Publications using the RZ6

Auditory neuropathies affect the spiral ganglion neurons of the auditory nerve or their synapses with the sensory hair cells, distorting the sound information transmitted from the ear to the brain. Deciphering the underlying pathophysiological mechanisms remains challenging owing to the diversity of spiral ganglion neuron subtypes and associated central auditory circuits. An auditory neuropathy mechanism is unraveled by investigating the origin of auditory hyperexcitability in a mouse model for hereditary congenital deafness. Otogl encodes the large Otogelin‐like protein, which is related to secreted epithelial mucins and is implicated in the mechanical stimulation of cochlear outer hair cells. Heterozygous Otogl +/− mutant mice display auditory hyperexcitability, highlighted by their susceptibility to audiogenic seizures induced by loud sounds. It is shown that Otogl is transiently expressed in a subpopulation of spiral ganglion neurons during cochlear development. Despite their apparently normal hearing, Otogl +/− mice display poor activation of the spiral ganglion neurons processing loud sounds and an elevation of the activation threshold of the middle the ear muscle reflex that attenuates loud sounds. The findings reveal how a neuropathy affecting spiral ganglion neurons specialized in loud sound processing and associated with the middle the ear muscle reflex can manifest itself as auditory hyperexcitability.

Kölliker's organ is a transient developmental structure in the mouse cochlea that undergoes significant remodeling postnatally. Utilizing an epithelial-specific conditional deletion mouse model of Prdm16 (marker and regulator of Kölliker's organ), we show that Prdm16 is required for interdental cell development, and thereby the development of the limbal domain of the tectorial membrane and its medial anchorage to the spiral limbus. Additionally, we show that Kölliker's organ is involved in normal tectorial membrane collagen fibril development and maturation. Interestingly, mesenchymal cells of the spiral limbus underneath Prdm16 -deficient Kölliker's organ failed to produce interstitial matrix proteins, resulting in a hypoplastic and truncated spiral limbus, indicating a non-cell autonomous role of Prdm16 in regulating spiral mesenchymal matrix development. Single-cell RNA sequencing identified differentially expressed genes in Prdm16 -deficient Kölliker's organ suggesting a role for connective tissue growth factor (CTGF) downstream Prdm16 in epithelial-mesenchymal signaling involved in spiral limbus matrix deposition. Prdm16 -deficient mice showed a hearing deficit, as indicated by elevated auditory brainstem response thresholds at most frequencies, consistent with the cochlear structural defects. Both sexes were studied. This work establishes Prdm16 as a deafness gene in mice through its role in regulating Kölliker's organ development. Such understanding recognizes Kölliker's organ as a developmental hub regulating multiple surrounding cochlear structures.

Alport syndrome (AS) is a genetic disorder marked by mutations in type IV collagen, leading to kidney glomerular dysfunction. AS also involves the cochlea, causing late-onset progressive hearing loss. Currently, there are no therapeutic drugs to protect hearing from AS. HDAC inhibitors (HDACis) are chemical compounds that block the activity of histone deacetylase and are known to exert diverse biologic effects. We investigated the effect of Trichostatin A (TSA), an HDACi, to assess its potential to inhibit hearing deterioration in AS. Col4a3 knockout (KO) mice were treated with TSA at 3 weeks of age, and hearing levels were measured using auditory brainstem response (ABR). The results demonstrate that TSA significantly protects the hearing of KO mice compared to the untreated group. The TSA-treated group exhibited a reduction in the levels of oxidative stress markers 4-Hydroxynonenal and 3-Nitrotyrosine, along with a decrease in inflammatory cytokines, in both the mouse cochlea and in vitro HEI-OC1 (House Ear Institute-Organ of Corti 1) cell and HEK (Human Embryonic Kidney)293T cells. AS demonstrated a thickening in the stria vascular vessels, a phenomenon that TSA attenuated. Col4α3 deficiency showed down-regulation of Hemeoxygenase-1 (HO-1), a key anti-inflammatory molecule. TSA treatment induced HO-1 signaling, which contributed to the inhibition of oxidative stress and inflammatory cytokines. These findings suggest that TSA represents a promising candidate molecule for mitigating the progression of hearing loss in AS.

AMPA-type glutamate receptors (AMPARs) mediate excitatory cochlear transmission. However, unique roles of AMPAR subunits are unresolved. Lack of subunit GluA3 (Gria3 KO ) in male mice reduced cochlear output by 8 postnatal weeks. Here, we studied the role of X-linked Gria3 in cochlear function and synapse anatomy in females. Auditory brainstem responses (ABRs) were similar in 3-week-old female Gria3 WT and Gria3 KO mice raised in quiet. However, after switching to ambient sound, ABR thresholds were elevated and wave-1 amplitudes were diminished at 5-week and older in Gria3 KO . A quiet vivarium precluded this effect. Paired synapses were similar in number, but lone ribbons and ribbonless synapses were more frequent, and swollen afferent terminals were observed only in female Gria3 KO mice in ambient sound. Synaptic GluA4:GluA2 ratios increased relative to Gria3 WT , particularly in ambient sound, suggesting an activity-dependent increase in calcium-permeable AMPARs in Gria3 KO . We propose that lack of GluA3 induces a sex-dependent vulnerability to AMPAR-mediated excitotoxicity.

Assembly of actin-based stereocilia is critical for cochlear hair cells to detect sound. To tune their mechanosensivity, stereocilia form bundles composed of graded rows of ascending height, necessitating the precise control of actin polymerization. Myosin 15 (MYO15A) drives hair bundle development by delivering critical proteins to growing stereocilia that regulate actin polymerization via an unknown mechanism. Here, we show that MYO15A is itself an actin nucleation-promoting factor. Moreover, a deafness-causing mutation in the MYO15A actin-binding interface inhibits nucleation activity but still preserves some movement on filaments in vitro and partial trafficking on stereocilia in vivo. Stereocilia fail to elongate correctly in this mutant mouse, providing evidence that MYO15A-driven actin nucleation contributes to hair bundle biogenesis. Our work shows that in addition to generating force and motility, the ATPase domain of MYO15A can directly regulate actin polymerization and that disrupting this activity can promote cytoskeletal disease, such as hearing loss.

Tinnitus, a widespread condition affecting numerous individuals worldwide, remains a significant challenge due to limited effective therapeutic interventions. Intriguingly, patients using cochlear implants (CIs) have reported significant relief from tinnitus symptoms, although the underlying mechanisms remain unclear and intracochlear implantation risks cochlear damage and hearing loss. This study demonstrates that targeted intracochlear electrical stimulation (ES) in guinea pigs with noise‐induced hearing loss reversed tinnitus‐related maladaptive plasticity in the cochlear nucleus (CN), characterized by reduced auditory innervation, increased somatosensory innervation, and diminished inhibitory neural networks. Additionally, a customized extracochlear ES delivered by a newly designed extracochlear electrode array to guinea pigs with salicylate‐induced tinnitus also reversed the aforementioned maladaptive plasticity and alleviated tinnitus without causing additional cochlear damage or hearing loss. These findings suggest that CI‐delivered ES may alleviate tinnitus by reversing maladaptive CN plasticity. Additionally, the extracochlear ES strategy offers a promising tinnitus treatment with minimal risk to hearing.

While soundscapes shape the structure and function of auditory systems over evolutionary timescales, there is limited information regarding the adaptation of wild fish populations to their natural acoustic environments. This is particularly relevant for freshwater ecosystems, which are extremely diverse and face escalating pressures from human activities and associated noise pollution. The Siamese fighting fish Betta splendens is one of the most important cultured species in the global ornamental fish market and is increasingly recognized as a model organism for genetics and behavioural studies. This air-breathing species (Anabantoidei), characterized by the presence of a suprabranchial labyrinth organ that enhances auditory sensitivity, is native to Southeast Asia and inhabits low flow freshwater ecosystems that are increasingly threatened due to habitat destruction and pollution. We characterized the underwater soundscape, along with various ecological parameters, across five marshland habitats of B. splendens , from lentic waterbodies to small canals near a lake in Chiang Rai province (Thailand). All habitats exhibited common traits of low dissolved oxygen and dense herbaceous vegetation. Soundscapes were relatively quiet with Sound Pressure Level (SPL) around 102–105 dB re 1 µPa and most spectral energy below 1,000 Hz. Sound recordings captured diverse biological sounds, including potential fish vocalizations, but primarily insect sounds. Hearing thresholds were determined using auditory evoked potential (AEP) recordings, revealing best hearing range within 100–400 Hz. Males exhibited lower hearing thresholds than females at 400 and 600 Hz. This low-frequency tuning highlights the potential susceptibility of B. splendens to anthropogenic noise activities. This study provides first characterization of the auditory sensitivity and natural soundscape of B. splendens, establishing an important ground for future hearing research in this species. The information provided on the auditory sensory adaptation of B. splendens emphasizes the importance of preserving quiet soundscapes from lentic freshwater ecosystems.

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.

Background Fragile X syndrome (FXS) is a leading known genetic cause of intellectual disability and autism spectrum disorders (ASD)-associated behaviors. A consistent and debilitating phenotype of FXS is auditory hypersensitivity that may lead to delayed language and high anxiety. Consistent with findings in FXS human studies, the mouse model of FXS, the Fmr1 knock out (KO) mouse, shows auditory hypersensitivity and temporal processing deficits. In electroencephalograph (EEG) recordings from humans and mice, these deficits manifest as increased N1 amplitudes in event-related potentials (ERP), increased gamma band single trial power (STP) and reduced phase locking to rapid temporal modulations of sound. In our previous study, we found that administration of the selective serotonin-1 A (5-HT 1A )receptor biased agonist, NLX-101, protected Fmr1 KO mice from auditory hypersensitivity-associated seizures. Here we tested the hypothesis that NLX-101 will normalize EEG phenotypes in developing Fmr1 KO mice. Methods To test this hypothesis, we examined the effect of NLX-101 on EEG phenotypes in male and female wildtype (WT) and Fmr1 KO mice. Using epidural electrodes, we recorded auditory event related potentials (ERP) and auditory temporal processing with a gap-in-noise auditory steady state response (ASSR) paradigm at two ages, postnatal (P) 21 and 30 days, from both auditory and frontal cortices of awake, freely moving mice, following NLX-101 (at 1.8 mg/kg i.p.) or saline administration. Results Saline-injected Fmr1 KO mice showed increased N1 amplitudes, increased STP and reduced phase locking to auditory gap-in-noise stimuli versus wild-type mice, reproducing previously published EEG phenotypes. An acute injection of NLX-101 did not alter ERP amplitudes at either P21 or P30, but significantly reduces STP at P30. Inter-trial phase clustering was significantly increased in both age groups with NLX-101, indicating improved temporal processing. The differential effects of serotonin modulation on ERP, background power and temporal processing suggest different developmental mechanisms leading to these phenotypes. Conclusions These results suggest that NLX-101 could constitute a promising treatment option for targeting post-synaptic 5-HT 1A receptors to improve auditory temporal processing, which in turn may improve speech and language function in FXS.

Mutations in the human genes encoding the endothelin ligand-receptor pair EDN3 and EDNRB cause Waardenburg-Shah syndrome (WS4), which includes congenital hearing impairment. The current explanation for auditory dysfunction is defective migration of neural crest-derived melanocytes to the inner ear. We explored the role of endothelin signaling in auditory development in mice using neural crest-specific and placode-specific Ednrb mutation plus related genetic resources. On an outbred strain background, we find a normal representation of melanocytes in hearing-impaired mutant mice. Instead, our results in neural crest-specific Ednrb mutants implicate a previously unrecognized role for glial support of synapse assembly between auditory neurons and cochlear hair cells. Placode-specific Ednrb mutation also caused impaired hearing, resulting from deficient synaptic transmission. Our observations demonstrate the significant influence of genetic modifiers in auditory development, and invoke independent and separable roles for endothelin signaling in the neural crest and placode lineages to create a functional auditory circuitry.

The ability of the brain to process auditory information across multiple temporal scales is crucial for perception. This study introduces an innovative experimental approach using nonlinguistic structured stimuli composed of click trains that alternate at different rates to investigate auditory processing across 3 distinct temporal scales: individual clicks (tens of milliseconds), click trains forming auditory objects (hundreds of milliseconds), and higher-order click trains in neuronal novelty detection (seconds) in both rhesus monkeys and humans. Electrocorticography recordings in the auditory cortex of rhesus monkeys unveil the primate brain’s remarkable ability to process intricate auditory temporal patterns at timescales of tens and hundreds of milliseconds. Furthermore, extracellular recordings in monkeys demonstrate pronounced responsiveness to deviant click trains at longer temporal scales in the primary auditory cortex (A1), accompanied by synchronization with both individual clicks and click trains. By contrast, neurons in the auditory thalamus prefer individual clicks only. Notably, neurons in A1 exhibit the ability to synchronize with individual clicks while simultaneously integrating these clicks into cohesive train objects, a phenomenon we term “temporal integration during synchronization”. Additional human electroencephalography recordings complement and support these findings, highlighting the paradigm’s potential for clinical applications. Our research offers novel insights into the neural mechanisms underpinning auditory information processing across various temporal scales at the neuronal level.

Objectives: The purinergic receptor P2X2 (P2RX2) encodes an ATP-gated ion channel permeable to Na+, K+, and especially Ca²⁺. Loss-of-function mutations in P2RX2 are known to cause autosomal dominant nonsyndromic deafness 41 (DFNA41), which manifests as high-frequency hearing loss, accelerated presbycusis, and increased susceptibility to noise-induced damage. Zebrafish, owing to their small size, rapid development, high fecundity, transparent embryos, and high gene conservation with humans, provide an ideal model for studying human diseases and developmental mechanisms. This study aims to generate a p2rx2 knockout zebrafish model using CRISPR/Cas9 gene editing system to investigate the effect of p2rx2 deficiency on the auditory system, providing a basis for understanding P2RX2-related hearing loss and developing gene therapy strategies. Methods: Two CRISPR targets (sgRNA1 and sgRNA2) spaced 47 bp apart were designed within the zebrafish p2rx2 gene. Synthesized sgRNAs and Cas9 protein were microinjected into single-cell stage Tübingen (TU)-strain zebrafish embryos. PCR and gel electrophoresis verified editing efficiency at 36 hours post-fertilization (hpf). Surviving embryos were raised to adulthood (F0), tail-clipped, genotyped, and screened for positive mosaics. F1 heterozygotes were generated by outcrossing, and F2 homozygous mutants were obtained by intercrossing. Polymerase chain reaction (PCR) combined with sequencing verified mutation type and heritability. At 5 days post-fertilization (dpf), YO-PRO-1 staining was used to examine hair cell morphology and count in lateral line neuromasts and the otolith region. Auditory evoked potential (AEP) thresholds at 600, 800, 1 000, and 2 000 Hz were measured in nine 4-month-old wild type and mutant zebrafish per group. Results: A stable p2rx2 knockout zebrafish line was successfully established. Sequencing revealed a 66 bp insertion at the first target site introducing a premature stop codon (TAA), leading to early termination of protein translation and loss of function. Embryos developed normally with no gross malformations. At 5 dpf, mutants exhibited significantly reduced hair cell density in the otolith region compared with wild type, although lateral line neuromasts were unaffected. AEP testing showed significantly elevated auditory thresholds at all 4 frequencies in homozygous mutants compared with wild type (all P<0.001), indicating reduced hearing sensitivity. Conclusions: We successfully generated a p2rx2 loss-of-function zebrafish model using CRISPR/Cas9 technology. p2rx2 deficiency caused hair cell defects in the otolith region and increased auditory thresholds across frequencies, indicating its key role in maintaining zebrafish auditory hair cell function and hearing perception. The phenotype's restriction to the otolith region suggests tissue-specific roles of p2rx2 in sensory organs. This model provides a valuable tool for elucidating the molecular mechanisms of P2RX2-related hearing loss and for screening otoprotective drugs and developing gene therapies.

GJB2 encodes connexin 26 (Cx26), the most commonly mutated gene causing hereditary non‐syndromic hearing loss. Cx26 is mainly expressed in supporting cells (SCs) and fibrocytes in the mammalian cochlea. Gene therapy is currently considered the most promising strategy for eradicating genetic diseases. However, there have been no significant effects of gene therapy for GJB2 gene mutation‐associated deafness because deficiency of Cx26 leads to expanded sensory epithelial damage. In this study, the AAV2.7m8 serotype combined with the gfaABC1D promoter targeted infection of SCs is identified. It is found that Gjb2 gene replacement therapy in wild‐type mice results in sensory hair cells (HCs) deficits, excessive inflammatory responses, and hearing loss. This may be one of the key factors contributing to the hardship of GJB2 gene replacement therapy. Dexamethasone (DEX) shows promising results in inhibiting macrophage recruitment, with a protective effect against HC damage. Further, the combination of AAV2.7m8‐ Gjb2 with DEX shows a synergistic effect and enhances the gene therapy effect in a conditional Cx26 null mice model. These results indicate that the combination of gene therapy and medication will provide a new strategy for the treatment of hereditary deafness associated with GJB2 defects.

Background Recent studies have identified hearing loss (HL) as a primary risk factor for Alzheimer’s disease (AD) onset. However, the mechanisms linking HL to AD are not fully understood. This study explored the effects of drug-induced hearing loss (DIHL) on the expression of proteins associated with AD progression in mouse models. Methods DIHL was induced in 5xFAD and Tg2576 mice aged 3 to 3.5 weeks using kanamycin (700 mg/kg, subcutaneous) and furosemide (600 mg/kg, intraperitoneal). The accumulation and expression of beta-amyloid (Aβ), ionized calcium-binding adaptor molecule 1 (Iba1), and glial fibrillary acidic protein (GFAP) were measured through immunohistochemistry and immunoblotting. Additionally, the expression of proteins involved in the mammalian target of rapamycin (mTOR) pathway, including downstream effectors p70 ribosomal S6 kinase (p70S6K) and S6, as well as proinflammatory cytokines, was analyzed. Results Compared to control conditions, HL led to a significant increase in the accumulation of Aβ in the hippocampus and cortex. Elevated levels of neuroinflammatory markers, including Iba1 and GFAP, as well as proinflammatory cytokines such as interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-alpha (TNF-α), were observed. Moreover, DIHL enhanced phosphorylation of mTOR, p70S6K, and S6, indicating activation of the mTOR pathway. Conclusions HL significantly increases Aβ accumulation in the brain. Furthermore, HL activates astrocytes and microglia, leading to increased neuroinflammation and thereby accelerating AD progression. These findings strongly suggest that HL contributes autonomously to neuroinflammation, highlighting the potential for early intervention in HL to reduce AD risk.

Exposure to loud noise is a common cause of acquired hearing loss. Disruption of subcellular calcium homeostasis and downstream stress pathways in the endoplasmic reticulum and mitochondria, including the unfolded protein response (UPR), have been implicated in the pathophysiology of noise-induced hearing loss. However, studies on the association between calcium homeostasis and stress pathways have been limited due to limited ability to measure calcium dynamics in mature-hearing, noise-exposed mice. We used a genetically encoded calcium indicator mouse model in which GCaMP6f is expressed specifically in hair cells or supporting cells under control of Myo15Cre or Sox2Cre, respectively. We performed live calcium imaging and UPR gene expression analysis in 8-week-old mice exposed to levels of noise that cause cochlear synaptopathy (98 db sound pressure level [SPL]) or permanent hearing loss (106 dB SPL). UPR activation occurred immediately after noise exposure, and the pattern of UPR activation was dependent on noise level, with the proapoptotic pathway upregulated only after 106 dB noise exposure. Spontaneous calcium transients in hair cells and intercellular calcium waves in supporting cells, which are present in neonatal cochleae, were quiescent in mature-hearing cochleae but reactivated upon noise exposure. Noise exposure of 106 dB was associated with more persistent and expansive intercellular Ca 2+ signaling wave activity. These findings demonstrate a strong and dose-dependent association between noise exposure, UPR activation, and changes in calcium homeostasis in hair cells and supporting cells, suggesting that targeting these pathways may be effective to develop treatments for noise-induced hearing loss.

In the mammalian cochlea, sensory hair cells are crucial for the transduction of acoustic stimuli into electrical signals, which are then relayed to the central auditory pathway via spiral ganglion neuron (SGN) afferent dendrites. The SGN output is directly modulated by inhibitory cholinergic axodendritic synapses from the efferent fibers originating in the superior olivary complex. When the adult cochlea is subjected to noxious stimuli or aging, the efferent system undergoes major rewiring, such that it reestablishes direct axosomatic contacts with the inner hair cells (IHCs), which occur only transiently during prehearing stages of development. The trigger, origin, and degree of efferent plasticity in the cochlea remains largely unknown. Using functional and morphological approaches, we demonstrate that efferent plasticity in the adult cochlea occurs as a direct consequence of mechanoelectrical transducer current dysfunction. We also show that, different from prehearing stages of development, the lateral olivocochlear — but not the medial olivocochlear — efferent fibers are those that form the axosomatic synapses with the IHCs. The study also demonstrates that in vivo restoration of IHC function using AAV- Myo7a rescue reestablishes the synaptic profile of adult IHCs and improves hearing, highlighting the potential of using gene-replacement therapy for progressive hearing loss.

The class C orphan G-protein-coupled receptor (GPCR) GPR156, which lacks the large extracellular region, plays a pivotal role in auditory function through Gi2/3. Here, we firstly demonstrate that GPR156 with high constitutive activity is essential for maintaining auditory function, and further reveal the structural basis of the sustained role of GPR156. We present the cryo-EM structures of human apo GPR156 and the GPR156-Gi3 complex, unveiling a small extracellular region formed by extracellular loop 2 (ECL2) and the N-terminus. The GPR156 dimer in both apo state and Gi3 protein-coupled state adopt a transmembrane (TM)5/6-TM5/6 interface, indicating the high constitutive activity of GPR156 in the apo state. Furthermore, C-terminus in G-bound subunit of GPR156 plays a dual role in promoting G protein binding within G-bound subunit while preventing the G-free subunit from binding to additional G protein. Together, these results explain how GPR156 constitutive activity is maintained through dimerization and provide a mechanistic insight into the sustained role of GPR156 in maintaining auditory function.

Background/Objectives: Tinnitus is a debilitating auditory disorder commonly described as a ringing in the ears in the absence of an external sound source. Sound trauma is considered a primary cause. Neuronal hyperactivity is one potential mechanism for the genesis of tinnitus and has been identified in the cochlear nucleus (CN) and the auditory cortex (AC), where there may be an imbalance of excitatory and inhibitory neurotransmissions. However, no study has directly correlated tinnitus with the extracellular levels of amino acids in the CN and the AC using microdialysis, which reflects the functions of these neurochemicals. In the present study, rats were exposed to acoustic trauma and then subjected to behavioural confirmation of tinnitus after one month, followed by microdialysis. Methods: Rats were divided into sham (aged, n = 6; young, n = 6); tinnitus-positive (aged, n = 7; young, n = 7); and tinnitus-negative (aged, n = 3; young, n = 3) groups. In vivo microdialysis was utilized to collect samples from the CN and the AC, simultaneously, in the same rat. Extracellular levels of amino acids were quantified using high-performance liquid chromatography (HPLC) coupled with an electrochemical detector (ECD). The effects of sound stimulation and age on neurochemical changes associated with tinnitus were also examined. Results: There were no significant differences in either the basal levels or the sound stimulation-evoked changes of any of the amino acids examined in the CN and the AC between the sham and tinnitus animals. However, the basal levels of serine and threonine exhibited age-related alterations in the AC, and significant differences in threonine and glycine levels were observed in the responses to 4 kHz and 16 kHz stimuli in the CN. Conclusions: These results demonstrate the lack of a direct link between extracellular levels of amino acids in the CN and the AC and tinnitus perception in a rat model of tinnitus.

Effective, reproducible, and safe delivery of therapeutics into the inner ear is required for the prevention and treatment of hearing loss. A commonly used delivery method is via the posterior semicircular canal (PSCC); however, its specific targeting within the cochlea remains unclear, impacting precision and reproducibility. To assess safety and target specificity, we conducted in vivo recordings of the pharmacological manipulations delivered through the PSCC. Measurements of auditory brainstem response (ABR), vibrometry, and vestibular behavioral and sensory-evoked potential (VsEP) revealed preserved hearing and vestibular functions after artificial perilymph injections. Injection of curare, a mechanoelectrical transducer (MET) channel blocker that affects hearing when in the endolymph, had no effect on ABR or VsEP thresholds. Conversely, injection of CNQX, an AMPA receptor blocker, or lidocaine, a Na + channel blocker, which affects hearing when in the perilymph, significantly increased both thresholds, indicating that PSCC injections selectively target the perilymphatic space. In vivo tracking of gold nanoparticles confirmed their exclusive distribution in the perilymph during PSCC injection, supporting the pharmacological finding. Together, PSCC injection is a safe method for inner ear delivery, specifically targeting the perilymphatic space. Our findings will allow for precise delivery of therapeutics within the inner ear for therapeutic and research purposes.

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.