ABR/DPOAE publications

Noise-induced hearing loss (NIHL) is a major public health problem caused by damage to cochlear hair cells, synapses, and spiral ganglion neurons (SGNs). Since effective treatments are lacking, we investigated cellular stress responses induced by moderate and loud noise in a mouse model of cochlear synaptopathy. RNA sequencing and spatial transcriptomics revealed that noise exposure elicited a robust but transient upregulation of endoplasmic reticulum chaperones and proteasome subunits in SGNs and their supporting cells. To target this response, we administered TRC051384, a small-molecule activator of the heat shock transcription factor Hsf1, prior to noise exposure. TRC051384 crossed the blood–labyrinth barrier and reached the cochlea, induced heat shock protein gene expression, and restored ubiquitin–proteasome function in SGNs. Notably, TRC051384 treatment enhanced auditory brainstem response threshold recovery, preserved Wave I amplitudes, and maintained ribbon synapse density. Together with the existing literature, these findings identify proteotoxic stress in spiral ganglion neurons as a contributor to noise-induced hearing loss and support pharmacological activation of HSF1 as a promising therapeutic strategy.

Hearing is the largest modifiable mid-life risk factor for Alzheimer's disease (AD), yet its link to dementia remains unclear. We identified a neurophysiological biomarker of AD risk using the non-invasive, rapidly acquired, and clinically translatable auditory brainstem response (ABR) in normal hearing knock-in rats (Swedish familial AD risk variant to Amyloid precursor protein, AppS; male and female). Human ABRs have been proposed as a biomarker for AD and related dementias. The novel metric reported here is derived from multidimensional parametric feature extraction on the distribution statistics of repeated single-trial ABR traces. We report accurate prediction of genetic risk for AD risk in young and aged rats: AppS separate clearly from healthy humanized (AppH) in sex- and age-dependent manners. Notably, auditory learning during young adulthood shifted the AppS ABR signature towards a healthy AppH-like state that maintained over time into older age. Altogether the findings support the utility of the ABR to track disease state, progression, and effects of intervention, and point to a central neural generator of auditory dysfunction related to AD risk. ABRs could provide a very early biomarker for detection of AD risk and used to test the synergy of auditory and cognitive functions in human dementia.

Proper orientation of the apical cytoskeleton in auditory and vestibular hair cells is essential for their sensory function. A recently identified regulator of hair cell orientation is the G protein-coupled receptor GPR156, which signals through inhibitory heterotrimeric G proteins. In hair cells expressing the transcription factor EMX2, GPR156 is apically enriched and polarized at cell junctions. There, GPR156 signaling reverses the interpretation of tissue-level core planar cell polarity cues, effectively reversing the orientation of Emx2 -positive compared to Emx2 -negative hair cells. This mechanism establishes key anatomical features, such as the correct alignment of auditory outer hair cells and the line of polarity reversal in the otolith organs of the vestibular system. Null mice with constitutive Gpr156 inactivation exhibit severe hearing loss, mirroring congenital hearing impairment in human patients with homozygous GPR156 variants. These null mutants also display impaired swimming and vestibulo-ocular reflexes, although the nature of these vestibular deficits differs from those reported in Emx2 mutants. Here, to determine the extent to which functional deficits arise from hair cell misorientation, we conditionally inactivated Gpr156 in postmitotic hair cells in the inner ear. This targeted deletion approach recapitulated the misorientation phenotype observed in null mutants. Notably, 30–40% of cochlear and utricular hair cells affected in the null background retained normal orientation in conditional mutants, likely due to the later timing of Gpr156 inactivation. Despite reduced efficiency, conditional mutants exhibited similar, albeit predictably milder, auditory and vestibular dysfunction. As hair cells can carry out mechano-electrical transduction without GPR156, we conclude that sensory deficits mainly result from its essential role in hair cell orientation.

Cisplatin-based chemotherapy causes hearing loss in 40-60 % of all patients, yet effective preventative options remain limited. Building on prior animal studies, we demonstrate that oral administration of AZD5438, a potent and selective CDK2 inhibitor, provides dose-dependent protection against hearing loss in a clinically relevant multi-dose cisplatin mouse model. Protective doses (4.7 and 9.4 mg/kg b.i.d.) fall within the human-equivalent maximum tolerated dose range established in AstraZeneca trials, and exhibit plasma pharmacokinetics comparable to those in humans. Importantly, AZD5438 at 9.4 mg/kg b.i.d. does not reduce cisplatin's anti-tumor efficacy in a testicular cancer xenograft model, consistent with in vitro findings. These results support AZD5438 as a promising candidate for clinical trials to prevent cisplatin-induced hearing loss while preserving cancer treatment efficacy.

Hearing loss is the most common sensory organ disorder, with genetic factors contributing substantially to the disease. Among the 87 genes responsible for autosomal recessive nonsyndromic hearing loss, mutations in MPZL2 have been frequently linked to mild-to-moderate autosomal recessive hearing loss (DFNB111). Here, we present multiple families whose hearing loss arose from biallelic mutations in the MPZL2 gene and found that the MPZL2 p.Q74∗ mutation may be a founder allele among East Asians. Furthermore, we generated an Mpzl2 p.Q74∗ knockin mouse model that exhibited autosomal recessive, progressive, ski-sloping hearing loss with Deiter's cell degeneration. Gene supplementation using AAV-DJ or AAV-PHP.eB to deliver human MPZL2 (hMPZL2) under control of the CAG promoter induced ototoxicity, whereas employing an alternative EF1α promoter with AAV-DJ (DJ-EF1α-hMPZL2) circumvented this issue, restoring long-term auditory function and Deiter's cell survival in Mpzl2Q74∗/Q74∗ mice for up to 24 weeks. Overall, this study provides the foundational steps for developing a safe and effective biological treatment for DFNB111 and underscores the importance of precise regulation of target cells and transgene expression in AAV-based treatments.

Objective: The purpose of this paper is to present a chronic, preclinical, pilot study of a multi-electrode auditory nerve implant. To date, the most successful auditory neuroprosthesis is the cochlear implant, but these devices have known limitations. Intraneural stimulation of the auditory nerve is a promising alternative, but preclinical investigations of multi-electrode devices have been limited to acute or semi-chronic studies. Methods: Six cats were chronically implanted in the right auditory nerve with a 15-channel Utah Slant ArrayTM. The array's position in the internal auditory canal was confirmed using computed tomography. Electrode impedance and evoked responses were recorded regularly during the indwelling period to evaluate performance. Results: Two of the six implants met endpoint criteria at 6 months, with stable electrode impedances and robust evoked responses on at least 50% of their channels. Another two implants exhibited stable electrode impedances, but activation thresholds increased over three months. Two implants failed within the first two weeks due to device migration caused by connector-related issues. Conclusion: This study reports the first successful chronic deployment of an multi-electrode auditory nerve implant in a preclinical model. Two implanted cats exhibited stable chronic performance, marked by a consistent set of electrodes capable of eliciting evoked responses. The failure modes of the remaining four cats are discussed and recommendations are made to stabilize implant placement. The study lays a solid foundation for future chronic safety and efficacy investigations as part of an effort to translate this new neuroprosthetic technology into the clinic.

The hippocampus is classically linked to memory, yet increasing evidence points to a broader role in perceptual inference and deviance detection. Predictive coding theories propose that perception minimizes mismatches between expected and actual sensory input, expressed in neural signatures such as mismatch negativity (MMN) and P300. Although MMN arises mainly from sensory and prefrontal cortices, the hippocampus is anatomically interconnected with both and may also contribute to prediction error processing. We recorded single- and multi-unit activity and local field potentials (LFPs) from DG and CA1 in urethane-anesthetized rats during an auditory oddball paradigm and a no-repetition control sequence to dissociate prediction error from repetition suppression. Approximately 20% of hippocampal neurons were sound responsive, and a subset showed deviant selectivity. Spiking activity predominantly reflected prediction errors, while LFPs revealed complementary contributions from repetition suppression and prediction error. Early LFP components were enhanced for randomly presented deviants, whereas later components within the P300 latency range were stronger for predictable deviants, indicating temporally distinct phases of error signaling and top-down modulation. These findings identify the hippocampus as an active contributor to auditory deviance detection and support a hierarchical model in which hippocampal circuits participate in predictive sensory processing beyond memory.

Hearing loss is the most common sensory disorder, with a substantial proportion caused by genetic mutations. KCNQ4, a voltage-gated potassium channel highly expressed in cochlear outer hair cells, is a common genetic etiology implicated in autosomal dominant progressive hearing loss (DFNA2). The dominant-negative KCNQ4 p.W276S (c.827G>C) mutation represents a mutational hotspot in DFNA2, yet no effective treatments exist. Here, we developed allele-preferential antisense oligonucleotides (ASOs) targeting this dominant-negative KCNQ4 mutation. In a systemic in vitro screen, ASO-123 demonstrated a knockdown of mutant Kcnq4 while preserving wild-type transcripts. In a Kcnq4 p.W277S knockin mouse model mimicking DFNA2, ASO-123 preferentially suppressed mutant transcripts, attenuated progressive hearing loss, and improved outer hair cell survival while enhancing their electrophysiologic function. Comprehensive transcriptomic analyses further validated the efficacy of ASO-123. Thus, our findings establish ASO-based therapy as a promising strategy for treating hereditary hearing loss caused by dominant-negative KCNQ4 mutations.

Outer hair cells (OHCs) in the cochlea contain specialized stereociliary structures essential for auditory function. These include horizontal top connectors (HTCs), linking adjacent stereocilia and tectorial membrane (TM)-attachment crowns (ACs), anchoring the tallest stereocilia to the TM. The known molecular components of these structures, such as stereocilin, otogelin, otogelin-like, and tubby, lack transmembrane domains, suggesting the existence of anchoring proteins. This study identifies TMEM145, a transmembrane protein with a Golgi dynamics domain, as a crucial OHC stereocilia component. TMEM145 is expressed in both OHCs and spiral ganglion neurons, with specific localization to TM-ACs and HTCs in OHCs. Tmem145 knockout (KO) mice exhibit profound hearing impairment at 3 weeks of age, with complete loss of distortion product otoacoustic emissions, indicating OHC dysfunction. Immunostaining and scanning electron microscopy reveal the absence of TM-ACs and HTCs in Tmem145 KO mice. In heterologous cell systems, TMEM145 interacts with stereocilin and tubby, facilitating their extracellular secretion. TMEM145 is undetectable in Stereocilin KO and tubby mutant mice, indicating interdependence among these proteins. These findings establish TMEM145 as an essential membrane protein for the structural integrity of OHC stereocilia, providing insights into the molecular architecture of cochlear hair cells and their role in auditory function.

Autism is an early‐onset neurodevelopmental disorder characterized by restricted, repetitive behaviors and atypical patterns of social communication and interaction. A considerable proportion of autistic individuals experience divergent auditory perception, which can interfere with their ability to navigate everyday sound environments. Auditory brainstem responses are electrophysiological potentials elicited by auditory stimuli that evaluate neural activity along the auditory nerve and brainstem. Importantly, the auditory brainstem response varies by sex, with females typically showing higher amplitudes and shorter latencies than males. This sex‐specific neurophysiological profile is especially relevant in autism research, where the male‐to‐female diagnosis ratio is approximately 3:1. Thus, exploring the neurobiological mechanisms underlying sex‐specific variations in autistic traits is essential. Furthermore, autism sensory profiles may vary based on the independent and mutual effects of environmental and genetic factors. To deepen this understanding, we examined auditory brainstem responses in two rat models of autism: the GRIN2B rare mutation model and the prenatal valproic acid induction model, alongside control animals. We assessed peak amplitudes and latencies (Waves I through V), inter‐peak intervals (I–III, I–V, and III–V), and amplitude ratios (III:I, V:I, and V:III). Female rats generally exhibited greater amplitudes and longer latencies across waveforms. Regarding rat models, control animals consistently showed larger amplitudes and shorter latencies compared to autism‐like models. Exploratory analyses further suggested pairwise interactions between sex and rat model, indicating modulation of auditory phenotypes linked to autism. Thus, our findings reveal key insights into the effects of sex and rat model, as well as their interactions.

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.

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.

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.

Presbycusis or age-related hearing loss (ARHL) affects millions of people worldwide, increasing their risk of cognitive decline and poor quality of life. However, ARHL remains an irreversible condition due to our inability to induce inner-ear hair cell regeneration. Nevertheless, multisession epidural stimulation of the auditory cortex (AC) at the onset of ARHL prevents hearing threshold elevation in naturally aging Wistar rats. Accordingly, we hypothesized that anodal direct current (DC) stimulation of the AC may also compensate for age-related maladaptive, activity-dependent changes. Here, we examined immunocytochemical markers in the AC, including early genes (c-fos and Arc), AMPA receptors (GluR2/3), parvalbumin (PV), and GAD67, along with auditory-evoked potentials (CAEPs) recorded in both auditory and visual (VC) cortices. When comparing 6 and 18.13-month-old rats without AC simulation, we observed loss of c-fos and Arc-positive neurons and decreased GluR2/3 expression, confirming altered AC neuronal network plasticity and activation. In addition, we noted changes in PV and decreased GAD67 immunoreactivity suggesting disrupted inhibition and significantly increased wave amplitudes in CAEPs, altered AC latencies, and decreased VC responses. By contrast, electrically stimulated rats showed no significant variations in early gene markers, GluR2/3, PV, or GAD67 with age, and the amplitudes and latencies of CAEPs recorded in their AC and VC resembled those of young rat. These findings indicate that anodal DC stimulation at the onset of ARHL delays AC aging by minimizing the loss of inhibition and preventing increases in cortical excitability in Wistar rats.

Epilepsy is a multifaceted neurological disorder characterized by recurrent seizures and associated with molecular and immune alterations in key brain regions. The GASH/Sal (Genetic Audiogenic Seizure Hamster, Salamanca), a genetic model for audiogenic epilepsy, provides a powerful tool to study seizure mechanisms and resistance in predisposed individuals. This study investigates the proteomic and immune responses triggered by audiogenic kindling in the inferior colliculus, comparing non-responder animals exhibiting reduced seizure severity following repeated stimulation versus GASH/Sal naïve hamsters. To assess auditory pathway functionality, Auditory Brainstem Responses (ABRs) were recorded, revealing reduced neuronal activity in the auditory nerve of non-responders, while central auditory processing remained unaffected. Cytokine profiling demonstrated increased levels of proinflammatory markers, including IL-1 alpha (Interleukin-1 alpha), IL-10 (Interleukin-10), and TGF-beta (Transforming Growth Factor beta), alongside decreased IGF-1 (Insulin-like Growth Factor 1) levels, highlighting systemic inflammation and its interplay with neuroprotection. Building on these findings, a proteomic analysis identified 159 differentially expressed proteins (DEPs). Additionally, bioinformatic approaches, including Gene Set Enrichment Analysis (GSEA) and Weighted Gene Co-expression Network Analysis (WGCNA), revealed disrupted pathways related to metabolic and inflammatory epileptic processes and a module potentially linked to a rise in the threshold of seizures, respectively. Differentially expressed genes, identified through bioinformatic and statistical analyses, were validated by RT-qPCR. This confirmed the upregulation of six genes (Gpc1—Glypican-1; Sdc3—Syndecan-3; Vgf—Nerve Growth Factor Inducible; Cpne5—Copine 5; Agap2—Arf-GAP with GTPase domain, ANK repeat, and PH domain-containing protein 2; and Dpp8—Dipeptidyl Peptidase 8) and the downregulation of two (Ralb—RAS-like proto-oncogene B—and S100b—S100 calcium-binding protein B), aligning with reduced seizure severity. This study may uncover key proteomic and immune mechanisms underlying seizure susceptibility, providing possible novel therapeutic targets for refractory epilepsy.

Humans and mice with mutations in genes encoding CIB2 and whirlin (WHRN) are deaf. We previously reported that CIB2 binds to WHRN and is essential for stereocilia staircase architecture of cochlear hair cells. Here, we refine the interaction domains of both proteins and show that these proteins play unique roles in stereocilia bundle formation and organization. We found that the EF2 domain of CIB2 binds to the HHD2 region of WHRN. AlphaFold2 multimer independently identified the same interacting regions and gave a thorough structural model. Next, we investigated genetic interaction between murine Cib2 and Whrn. Hearing in mice double heterozygous for functionally null alleles (Cib2KO/+;Whrnwi/+) was similar to that in age-matched wild-type mice, indicating that partial deficiency for both Cib2 and Whrn does not impair hearing. Double homozygous mutant mice (Cib2KO/KO;Whrnwi/wi) were deaf, and their cochlear stereocilia exhibited a predominant phenotype seen in single Whrnwi/wi mutants. Overexpression of WHRN in Cib2KO/KO mice did not rescue the stereocilia morphology. These data suggest that CIB2 is multifunctional, with key independent functions in the development and/or maintenance of the stereocilia staircase pattern in auditory hair cells.

It is crucial to manage hearing loss and its associated public health impacts. In this study, we aimed to understand the role of Sema3f in the development and maintenance of the auditory system. Inner ear-specific Sema3f knockout mice exhibited hearing loss at 8 weeks with an elevated threshold for auditory brainstem response and an absent threshold for distortion product optoacoustic emission tests. Additionally, an increased number of outer hair cells and abnormal patterns of spiral ganglion neuron projections in the outer hair cell regions were observed. Through the analyses of sequencing data from 558 families with hearing loss, we identified biallelic variants of SEMA3F, which encodes semaphorin-3F, in one of the families. In the family, the proband showed profound progressive nonsyndromic hearing loss with congenital onset. In vitro analysis revealed that the identified missense variants decreased the furin-mediated processing of SEMA3F and abolished the cellular abilities of SEMA3F, which collapsed the filamentous actin cytoskeleton in human umbilical vein-derived endothelial cells. Our data suggest that SEMA3F is essential for normal hearing and is associated with nonsyndromic hearing loss in humans.

CRISPR-Cas13 systems have therapeutic promise for the precise correction of point mutations in RNA. Using adenosine deaminase acting on RNA (ADAR) effectors, A-I base conversions can be targeted using guide RNAs (gRNAs). We compare the Cas13 effectors PspCas13b and Cas13bt3 for the repair of the gene USH2A , a common cause of inherited retinal disease and Usher syndrome. In cultured cells, we demonstrate up to 80% efficiency for the repair of the common c.11864 G > A and its murine equivalent c.11840 G > A, across different gRNAs and promoters. We develop and characterize a mouse model of Usher syndrome carrying the c.11840 G > A mutation designed for the evaluation of base editors for inherited retinal disease. Finally, we compare Cas13 effectors delivered via AAV for the repair of Ush2a in photoreceptors. Mean RNA editing rates in photoreceptors across different constructs ranged from 0.32% to 2.04%, with greater efficiency in those injected with PspCas13b compared to Cas13bt3 constructs. In mice injected with PspCas13b constructs, usherin protein was successfully restored and correctly localized to the connecting cilium following RNA editing. These results support the development of transcriptome targeting gene editing therapies for retinal disease.

Calcium and integrin-binding protein 2 (CIB2) and CIB3 bind to transmembrane channel-like 1 (TMC1) and TMC2, the pore-forming subunits of the inner-ear mechano-electrical transduction (MET) apparatus. These interactions have been proposed to be functionally relevant across mechanosensory organs and vertebrate species. Here, we show that both CIB2 and CIB3 can form heteromeric complexes with TMC1 and TMC2 and are integral for MET function in mouse cochlea and vestibular end organs as well as in zebrafish inner ear and lateral line. Our AlphaFold 2 models suggest that vertebrate CIB proteins can simultaneously interact with at least two cytoplasmic domains of TMC1 and TMC2 as validated using nuclear magnetic resonance spectroscopy of TMC1 fragments interacting with CIB2 and CIB3. Molecular dynamics simulations of TMC1/2 complexes with CIB2/3 predict that TMCs are structurally stabilized by CIB proteins to form cation channels. Overall, our work demonstrates that intact CIB2/3 and TMC1/2 complexes are integral to hair-cell MET function in vertebrate mechanosensory epithelia.

Hearing mediates many behaviours critical for survival in echolocating bats, including foraging and navigation. Although most mammals are susceptible to progressive age-related hearing loss, the evolution of biosonar, which requires the ability to hear low-intensity echoes from outgoing sonar signals, may have selected against the development of hearing deficits in bats. Many echolocating bats exhibit exceptional longevity and rely on acoustic behaviours for survival to old age; however, relatively little is known about the ageing bat auditory system. In this study, we used DNA methylation to estimate the ages of wild-caught big brown bats ( Eptesicus fuscus ) and measured hearing sensitivity in young and ageing bats using auditory brainstem responses (ABRs) and distortion product otoacoustic emissions (DPOAEs). We found no evidence for hearing deficits in bats up to 12.5 years of age, demonstrated by comparable thresholds and similar ABR and DPOAE amplitudes across age groups. We additionally found no significant histological evidence for cochlear ageing, with similar hair cell counts, afferent and efferent innervation patterns in young and ageing bats. Here, we demonstrate that big brown bats show minimal evidence for age-related hearing loss and therefore represent informative models for investigating mechanisms that may preserve hearing function over a long lifetime.