Letting the calcium flow.

Two calcium-binding proteins, CaBP1 and CaBP2, cooperate to keep calcium channels in the hair cells of the inner ear open.

Dopamine D2 receptors in WFS1-neurons regulate food-seeking and avoidance behaviors.

The selection and optimization of appropriate adaptive responses depends on interoceptive and exteroceptive stimuli as well as on the animal's ability to switch from one behavioral strategy to another. Although growing evidence indicate that dopamine D2R-mediated signaling events ensure the selection of the appropriate strategy for each specific situation, the underlying neural circuits through which they mediate these effects are poorly characterized. Here, we investigated the role of D2R signaling in a mesolimbic neuronal subpopulation expressing the Wolfram syndrome 1 (Wfs1) gene. This subpopulation is located within the nucleus accumbens, the central amygdala, the bed nucleus of the stria terminalis, and the tail of the striatum, all brain regions critical for the regulation of emotions and motivated behaviors. Using a mouse model carrying a temporally controlled deletion of D2R in WFS1-neurons, we demonstrate that intact D2R signaling in this neuronal population is necessary to regulate homeostasis-dependent food-seeking behaviors in both male and female mice. In addition, we found that reduced D2R signaling in WFS1-neurons impaired active avoidance learning and innate escape responses. Collectively, these findings identify a yet undocumented role for D2R signaling in WFS1-neurons as a novel effector through which dopamine optimizes appetitive behaviors and regulates defensive behaviors.

Abnormal outer hair cell efferent innervation in Hoxb1-dependent sensorineural hearing loss.

Autosomal recessive mutation of HOXB1 and Hoxb1 causes sensorineural hearing loss in patients and mice, respectively, characterized by the presence of higher auditory thresholds; however, the origin of the defects along the auditory pathway is still unknown. In this study, we assessed whether the abnormal auditory threshold and malformation of the sensory auditory cells, the outer hair cells, described in Hoxb1null mutants depend on the absence of efferent motor innervation, or alternatively, is due to altered sensory auditory components. By using a whole series of conditional mutant mice, which inactivate Hoxb1 in either rhombomere 4-derived sensory cochlear neurons or efferent motor neurons, we found that the hearing phenotype is mainly reproduced when efferent motor neurons are specifically affected. Our data strongly suggest that the interactions between olivocochlear motor neurons and outer hair cells during a critical postnatal period are crucial for both hair cell survival and the establishment of the cochlear amplification of sound.

Peristimulus Time Responses Predict Adaptation and Spontaneous Firing of Auditory-Nerve Fibers: From Rodents Data to Humans.

Sound-level coding in the auditory nerve is achieved through the progressive recruitment of auditory nerve fibers (ANFs) that differ in threshold of activation and in the stimulus level at which the spike rate saturates. To investigate the functional state of the ANFs, the electrophysiological tests routinely used in clinics only capture the first action potentials firing in synchrony at the onset of the acoustic stimulation. Assessment of other properties (e.g., spontaneous rate and adaptation time constants) requires single-fiber recordings directly from the nerve, which for ethical reasons is not allowed in humans. By combining neuronal activity measurements at the round window and signal-processing algorithms, we constructed a peristimulus time response (PSTR), with a waveform similar to the peristimulus time histograms (PSTHs) derived from single-fiber recordings in young adult female gerbils. Simultaneous recordings of round-window PSTR and single-fiber PSTH provided models to predict the adaptation kinetics and spontaneous rate of the ANFs tuned at the PSTR probe frequency. The predictive model derived from gerbils was then validated in female mice and finally applied to humans by recording PSTRs from the auditory nerve in normal-hearing patients who underwent cerebellopontine angle surgeries. A rapid adaptation time constant of ∼3 ms and a mean spontaneous rate of ∼22 spikes/s in the 4 kHz frequency range were found. This study offers a promising diagnostic tool to map the human auditory nerve, thus opening new avenues to better understanding auditory neuropathies, tinnitus, and hyperacusis.SIGNIFICANCE STATEMENT Neural adaptation in auditory nerve fibers corresponds to the reduction in the neuronal activity to prolonged or repeated sound stimulation. For obvious ethical reasons, single-fiber recordings from the auditory nerve are not feasible in humans, creating a critical gap in extending data obtained using animal models to humans. Using electrocochleography in rodents, we inferred adaptation kinetics and spontaneous discharge rates of the auditory nerve fibers in humans. Routinely used in basic and clinical laboratories, this tool will provide a better understanding of auditory disorders such as neuropathies, tinnitus, and hyperacusis, and will help to improve hearing-aid fittings.

VGLUT3-p.A211V variant fuses stereocilia bundles and elongates synaptic ribbons.

DFNA25 is an autosomal-dominant and progressive form of human deafness caused by mutations in the SLC17A8 gene, which encodes the vesicular glutamate transporter type 3 (VGLUT3). To resolve the mechanisms underlying DFNA25, we studied phenotypes of mice harbouring the p.A221V mutation in humans (corresponding to p.A224V in mice). Using auditory brainstem response and distortion product otoacoustic emissions, we showed progressive hearing loss with intact cochlear amplification in the VGLUT3A224V/A224V mouse. The summating potential was reduced, indicating the alteration of inner hair cell (IHC) receptor potential. Scanning electron microscopy examinations demonstrated the collapse of stereocilia bundles in IHCs, leaving those from outer hair cells unaffected. In addition, IHC ribbon synapses underwent structural and functional modifications at later stages. Using super-resolution microscopy, we observed oversized synaptic ribbons and patch-clamp membrane capacitance measurements showed an increase in the rate of the sustained releasable pool exocytosis. These results suggest that DFNA25 stems from a failure in the mechano-transduction followed by a change in synaptic transfer. The VGLUT3A224V/A224V mouse model opens the way to a deeper understanding and to a potential treatment for DFNA25. KEY POINTS: The vesicular glutamate transporter type 3 (VGLUT3) loads glutamate into the synaptic vesicles of auditory sensory cells, the inner hair cells (IHCs). The VGLUT3-p.A211V variant is associated with human deafness DFNA25. Mutant mice carrying the VGLUT3-p.A211V variant show progressive hearing loss. IHCs from mutant mice harbour distorted stereocilary bundles, which detect incoming sound stimulation, followed by oversized synaptic ribbons, which release glutamate onto the afferent nerve fibres. These results suggest that DFNA25 stems from the failure of auditory sensory cells to faithfully transduce acoustic cues into neural messages.

Spiking Pattern of the Mouse Developing Inner Hair Cells Is Mostly Invariant Along the Tonotopic Axis.

During development, the sensory cells of the cochlea, the inner hair cells (IHCs), fire spontaneous calcium action potentials. This activity at the pre-hearing stage allows the IHCs to autonomously excite the auditory nerve fibers and hence, represents an efficient mechanism to shape the tonotopic organization along the ascending auditory pathway. Using calcium imaging, we show that the activity in the developing cochlea consists of calcium waves that propagate across the supporting and sensory cells. Both basal and apical IHCs were characterized by similar spontaneous calcium transients interspaced with silent periods, consistent with bursts of action potentials recorded in patch-clamp. In addition, adjacent auditory hair cells tend to have a synchronized [Ca2+]i activity, irrespective of their location along the base-to-apex gradient of the cochlea. Finally, we show that the mechanical ablation of the inner phalangeal cells (IPCs), a class of supporting cells, reduces the synchronized [Ca2+]i activity between neighboring sensory cells. These findings support the hypothesis that the tonotopic map refinement in higher auditory centers would depend on the synchronization of a discrete number of auditory sensory cells.

The Interplay Between Spike-Time and Spike-Rate Modes in the Auditory Nerve Encodes Tone-In-Noise Threshold.

Auditory nerve fibers (ANFs) encode pure tones through two modes of coding, spike time and spike rate, depending on the tone frequency. In response to a low-frequency tone, ANF firing is phase locked to the sinusoidal waveform. Because time coding vanishes with an increase in the tone frequency, high-frequency tone coding relies on the spike rate of the ANFs. Adding a continuous broadband noise to a tone compresses the rate intensity function of ANFs and shifts its dynamic range toward higher intensities. Therefore, the ANFs with high-threshold/low-spontaneous rate (SR) are thought to contribute to behavioral tone detection in noise. However, this theory relies on the discharge rate of the ANFs. The direct comparison with the masking threshold through spike timing, irrespective of the spontaneous rate, has not so far been investigated. Taking advantage of a unique proxy to quantify the spike synchrony (i.e., the shuffle autocorrelogram), we show in female gerbils that high-SR ANFs are more adapted to encode low-frequency thresholds through temporal code, giving them a strong robustness in noise. By comparing behavioral thresholds measured using prepulse inhibition of the acoustical startle reflex with population thresholds calculated from ANFs pooled per octave band, we show that threshold-based spike timing provides a better estimate of behavioral thresholds in the low-frequency range, whereas the high-frequency behavioral thresholds rely on the spiking rate, particularly in noise. This emphasizes the complementarity of temporal and rate modes to code tone-in-noise thresholds over a large range of frequencies.SIGNIFICANCE STATEMENT There is a general agreement that high-threshold/low-spontaneous rate (SR) auditory nerve fibers (ANFs) are of prime importance for tone detection in noise. However, this theory is based on the discharge rate of the fibers. Comparing the behavioral thresholds and single ANF thresholds shows that this is only true in the high-frequency range of tone stimulations. In the low-frequency range of tones (up to 2.7 kHz in the gerbil), the most sensitive ANFs (high-SR fibers) carry neural information through a spike-timing mode, even for noise in which tones do not induce a noticeable increment in the spike rate. This emphasizes the interplay between spike-time and spike-rate modes in the auditory nerve to encode tone-in-noise threshold over a large range of tone frequencies.

Mass Potentials Recorded at the Round Window Enable the Detection of Low Spontaneous Rate Fibers in Gerbil Auditory Nerve.

Auditory nerve fibers (ANFs) transmit acoustic information from the sensory hair cells to the cochlear nuclei. In experimental and clinical audiology, probing the whole ANF population remains a difficult task, as the ANFs differ greatly in their threshold and onset response to sound. Thus, low spontaneous rate (SR) fibers, which have rather higher thresholds, delay and larger jitter in their first spike latency are not detectable in the far-field compound action potential of the auditory nerve. Here, we developed a new protocol of acoustic stimulation together with electrophysiological signal processing to track the steady state activity of ANFs. Mass potentials at the round window were recorded in response to repetitive 300-ms bursts of 1/3 octave band noise centered on a frequency probe. Analysis was assessed during the last 200-ms of the response to capture the steady-state response of ANFs. To eliminate the microphonic component reflecting the sensory cells activity, repetitive pairs of sounds of opposite polarities were used. The spectral analysis was calculated on the average of two consecutive responses, and the neural gain was calculated by dividing point-by-point the spectrum to sound over unstimulated condition. In response to low-sound-level stimulation, neural gain predominated in the low-frequency cochlear regions, while a second component of responses centered on higher cochlear frequency regions appeared beyond 30 dB SPL. At 60 dB SPL, neural gain showed a bimodal shape, with a notch near 5.6 kHz. In addition to correlate with the functional mapping of ANFs along the tonotopic axis, the deletion of low-SR fibers leads to a reduction in the high-frequency response, where the low-SR fibers are preferentially located. Thus, mass potentials at the round window may provide a useful tool to probe the SR-based distribution of ANFs in humans and in other species in which direct single-unit recordings are difficult to achieve or not feasible.

Reversible p53 inhibition prevents cisplatin ototoxicity without blocking chemotherapeutic efficacy.

Cisplatin is a widely used chemotherapy drug, despite its significant ototoxic side effects. To date, the mechanism of cisplatin-induced ototoxicity remains unclear, and hearing preservation during cisplatin-based chemotherapy in patients is lacking. We found activation of the ATM-Chk2-p53 pathway to be a major determinant of cisplatin ototoxicity. However, prevention of cisplatin-induced ototoxicity is hampered by opposite effects of ATM activation upon sensory hair cells: promoting both outer hair cell death and inner hair cell survival. Encouragingly, however, genetic or pharmacological ablation of p53 substantially attenuated cochlear cell apoptosis, thus preserving hearing. Importantly, systemic administration of a p53 inhibitor in mice bearing patient-derived triple-negative breast cancer protected auditory function, without compromising the anti-tumor efficacy of cisplatin. Altogether, these findings highlight a novel and effective strategy for hearing protection in cisplatin-based chemotherapy.

Actin Filaments Regulate Exocytosis at the Hair Cell Ribbon Synapse.

Exocytosis at the inner hair cell ribbon synapse is achieved through the functional coupling between calcium channels and glutamate-filled synaptic vesicles. Using membrane capacitance measurements, we investigated whether the actin network regulates the exocytosis of synaptic vesicles at the mouse auditory hair cell. Our results suggest that actin network disruption increases exocytosis and that actin filaments may spatially organize a subfraction of synaptic vesicles with respect to the calcium channels. Significance statement: Inner hair cells (IHCs), the auditory sensory cells of the cochlea, release glutamate onto the afferent auditory nerve fibers to encode sound stimulation. To achieve this task, the IHC relies on the recruitment of glutamate-filled vesicles that can be located in close vicinity to the calcium channels or more remotely from them. The molecular determinants responsible for organizing these vesicle pools are not fully identified. Using pharmacological tools in combination with membrane capacitance measurements, we show that actin filament disruption increases exocytosis in IHCs and that actin filaments most likely position a fraction of vesicles away from the calcium channels.

Remodeling of the Inner Hair Cell Microtubule Meshwork in a Mouse Model of Auditory Neuropathy AUNA1.

Auditory neuropathy 1 (AUNA1) is a form of human deafness resulting from a point mutation in the 5' untranslated region of the Diaphanous homolog 3 (DIAPH3) gene. Notably, the DIAPH3 mutation leads to the overexpression of the DIAPH3 protein, a formin family member involved in cytoskeleton dynamics. Through study of diap3-overexpressing transgenic (Tg) mice, we examine in further detail the anatomical, functional, and molecular mechanisms underlying AUNA1. We identify diap3 as a component of the hair cells apical pole in wild-type mice. In the diap3-overexpressing Tg mice, which show a progressive threshold shift associated with a defect in inner hair cells (IHCs), the neurotransmitter release and potassium conductances are not affected. Strikingly, the overexpression of diap3 results in a selective and early-onset alteration of the IHC cuticular plate. Molecular dissection of the apical components revealed that the microtubule meshwork first undergoes aberrant targeting into the cuticular plate of Tg IHCs, followed by collapse of the stereociliary bundle, with eventual loss of the IHC capacity to transmit incoming auditory stimuli.

Cochlear efferents in developing adult and pathological conditions.

Cochlear activity is regulated by the olivo-cochlear bundle, which originates from the brainstem and projects onto the hair cells and auditory nerve fibers. Two efferent components can be distinguished: the medial and lateral olivo-cochlear efferent originating from the medial, and the lateral nuclei of the superior olivary complex. The input of the efferent systems on hair cells occurs during development and persists in the adult cochlea. Recent studies have shown that the efferent innervations are required to set the activity pattern in developing hair cells and auditory nerve fibers and to protect the synaptic structures in adult cochlea. In addition, efferent innervations undergo plasticity during pathological conditions such as noise-trauma or aging. This review discusses the mechanisms underlying the control of the hair cells and afferent fibers excitability by efferent neurons and their putative role in developing adult and pathological conditions.

Contribution of auditory nerve fibers to compound action potential of the auditory nerve.

Sound-evoked compound action potential (CAP), which captures the synchronous activation of the auditory nerve fibers (ANFs), is commonly used to probe deafness in experimental and clinical settings. All ANFs are believed to contribute to CAP threshold and amplitude: low sound pressure levels activate the high-spontaneous rate (SR) fibers, and increasing levels gradually recruit medium- and then low-SR fibers. In this study, we quantitatively analyze the contribution of the ANFs to CAP 6 days after 30-min infusion of ouabain into the round window niche. Anatomic examination showed a progressive ablation of ANFs following increasing concentration of ouabain. CAP amplitude and threshold plotted against loss of ANFs revealed three ANF pools: 1) a highly ouabain-sensitive pool, which does not participate in either CAP threshold or amplitude, 2) a less sensitive pool, which only encoded CAP amplitude, and 3) a ouabain-resistant pool, required for CAP threshold and amplitude. Remarkably, distribution of the three pools was similar to the SR-based ANF distribution (low-, medium-, and high-SR fibers), suggesting that the low-SR fiber loss leaves the CAP unaffected. Single-unit recordings from the auditory nerve confirmed this hypothesis and further showed that it is due to the delayed and broad first spike latency distribution of low-SR fibers. In addition to unraveling the neural mechanisms that encode CAP, our computational simulation of an assembly of guinea pig ANFs generalizes and extends our experimental findings to different species of mammals. Altogether, our data demonstrate that substantial ANF loss can coexist with normal hearing threshold and even unchanged CAP amplitude.

Spatiotemporal pattern of action potential firing in developing inner hair cells of the mouse cochlea.

Inner hair cells (IHCs) are the primary transducer for sound encoding in the cochlea. In contrast to the graded receptor potential of adult IHCs, immature hair cells fire spontaneous calcium action potentials during the first postnatal week. This spiking activity has been proposed to shape the tonotopic map along the ascending auditory pathway. Using perforated patch-clamp recordings, we show that developing IHCs fire spontaneous bursts of action potentials and that this pattern is indistinguishable along the basoapical gradient of the developing cochlea. In both apical and basal IHCs, the spiking behavior undergoes developmental changes, where the bursts of action potential tend to occur at a regular time interval and have a similar length toward the end of the first postnatal week. Although disruption of purinergic signaling does not interfere with the action potential firing pattern, pharmacological ablation of the α9α10 nicotinic receptor elicits an increase in the discharge rate. We therefore suggest that in addition to carrying place information to the ascending auditory nuclei, the IHCs firing pattern controlled by the α9α10 receptor conveys a temporal signature of the cochlear development.

Modes and regulation of endocytic membrane retrieval in mouse auditory hair cells.

Synaptic vesicle recycling sustains high rates of neurotransmission at the ribbon-type active zones (AZs) of mouse auditory inner hair cells (IHCs), but its modes and molecular regulation are poorly understood. Electron microscopy indicated the presence of clathrin-mediated endocytosis (CME) and bulk endocytosis. The endocytic proteins dynamin, clathrin, and amphiphysin are expressed and broadly distributed in IHCs. We used confocal vglut1-pHluorin imaging and membrane capacitance (Cm) measurements to study the spatial organization and dynamics of IHC exocytosis and endocytosis. Viral gene transfer expressed vglut1-pHluorin in IHCs and targeted it to synaptic vesicles. The intravesicular pH was ∼6.5, supporting only a modest increase of vglut1-pHluorin fluorescence during exocytosis and pH neutralization. Ca(2+) influx triggered an exocytic increase of vglut1-pHluorin fluorescence at the AZs, around which it remained for several seconds. The endocytic Cm decline proceeded with constant rate (linear component) after exocytosis of the readily releasable pool (RRP). When exocytosis exceeded three to four RRP equivalents, IHCs additionally recruited a faster Cm decline (exponential component) that increased with the amount of preceding exocytosis and likely reflects bulk endocytosis. The dynamin inhibitor Dyngo-4a and the clathrin blocker pitstop 2 selectively impaired the linear component of endocytic Cm decline. A missense mutation of dynamin 1 (fitful) inhibited endocytosis to a similar extent as Dyngo-4a. We propose that IHCs use dynamin-dependent endocytosis via CME to support vesicle cycling during mild stimulation but recruit bulk endocytosis to balance massive exocytosis.

Tmprss3 loss of function impairs cochlear inner hair cell Kcnma1 channel membrane expression.

Before acquiring their mature state, cochlear hair cells undergo a series of changes in expression of ion channels. How this complex mechanism is achieved is not fully understood. Tmprss3, a type II serine protease expressed in hair cells, is required for their proper functioning at the onset of hearing. To unravel the role of Tmprss3 in the acquisition of mature K(+) currents, we compared their function by patch-clamp technique in wild-type Tmprss3(WT) and Tmprss3(Y260X)-mutant mice. Interestingly, only outward K(+) currents were altered in Tmprss3(Y260X)-mutant mice. To determine by which mechanism this occurred, we compared the protein network of Tmprss3(WT) and Tmprss3(Y260X)-mutant mice using proteomic analysis. This led to the identification of a pathway related to potassium Kcnma1 channels. This pathway was validated by immunohistochemistry, focusing on the most downregulated protein that was identified as a cochlear Kcnma1-associated protein, APOA1. Finally, we show that, in contrast to Tmprss3(WT), Kcnma1 channels were absent at the neck of inner hair cells (IHCs) in Tmprss3(Y260X)-mutant mice. In conclusion, our data suggest that lack of Tmprss3 leads to a decrease in Kcnma1 potassium channels expression in (IHCs).

Probing the functional equivalence of otoferlin and synaptotagmin 1 in exocytosis.

Cochlear inner hair cells (IHCs) use Ca(2+)-dependent exocytosis of glutamate to signal sound information. Otoferlin (Otof), a C(2) domain protein essential for IHC exocytosis and hearing, may serve as a Ca(2+) sensor in vesicle fusion in IHCs that seem to lack the classical neuronal Ca(2+) sensors synaptotagmin 1 (Syt1) and Syt2. Support for the Ca(2+) sensor of fusion hypothesis for otoferlin function comes from biochemical experiments, but additional roles in late exocytosis upstream of fusion have been indicated by physiological studies. Here, we tested the functional equivalence of otoferlin and Syt1 in three neurosecretory model systems: auditory IHCs, adrenal chromaffin cells, and hippocampal neurons. Long-term and short-term ectopic expression of Syt1 in IHCs of Otof (-/-) mice by viral gene transfer in the embryonic inner ear and organotypic culture failed to rescue their Ca(2+) influx-triggered exocytosis. Conversely, virally mediated overexpression of otoferlin did not restore phasic exocytosis in Syt1-deficient chromaffin cells or neurons but enhanced asynchronous release in the latter. We further tested exocytosis in Otof (-/-) hippocampal neurons and in Syt1(-/-) IHCs but found no deficits in vesicle fusion. Expression analysis of different synaptotagmin isoforms indicated that Syt1 and Syt2 are absent from mature IHCs. Our data argue against a simple functional equivalence of the two C(2) domain proteins in exocytosis of IHC ribbon synapses, chromaffin cells, and hippocampal synapses.

Exocytosis at the hair cell ribbon synapse apparently operates without neuronal SNARE proteins.

SNARE proteins mediate membrane fusion. Neurosecretion depends on neuronal soluble NSF attachment protein receptors (SNAREs; SNAP-25, syntaxin-1, and synaptobrevin-1 or synaptobrevin-2) and is blocked by neurotoxin-mediated cleavage or genetic ablation. We found that exocytosis in mouse inner hair cells (IHCs) was insensitive to neurotoxins and genetic ablation of neuronal SNAREs. mRNA, but no synaptically localized protein, of neuronal SNAREs was present in IHCs. Thus, IHC exocytosis is unconventional and may operate independently of neuronal SNAREs.

Impairment of SLC17A8 encoding vesicular glutamate transporter-3, VGLUT3, underlies nonsyndromic deafness DFNA25 and inner hair cell dysfunction in null mice.

Autosomal-dominant sensorineural hearing loss is genetically heterogeneous, with a phenotype closely resembling presbycusis, the most common sensory defect associated with aging in humans. We have identified SLC17A8, which encodes the vesicular glutamate transporter-3 (VGLUT3), as the gene responsible for DFNA25, an autosomal-dominant form of progressive, high-frequency nonsyndromic deafness. In two unrelated families, a heterozygous missense mutation, c.632C-->T (p.A211V), was found to segregate with DFNA25 deafness and was not present in 267 controls. Linkage-disequilibrium analysis suggested that the families have a distant common ancestor. The A211 residue is conserved in VGLUT3 across species and in all human VGLUT subtypes (VGLUT1-3), suggesting an important functional role. In the cochlea, VGLUT3 accumulates glutamate in the synaptic vesicles of the sensory inner hair cells (IHCs) before releasing it onto receptors of auditory-nerve terminals. Null mice with a targeted deletion of Slc17a8 exon 2 lacked auditory-nerve responses to acoustic stimuli, although auditory brainstem responses could be elicited by electrical stimuli, and robust otoacoustic emissions were recorded. Ca(2+)-triggered synaptic-vesicle turnover was normal in IHCs of Slc17a8 null mice when probed by membrane capacitance measurements at 2 weeks of age. Later, the number of afferent synapses, spiral ganglion neurons, and lateral efferent endings below sensory IHCs declined. Ribbon synapses remaining by 3 months of age had a normal ultrastructural appearance. We conclude that deafness in Slc17a8-deficient mice is due to a specific defect of vesicular glutamate uptake and release and that VGLUT3 is essential for auditory coding at the IHC synapse.

Salicylate enables cochlear arachidonic-acid-sensitive NMDA receptor responses.

Currently, many millions of people treated for various ailments receive high doses of salicylate. Consequently, understanding the mechanisms by which salicylate induces tinnitus is an important issue for the research community. Behavioral testing in rats have shown that tinnitus induced by salicylate or mefenamate (both cyclooxygenase blockers) are mediated by cochlear NMDA receptors. Here we report that the synapses between the sensory inner hair cells and the dendrites of the cochlear spiral ganglion neurons express NMDA receptors. Patch-clamp recordings and two-photon calcium imaging demonstrated that salicylate and arachidonate (a substrate of cyclooxygenase) enabled the calcium flux and the neural excitatory effects of NMDA on cochlear spiral ganglion neurons. Salicylate also increased the arachidonate content of the whole cochlea in vivo. Single-unit recordings of auditory nerve fibers in adult guinea pig confirmed the neural excitatory effect of salicylate and the blockade of this effect by NMDA antagonist. These results suggest that salicylate inhibits cochlear cyclooxygenase, which increased levels of arachidonate. The increased levels of arachidonate then act on NMDA receptors to enable NMDA responses to glutamate that inner hair cells spontaneously release. This new pharmacological profile of salicylate provides a molecular mechanism for the generation of tinnitus at the periphery of the auditory system.