompose sounds into complementary neural codes.

For sounds of a given frequency, spiral ganglion neurons (SGNs) with different thresholds and dynamic ranges collectively encode the wide range of audible sound pressures. Heterogeneity of synapses between inner hair cells (IHCs) and SGNs is an attractive candidate mechanism for generating complementary neural codes covering the entire dynamic range. Here, we quantified active zone (AZ) properties as a function of AZ position within mouse IHCs by combining patch clamp and imaging of presynaptic Ca(2+) influx and by immunohistochemistry. We report substantial AZ heterogeneity whereby the voltage of half-maximal activation of Ca(2+) influx ranged over ∼20 mV. Ca(2+) influx at AZs facing away from the ganglion activated at weaker depolarizations. Estimates of AZ size and Ca(2+) channel number were correlated and larger when AZs faced the ganglion. Disruption of the deafness gene GIPC3 in mice shifted the activation of presynaptic Ca(2+) influx to more hyperpolarized potentials and increased the spontaneous SGN discharge. Moreover, Gipc3 disruption enhanced Ca(2+) influx and exocytosis in IHCs, reversed the spatial gradient of maximal Ca(2+) influx in IHCs, and increased the maximal firing rate of SGNs at sound onset. We propose that IHCs diversify Ca(2+) channel properties among AZs and thereby contribute to decomposing auditory information into complementary representations in SGNs.

Optogenetic stimulation of the auditory pathway.

Auditory prostheses can partially restore speech comprehension when hearing fails. Sound coding with current prostheses is based on electrical stimulation of auditory neurons and has limited frequency resolution due to broad current spread within the cochlea. In contrast, optical stimulation can be spatially confined, which may improve frequency resolution. Here, we used animal models to characterize optogenetic stimulation, which is the optical stimulation of neurons genetically engineered to express the light-gated ion channel channelrhodopsin-2 (ChR2). Optogenetic stimulation of spiral ganglion neurons (SGNs) activated the auditory pathway, as demonstrated by recordings of single neuron and neuronal population responses. Furthermore, optogenetic stimulation of SGNs restored auditory activity in deaf mice. Approximation of the spatial spread of cochlear excitation by recording local field potentials (LFPs) in the inferior colliculus in response to suprathreshold optical, acoustic, and electrical stimuli indicated that optogenetic stimulation achieves better frequency resolution than monopolar electrical stimulation. Virus-mediated expression of a ChR2 variant with greater light sensitivity in SGNs reduced the amount of light required for responses and allowed neuronal spiking following stimulation up to 60 Hz. Our study demonstrates a strategy for optogenetic stimulation of the auditory pathway in rodents and lays the groundwork for future applications of cochlear optogenetics in auditory research and prosthetics.

Concurrent maturation of inner hair cell synaptic Ca2+ influx and auditory nerve spontaneous activity around hearing onset in mice.

Hearing over a wide range of sound intensities is thought to require complementary coding by functionally diverse spiral ganglion neurons (SGNs), each changing activity only over a subrange. The foundations of SGN diversity are not well understood but likely include differences among their inputs: the presynaptic active zones (AZs) of inner hair cells (IHCs). Here we studied one candidate mechanism for causing SGN diversity-heterogeneity of Ca(2+) influx among the AZs of IHCs-during postnatal development of the mouse cochlea. Ca(2+) imaging revealed a change from regenerative to graded synaptic Ca(2+) signaling after the onset of hearing, when in vivo SGN spike timing changed from patterned to Poissonian. Furthermore, we detected the concurrent emergence of stronger synaptic Ca(2+) signals in IHCs and higher spontaneous spike rates in SGNs. The strengthening of Ca(2+) signaling at a subset of AZs primarily reflected a gain of Ca(2+) channels. We hypothesize that the number of Ca(2+) channels at each IHC AZ critically determines the firing properties of its corresponding SGN and propose that AZ heterogeneity enables IHCs to decompose auditory information into functionally diverse SGNs.

Onset coding is degraded in auditory nerve fibers from mutant mice lacking synaptic ribbons.

Synaptic ribbons, found at the presynaptic membrane of sensory cells in both ear and eye, have been implicated in the vesicle-pool dynamics of synaptic transmission. To elucidate ribbon function, we characterized the response properties of single auditory nerve fibers in mice lacking Bassoon, a scaffolding protein involved in anchoring ribbons to the membrane. In bassoon mutants, immunohistochemistry showed that fewer than 3% of the hair cells' afferent synapses retained anchored ribbons. Auditory nerve fibers from mutants had normal threshold, dynamic range, and postonset adaptation in response to tone bursts, and they were able to phase lock with normal precision to amplitude-modulated tones. However, spontaneous and sound-evoked discharge rates were reduced, and the reliability of spikes, particularly at stimulus onset, was significantly degraded as shown by an increased variance of first-spike latencies. Modeling based on in vitro studies of normal and mutant hair cells links these findings to reduced release rates at the synapse. The degradation of response reliability in these mutants suggests that the ribbon and/or Bassoon normally facilitate high rates of exocytosis and that its absence significantly compromises the temporal resolving power of the auditory system.