ABR/DPOAE publications

Mutations in MYO7A , the gene encoding the unconventional myosin 7a, cause hereditary deafness in mice and humans. In the cochlea, MYO7A is present in the sensory hair cells from embryonic stages of development, and plays a critical role in the development and maintenance of the mechanosensitive hair bundles composed of actin‐rich stereocilia. Shaker‐1 mutant mice ( Myo7a Sh1/Sh1 ), the murine model of Usher 1B syndrome, exhibit a progressive loss of the stereocilia, subsequent degeneration of the sensory epithelium and ultimately profound deafness. In addition to the hair bundle defects, we found that the shaker‐1 mutation prevented both inner hair cells (IHCs) and outer hair cells (OHCs) from acquiring their fully mature basolateral current profile. Delivering exogenous Myo7a to newborn Myo7a Sh1/Sh1 mice using dual‐adeno‐associated virus 8 (AAV8)‐ Myo7a or dual‐AAV9‐PhP.eB‐ Myo7a , which primarily target IHCs, led to a substantial rescue of their hair bundle structure. The rescued bundles regained their ability to generate mechanoelectrical transducer (MET) currents in response to fluid jet displacement. Although the average MET current was smaller than in control IHCs, the normal resting open probability of the MET channel was fully restored. The IHCs of the treated cochlea also regained a mature basolateral membrane current profile. Functionally, rescue of the IHC structure and function, but not that of OHCs, leads to an average improvement of 20–30 dB in hearing thresholds across most frequencies. These results support dual AAV‐induced gene replacement therapy as an effective strategy to recover hair‐cell function in Myo7a Sh1/Sh1 mice. image Key points Shaker‐1 mutant mice ( Myo7a Sh1/Sh1 ), which carry a mutation in the unconventional myosin MYO7A and are the murine model of Usher 1B syndrome, become profoundly deaf at 1 month of age or soon after. In the mammalian cochlea, MYO7A is expressed in the hair cells, including within their actin‐rich stereociliary bundles. We show that hair cells of Myo7a Sh1/Sh1 mice progressively lose their transducing stereocilia and mechanoelectrical transduction, and fail to acquire their fully mature basolateral current profile. Delivering exogenous Myo7a to newborn Myo7a Sh1/Sh1 mice using dual‐adeno‐associated virus (AAVs) led to a substantial rescue of the bundle structure and function of inner hair cells, including mechanoelectrical transduction. This functional rescue led to a 20–30 dB improvement in hearing thresholds across most frequencies. These results support dual AAV‐induced gene replacement therapy as an effective strategy to recover the hair‐cell function in Myo7a Sh1/Sh1 mice.

Cisplatin is a widely used and effective anticancer drug. However, it causes permanent sensorineural hearing loss in over 50% of treated patients. There are no FDA-approved therapies to prevent cisplatin-induced hearing loss (CIHL) in adults, highlighting a critical unmet clinical need. Previous studies suggest that statins, commonly prescribed cholesterol-lowering drugs, are associated with reduced incidence and severity of cisplatin-induced hearing loss. Statins are primarily used to lower cholesterol, but they also exert several pleiotropic effects, making the mechanism(s) underlying this protection unclear. Here we examine whether reduced plasma cholesterol confers protection against CIHL independent of statin treatment. We utilized mice lacking serine protease proprotein convertase subtilisin/kexin type 9 ( Pcsk9 knockout (KO) mice) as a genetic model of reduced plasma cholesterol. We find that Pcsk9 KO mice are protected against cisplatin-induced hearing loss, as reflected by significantly lower ABR and DPOAE threshold shifts relative to wild type (WT) mice following treatment. Histological analyses confirmed preservation of cochlear outer hair cells (OHCs) in Pcsk9 KO mice treated with cisplatin, whereas WT mice showed significant OHC loss in the high-frequency cochlear regions. Finally, hearing loss positively correlated with baseline plasma cholesterol levels. Together our data demonstrate that systemic cholesterol reduction provides significant protection against CIHL, and they suggest that the protective effect of statins against CIHL is mediated by cholesterol reduction.

Genetic mutations cause hereditary deafness, in which mutations in the POU4 transcription factor 3 gene (POU4F3) lead to autosomal dominant non-syndromic deafness 15 (DFNA15), for which no effective clinical treatment currently exists. Gene editing holds promise for precisely repairing mutated nucleotides, thus offering a potential cure for hereditary hearing loss. Here, we establish a Pou4f3WT/Q113* mutant mouse model mimicking DFNA15. We develop and screen adenine base editors (ABEs) targeting the Pou4f3Q113* allele by fusing diverse adenine deaminases to Cas9 we discovered before. SchABE8e accomplishes highly precise and efficient editing (up to 48.5%) at sgRNA3 in vitro. Neonatal Pou4f3WT/Q113* mice are treated via synthetic AAV (Anc80L65)-delivered SchABE8e-sgRNA3, resulting in near-complete hearing recovery, with the effect persisting for at least four months. Biosafety analyses further support the feasibility of base editing, providing a therapeutic strategy for DFNA15.

Noise‐induced hearing loss (NIHL), caused by irreversible cochlear hair cell (HC) damage, lacks effective therapies due to a limited understanding of endogenous protective mechanisms. The echolocating bats exhibit natural resistance to intense noise, and this suggested novel insights into methods to protect against NIHL. Here, through comparative transcriptomic analysis of noise‐exposed cochleae from the eastern bent‐winged bats ( Miniopterus fuliginosus ) and mice, the specific transcriptional dynamics in noise‐resistant Miniopterus fuliginosus are revealed, thus highlighting potential mechanisms for preventing cochlear damage that mouse models cannot replicate, with Hras emerging as the most significant hub upregulator. Functional validation in mice demonstrates that HC‐specific Hras overexpression significantly attenuates noise‐induced HC death, synaptic loss, and auditory threshold shifts. Mechanistically, Hras confers protection by activating the PI3K/Akt signaling pathway, a critical pro‐survival cascade. The findings further disentangle the mechanisms of cochlear resistance to intense noise in echolocating bats and suggest that targeting Hras expression may be a potential therapeutic intervention against NIHL.

The treatment of inner and middle ear diseases remains a significant challenge, often requiring surgical intervention as the only option. In this study, we investigated porphysomes, self-assembled porphyrin-based nanoparticles, as a minimally invasive drug delivery platform for inner ear applications and as photothermal agents for cholesteatoma ablation. Three porphysome formulations were evaluated: parent porphysomes (PS), porphyrin-stabilized nanoemulsions (nPS), and EDTA-lipid incorporated porphysomes (ePS). Rats received intratympanic injections of each formulation, and fluorescence imaging performed 1 h postinjection demonstrated concentration-dependent inner ear penetration for all formulations. Notably, no signs of ototoxicity were observed based on histological and functional assessments of hearing and balance. Among ePS-treated rats, the 250 and 1,000 μM groups showed significantly higher inner ear fluorescence compared with the 50 μM group. Auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE) tests were performed at baseline and 2 and 6 weeks postinjection. No significant threshold shifts were detected in PS and ePS groups compared with controls. In contrast, nPS-treated rats exhibited a significant ABR threshold elevation at 6 weeks ( p < 0.02). At 6 weeks, minor yet statistically significant DPOAE threshold differences were observed at 16 kHz (PS group) and 32 kHz (all porphysome groups); however, all shifts remained below 0 dB, indicating no functional hearing loss. Vestibular assessments, including swim and beam tests, revealed no significant impairments. Upon laser activation, all porphysome formulations induced substantial temperature elevation (Δ T = 31 ± 3.76°C, p < 0.05) and histological burn effects in cholesteatoma tissues. These findings support the potential of porphysomes as a safe, minimally invasive drug delivery system and photothermal agent for the treatment of inner and middle ear disorders.

Here, we present a protocol for evaluating auditory function and synaptic molecular-structural integrity in mice, specifically female GluA3-knockout (GluA3KO) mice raised in quiet or ambient sound levels. We describe steps for combining auditory brainstem response recordings, immunofluorescence, and electron microscopy. We detail procedures for assessing hearing function, the expression and localization of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptor subunits, and the ultrastructure of cochlear-ribbon synapses. This protocol provides a reproducible framework for studying molecular-structural synaptopathy in mouse models of cochlear dysfunction. For complete details on the use and execution of this protocol, please refer to Pal et al.1.

Adeno-associated virus-based gene therapy offers a promising treatment paradigm for inner ear diseases; however, the genetic heterogeneity of hereditary deafness requires gene-specific strategies and optimization of current approaches to identify the range of treatable conditions and improve therapeutic outcomes. To consider the therapeutic potential for a hearing loss gene not previously explored, we investigated the gene encoding the chloride intracellular channel protein CLIC5, mutations in which lead to DFNB103 in humans and deafness and circling behavior in a Clic5 -deficient mouse model. In this study, we utilized two constructs to deliver the wild-type Clic5 coding sequence into Clic5 -deficient mice: single-stranded and self-complementary adeno-associated virus, the latter known for rapid onset of transgene expression. We report a robust restoration of CLIC5 expression using either construct, including prevention of morphological degeneration and preserving auditory and vestibular function. Interestingly, the self-complementary construct achieved comparable functional recovery to single-stranded construct but at a lower titer. These findings highlight the potential of self-complementary adeno-associated virus to reduce dose requirements, minimize toxicity and broaden clinical utility for inner ear therapies.

Background: Hearing loss is known to cause structural and functional abnormalities in the central auditory pathways. Interventions with hearing aids that amplify acoustic signals have been developed to combat hearing loss. However, little is known about how such devices may affect the brain and mitigate the progression of hearing loss. We hypothesized that timely intervention that amplifies acoustic signals would delay further progression of hearing loss by maintaining central auditory activity and neural structure. Method: To that end, we provided eight weeks of acoustic stimulation tailored to compensate for subject-specific patterns of frequency loss in two mouse models of progressive hearing loss. We evaluated the effects of sound amplification on endbulb of Held anatomy at different ages of intervention in mice with early-onset (DBA/2) and late-onset (C57Bl/6) hearing loss. Results: We observed in both strains that endbulbs undergo rapid and progressive atrophy in untreated control subjects exposed to a baseline, unamplified, sound environment. In contrast, endbulb atrophy was significantly slowed in treated mice (p < 0.05). Conclusions: These data provide a possible explanation for how the brain benefits from sound amplification via hearing aid devices.

Objective Cisplatin is a chemotherapeutic agent with the undesirable side effect of ototoxicity. Transtympanic injections of antioxidant formulations may provide local otoprotection. We tested a novel antioxidant‐eluting microparticle for its otoprotective capability from systemic cisplatin as measured by cochlear electrophysiology. Study Design Basic science. Setting Translational research laboratory. Methods Eighteen mice were assigned to three groups. All mice underwent baseline click‐evoked auditory brainstem response (ABR) audiometry and right ear microparticle injections before beginning 42‐day intraperitoneal administration regimens of either saline (healthy control empty microparticle [HCEMP] group) or cisplatin (cisplatin empty microparticle [CEMP] group and cisplatin N ‐acetylcysteine microparticle [CNAC] group). These regimens consisted of three 4‐day cycles of intraperitoneal saline or cisplatin administration followed by 10 rest days. HCEMP and CEMP received right‐sided transtympanic empty microparticles, and CNAC received transtympanic N ‐acetylcysteine eluting microparticles. On day 43, all mice underwent posttreatment ABR. ABR thresholds and threshold shifts were analyzed with mixed‐effects models and Tukey's post hoc tests and were compared across pretreatment/posttreatment ears, treatment groups, and injected and non‐injected ears. Results We found that threshold shifts in the ears that received a transtympanic injection of N ‐acetylcysteine and three cycles of intraperitoneal cisplatin were similar to the paired ears of mice that received no cisplatin. Mice that received a transtympanic injection without N ‐acetylcysteine and intraperitoneal cisplatin had increased thresholds compared to mice that received a transtympanic injection of N ‐acetylcysteine and cisplatin. Conclusion Transtympanic N ‐acetylcysteine microparticle injections provided functional otoprotection in cisplatin‐exposed mice.

Diverse biological factors, such as sex and age, confer heterogeneity on sensory processing challenges in autism. These factors result in major difficulties in the processing of contextual information in social and non-social situations. To assess divergence in autistic traits, it is critical to consider sex- and age-related variability. Nevertheless, these differences remain largely elusive. Animal models of autism offer the possibility to examine contextual processing at the single-neuron level. Here, we investigated predictive processing of contextual auditory cues in the auditory midbrain of control and prenatally valproic acid-induced rats, a well-established animal model of autism. The rats were prepubertal and adult female and male animals. We performed single-unit recordings in the inferior colliculus of control and prenatally, or in utero , exposed rats under the classical oddball paradigm and non-repetitive cascade control sequences to study neuronal mismatch. This is the neuronal correlate of mismatch negativity, the brain’s automatic response to interruptions in environmental regularity. When comparing control and exposed rats, our results demonstrated a reduction in neuronal mismatch in rats exposed to valproic acid. However, exposed adult females exhibited an increased neuronal mismatch compared to their control counterparts. With respect to sex distinctions, valproic acid induced sex differences in neuronal mismatch of prepubertal and adult rats that are not observable in control animals. Moreover, we detected an age-dependent refinement in prediction error that is not affected by the drug. But valproic acid altered typical developmental trajectory of neuronal mismatch in both sexes. Such observations support sex- and age-related effects of in utero valproic acid exposure in contextual auditory processing at the neural level of the inferior colliculus. In autism, atypical predictive processing of environmental regularities underlies unusual responses to novel experiences. The present study highlights the importance of sex and age, that confer heterogeneity to these challenges.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by complex sensory processing deficits, which continue to elude comprehensive mechanistic understanding. A key unresolved question is how alterations in neural connectivity and communication translate into the behavioral manifestations seen in ASD. Here, we investigate how oligodendrocyte dysfunction alters myelin plasticity and neuronal activity, leading to auditory processing disorder associated with ASD. We focus on the SCN2A gene, an ASD-risk factor, to understand its role in myelination and neural processing within the auditory nervous system. Transcriptional profiling suggests alterations in the expression of myelin-associated genes in Scn2a conditional knockout mice, highlighting the cellular consequences engendered by Scn2a deletion in oligodendrocytes. The results reveal a nuanced interplay between oligodendrocytes and axons, where Scn2a deletion causes alterations in the intricate process of myelination. This disruption instigates changes in axonal properties, presynaptic excitability, and synaptic plasticity at the single cell level. Furthermore, oligodendrocyte-specific Scn2a deletion compromises the integrity of neural circuitry within auditory pathways, leading to auditory hypersensitivity. Our findings reveal a pathway linking myelin deficits to synaptic activity and sensory abnormalities in ASD.

Machine learning (ML) techniques are increasingly being used to improve disease diagnosis and treatment. However, the application of these computational approaches to the early diagnosis of age-related hearing loss (ARHL), the most common sensory deficit in adults, remains underexplored. Here, we demonstrate the potential of ML for identifying early signs of ARHL in adult mice. We used auditory brainstem responses (ABRs), which are non-invasive electrophysiological recordings that can be performed in both mice and humans, as a readout of hearing function. We recorded ABRs from C57BL/6N mice (6N), which develop early-onset ARHL due to a hypomorphic allele of Cadherin23 (Cdh23ahl), and from co-isogenic C57BL/6NTacCdh23+ mice (6N-Repaired), which do not harbour the Cdh23ahl allele and maintain good hearing until later in life. We evaluated several ML classifiers across different metrics for their ability to distinguish between the two mouse strains based on ABRs. Remarkably, the models accurately identified mice carrying the Cdh23ahl allele even in the absence of obvious signs of hearing loss at 1 month of age, surpassing the classification accuracy of human experts. Feature importance analysis using Shapley values indicated that subtle differences in ABR wave 1 were critical for distinguishing between the two genotypes. This superior performance underscores the potential of ML approaches in detecting subtle phenotypic differences that may elude manual classification. Additionally, we successfully trained regression models capable of predicting ARHL progression rate at older ages from ABRs recorded in younger mice. We propose that ML approaches are suitable for the early diagnosis of ARHL and could potentially improve the success of future treatments in humans by predicting the progression of hearing dysfunction.

Middle age represents a critical period of accelerated brain changes and provides a window for early detection and intervention in age-related neurological decline. Hearing loss is a key early marker of such decline and is linked to numerous comorbidities in older adults. Yet, ~10% of middle-aged individuals who report hearing difficulties show normal audiograms. Cochlear neural degeneration (CND) could contribute to these hidden hearing deficits, though its role remains unclear due to a lack of objective diagnostics and uncertainty regarding its perceptual outcomes. Here, we employed a cross-species design to examine neural and behavioral signatures of CND. We measured envelope following responses (EFRs) – neural ensemble responses to sound originating from the peripheral auditory pathway – in young and middle-aged adults with normal audiograms and compared these responses to young and middle-aged Mongolian gerbils, where CND was histologically confirmed. We observed near-identical changes in EFRs across species that were associated with CND. Behavioral assessments revealed age-related speech-in-noise deficits under challenging conditions, while pupil-indexed listening effort increased with age even when behavioral performance was matched. Together, these results demonstrate that CND contributes to speech perception difficulties and elevated listening effort in midlife, which may ultimately lead to listening fatigue and social withdrawal.

Circadian sensitivity significantly influences the severity of noise-induced hearing loss (NIHL), but the underlying mechanisms remain unclear. Here, we applied single-cell RNA sequencing to 97,043 cochlear cells, identifying macrophages as the primary immune responders to acoustic trauma, with a notable increase in their proportion in the cochlea. Immunofluorescence confirmed significant recruitment and activation of cochlear macrophages following noise exposure, while in vivo macrophage depletion resulted in the recovery of hearing. Furthermore, analyses of differentially-expressed genes and pathways revealed pronounced activation of NLRP3 inflammasome signaling in macrophages during night-time noise exposure. Measurements of elevated IL-1β and IL-18 expression in cochlear macrophages by multiplex immunohistochemistry correlated with heightened inflammation in the night-time exposure group. These findings were further confirmed by the administration of the selective NLRP3 inhibitor CY-09, which mitigated inflammasome activation, preserved synaptic integrity, and protect against hearing loss. In conclusion, our findings underscore the role of macrophage-driven NLRP3 inflammasome activation in mediating circadian variations in cochlear damage, offering a potential therapeutic target for mitigating NIHL.

Aminoglycoside antibiotics are essential in managing many life‐threatening diseases. However, their derivatives, such as neomycin, are associated with severe side effects such as persistent sensorineural hearing loss. Therefore, it is essential to elucidate the molecular and biochemical mechanisms of aminoglycoside‐induced ototoxicity and identify targets for alleviating ototoxic injury. Here, we provide a detailed cochlear cell atlas of neomycin‐induced acute and chronic ototoxicity‐related changes through single‐nucleus RNA sequencing profiling. Utilising this cochlear cell atlas, we used the Augur and scDist algorithms to evaluate cell‐type‐specific susceptibility to neomycin injury. We observed aberrant expression of X‐linked inhibitor of apoptosis (Xiap)–associated factor 1 (Xaf1) in neomycin‐exposed cochleae using the cochlear cell atlas, and we identified a novel role for Xaf1 in facilitating PANoptosis through overexpression and knockdown assays in vitro. Finally, we assessed the protective role of Xaf1 against neomycin‐induced ototoxicity by Xaf1 knockdown in cochlear hair cells using adeno‐associated virus‐based gene delivery. Mechanistically, Xaf1 orchestrates PANoptosis activation through direct interaction with and transcriptional regulation of ZBP1, establishing its hierarchical position upstream in the signalling cascade. This study presents detailed cochlear cellular maps of neomycin‐induced ototoxicity and serves as a valuable resource for identifying transcriptome‐wide disease‐driving perturbations at the single‐cell level. More importantly, we identified Xaf1 as a critical target for modulating the PANoptosis pathway, offering a promising treatment strategy for aminoglycoside‐induced ototoxicity.

Noise-induced hearing loss (NIHL) is a widespread auditory disorder in industrialized nations, posing significant global and domestic health concerns. Despite its prevalence, the mechanisms underlying NIHL remain poorly understood, hindering the development of effective therapeutic interventions. Emerging evidence from clinical and animal studies suggests a potential relationship between altered gut microbiota and hearing loss, although causality remains unclear. In this study, we investigated the role of postnatal microbial signals in NIHL pathogenesis using acute noise exposure models in C57BL/6 mice. Our findings indicate that acute noise exposure does not significantly alter gut microbiota composition in this strain and that microbiota depletion does not confer protection against hearing loss following acute acoustic trauma. These results suggest no link between gut microbiota and acute NIHL in C57BL/6 mice.

Most patients with sensorineural hearing loss (SNHL) may initially experience high‐frequency hearing loss linked to greater vulnerability of outer hair cells (OHCs) in the cochlear basal turn. However, the molecular mechanism underlying this susceptibility in the high‐frequency region remains unclear. Here, NOX2 is first identified as a differentially expressed gene related to oxidative damage in the apical and basal turns through single‐cell RNA sequencing. In mouse cochleae damaged by neomycin and noise, Nox2‐positive OHCs increase progressively from the apex to base, indicating that NOX2 expression clearly differed significantly between the apical and basal OHCs. In NOX2 knockout mice, OHC damage caused by neomycin and noise exposure is significantly reduced. Furthermore, knocking out NOX2 increases the expression of Nrf2 in the cytoplasm and Nrf2 nuclear translocation in OHCs. In addition, Nrf2 inhibitors counteract the protective effect of NOX2 knockout against neomycin. Finally, when Nox2 is used as the target, ginsenoside Rg1 is identified as a compound with protective effects against neomycin‐induced hearing loss. In summary, these results indicate NOX2 is highly correlated with high‐frequency OHCs vulnerability in the cochlea and is a potential intervention target for the clinical treatment of SNHL.

Stereocilia are rod-like mechanosensory projections consisting of unidirectionally oriented actin filaments that extend into the inner ear hair cell cytoskeleton, forming dense rootlets. Taperin (TPRN) localizes to the narrowed-down base of stereocilia, where they pivot in response to sound and gravity. We show that TPRN-deficient mice have progressive deafness characterized by gradual asynchronous retraction and fusion of outer and inner hair cell stereocilia, followed by synaptic abnormalities. Stereocilia that lack TPRN develop warped rootlets with gradual loss of TRIOBP-5 and ANKRD24 from mechanosensory rows starting postnatally. In contrast, TPRN overexpression causes excessive F-actin bundling, extra rows, and over-elongation of stereocilia during development. Purified full-length mouse TPRN cross-links F-actin into bendable bundles reflecting in vivo data. This F-actin–bundling ability is attributed to the TPRN N-terminal region. TPRN interacts with the membrane receptor PTPRQ, connecting the F-actin core to the plasma membrane, stabilizing stereocilia. Thus, TPRN is a specialized F-actin bundler strategically located to augment stereocilia rootlet formation and their pivot point flexibility for sustained sound-induced deflections.

Aging is associated with deficits in auditory functioning. Characterization of auditory deficits that originate in middle-age is crucial for understanding the initial age-related functional impairments and the spatio-temporal progression of age-related auditory pathophysiology. Early age-related deficits in auditory processing are evident in difficult listening conditions, such as background noise, before becoming evident in quiet. To investigate the effect of noise on age-related auditory dysfunction, we collected suprathreshold auditory brainstem responses (ABRs) from young, middle-aged, and aged CBA/CaJ mice in quiet and broad-band background noise. We utilized multiple ABR metrics, including phase locking value (PLV), a measure of neural synchrony correlated to speech-in-noise understanding in humans. Despite no differences in auditory processing in quiet between young and middle-aged mice, middle-aged mice exhibited a distinct auditory phenotype from both young and aged mice in background noise conditions. We found that noise significantly decreased amplitude in middle-aged mice more than in young and aged mice. Noise significantly increased latencies for wave I and V in young mice, but only affected wave V in middle-aged mice and did not affect aged latencies. Noise significantly decreased PLV in middle-aged mice to a greater extent than in young mice, but to a lesser extent in aged mice. These results show that middle-aged mice have a distinct, auditory dysfunction phenotype evident in background noise. Our data show that suprathreshold auditory function in noise can identify early age-related hearing loss and can be used as a sensitive tool for detecting auditory dysfunction in normal hearing animal models.

Noise-induced hearing loss (NIHL) poses an emerging global health problem with only ear protection or sound avoidance as preventive strategies. The cochlea receives some protection from medial olivocochlear efferent neurons, providing a potential target for therapeutic enhancement. Cholinergic efferents release acetylcholine (ACh) to hyperpolarize and shunt the outer hair cells (OHCs), reducing sound-evoked activation. The (α9)2(α10)3 nicotinic ACh receptor (nAChR) on the OHCs mediates this effect. Transgenic knockin mice with a gain-of-function nAChR (α9L9'T) suffer less NIHL. α9 knockout mice are more vulnerable to NIHL but can be rescued by viral transduction of the α9L9'T subunit. In this study, an HA-tagged gain-of-function α9 isoform was expressed in wild-type mice to reduce NIHL. Synaptic integration of the virally expressed nAChR subunit was confirmed by HA immunopuncta localized to the postsynaptic membrane of OHCs. After noise exposure, AAV2.7m8-CAG-α9L9'T-HA (α9L9'T-HA)-injected mice had less hearing loss (auditory brainstem response [ABR] thresholds and threshold shifts) than did control mice. ABRs of α9L9'T-HA-injected mice also had larger wave-1 amplitudes and better recovery of wave-1 amplitudes post noise exposure. Thus, virally expressed α9L9'T combines effectively with native α9 and α10 subunits to mitigate NIHL in wild-type cochleas.