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For normal cochlear function, outer hair cells (OHCs) require a precise control of intracellular Ca2+ levels. In the absence of regulatory elements such as proteinaceous buffers or extrusion pumps, OHCs degenerate, leading to profound hearing impairment. Influx of Ca2+ occurs both at the stereocilia tips and the basolateral membrane. In this latter compartment, two different origins for Ca2+ influx have been poorly explored: voltage-gated L-type Ca2+ channels (VGCCs) at synapses with Type II afferent neurons, and a9a10 cholinergic nicotinic receptors at synapses with medio-olivochlear complex (MOC) neurons. Using functional imaging in mouse OHCs, we dissected Ca2+ influx individually through each of these sources, either by applying step depolarizations to activate VGCC, or stimulating MOC axons. Ca2+ ions originated in MOC synapses, but not by VGCC activation, was confined by Ca2+-ATPases most likely present in nearby synaptic cisterns. Although Ca2+ currents in OHCs are small, VGCC Ca2+ signals were comparable in size to those elicited by a9a10 receptors, and were potentiated by ryanodine receptors (RyRs). In contrast, no evidence of potentiation by RyRs was found for MOC Ca2+ signals over a wide range of presynaptic stimulation strengths. Our study shows that despite the fact that these two Ca2+ entry sites are closely positioned, they differ in their regulation by intracellular cisterns and/or organelles, suggesting the existence of well-tuned mechanisms to separate the two different OHC synaptic functions. For normal cochlear function, outer hair cells (OHCs) require a precise control of intracellular Ca2+ levels. In the absence of regulatory elements such as proteinaceous buffers or extrusion pumps, OHCs degenerate, leading to profound hearing impairment. Influx of Ca2+ occurs both at the stereocilia tips and the basolateral membrane. In this latter compartment, two different origins for Ca2+ influx have been poorly explored: voltage-gated L-type Ca2+ channels (VGCCs) at synapses with Type II afferent neurons, and a9a10 cholinergic nicotinic receptors at synapses with medio-olivochlear complex (MOC) neurons. Using functional imaging in mouse OHCs, we dissected Ca2+ influx individually through each of these sources, either by applying step depolarizations to activate VGCC, or stimulating MOC axons. Ca2+ ions originated in MOC synapses, but not by VGCC activation, was confined by Ca2+-ATPases most likely present in nearby synaptic cisterns. Although Ca2+ currents in OHCs are small, VGCC Ca2+ signals were comparable in size to those elicited by a9a10 receptors, and were potentiated by ryanodine receptors (RyRs). In contrast, no evidence of potentiation by RyRs was found for MOC Ca2+ signals over a wide range of presynaptic stimulation strengths. Our study shows that despite the fact that these two Ca2+ entry sites are closely positioned, they differ in their regulation by intracellular cisterns and/or organelles, suggesting the existence of well-tuned mechanisms to separate the two different OHC synaptic functions. [ABSTRACT FROM AUTHOR]

The lateral line (LL) is a sensory system that allows fish and amphibians to detect water currents. LL responsiveness is modulated by efferent neurons that aid in distinguishing between external and self-generated stimuli, maintaining sensitivity to relevant cues. One component of the efferent system is cholinergic, the activation of which inhibits afferent activity. LL hair cells (HCs) share structural, functional, and molecular similarities with those of the cochlea, making them a popular model for studying human hearing and balance disorders. Because of these commonalities, one could propose that the receptor at the LL efferent synapse is a α9α10 nicotinic acetylcholine receptor (nAChR). However, the identities of the molecular players underlying ACh-mediated inhibition in the LL remain unknown. Surprisingly, through the analysis of single-cell expression studies and in situ hybridization, we describe that α9, but not the α10, subunits are enriched in zebrafish HCs. Moreover, the heterologous expression of zebrafish α9 subunits indicates that homomeric receptors are functional and exhibit robust AChgated currents blocked by a-bungarotoxin and strychnine. In addition, in vivo Ca21 imaging on mechanically stimulated zebrafish LL HCs show that ACh elicits a decrease in evoked Ca21 signals, regardless of HC polarity. This effect is blocked by both a-bungarotoxin and apamin, indicating coupling of ACh-mediated effects to small-conductance Ca21-activated potassium (SKs) channels. Our results indicate that an α9-containing (α9p) nAChR operates at the zebrafish LL efferent synapse. Moreover, the activation of α9p nAChRs most likely leads to LL HC hyperpolarization served by SK channels. [ABSTRACT FROM AUTHOR]

The auditory system processes time and intensity through separate brainstem pathways to derive spatial location as well as other salient features of sound. The independent coding of time and intensity begins in the cochlea, where afferent neurons can fire action potentials at constant phase throughout a wide range of stimulus intensities. We have investigated time and intensity coding by simultaneous presynaptic and postsynaptic recording at the hair cell-afferent synapse from rats. Trains of depolarizing steps to die hair cell were used to elicit postsynaptic currents that occurred at constant phase for a range of membrane potentials over which release probability varied significandy. To probe the underlying mechanisms, release was examined using single steps to various command voltages. As expected for vesicular release, first synaptic events occurred earlier as presynaptic calcium influx grew larger. However, synaptic depression produced smaller responses with longer first latencies. Thus, during repetitive hair cell stimulation, as the hair cell is more strongly depolarized, increased calcium channel gating hurries transmitter release, but the resulting vesicular depletion produces a compensatory slowing. Quantitative simulation of ribbon function shows that these two factors varied reciprocally with hair cell depolarization (stimulus intensity) to produce constant synaptic phase. Finally, we propose that the observed rapid vesicle replenishment would help maintain the vesicle pool, which in turn would equilibrate with the stimulus intensity (and therefore the number of open Ca2+ channels), so that for trains of different levels the average phase will be conserved. [ABSTRACT FROM AUTHOR]

Inner hair cells (IHCs) in the mammalian cochlea are able to continuously release neurotransmitter in the presence of constant stimuli. Nonetheless, strong synaptic depression is observed over the first few milliseconds of stimulation. This process most likely underlies adaptation in the auditory nerve. In the present study we demonstrate that under certain conditions of stimulation, facilitation can occur at the IHC ribbon synapse. Using simultaneous whole-cell, voltage-clamp recordings from IHCs and afferent fiber endings in excised postnatal rat cochleae, we stimulated IHCs with 2 ms long test depolarizations from a holding potential of −89 mV. Synaptic currents in afferent fibers occurred with high failure rates of ∼50%. However, when a pre-depolarization to values of −55 to −49 mV was implemented before the test pulse, success rates of the synaptic response increased to 100%, the strength of the synaptic response increased ∼2.8-fold, and synaptic latency was reduced by ∼50%. When calcium influx was minimized during pre-depolarization, none of these effects were found, suggesting that calcium influx during pre-depolarizations is required for synaptic conditioning. Similarly, in response to paired-pulse protocols, short term facilitation occurred. The response to the second stimulus increased up to ∼5-fold, and its latency was reduced by up to 35% compared to the response to the first stimulus. We propose that at the IHC resting membrane potential, the ribbon synapse operates in a constantly facilitated mode caused by Ca2+ influx, optimizing the size and timing of the postsynaptic response in auditory nerve fibers. [ABSTRACT FROM AUTHOR]

For normal cochlear function, outer hair cells (OHCs) require a precise control of intracellular Ca 2+ levels. In the absence of regulatory elements such as proteinaceous buffers or extrusion pumps, OHCs degenerate, leading to profound hearing impairment. Influx of Ca 2+ occurs both at the stereocilia tips and the basolateral membrane. In this latter compartment, two different origins for Ca 2+ influx have been poorly explored: voltage-gated L-type Ca 2+ channels (VGCCs) at synapses with Type II afferent neurons, and α9α10 cholinergic nicotinic receptors at synapses with medio-olivochlear complex (MOC) neurons. Using functional imaging in mouse OHCs, we dissected Ca 2+ influx individually through each of these sources, either by applying step depolarizations to activate VGCC, or stimulating MOC axons. Ca 2+ ions originated in MOC synapses, but not by VGCC activation, was confined by Ca 2+ -ATPases most likely present in nearby synaptic cisterns. Although Ca 2+ currents in OHCs are small, VGCC Ca 2+ signals were comparable in size to those elicited by α9α10 receptors, and were potentiated by ryanodine receptors (RyRs). In contrast, no evidence of potentiation by RyRs was found for MOC Ca 2+ signals over a wide range of presynaptic stimulation strengths. Our study shows that despite the fact that these two Ca 2+ entry sites are closely positioned, they differ in their regulation by intracellular cisterns and/or organelles, suggesting the existence of well-tuned mechanisms to separate the two different OHC synaptic functions. SIGNIFICANCE STATEMENT Outer hair cells (OHCs) are sensory cells in the inner ear operating under very special constraints. Acoustic stimulation leads to fast changes both in membrane potential and in the intracellular concentration of metabolites such as Ca 2+ Tight mechanisms for Ca 2+ control in OHCs have been reported. Interestingly, Ca 2+ is crucial for two important synaptic processes: inhibition by efferent cholinergic neurons, and glutamate release onto Type II afferent fibers. In the current study we functionally imaged Ca 2+ at these two different synapses, showing close positioning within the basolateral compartment of OHCs. In addition, we show differential regulation of these two Ca 2+ sources by synaptic cisterns and/or organelles, which could result crucial for functional segregation during normal hearing., (Copyright © 2021 the authors.)

The lateral line (LL) is a sensory system that allows fish and amphibians to detect water currents. LL responsiveness is modulated by efferent neurons that aid in distinguishing between external and self-generated stimuli, maintaining sensitivity to relevant cues. One component of the efferent system is cholinergic, the activation of which inhibits afferent activity. LL hair cells (HCs) share structural, functional, and molecular similarities with those of the cochlea, making them a popular model for studying human hearing and balance disorders. Because of these commonalities, one could propose that the receptor at the LL efferent synapse is a α9α10 nicotinic acetylcholine receptor (nAChR). However, the identities of the molecular players underlying ACh-mediated inhibition in the LL remain unknown. Surprisingly, through the analysis of single-cell expression studies and in situ hybridization, we describe that α9, but not the α10, subunits are enriched in zebrafish HCs. Moreover, the heterologous expression of zebrafish α9 subunits indicates that homomeric receptors are functional and exhibit robust ACh-gated currents blocked by α-bungarotoxin and strychnine. In addition, in vivo Ca 2+ imaging on mechanically stimulated zebrafish LL HCs show that ACh elicits a decrease in evoked Ca 2+ signals, regardless of HC polarity. This effect is blocked by both α-bungarotoxin and apamin, indicating coupling of ACh-mediated effects to small-conductance Ca 2+ -activated potassium (SKs) channels. Our results indicate that an α9-containing (α9*) nAChR operates at the zebrafish LL efferent synapse. Moreover, the activation of α9* nAChRs most likely leads to LL HC hyperpolarization served by SK channels. SIGNIFICANCE STATEMENT The fish lateral line (LL) mechanosensory system shares structural, functional, and molecular similarities with those of the mammalian cochlea. Thus, it has become an accessible model for studying human hearing and balance disorders. However, the molecular players serving efferent control of LL hair cell (HC) activity have not been identified. Here we demonstrate that, different from the hearing organ of vertebrate species, a nicotinic acetylcholine receptor composed only of α9 subunits operates at the LL efferent synapse. Activation of α9-containing receptors leads to LL HC hyperpolarization because of the opening of small-conductance Ca 2+ -activated potassium channels. These results will further aid in the interpretation of data obtained from LL HCs as a model for cochlear HCs., (Copyright © 2021 the authors.)