GABAergic synapses between auditory efferent neurons and type II spiral ganglion afferent neurons in the mouse cochlea.

Cochlear outer hair cells (OHCs) are electromotile and are implicated in mechanisms of amplification of responses to sound that enhance sound sensitivity and frequency tuning. They send information to the brain through glutamatergic synapses onto a small subpopulation of neurons of the ascending auditory nerve, the type II spiral ganglion neurons (SGNs). The OHC synapses onto type II SGNs are sparse and weak, suggesting that type II SGNs respond primarily to loud and possibly damaging levels of sound. OHCs also receive innervation from the brain through the medial olivocochlear (MOC) efferent neurons. MOC neurons are cholinergic yet exert an inhibitory effect on auditory function as they are coupled to alpha9/alpha10 nicotinic acetylcholine receptors (nAChRs) on OHCs, which leads to calcium influx that gates SK potassium channels. The net hyperpolarization exerted by this efferent synapse reduces OHC activity-evoked electromotility and is implicated in cochlear gain control, protection against acoustic trauma, and attention. MOC neurons also label for markers of gamma-aminobutyric acid (GABA) and GABA synthesis. GABAB autoreceptor (GABABR) activation by GABA released from MOC terminals has been demonstrated to reduce ACh release, confirming important negative feedback roles for GABA. However, the full complement of GABAergic activity in the cochlea is not currently understood, including the mechanisms that regulate GABA release from MOC axon terminals, whether GABA diffuses from MOC axon terminals to other postsynaptic cells, and the location and function of GABAA receptors (GABAARs). Previous electron microscopy studies suggest that MOC neurons form contacts onto several other cell types in the cochlea, but whether these contacts form functional synapses, and what neurotransmitters are employed, are unknown. Here we use immunohistochemistry, optical neurotransmitter imaging and patch-clamp electrophysiology from hair cells, afferent dendrites, and efferent axons to demonstrate that in addition to presynaptic GABABR autoreceptor activation, MOC efferent axon terminals release GABA onto type II SGN afferent dendrites with postsynaptic activity mediated by GABAARs. This synapse may have multiple roles including developmental regulation of cochlear innervation, fine tuning of OHC activity, or providing feedback to the brain about MOC and OHC activity.

Functional Postnatal Maturation of the Medial Olivocochlear Efferent-Outer Hair Cell Synapse.

The organ of Corti, the auditory mammalian sensory epithelium, contains two types of mechanotransducer cells, inner hair cells (IHCs) and outer hair cells (OHCs). IHCs are involved in conveying acoustic stimuli to the CNS, while OHCs are implicated in the fine tuning and amplification of sounds. OHCs are innervated by medial olivocochlear (MOC) cholinergic efferent fibers. The functional characteristics of the MOC-OHC synapse during maturation were assessed by electrophysiological and pharmacological methods in mouse organs of Corti at postnatal day 11 (P11)-P13, hearing onset in altricial rodents, and at P20-P22 when the OHCs are morphologically and functionally mature. Synaptic currents were recorded in whole-cell voltage-clamped OHCs while electrically stimulating the MOC fibers. A progressive increase in the number of functional MOC-OHC synapses, as well as in their strength and efficacy, was observed between P11-13 and P20-22. At hearing onset, the MOC-OHC synapse presented facilitation during MOC fibers high-frequency stimulation that disappeared at mature stages. In addition, important changes were found in the VGCC that are coupled to transmitter release. Ca2+ flowing in through L-type VGCCs contribute to trigger ACh release together with P/Q- and R-type VGCCs at P11-P13, but not at P20-P22. Interestingly, N-type VGCCs were found to be involved in this process at P20-P22, but not at hearing onset. Moreover, the degree of compartmentalization of calcium channels with respect to BK channels and presynaptic release components significantly increased from P11-P13 to P20-P22. These results suggest that the MOC-OHC synapse is immature at the onset of hearing.SIGNIFICANCE STATEMENT The functional expression of both VGCCs and BK channels, as well as their localization with respect to the presynaptic components involved in transmitter release, are key elements in determining synaptic efficacy. In this work, we show dynamic changes in the expression of VGCCs and Ca2+-dependent BK K+ channels coupled to ACh release at the MOC-OHC synapse and their shift in compartmentalization during postnatal maturation. These processes most likely set the short-term plasticity pattern and reliability of the MOC-OHC synapse on high-frequency activity.

Developmental Synaptic Changes at the Transient Olivocochlear-Inner Hair Cell Synapse.

In the mature mammalian cochlea, inner hair cells (IHCs) are mainly innervated by afferent fibers that convey sound information to the CNS. During postnatal development, however, medial olivocochlear (MOC) efferent fibers transiently innervate the IHCs. The MOC-IHC synapse, functional from postnatal day 0 (P0) to hearing onset (P12), undergoes dramatic changes in the sensitivity to acetylcholine (ACh) and in the expression of key postsynaptic proteins. To evaluate whether there are associated changes in the properties of ACh release during this period, we used a cochlear preparation from mice of either sex at P4, P6-P7, and P9-P11 and monitored transmitter release from MOC terminals in voltage-clamped IHCs in the whole-cell configuration. The quantum content increased 5.6× from P4 to P9-P11 due to increases in the size and replenishment rate of the readily releasable pool of synaptic vesicles without changes in their probability of release or quantum size. This strengthening in transmission was accompanied by changes in short-term plasticity properties, which switched from facilitation at P4 to depression at P9-P11. We have previously shown that at P9-P11, ACh release is supported by P/Q- and N-type voltage-gated calcium channels (VGCCs) and negatively regulated by BK potassium channels activated by Ca2+ influx through L-type VGCCs. We now show that at P4 and P6-P7, release is mediated by P/Q-, R- and L-type VGCCs. Interestingly, L-type VGCCs have a dual role: they both support release and fuel BK channels, suggesting that at immature stages presynaptic proteins involved in release are less compartmentalized.SIGNIFICANCE STATEMENT During postnatal development before the onset of hearing, cochlear inner hair cells (IHCs) present spontaneous Ca2+ action potentials that release glutamate at the first auditory synapse in the absence of sound stimulation. The IHC Ca2+ action potential frequency pattern, which is crucial for the correct establishment and function of the auditory system, is regulated by the efferent medial olivocochlear (MOC) system that transiently innervates IHCs during this period. We show here that developmental changes in synaptic strength and synaptic plasticity properties at the MOC-IHC synapse upon MOC fiber activation at different frequencies might be crucial for tightly shaping the pattern of afferent activity during this critical period.

A Gain-of-Function Mutation in the α9 Nicotinic Acetylcholine Receptor Alters Medial Olivocochlear Efferent Short-Term Synaptic Plasticity.

Gain control of the auditory system operates at multiple levels. Cholinergic medial olivocochlear (MOC) fibers originate in the brainstem and make synaptic contacts at the base of the outer hair cells (OHCs), the final targets of several feedback loops from the periphery and higher-processing centers. Efferent activation inhibits OHC active amplification within the mammalian cochlea, through the activation of a calcium-permeable α9α10 ionotropic cholinergic nicotinic receptor (nAChR), functionally coupled to calcium activated SK2 potassium channels. Correct operation of this feedback requires careful matching of acoustic input with the strength of cochlear inhibition (Galambos, 1956; Wiederhold and Kiang, 1970; Gifford and Guinan, 1987), which is driven by the rate of MOC activity and short-term facilitation at the MOC-OHC synapse (Ballestero et al., 2011; Katz and Elgoyhen, 2014). The present work shows (in mice of either sex) that a mutation in the α9α10 nAChR with increased duration of channel gating (Taranda et al., 2009) greatly elongates hair cell-evoked IPSCs and Ca2+ signals. Interestingly, MOC-OHC synapses of L9'T mice presented reduced quantum content and increased presynaptic facilitation. These phenotypic changes lead to enhanced and sustained synaptic responses and OHC hyperpolarization upon high-frequency stimulation of MOC terminals. At the cochlear physiology level these changes were matched by a longer time course of efferent MOC suppression. This indicates that the properties of the MOC-OHC synapse directly determine the efficacy of the MOC feedback to the cochlea being a main player in the "gain control" of the auditory periphery.SIGNIFICANCE STATEMENT Plasticity can involve reciprocal signaling across chemical synapses. An opportunity to study this phenomenon occurs in the mammalian cochlea whose sensitivity is regulated by efferent olivocochlear neurons. These release acetylcholine to inhibit sensory hair cells. A point mutation in the hair cell's acetylcholine receptor that leads to increased gating of the receptor greatly elongates IPSCs. Interestingly, efferent terminals from mutant mice present a reduced resting release probability. However, upon high-frequency stimulation transmitter release facilitates strongly to produce stronger and far longer-lasting inhibition of cochlear function. Thus, central neuronal feedback on cochlear hair cells provides an opportunity to define plasticity mechanisms in cholinergic synapses other than the highly studied neuromuscular junction.

Short-term plasticity and modulation of synaptic transmission at mammalian inhibitory cholinergic olivocochlear synapses.

The organ of Corti, the mammalian sensory epithelium of the inner ear, has two types of mechanoreceptor cells, inner hair cells (IHCs) and outer hair cells (OHCs). In this sensory epithelium, vibrations produced by sound waves are transformed into electrical signals. When depolarized by incoming sounds, IHCs release glutamate and activate auditory nerve fibers innervating them and OHCs, by virtue of their electromotile property, increase the amplification and fine tuning of sound signals. The medial olivocochlear (MOC) system, an efferent feedback system, inhibits OHC activity and thereby reduces the sensitivity and sharp tuning of cochlear afferent fibers. During neonatal development, IHCs fire Ca(2+) action potentials which evoke glutamate release promoting activity in the immature auditory system in the absence of sensory stimuli. During this period, MOC fibers also innervate IHCs and are thought to modulate their firing rate. Both the MOC-OHC and the MOC-IHC synapses are cholinergic, fast and inhibitory and mediated by the α9α10 nicotinic cholinergic receptor (nAChR) coupled to the activation of calcium-activated potassium channels that hyperpolarize the hair cells. In this review we discuss the biophysical, functional and molecular data which demonstrate that at the synapses between MOC efferent fibers and cochlear hair cells, modulation of transmitter release as well as short term synaptic plasticity mechanisms, operating both at the presynaptic terminal and at the postsynaptic hair-cell, determine the efficacy of these synapses and shape the hair cell response pattern.

Tracking the molecular evolution of calcium permeability in a nicotinic acetylcholine receptor.

Nicotinic acetylcholine receptors are a family of ligand-gated nonselective cationic channels that participate in fundamental physiological processes at both the central and the peripheral nervous system. The extent of calcium entry through ligand-gated ion channels defines their distinct functions. The α9α10 nicotinic cholinergic receptor, expressed in cochlear hair cells, is a peculiar member of the family as it shows differences in the extent of calcium permeability across species. In particular, mammalian α9α10 receptors are among the ligand-gated ion channels which exhibit the highest calcium selectivity. This acquired differential property provides the unique opportunity of studying how protein function was shaped along evolutionary history, by tracking its evolutionary record and experimentally defining the amino acid changes involved. We have applied a molecular evolution approach of ancestral sequence reconstruction, together with molecular dynamics simulations and an evolutionary-based mutagenesis strategy, in order to trace the molecular events that yielded a high calcium permeable nicotinic α9α10 mammalian receptor. Only three specific amino acid substitutions in the α9 subunit were directly involved. These are located at the extracellular vestibule and at the exit of the channel pore and not at the transmembrane region 2 of the protein as previously thought. Moreover, we show that these three critical substitutions only increase calcium permeability in the context of the mammalian but not the avian receptor, stressing the relevance of overall protein structure on defining functional properties. These results highlight the importance of tracking evolutionarily acquired changes in protein sequence underlying fundamental functional properties of ligand-gated ion channels.

Activation of presynaptic GABA(B(1a,2)) receptors inhibits synaptic transmission at mammalian inhibitory cholinergic olivocochlear-hair cell synapses.

The synapse between olivocochlear (OC) neurons and cochlear mechanosensory hair cells is cholinergic, fast, and inhibitory. The inhibitory sign of this cholinergic synapse is accounted for by the activation of Ca(2+)-permeable postsynaptic α9α10 nicotinic receptors coupled to the opening of hyperpolarizing Ca(2+)-activated small-conductance type 2 (SK2)K(+) channels. Acetylcholine (ACh) release at this synapse is supported by both P/Q- and N-type voltage-gated calcium channels (VGCCs). Although the OC synapse is cholinergic, an abundant OC GABA innervation is present along the mammalian cochlea. The role of this neurotransmitter at the OC efferent innervation, however, is for the most part unknown. We show that GABA fails to evoke fast postsynaptic inhibitory currents in apical developing inner and outer hair cells. However, electrical stimulation of OC efferent fibers activates presynaptic GABA(B(1a,2)) receptors [GABA(B(1a,2))Rs] that downregulate the amount of ACh released at the OC-hair cell synapse, by inhibiting P/Q-type VGCCs. We confirmed the expression of GABA(B)Rs at OC terminals contacting the hair cells by coimmunostaining for GFP and synaptophysin in transgenic mice expressing GABA(B1)-GFP fusion proteins. Moreover, coimmunostaining with antibodies against the GABA synthetic enzyme glutamic acid decarboxylase and synaptophysin support the idea that GABA is directly synthesized at OC terminals contacting the hair cells during development. Thus, we demonstrate for the first time a physiological role for GABA in cochlear synaptic function. In addition, our data suggest that the GABA(B1a) isoform selectively inhibits release at efferent cholinergic synapses.

Phylogenetic differences in calcium permeability of the auditory hair cell cholinergic nicotinic receptor.

The α9 and α10 cholinergic nicotinic receptor subunits assemble to form the receptor that mediates efferent inhibition of hair cell function within the auditory sensory organ, a mechanism thought to modulate the dynamic range of hearing. In contrast to all nicotinic receptors, which serve excitatory neurotransmission, the activation of α9α10 produces hyperpolarization of hair cells. An evolutionary analysis has shown that the α10 subunit exhibits signatures of positive selection only along the mammalian lineage, strongly suggesting the acquisition of a unique function. To establish whether mammalian α9α10 receptors have acquired distinct functional properties as a consequence of this evolutionary pressure, we compared the properties of rat and chicken recombinant and native α9α10 receptors. Our main finding in the present work is that, in contrast to the high (pCa(2+)/pMonovalents ∼10) Ca(2+) permeability reported for rat α9α10 receptors, recombinant and native chicken α9α10 receptors have a much lower permeability (∼2) to this cation, comparable to that of neuronal α4ß2 receptors. Moreover, we show that, in contrast to α10, α7 as well as α4 and ß2 nicotinic subunits are under purifying selection in vertebrates, consistent with the conserved Ca(2+) permeability reported across species. These results have important consequences for the activation of signaling cascades that lead to hyperpolarization of hair cells after α9α10 gating at the cholinergic-hair cell synapse. In addition, they suggest that high Ca(2+) permeability of the α9α10 cholinergic nicotinic receptor might have evolved together with other features that have given the mammalian ear an expanded high-frequency sensitivity.

The efferent medial olivocochlear-hair cell synapse.

Amplification of incoming sounds in the inner ear is modulated by an efferent pathway which travels back from the brain all the way to the cochlea. The medial olivocochlear system makes synaptic contacts with hair cells, where the neurotransmitter acetylcholine is released. Synaptic transmission is mediated by a unique nicotinic cholinergic receptor composed of α9 and α10 subunits, which is highly Ca2+ permeable and is coupled to a Ca2+-activated SK potassium channel. Thus, hyperpolarization of hair cells follows efferent fiber activation. In this work we review the literature that has enlightened our knowledge concerning the intimacies of this synapse.

Short-term synaptic plasticity regulates the level of olivocochlear inhibition to auditory hair cells.

In the mammalian inner ear, the gain control of auditory inputs is exerted by medial olivocochlear (MOC) neurons that innervate cochlear outer hair cells (OHCs). OHCs mechanically amplify the incoming sound waves by virtue of their electromotile properties while the MOC system reduces the gain of auditory inputs by inhibiting OHC function. How this process is orchestrated at the synaptic level remains unknown. In the present study, MOC firing was evoked by electrical stimulation in an isolated mouse cochlear preparation, while OHCs postsynaptic responses were monitored by whole-cell recordings. These recordings confirmed that electrically evoked IPSCs (eIPSCs) are mediated solely by α9α10 nAChRs functionally coupled to calcium-activated SK2 channels. Synaptic release occurred with low probability when MOC-OHC synapses were stimulated at 1 Hz. However, as the stimulation frequency was raised, the reliability of release increased due to presynaptic facilitation. In addition, the relatively slow decay of eIPSCs gave rise to temporal summation at stimulation frequencies >10 Hz. The combined effect of facilitation and summation resulted in a frequency-dependent increase in the average amplitude of inhibitory currents in OHCs. Thus, we have demonstrated that short-term plasticity is responsible for shaping MOC inhibition and, therefore, encodes the transfer function from efferent firing frequency to the gain of the cochlear amplifier.

Ca(2+) and Ca(2+)-activated K(+) channels that support and modulate transmitter release at the olivocochlear efferent-inner hair cell synapse.

In the mammalian auditory system, the synapse between efferent olivocochlear (OC) neurons and sensory cochlear hair cells is cholinergic, fast, and inhibitory. This efferent synapse is mediated by the nicotinic alpha9alpha10 receptor coupled to the activation of SK2 Ca(2+)-activated K(+) channels that hyperpolarize the cell. So far, the ion channels that support and/or modulate neurotransmitter release from the OC terminals remain unknown. To identify these channels, we used an isolated mouse cochlear preparation and monitored transmitter release from the efferent synaptic terminals in inner hair cells (IHCs) voltage clamped in the whole-cell recording configuration. Acetylcholine (ACh) release was evoked by electrically stimulating the efferent fibers that make axosomatic contacts with IHCs before the onset of hearing. Using the specific antagonists for P/Q- and N-type voltage-gated calcium channels (VGCCs), omega-agatoxin IVA and omega-conotoxin GVIA, respectively, we show that Ca(2+) entering through both types of VGCCs support the release process at this synapse. Interestingly, we found that Ca(2+) entering through the dihydropiridine-sensitive L-type VGCCs exerts a negative control on transmitter release. Moreover, using immunostaining techniques combined with electrophysiology and pharmacology, we show that BK Ca(2+)-activated K(+) channels are transiently expressed at the OC efferent terminals contacting IHCs and that their activity modulates the release process at this synapse. The effects of dihydropiridines combined with iberiotoxin, a specific BK channel antagonist, strongly suggest that L-type VGCCs negatively regulate the release of ACh by fueling BK channels that are known to curtail the duration of the terminal action potential in several types of neurons.

The nicotinic receptor of cochlear hair cells: a possible pharmacotherapeutic target?

Mechanosensory hair cells of the organ of Corti transmit information regarding sound to the central nervous system by way of peripheral afferent neurons. In return, the central nervous system provides feedback and modulates the afferent stream of information through efferent neurons. The medial olivocochlear efferent system makes direct synaptic contacts with outer hair cells and inhibits amplification brought about by the active mechanical process inherent to these cells. This feedback system offers the potential to improve the detection of signals in background noise, to selectively attend to particular signals, and to protect the periphery from damage caused by overly loud sounds. Acetylcholine released at the synapse between efferent terminals and outer hair cells activates a peculiar nicotinic cholinergic receptor subtype, the alpha9alpha10 receptor. At present no pharmacotherapeutic approaches have been designed that target this cholinergic receptor to treat pathologies of the auditory system. The potential use of alpha9alpha10 selective drugs in conditions such as noise-induced hearing loss, tinnitus and auditory processing disorders is discussed.

Constitutive expression of the alpha10 nicotinic acetylcholine receptor subunit fails to maintain cholinergic responses in inner hair cells after the onset of hearing.

Efferent inhibition of cochlear hair cells is mediated by alpha9alpha10 nicotinic cholinergic receptors (nAChRs) functionally coupled to calcium-activated, small conductance (SK2) potassium channels. Before the onset of hearing, efferent fibers transiently make functional cholinergic synapses with inner hair cells (IHCs). The retraction of these fibers after the onset of hearing correlates with the cessation of transcription of the Chrna10 (but not the Chrna9) gene in IHCs. To further analyze this developmental change, we generated a transgenic mice whose IHCs constitutively express alpha10 into adulthood by expressing the alpha10 cDNA under the control of the Pou4f3 gene promoter. In situ hybridization showed that the alpha10 mRNA is expressed in IHCs of 8-week-old transgenic mice, but not in wild-type mice. Moreover, this mRNA is translated into a functional protein, since IHCs from P8-P10 alpha10 transgenic mice backcrossed to a Chrna10(-/-) background (whose IHCs have no cholinergic function) displayed normal synaptic and acetylcholine (ACh)-evoked currents in patch-clamp recordings. Thus, the alpha10 transgene restored nAChR function. However, in the alpha10 transgenic mice, no synaptic or ACh-evoked currents were observed in P16-18 IHCs, indicating developmental down-regulation of functional nAChRs after the onset of hearing, as normally observed in wild-type mice. The lack of functional ACh currents correlated with the lack of SK2 currents. These results indicate that multiple features of the efferent postsynaptic complex to IHCs, in addition to the nAChR subunits, are down-regulated in synchrony after the onset of hearing, leading to lack of responses to ACh.

Electrical properties and functional expression of ionic channels in cochlear inner hair cells of mice lacking the alpha10 nicotinic cholinergic receptor subunit.

Cochlear inner hair cells (IHCs) release neurotransmitter onto afferent auditory nerve fibers in response to sound stimulation. During early development, synaptic transmission is triggered by spontaneous Ca2+ spikes which are modulated by an efferent cholinergic innervation to IHCs. This synapse is inhibitory and mediated by the alpha9alpha10 nicotinic cholinergic receptor (nAChR). After the onset of hearing, large-conductance Ca2+-activated K+ channels are acquired and both the spiking activity and the efferent innervation disappear from IHCs. In this work, we studied the developmental changes in the membrane properties of cochlear IHCs from alpha10 nAChR gene (Chrna10) "knockout" mice. Electrophysiological properties of IHCs were studied by whole-cell recordings in acutely excised apical turns of the organ of Corti from developing mice. Neither the spiking activity nor the developmental functional expression of voltage-gated and/or calcium-sensitive K+ channels is altered in the absence of the alpha10 nAChR subunit. The present results show that the alpha10 nAChR subunit is not essential for the correct establishment of the intrinsic electrical properties of IHCs during development.

A point mutation in the hair cell nicotinic cholinergic receptor prolongs cochlear inhibition and enhances noise protection.

The transduction of sound in the auditory periphery, the cochlea, is inhibited by efferent cholinergic neurons projecting from the brainstem and synapsing directly on mechanosensory hair cells. One fundamental question in auditory neuroscience is what role(s) this feedback plays in our ability to hear. In the present study, we have engineered a genetically modified mouse model in which the magnitude and duration of efferent cholinergic effects are increased, and we assess the consequences of this manipulation on cochlear function. We generated the Chrna9L9'T line of knockin mice with a threonine for leucine change (L9'T) at position 9' of the second transmembrane domain of the alpha9 nicotinic cholinergic subunit, rendering alpha9-containing receptors that were hypersensitive to acetylcholine and had slower desensitization kinetics. The Chrna9L9'T allele produced a 3-fold prolongation of efferent synaptic currents in vitro. In vivo, Chrna9L9'T mice had baseline elevation of cochlear thresholds and efferent-mediated inhibition of cochlear responses was dramatically enhanced and lengthened: both effects were reversed by strychnine blockade of the alpha9alpha10 hair cell nicotinic receptor. Importantly, relative to their wild-type littermates, Chrna9(L9'T/L9'T) mice showed less permanent hearing loss following exposure to intense noise. Thus, a point mutation designed to alter alpha9alpha10 receptor gating has provided an animal model in which not only is efferent inhibition more powerful, but also one in which sound-induced hearing loss can be restrained, indicating the ability of efferent feedback to ameliorate sound trauma.

Properties of mutated murine alpha4beta2 nicotinic receptors linked to partial epilepsy.

We characterized, by electrophysiological methods, two biophysical properties of murine recombinant alpha4beta2 nicotinic acetylcholine receptors (nAChR) bearing a mutation (alpha4:+L264alpha4:beta2 or alpha4:S252Falpha4:beta2) linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). Sensitivity to acetylcholine (ACh) was increased by the S252F substitution expressed in heterozygosis (alpha4:S252Falpha4:beta2) but was markedly reduced when this mutation was expressed in homozygosis (S252Falpha4:beta2). ACh sensitivity was not altered by the +L264 insertion. Moreover, receptor desensitization was significantly increased by both mutations expressed in heterozygosis. These results are in general agreement to those of rat and human recombinant receptors bearing the same mutations, thus contributing to validate the use of knock-in mice harboring ADNFLE mutations as models to study this pathology.

The alpha10 nicotinic acetylcholine receptor subunit is required for normal synaptic function and integrity of the olivocochlear system.

Although homomeric channels assembled from the alpha9 nicotinic acetylcholine receptor (nAChR) subunit are functional in vitro, electrophysiological, anatomical, and molecular data suggest that native cholinergic olivocochlear function is mediated via heteromeric nAChRs composed of both alpha9 and alpha10 subunits. To gain insight into alpha10 subunit function in vivo, we examined olivo cochlear innervation and function in alpha10 null-mutant mice. Electrophysiological recordings from postnatal (P) days P8-9 inner hair cells revealed ACh-gated currents in alpha10(+/+) and alpha10(+/-) mice, with no detectable responses to ACh in alpha10(-/-) mice. In contrast, a proportion of alpha10(-/-) outer hair cells showed small ACh-evoked currents. In alpha10(-/-) mutant mice, olivocochlear fiber stimulation failed to suppress distortion products, suggesting that the residual alpha9 homomeric nAChRs expressed by outer hair cells are unable to transduce efferent signals in vivo. Finally, alpha10(-/-) mice exhibit both an abnormal olivocochlear morphology and innervation to outer hair cells and a highly disorganized efferent innervation to the inner hair cell region. Our results demonstrate that alpha9(-/-) and alpha10(-/-) mice have overlapping but nonidentical phenotypes. Moreover, alpha10 nAChR subunits are required for normal olivocochlear activity because alpha9 homomeric nAChRs do not support maintenance of normal olivocochlear innervation or function in alpha10(-/-) mutant mice.

Ryanodine is a positive modulator of acetylcholine receptor gating in cochlear hair cells.

The efferent synaptic specialization of hair cells includes a near-membrane synaptic cistern, whose presence suggests a role for internal calcium stores in cholinergic inhibition. Calcium release channels from internal stores include 'ryanodine receptors', whose participation is usually demonstrated by sensitivity to the eponymous plant alkaloid, ryanodine. However, use of this and other store-active compounds on hair cells could be confounded by the unusual pharmacology of the alpha9alpha10-containing hair cell nicotinic cholinergic receptor (nAChR), which has been shown to be antagonized by a broad spectrum of compounds. Surprisingly, we found that ryanodine, rather than antagonizing, is a positive modulator of the alpha9alpha10 nAChR expressed in Xenopus oocytes, the first such compound to be found. The effect of ryanodine was to increase the apparent affinity and efficacy for acetylcholine (ACh). Correspondingly, ACh-evoked currents through the isolated cholinergic receptors of inner hair cells in excised mouse cochleas were approximately doubled by 200 microM ryanodine, a concentration that inhibits gating of the ryanodine receptor itself. This unusual positive modulation was not unique to the mammalian receptor. The response to ACh of chicken 'short' hair cells likewise was enhanced in the presence of 100 microM ryanodine. This facilitatory effect on current through the AChR could enhance brief ( approximately 1 s) activation of associated calcium-dependent K(+) (SK) channels in both chicken short hair cells and rat outer hair cells. This novel effect of ryanodine provides new opportunities for the design of compounds that potentiate alpha9alpha10-mediated responses and for potential inner ear therapeutics based on this interaction.

Inhibition of the alpha9alpha10 nicotinic cholinergic receptor by neramexane, an open channel blocker of N-methyl-D-aspartate receptors.

In this study we report the effects of neramexane, a novel amino-alkyl-cyclohexane derivative that is a non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist, on recombinant rat alpha9alpha10 nicotinic acetylcholine receptors expressed in Xenopus laevis oocytes. We compared its effects with those of memantine, a well-studied pore blocker of NMDA receptors, currently used in therapeutics for the treatment of Alzheimer's disease. Our results indicate that both compounds block acetylcholine-evoked responses at micromolar concentrations with a rank order of potency of neramexane>memantine, P<0.05. Block by neramexane of acetylcholine responses was not overcome at high concentrations of the agonist, indicative of a non-competitive inhibition. The lack of interaction of neramexane with the ligand binding domain was confirmed by radioligand binding experiments in transfected tsA201 cells. Moreover, block did not involve an increase in desensitization kinetics, it was independent of the resting potential of the membrane at low concentrations of neramexane and slightly voltage-dependent at concentrations higher than 1 microM. Finally, clinically-relevant concentrations of neramexane blocked native alpha9alpha10-containing nicotinic acetylcholine receptors of rat inner hair cells, thus demonstrating a possible in vivo relevance in potentially unexplored therapeutic areas.

Stoichiometry of the alpha9alpha10 nicotinic cholinergic receptor.

The alpha9 and alpha10 nicotinic cholinergic subunits assemble to form the receptor that mediates synaptic transmission between efferent olivocochlear fibers and hair cells of the cochlea. They are the latest vertebrate nicotinic cholinergic receptor (nAChR) subunits that have been cloned, and their identification has established a distant early divergent branch within the nAChR gene family. The alpha10 subunit serves as a "structural" component leading to heteromeric alpha9alpha10 nAChRs with distinct properties. We now have probed the stoichiometry of recombinant alpha9alpha10 nAChRs expressed in Xenopus oocytes. We have made use of the analysis of the population of receptors assembled from a wild-type subunit and its partner alpha9 or alpha10 subunit bearing a reporter mutation of a valine to threonine at position 13' of the second transmembrane domain (TM2). Because the mutation increased the sensitivity of the receptor for acetylcholine (ACh) but mutations at different subunits were not equivalent, the number of alpha9 and alpha10 subunits could be inferred from the number of components in compound concentration-response curves to ACh. The results were confirmed via the analysis of the effects of a mutation to threonine at position 17' of TM2. Because at this position the mutations at different subunits were equivalent, the stoichiometry was inferred directly from the shifts in the ACh EC50 values. We conclude that the recombinant alpha9alpha10 receptor is a pentamer with a (alpha9)2(alpha10)3 stoichiometry.