Although homomeric channels assembled from the ␣9 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 ␣9 and ␣10 subunits. To gain insight into ␣10 subunit function in vivo, we examined olivocochlear innervation and function in ␣10 null-mutant mice. Electrophysiological recordings from postnatal (P) days P8 -9 inner hair cells revealed ACh-gated currents in ␣10 ؉/؉ and ␣10 ؉/؊ mice, with no detectable responses to ACh in ␣10 ؊/؊ mice. In contrast, a proportion of ␣10 ؊/؊ outer hair cells showed small ACh-evoked currents. In ␣10 ؊/؊ mutant mice, olivocochlear fiber stimulation failed to suppress distortion products, suggesting that the residual ␣9 homomeric nAChRs expressed by outer hair cells are unable to transduce efferent signals in vivo. Finally, ␣10 ؊/؊ 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 ␣9 ؊/؊ and ␣10 ؊/؊ mice have overlapping but nonidentical phenotypes. Moreover, ␣10 nAChR subunits are required for normal olivocochlear activity because ␣9 homomeric nAChRs do not support maintenance of normal olivocochlear innervation or function in ␣10 ؊/؊ mutant mice.cochlea ͉ electrophysiology ͉ inner hair cells ͉ outer hair cells T he sensory epithelia responsible for hearing (cochlea) and balance (saccule, utricle, and cristae ampullaris) share a unique subset of cells that respond to mechanical cues. These hair cells possess apical mechanoreceptors and specialized basolateral membranes that act in concert to transduce mechanical stimuli into electrical signals (1). In mammals, cochlear hair cells are anatomically and functionally divided into inner and outer hair cells (IHCs and OHCs, respectively). IHCs are responsible for transducing acoustic stimuli and exciting the fibers of the cochlear nerve, whereas OHC are involved in the mechanical amplification and fine tuning of cochlear vibrations via their electromotile response (2, 3).Both OHCs and type-I spiral ganglion cell processes receive descending cholinergic innervation, which originates in the superior olivary complex (4). Although the precise role of the olivocochlear (OC) system in hearing remains uncertain, the effects of activating efferent terminals forming synapses with OHCs have been well described (5-7). Acetylcholine (ACh), the principal neurotransmitter released by OC terminals (8), binds to postsynaptic nicotinic acetylcholine receptors (nAChRs) leading to calcium influx, activation of small-conductance calciumactivated potassium channels, and subsequent hair cell hyperpolarization (9 -17). As with electrical stimulation of the olivocochlear bundle (10), the result of OHC hyperpolarization is to reduce auditory afferent output via suppression of basilar membrane motion (18).Combined immunohistochemical (19,20),...

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 α9 nicotinic cholinergic subunit, rendering α9-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 α9α10 hair cell nicotinic receptor. Importantly, relative to their wild-type littermates, Chrna9L9′T/L9′T mice showed less permanent hearing loss following exposure to intense noise. Thus, a point mutation designed to alter α9α10 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.

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 OHCs 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 inhibitory postsynaptic currents (eIPSCs) are mediated solely by α9α10 nicotinic acetylcholine receptors (nAChRs) functionally coupled to calcium-activated SK2 channels. Synaptic release occurred with low probability when MOC-OHC synapses were stimulated at 1Hz. 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 above 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.

Neurons in the medial superior olive (MSO) and lateral superior olive (LSO) of the auditory brainstem code for sound-source location in the horizontal plane, extracting interaural time differences (ITDs) from the stimulus fine structure and interaural level differences (ILDs) from the stimulus envelope. Here, we demonstrate a postsynaptic gradient in temporal processing properties across the presumed tonotopic axis; neurons in the MSO and the low-frequency limb of the LSO exhibit fast intrinsic electrical resonances and low input impedances, consistent with their processing of ITDs in the temporal fine structure. Neurons in the high-frequency limb of the LSO show low-pass electrical properties, indicating they are better suited to extracting information from the slower, modulated envelopes of sounds. Using a modeling approach, we assess ITD and ILD sensitivity of the neural filters to natural sounds, demonstrating that the transformation in temporal processing along the tonotopic axis contributes to efficient extraction of auditory spatial cues.auditory system | superior olivary nucleus | spatial listening T he auditory system analyzes sounds over different time scales to extract ecologically relevant information, including the identity and location of a sound source ( Fig. 1A; also see ref. 1). In particular, sensitivity to rapidly fluctuating signals in the temporal fine structure (TFS; the sound-pressure waveform) of sounds enables the extraction of spatial information in the form of interaural time differences (ITDs), i.e., the time difference in the arrival of the stimulus to both ears. For frequencies below about 1,500 Hz, human listeners can discriminate ITDs of just a few tens of microseconds (2-4), corresponding to a spatial resolution of about two degrees for sources located to the front. Such exquisite sensitivity relies on the ability of cochlear hair cells to generate action potentials in auditory nerve fibers that are phase-locked to the instantaneous sound-pressure waveform at each eardrum (Fig. 1A, bottom right). Phase-locking to the TFS in nerve fibers extends to at least 4 kHz in many mammalian species but starts to degrade from about 1 kHz as the result of low-pass filtering by the sensory hair cells (5). Postsynaptic specializations in subsequent stages of the ascending pathway-such as the cochlear nucleus-may improve temporal locking of action potentials to the TFS, at least for frequencies below 1 kHz (6). Phase-locked excitatory (and potentially inhibitory) inputs from each ear ("EE" input) ultimately converge on neurons in the medial superior olive (MSO) of the brainstem, where ITDs are explicitly computed (Fig. 1B).For sounds above a few kilohertz in frequency, differences in the intensity of the sound at each ear (interaural level differences, ILDs)-generated by the head "shadowing" the ear further from the source-become increasingly important as localization cues. Although phase-locking of action potentials to the TFS typically is absent in this frequency range, the spiking activity do...

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 ␣9␣10 receptor coupled to the activation of SK2 Ca 2ϩ -activated K ϩ 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), -agatoxin IVA and -conotoxin GVIA, respectively, we show that Ca 2ϩ entering through both types of VGCCs support the release process at this synapse. Interestingly, we found that Ca 2ϩ 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 2ϩ -activated K ϩ 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.

A continuous isoelectric electroencephalogram reflects an interruption of endogenously-generated activity in cortical networks and systematically results in a complete dissolution of conscious processes. This electro-cerebral inactivity occurs during various brain disorders, including hypothermia, drug intoxication, long-lasting anoxia and brain trauma. It can also be induced in a therapeutic context, following the administration of high doses of barbiturate-derived compounds, to interrupt a hyper-refractory status epilepticus. Although altered sensory responses can be occasionally observed on an isoelectric electroencephalogram, the electrical membrane properties and synaptic responses of individual neurons during this cerebral state remain largely unknown. The aim of the present study was to characterize the intracellular correlates of a barbiturate-induced isoelectric electroencephalogram and to analyse the sensory-evoked synaptic responses that can emerge from a brain deprived of spontaneous electrical activity. We first examined the sensory responsiveness from patients suffering from intractable status epilepticus and treated by administration of thiopental. Multimodal sensory responses could be evoked on the flat electroencephalogram, including visually-evoked potentials that were significantly amplified and delayed, with a high trial-to-trial reproducibility compared to awake healthy subjects. Using an analogous pharmacological procedure to induce prolonged electro-cerebral inactivity in the rat, we could describe its cortical and subcortical intracellular counterparts. Neocortical, hippocampal and thalamo-cortical neurons were all silent during the isoelectric state and displayed a flat membrane potential significantly hyperpolarized compared with spontaneously active control states. Nonetheless, all recorded neurons could fire action potentials in response to intracellularly injected depolarizing current pulses and their specific intrinsic electrophysiological features were preserved. Manipulations of the membrane potential and intracellular injection of chloride in neocortical neurons failed to reveal an augmented synaptic inhibition during the isoelectric condition. Consistent with the sensory responses recorded from comatose patients, large and highly reproducible somatosensory-evoked potentials could be generated on the inactive electrocorticogram in rats. Intracellular recordings revealed that the underlying neocortical pyramidal cells responded to sensory stimuli by complex synaptic potentials able to trigger action potentials. As in patients, sensory responses in the isoelectric state were delayed compared to control responses and exhibited an elevated reliability during repeated stimuli. Our findings demonstrate that during prolonged isoelectric brain state neurons and synaptic networks are dormant rather than excessively inhibited, conserving their intrinsic properties and their ability to integrate and propagate environmental stimuli.

In this study, we report the effects of the quinoline derivatives quinine, its optical isomer quinidine, and chloroquine on ␣9␣10-containing nicotinic acetylcholine receptors (nAChRs). The compounds blocked acetylcholine (ACh)-evoked responses in ␣9␣10-injected Xenopus laevis oocytes in a concentration-dependent manner, with a rank order of potency of chloroquine (IC 50 ϭ 0.39 M) Ͼ quinine (IC 50 ϭ 0.97 M) ϳ quinidine (IC 50 ϭ 1.37 M). Moreover, chloroquine blocked ACh-evoked responses on rat cochlear inner hair cells with an IC 50 value of 0.13 M, which is within the same range as that observed for recombinant receptors. Block by chloroquine was purely competitive, whereas quinine inhibited ACh currents in a mixed competitive and noncompetitive manner. The competitive nature of the blockage produced by the three compounds was confirmed by equilibrium binding experiments using [ 3 H]methyllycaconitine. Binding affinities (K i values) were 2.3, 5.5, and 13.0 M for chloroquine, quinine, and quinidine, respectively. Block by quinine was found to be only slightly voltage-dependent, thus precluding open-channel block as the main mechanism of interaction of quinine with ␣9␣10 nAChRs. The present results add to the pharmacological characterization of ␣9␣10-containing nicotinic receptors and indicate that the efferent olivocochlear system that innervates the cochlear hair cells is a target of these ototoxic antimalarial compounds.

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 Ca signals. Interestingly, MOC-OHC synapses of ' 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. 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.