Publications using the RZ6

The therapeutic effects of pharmaceuticals depend on their drug concentrations in the cochlea. Efficient drug delivery from the systemic circulation into the inner ear is limited by the blood‐labyrinth‐barrier (BLB). This study investigated a novel noninvasive sound conditioning (SC) strategy (90 dB SPL, 8–16 kHz, 2 h sound exposure) to temporally enhance BLB permeability in a controllable way, contributing to maximizing the penetration of pharmaceuticals from blood circulation into the cochlea. Trafficking of Fluorescein Isothiocyanate conjugated dextran and bovine serum albumin (FITC‐dextran and FITC‐BSA) demonstrated that paracellular leakage of BLB sustained for 6 h after SC, providing a controllable time window for systemic administration. Cochlear concentrations of dexamethasone (DEX) and dexamethasone phosphate (DEX‐P), respectively transported by transcellular and paracellular pathways, showed a higher content of the latter one after SC, further confirming the key role of paracellular pathway in the SC‐induced hyperpermeability. Results of high‐throughput RNA‐sequencing identified a series of tight junction (TJ)‐associated genes after SC. The expressions of TJ (ZO‐1) were reduced and irregular rearrangements of the junction were observed by transmission electron microscopy after SC. We further determined the inhibiting role of Rab13 in the recruitment of ZO‐1 and later in the regulation of cellular permeability. Meanwhile, no significant change in the quantifications of endothelial caveolae vesicles after SC indicated that cellular transcytosis accounted little for the temporary hyperpermeability after SC. Based on these results, SC enhances the BLB permeability within 6 h and allows systemically applied drugs which tend to be transported by paracellular pathway to readily enter the inner ear, contributing to guiding the clinical medications on hearing loss.

Background Aminoglycoside antibiotics are used for treating certain acute infections. However, these drugs cause ototoxicity by inducing inner ear hair cell death. Aims/Objectives We investigated the protective effect of a nanoemulsion of the carotenoid astaxanthin on mammalian inner ear hair cells against neomycin-induced ototoxicity. Material and Methods Dose-response relationship, quantification of hair cell loss, and reactive oxygen species production were assayed in response to neomycin with and without astaxanthin in cultured utricles of CBA/N mice. In addition, auditory brain response (ABR) and hair cell loss after exposure to the nanoformulation and loud noise were examined in vivo in guinea pigs. Results Astaxanthin suppressed neomycin-induced reduction of hair cells by reducing the production of hydroxy radicals. Furthermore, hair cell loss in the second rotation of the cochlea was significantly lower in the astaxanthin group than in the noise-only group. Conclusions and Significance The blood-labyrinth barrier limits the successful delivery of drugs for inner ear complications. However, in the nanoemulsion form, astaxanthin can penetrate the round window (fenestra ovale) membrane, enabling topical administration. Thus, astaxanthin nanoemulsion could be useful in treating ototoxicity in individuals with inner ear complications.

Introduction Neuronal hyperactivity has been associated with many brain diseases. In the auditory system, hyperactivity has been linked to hyperacusis and tinnitus. Previous research demonstrated the development of hyperactivity in inferior colliculus (IC) neurons after sound overexposure, but the underlying mechanism of this hyperactivity remains unclear. The main goal of this study was to determine the mechanism of this hyperactivity. Methods Experiments were performed on CBA/CaJ mice in a restrained, unanesthetized condition using intracellular recordings with sharp microelectrodes. Recordings were obtained from control (unexposed) and unilaterally sound overexposed groups of mice. Results Our data suggest that sound exposure-induced hyperactivity was due to a depolarizing shift of the resting membrane potential (RMP) in the hyperactive neurons. The half width of action potentials in these neurons was also decreased after sound exposure. Surprisingly, we also found an RMP gradient in which neurons have more hyperpolarized RMPs with increasing depth in the IC. This gradient was altered in the overexposed animals.

Usher syndrome is the most common cause of deafness-blindness in the world. Usher syndrome type 1B (USH1B) is associated with mutations in MYO7A. Patients with USH1B experience deafness, blindness, and vestibular dysfunction. In this study, we applied adeno-associated virus (AAV)-mediated gene therapy to the shaker-1 (Myo7a4626SB/4626SB) mouse, a model of USH1B. The shaker-1 mouse has a nonsense mutation in Myo7a, is profoundly deaf throughout life, and has significant vestibular dysfunction. Because of the ∼6.7-kb size of the MYO7A cDNA, a dual-AAV approach was used for gene delivery, which involves splitting human MYO7A cDNA into 5' and 3' halves and cloning them into two separate AAV8(Y733F) vectors. When MYO7A cDNA was delivered to shaker-1 inner ears using the dual-AAV approach, cochlear hair cell survival was improved. However, stereocilium organization and auditory function were not improved. In contrast, in the vestibular system, dual-AAV-mediated MYO7A delivery significantly rescued hair cell stereocilium morphology and improved vestibular function, as reflected in a reduction of circling behavior and improved vestibular sensory-evoked potential (VsEP) thresholds. Our data indicate that dual-AAV-mediated MYO7A expression improves vestibular function in shaker-1 mice and supports further development of this approach for the treatment of disabling dizziness from vestibular dysfunction in USH1B patients.

mTOR broadly controls cell growth, but little is known about the role of mTOR complex 2 (mTORC2) in the inner ear. To investigate the role of mTORC2 in sensory hair cells (HCs), we generated HC-specific Rictor knockout (HC-RicKO) mice. HC-RicKO mice exhibited early-onset, progressive, and profound hearing loss. Increased DPOAE thresholds indicated outer HC dysfunction. HCs are lost, but this occurs after hearing loss. Ultrastructural analysis revealed stunted and absent stereocilia in outer HCs. In inner HCs, the number of synapses was significantly decreased and the remaining synapses displayed a disrupted actin cytoskeleton and disorganized Ca2+ channels. Thus, the mTORC2 signaling pathway plays an important role in regulating auditory HC structure and function via regulation of the actin cytoskeleton. These results provide molecular insights on a central regulator of cochlear HCs and thus hearing.

Hearing loss is a common disorder affecting nearly 20% of the world's population. Recently, studies have shown that inner ear gene therapy can improve auditory function in several mouse models of hereditary hearing loss. In most of these studies, the underlying mutations affect only a small number of cell types of the inner ear (e.g., sensory hair cells). Here, we applied inner ear gene therapy to the Ildr1Gt(D178D03)Wrst (Ildr1w-/-) mouse, a model of human DFNB42, non-syndromic autosomal recessive hereditary hearing loss associated with ILDR1 variants. ILDR1 is an integral protein of the tricellular tight junction complex and is expressed by diverse inner ear cell types in the organ of Corti and the cochlear lateral wall. We simultaneously applied two synthetic adeno-associated viruses (AAVs) with different tropism to deliver Ildr1 cDNA to the Ildr1w-/- mouse inner ear: one targeting the organ of Corti (AAV2.7m8) and the other targeting the cochlear lateral wall (AAV8BP2). We showed that combined AAV2.7m8/AAV8BP2 gene therapy improves cochlear structural integrity and auditory function in Ildr1w-/- mice.

Background: Transcranial ultrasound stimulation (TUS) is a promising noninvasive neuromodulation modality. The inadvertent and unpredictable activation of the auditory system in response to TUS obfuscates the interpretation of non-auditory neuromodulatory responses. Objective: The objective was to develop and validate a computational metric to quantify the susceptibility to unintended auditory brainstem response (ABR) in mice premised on time frequency analyses of TUS signals and auditory sensitivity. Methods: Ultrasound pulses with varying amplitudes, pulse repetition frequencies (PRFs), envelope smoothing profiles, and sinusoidal modulation frequencies were selected. Each pulse's time-varying frequency spectrum was differentiated across time, weighted by the mouse hearing sensitivity, then summed across frequencies. The resulting time-varying function, computationally predicting the ABR, was validated against experimental ABR in mice during TUS with the corresponding pulse. Results: There was a significant correlation between experimental ABRs and the computational predictions for 19 TUS signals (R2 = 0.97). Conclusions: To reduce ABR in mice during in vivo TUS studies, 1) reduce the amplitude of a rectangular continuous wave envelope, 2) increase the rise/fall times of a smoothed continuous wave envelope, and/or 3) change the PRF and/or duty cycle of a rectangular or sinusoidal pulsed wave to reduce the gap between pulses and increase the rise/fall time of the overall envelope. This metric can aid researchers performing in vivo mouse studies in selecting TUS signal parameters that minimize unintended ABR. The methods for developing this metric can be adapted to other animal models.

Introduction Conductive hearing loss (CHL) attenuates the ability to transmit air conducted sounds to the ear. In humans, severe hearing loss is often accompanied by alterations to other neural systems, such as the vestibular system; however, the inter-relations are not well understood. The overall goal of this study was to assess vestibular-related functioning proxies in a rat CHL model. Methods Male Sprague–Dawley rats ( N =134, 250g, 2months old) were used in a CHL model which produced a >20dB threshold shift induced by tympanic membrane puncture. Auditory brainstem response (ABRs) recordings were used to determine threshold depth at different times before and after CHL. ABR threshold depths were assessed both manually and by an automated ABR machine learning algorithm. Vestibular-related functioning proxy assessment was performed using the rotarod, balance beam, elevator vertical motion (EVM) and Ferris-wheel rotation (FWR) assays. Results The Pre-CHL (control) threshold depth was 27.92dB±11.58dB compared to the Post-CHL threshold depth of 50.69dB±13.98dB (mean±SD) across the frequencies tested. The automated ABR machine learning algorithm determined the following threshold depths: Pre-CHL=24.3dB, Post-CHL same day=56dB, Post-CHL 7 days=41.16dB, and Post-CHL 1 month=32.5dB across the frequencies assessed (1, 2, 4, 8, 16, and 32kHz). Rotarod assessment of motor function was not significantly different between pre and post-CHL (~1week) rats for time duration (sec) or speed (RPM), albeit the former had a small effect size difference. Balance beam time to transverse was significantly longer for post-CHL rats, likely indicating a change in motor coordination. Further, failure to cross was only noted for CHL rats. The defection count was significantly reduced for CHL rats compared to control rats following FWR, but not EVM. The total distance traveled during open-field examination after EVM was significantly different between control and CHL rats, but not for FWR. The EVM is associated with linear acceleration (acting in the vertical plane: up-down) stimulating the saccule, while the FWR is associated with angular acceleration (centrifugal rotation about a circular axis) stimulating both otolith organs and semicircular canals; therefore, the difference in results could reflect the specific vestibular-organ functional role. Discussion Less movement (EVM) and increase time to transverse (balance beam) may be associated with anxiety and alterations to defecation patterns (FWR) may result from autonomic disturbances due to the impact of hearing loss. In this regard, vestibulomotor deficits resulting in changes in balance and motion could be attributed to comodulation of auditory and vestibular functioning. Future studies should manipulate vestibular functioning directly in rats with CHL.

In Parkinson’s disease (PD), pathomechanisms such as aberrant network dysfunctions can be elucidated by conducting multiscale explorations in animal models. However, the lack of specificity in the existing models limits a restricted targeting of individual network elements and characterization of PD as a “circuitopathy”. We therefore developed a cell-type specific viral vector (AAV2/9-Cre ON -A53T-αSyn) mouse model that allows to induce synucleinopathy within individual circuit elements in vivo . When specifically targeted to dopaminergic (DA) neurons of the substantia nigra pars compacta (SNc), our approach recapitulates the main hallmarks of the disease, namely Lewy-body-like aggregation, progressive cellular and nigrostriatal projections loss, together with locomotor impairment. Our strategy is supported by new state-of-the-art analytical approaches for cell quantification and behavior characterization. Altogether, we provide a novel model of synucleinopathy, which offers new opportunities to study the contribution of individual network elements to disease pathomechanisms.

Introduction The leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) is a tissue resident stem cell marker, which it is expressed in supporting cells (SCs) in the organ of Corti in the mammalian inner ear. These LGR5+ SCs can be used as an endogenous source of progenitor cells for regeneration of hair cells (HCs) to treat hearing loss and deafness. We have recently reported that LGR5+ SCs survive 1 week after ototoxic trauma. Here, we evaluated Lgr5 expression in the adult cochlea and long-term survival of LGR5+ SCs following severe hearing loss. Methods Lgr5GFP transgenic mice and wild type mice aged postnatal day 30 (P30) and P200 were used. P30 animals were deafened with a single dose of furosemide and kanamycin. Seven and 28 days after deafening, auditory brainstem responses (ABRs) were recorded. Cochleas were harvested to characterize mature HCs and LGR5+ SCs by immunofluorescence microscopy and quantitative reverse transcription PCR (q-RT-PCR). Results There were no significant age-related changes in Lgr5 expression when comparing normal-hearing (NH) mice aged P200 with P30. Seven and 28 days after ototoxic trauma, there was severe outer HC loss and LGR5 was expressed in the third row of Deiters’ cells and in inner pillar cells. Seven days after induction of ototoxic trauma there was an up-regulation of the mRNA expression of Lgr5 compared to the NH condition; 28 days after ototoxic trauma Lgr5 expression was similar to NH levels. Discussion The presence of LGR5+ SCs in the adult mouse cochlea, which persists after severe HC loss, suggests potential regenerative capacity of endogenous cochlear progenitor cells in adulthood. To our knowledge, this is the first study showing not only long-term survival of LGR5+ SCs in the normal and ototoxically damaged cochlea, but also increased Lgr5 expression in the adult mouse cochlea after deafening, suggesting long-term availability of potential target cells for future regenerative therapies.

Objectives This study investigated the effect of synchronized activation of automatic gain control (AGC) on sound-localization and speech-recognition performance in cochlear implant users who use a contralateral hearing aid (bimodal listeners). Bimodal listeners often have poor spatial-hearing abilities, which could be partly due to perturbed interaural spatial acoustic cues from the two devices, as these act independently and provide incongruent or mismatched information. Synchronized AGC activation may remove this perturbation and improve spatial hearing. Design Ten bimodal listeners were fitted with a bimodal prototype allowing real-time communication between hearing aid and CI. They were tested for their horizontal localization ability of brief (150 ms) noise bursts presented in the frontal hemisphere. To test for frequency- and level-specific effects, sounds were band-passed (broadband 0.25-6 kHz, high-pass 1.5-6 kHz, and low-pass 0.25-0.85 kHz) and presented at levels of 50 and 80 dBA (i.e., below and above AGC activation threshold). Speech-in-noise recognition was assessed for short sentences presented at 75 dBA with a noise-adaptive algorithm. Sentences were presented from the front, with the noise presented either from the front or at a horizontal angle of ±70 deg. The sound-localization and speech-recognition tests were performed both with independently acting and synchronized AGC settings. Results Localization bias (mean error [95% confidence interval lower, upper range]) varied with sound type: -46 [-53, -38], -31 [-37, -23] and -2 [-16, 10] deg for high-pass, broadband and low-pass sounds, respectively. AGC synchronization did not systematically affect bias (paired difference in mean error with independent and synchronized AGCs: 6 [-3, 20] deg). However, AGC synchronization did reduce sound-localization error variability for 80-dB broadband sounds by 15 [6, 26] deg from an error standard deviation of 67 [63, 77] to 52 [45, 59] deg for independent and synchronized AGCs, respectively (no systematic difference was found for 50-dB and the high-pass and low-pass sounds). Speech-in-noise thresholds differed for noise presented from the front (0.3 [-1.3, 1.1] dB), implanted side (0.6 [-1.3, 2.3] dB), and non-implanted side (-2.4 [-4.6, 0.1] dB), but synchronization did not have an effect in these conditions. However, spatial release from masking thresholds did improve with synchronization when the noise was presented at the hearing-aid side: -2.7 [-4.5, -0.6] dB. Conclusions AGC synchronization improved the localization of broadband sounds presented above the AGC activation threshold in bimodal listeners. This is in line with the hypothesis that bimodal interaural level differences can be preserved for loud broadband sounds through AGC synchronization. Synchronization did not compromise the listener’s speech perception abilities, despite the reduction of the level of speech when AGC kicks in. Moreover, spatial release from masking was enhanced, suggesting that improved spatial hearing benefits speech recognition in noise

Background Autism spectrum disorders (ASD) encompass a wide array of debilitating symptoms, including sensory dysfunction and delayed language development. Auditory temporal processing is crucial for speech perception and language development. Abnormal development of temporal processing may account for the language impairments associated with ASD. Very little is known about the development of temporal processing in any animal model of ASD. Methods In the current study, we quantify auditory temporal processing throughout development in the Fmr1 knock-out (KO) mouse model of Fragile X Syndrome (FXS), a leading genetic cause of intellectual disability and ASD-associated behaviors. Using epidural electrodes in awake and freely moving wildtype (WT) and KO mice, we recorded auditory event related potentials (ERP) and auditory temporal processing with a gap-in-noise auditory steady state response (gap-ASSR) paradigm. Mice were recorded at three different ages in a cross sectional design: postnatal (p)21, p30 and p60. Recordings were obtained from both auditory and frontal cortices. The gap-ASSR requires underlying neural generators to synchronize responses to gaps of different widths embedded in noise, providing an objective measure of temporal processing across genotypes and age groups. Results We present evidence that the frontal, but not auditory, cortex shows significant temporal processing deficits at p21 and p30, with poor ability to phase lock to rapid gaps in noise. Temporal processing was similar in both genotypes in adult mice. ERP amplitudes were larger in Fmr1 KO mice in both auditory and frontal cortex, consistent with ERP data in humans with FXS. Conclusions These data indicate cortical region-specific delays in temporal processing development in Fmr1 KO mice. Developmental delays in the ability of frontal cortex to follow rapid changes in sounds may shape language delays in FXS, and more broadly in ASD.

Hyperacusis, i.e., an increased sensitivity to sounds, is described in several neurodevelopmental disorders (NDDs), including Fragile X Syndrome (FXS). The mechanisms underlying hyperacusis in FXS are still largely unknown and effective therapies are lacking. Big conductance calcium-activated potassium (BKCa) channels were proposed as a therapeutic target to treat several behavioral disturbances in FXS preclinical models, but their role in mediating their auditory alterations was not specifically addressed. Furthermore, studies on the acoustic phenotypes of FXS animal models mostly focused on central rather than peripheral auditory pathways. Here, we provided an extensive characterization of the peripheral auditory phenotype of the Fmr1-knockout (KO) mouse model of FXS at adulthood. We also assessed whether the acute administration of Chlorzoxazone, a BKCa agonist, could rescue the auditory abnormalities of adult mutant mice. Fmr1-KO mice both at 3 and 6 months showed a hyperacusis-like startle phenotype with paradoxically reduced auditory brainstem responses associated with a loss of ribbon synapses in the inner hair cells (IHCs) compared to their wild-type (WT) littermates. BKCa expression was markedly reduced in the IHCs of KOs compared to WT mice, but only at 6 months, when Chlorzoxazone rescued mutant auditory dysfunction. Our findings highlight the age-dependent and progressive contribution of peripheral mechanisms and BKCa channels to adult hyperacusis in FXS, suggesting a novel therapeutic target to treat auditory dysfunction in NDDs.

Cortical representations supporting many cognitive abilities emerge from underlying circuits comprised of several different cell types. However, cell type-specific contributions to rate and timing-based cortical coding are not well-understood. Here, we investigated the role of parvalbumin neurons in cortical complex scene analysis. Many complex scenes contain sensory stimuli which are highly dynamic in time and compete with stimuli at other spatial locations. Parvalbumin neurons play a fundamental role in balancing excitation and inhibition in cortex and sculpting cortical temporal dynamics; yet their specific role in encoding complex scenes via timing-based coding, and the robustness of temporal representations to spatial competition, has not been investigated. Here, we address these questions in auditory cortex of mice using a cocktail party-like paradigm, integrating electrophysiology, optogenetic manipulations, and a family of spike-distance metrics, to dissect parvalbumin neurons’ contributions towards rate and timing-based coding. We find that suppressing parvalbumin neurons degrades cortical discrimination of dynamic sounds in a cocktail party-like setting via changes in rapid temporal modulations in rate and spike timing, and over a wide range of time-scales. Our findings suggest that parvalbumin neurons play a critical role in enhancing cortical temporal coding and reducing cortical noise, thereby improving representations of dynamic stimuli in complex scenes.

Peripheral sensory organ damage leads to compensatory cortical plasticity that is associated with a remarkable recovery of cortical responses to sound. The precise mechanisms that explain how this plasticity is implemented and distributed over a diverse collection of excitatory and inhibitory cortical neurons remain unknown. After noise trauma and persistent peripheral deficits, we found recovered sound-evoked activity in mouse A1 excitatory principal neurons (PNs), parvalbumin- and vasoactive intestinal peptide-expressing neurons (PVs and VIPs), but reduced activity in somatostatin-expressing neurons (SOMs). This cell-type-specific recovery was also associated with cell-type-specific intrinsic plasticity. These findings, along with our computational modelling results, are consistent with the notion that PV plasticity contributes to PN stability, SOM plasticity allows for increased PN and PV activity, and VIP plasticity enables PN and PV recovery by inhibiting SOMs.

The auditory brainstem response (ABR) is a scalp recording of potentials produced by sound stimulation, and is commonly used as an indicator of auditory function. However, the ABR threshold, which is the lowest audible sound pressure, cannot be objectively determined since it is determined visually using a measurer, and this has been a problem for several decades. Although various algorithms have been developed to objectively determine ABR thresholds, they remain lacking in terms of accuracy, efficiency, and convenience. Accordingly, we proposed an improved algorithm based on the mutual covariance at adjacent sound pressure levels. An ideal ABR waveform with clearly defined waves I–V was created; moreover, using this waveform as a standard template, the experimentally obtained ABR waveform was inspected for disturbances based on mutual covariance. The ABR testing was repeated if the value was below the established cross-covariance reference value. Our proposed method allowed more efficient objective determination of ABR thresholds and a smaller burden on experimental animals.

Introduction Noise-induced calcium overload in sensory hair cells has been well documented as an early step in the pathogenesis of noise-induced hearing loss (NIHL). Alterations in cellular calcium homeostasis mediate a series of cellular events, including activation of calcium-dependent protein kinases and phosphatases. Using cell-membrane- and blood-brain-barrier-permeable calpain-1 (μ-calpain) and calpain-2 (m-calpain) inhibitor MDL-28170, we tested the involvement of calpains, a family of calcium-dependent cysteine proteases, and the potential of MDL-28170 in preventing NIHL. Methods CBA/J mice at the age of 12 weeks were exposed to broadband noise with a frequency spectrum from 2–20 kHz for 2 h at 101 dB sound pressure level to induce permanent hearing loss as measured by auditory brainstem response and distortion product otoacoustic emissions. Morphological damage was assessed by quantification of remaining sensory hair cells and inner hair cell synapses 2 weeks after the exposure. Results MDL-28170 treatment by intraperitoneal injection significantly attenuated noise-induced functional deficits and cochlear pathologies. MDL-28170 treatment also prevented noise-induced cleavage of alpha-fodrin, a substrate for calpain-1. Furthermore, MDL-28170 treatment prevented reduction of PI3K/Akt signaling after exposure to noise and upregulated p85α and p-Akt (S473) in outer hair cells. Discussion These results indicate that noise-induced calpain activation negatively regulates PI3K/Akt downstream signaling, and that prevention of NIHL by treatment with MDL-28170 is associated with upregulation of PI3K/Akt survival signaling pathways.

Intrinsically generated neural activity propagates through the developing auditory system to promote maturation and refinement of sound processing circuits prior to hearing onset. This early patterned activity is induced by non-sensory supporting cells in the organ of Corti, which are highly interconnected through gap junctions containing connexin 26 ( Gjb2 ). Although loss of function mutations in Gjb2 impair cochlear development and are the most common cause of congenital deafness, it is not known if these variants disrupt spontaneous activity and the developmental trajectory of sound processing circuits in the brain. Here, we show in a new mouse model of Gjb2- mediated congenital deafness that cochlear supporting cells adjacent to inner hair cells (IHCs) unexpectedly retain intercellular coupling and the capacity to generate spontaneous activity, exhibiting only modest deficits prior to hearing onset. Supporting cells lacking Gjb2 elicited coordinated activation of IHCs, leading to coincident bursts of activity in central auditory neurons that will later process similar frequencies of sound. Despite alterations in the structure of the sensory epithelium, hair cells within the cochlea of Gjb2 -deficient mice were intact and central auditory neurons could be activated within appropriate tonotopic domains by loud sounds at hearing onset, indicating that early maturation and refinement of auditory circuits was preserved. Only after cessation of spontaneous activity following hearing onset did progressive hair cell degeneration and enhanced auditory neuron excitability manifest. This preservation of cochlear spontaneous neural activity in the absence of connexin 26 may increase the effectiveness of early therapeutic interventions to restore hearing.

Prediction provides key advantages for survival, and cognitive studies have demonstrated that the brain computes multilevel predictions. Evidence for predictions remains elusive at the neuronal level because of the complexity of separating neural activity into predictions and stimulus responses. We overcome this challenge by recording from single neurons from cortical and subcortical auditory regions in anesthetized and awake preparations, during unexpected stimulus omissions interspersed in a regular sequence of tones. We find a subset of neurons that responds reliably to omitted tones. In awake animals, omission responses are similar to anesthetized animals, but larger and more frequent, indicating that the arousal and attentional state levels affect the degree to which predictions are neuronally represented. Omission-sensitive neurons also responded to frequency deviants, with their omission responses getting emphasized in the awake state. Because omission responses occur in the absence of sensory input, they provide solid and empirical evidence for the implementation of a predictive process.

Isoforms of a gene may contribute to diverse biological functions. In the cochlea, the repertoire of alternative isoforms remains unexplored. We integrated single-cell short-read and long-read RNA sequencing techniques and identified 236,012 transcripts, 126,612 of which were unannotated in the GENCODE database. Then we analyzed and verified the unannotated transcripts using RNA-seq, RT-PCR, Sanger sequencing, and MS-based proteomics approaches. To illustrate the importance of identifying spliced isoforms, we investigated otoferlin, a key protein involved in synaptic transmission in inner hair cells (IHCs). Upon deletion of the canonical otoferlin isoform, the identified short isoform is able to support normal hearing thresholds but with reduced sustained exocytosis of IHCs, and further revealed otoferlin functions in endocytic membrane retrieval that was not well-addressed previously. Furthermore, we found that otoferlin isoforms are associated with IHC functions and auditory phenotypes. This work expands our mechanistic understanding of auditory functions at the level of isoform resolution.