ABR/DPOAE publications

Background Obesity is an independent risk factor for hearing loss. Although attention has focused on major obesity comorbidities such as cardiovascular disease, stroke, and type 2 diabetes, the impact of obesity on sensorineural organs, including the auditory system, is unclear. Using a high-fat diet (HFD)-induced obese mouse model, we investigated the impact of diet-induced obesity on sexual dimorphism in metabolic alterations and hearing sensitivity. Methods Male and female CBA/Ca mice were randomly assigned to three diet groups and fed, from weaning (at 28 days) to 14 weeks of age, a sucrose-matched control diet (10 kcal% fat content diet), or one of two HFDs (45 or 60 kcal% fat content diets). Auditory sensitivity was evaluated based on the auditory brainstem response (ABR), distortion product otoacoustic emission (DPOAE), and ABR wave 1 amplitude at 14 weeks of age, followed by biochemical analyses. Results We found significant sexual dimorphism in HFD-induced metabolic alterations and obesity-related hearing loss. Male mice exhibited greater weight gain, hyperglycemia, increased ABR thresholds at low frequencies, elevated DPOAE, and lower ABR wave 1 amplitude compared to female mice. The hair cell (HC) ribbon synapse (CtBP2) puncta showed significant sex differences. The serum concentration of adiponectin, an otoprotective adipokine, was significantly higher in female than in male mice; cochlear adiponectin levels were elevated by HFD in female but not male mice. Adiponectin receptor 1 (AdipoR1) was widely expressed in the inner ear, and cochlear AdipoR1 protein levels were increased by HFD, in female but not male mice. Stress granules (G3BP1) were significantly induced by the HFD in both sexes; conversely, inflammatory (IL-1β) responses were observed only in the male liver and cochlea, consistent with phenotype HFD-induced obesity. Conclusions Female mice are more resistant to the negative effects of an HFD on body weight, metabolism, and hearing. Females showed increased peripheral and intra-cochlear adiponectin and AdipoR1 levels, and HC ribbon synapses. These changes may mediate resistance to HFD-induced hearing loss seen in female mice.

The cochlear nuclei (CNs) receive sensory information from the ear and perform fundamental computations before relaying this information to higher processing centers. These computations are performed by distinct types of neurons interconnected in circuits dedicated to the specialized roles of the auditory system. In the present study, we explored the use of voltage imaging to investigate CN circuitry. We tested two approaches based on fundamentally different voltage sensing technologies. Using a voltage-sensitive dye we recorded glutamate receptor-independent signals arising predominantly from axons. The mean conduction velocity of these fibers of 0.27 m/s was rapid but in range with other unmyelinated axons. We then used a genetically-encoded hybrid voltage sensor (hVOS) to image voltage from a specific population of neurons. Probe expression was controlled using Cre recombinase linked to c-fos activation. This activity-induced gene enabled targeting of neurons that are activated when a mouse hears a pure 15-kHz tone. In CN slices from these animals auditory nerve fiber stimulation elicited a glutamate receptor-dependent depolarization in hVOS probe-labeled neurons. These cells resided within a band corresponding to an isofrequency lamina, and responded with a high degree of synchrony. In contrast to the axonal origin of voltage-sensitive dye signals, hVOS signals represent predominantly postsynaptic responses. The introduction of voltage imaging to the CN creates the opportunity to investigate auditory processing circuitry in populations of neurons targeted on the basis of their genetic identity and their roles in sensory processing.

In an effort to help elucidate the neural mechanisms underlying tinnitus in humans, researchers have often relied on animal models; a preclinical approach which ultimately required that behavioral paradigms be designed to reliably screen animals for tinnitus. Previously, we developed a two-alternative forced-choice (2AFC) paradigm for rats that allowed for the simultaneous recording of neural activity at the very moments when they were reporting the presence/absence of tinnitus. Because we first validated our paradigm in rats experiencing transient tinnitus following a high-dose of sodium salicylate, the present study now sought to evaluate its utility to screen for tinnitus caused by intense sound exposure; a common tinnitus-inducer in humans. More specifically, through a series of experimental protocols, we aimed to (1) conduct sham experiments to ensure that the paradigm was able to correctly classify control rats as not having tinnitus, (2) confirm the time course over which the behavioral testing could reliably be performed post-exposure to assess chronic tinnitus, and (3) determine if the paradigm was sensitive to the variable outcomes often observed after intense sound exposure (e.g., hearing loss with our without tinnitus). Ultimately, in accordance with our predictions, the 2AFC paradigm was indeed resistant to false-positive screening of rats for intense sound-induced tinnitus, and it was able to reveal variable tinnitus and hearing loss profiles in individual rats following intense sound exposure. Taken together, the present study documents the utility of our appetitive operant conditioning paradigm to assess acute and chronic sound-induced tinnitus in rats. Finally, based on our findings, we discuss important experimental considerations that will help ensure that our paradigm is able to provide a suitable platform for future investigations into the neural basis of tinnitus.

Introduction Electrocochleography (ECochG) is increasingly used in cochlear implant (CI) surgery, in order to monitor the effect of insertion of the electrode array aiming to preserve residual hearing. However, obtained results are often difficult to interpret. Here we aim to relate changes in ECochG responses to acute trauma induced by different stages of cochlear implantation by performing ECochG at multiple time points during the procedure in normal-hearing guinea pigs. Materials and methods Eleven normal-hearing guinea pigs received a gold-ball electrode that was fixed in the round-window niche. ECochG recordings were performed during the four steps of cochlear implantation using the gold-ball electrode: (1) Bullostomy to expose the round window, (2) hand-drilling of 0.5–0.6 mm cochleostomy in the basal turn near the round window, (3) insertion of a short flexible electrode array, and (4) withdrawal of electrode array. Acoustical stimuli were tones varying in frequency (0.25–16 kHz) and sound level. The ECochG signal was primarily analyzed in terms of threshold, amplitude, and latency of the compound action potential (CAP). Midmodiolar sections of the implanted cochleas were analyzed in terms of trauma to hair cells, modiolar wall, osseous spiral lamina (OSL) and lateral wall. Results Animals were assigned to cochlear trauma categories: minimal ( n = 3), moderate ( n = 5), or severe ( n = 3). After cochleostomy and array insertion, CAP threshold shifts increased with trauma severity. At each stage a threshold shift at high frequencies (4–16 kHz) was accompanied with a threshold shift at low frequencies (0.25–2 kHz) that was 10–20 dB smaller. Withdrawal of the array led to a further worsening of responses, which probably indicates that insertion and removal trauma affected the responses rather than the mere presence of the array. In two instances, CAP threshold shifts were considerably larger than threshold shifts of cochlear microphonics, which could be explained by neural damage due to OSL fracture. A change in amplitudes at high sound levels was strongly correlated with threshold shifts, which is relevant for clinical ECochG performed at one sound level. Conclusion Basal trauma caused by cochleostomy and/or array insertion should be minimized in order to preserve the low-frequency residual hearing of CI recipients.

Cochlear sound encoding depends on α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs), but reliance on specific pore-forming subunits is unknown. With 5-week-old male C57BL/6J Gria3 -knockout mice (i.e., subunit GluA3 KO ) we determined cochlear function, synapse ultrastructure, and AMPAR molecular anatomy at ribbon synapses between inner hair cells (IHCs) and spiral ganglion neurons. GluA3 KO and wild-type (GluA3 WT ) mice reared in ambient sound pressure level (SPL) of 55–75 dB had similar auditory brainstem response (ABR) thresholds, wave-1 amplitudes, and latencies. Postsynaptic densities (PSDs), presynaptic ribbons, and synaptic vesicle sizes were all larger on the modiolar side of the IHCs from GluA3 WT , but not GluA3 KO , demonstrating GluA3 is required for modiolar–pillar synapse differentiation. Presynaptic ribbons juxtaposed with postsynaptic GluA2/4 subunits were similar in quantity, however, lone ribbons were more frequent in GluA3 KO and GluA2-lacking synapses were observed only in GluA3 KO . GluA2 and GluA4 immunofluorescence volumes were smaller on the pillar side than the modiolar side in GluA3 KO , despite increased pillar-side PSD size. Overall, the fluorescent puncta volumes of GluA2 and GluA4 were smaller in GluA3 KO than GluA3 WT . However, GluA3 KO contained less GluA2 and greater GluA4 immunofluorescence intensity relative to GluA3 WT (threefold greater mean GluA4:GluA2 ratio). Thus, GluA3 is essential in development, as germline disruption of Gria3 caused anatomical synapse pathology before cochlear output became symptomatic by ABR. We propose the hearing loss in older male GluA3 KO mice results from progressive synaptopathy evident in 5-week-old mice as decreased abundance of GluA2 subunits and an increase in GluA2-lacking, GluA4-monomeric Ca 2+ -permeable AMPARs.

The ability to localize a sound source in complex environments is essential for communication and navigation. Spatial hearing relies predominantly on the comparison of differences in the arrival time of sound between the two ears, the interaural time differences (ITDs). Hearing impairments are highly detrimental to sound localization. While cochlear implants (CIs) have been successful in restoring many crucial hearing capabilities, sound localization via ITD detection with bilateral CIs remains poor. The underlying reasons are not well understood. Neuronally, ITD sensitivity is generated by coincidence detection between excitatory and inhibitory inputs from the two ears performed by specialized brainstem neurons. Due to the lack of electrophysiological brainstem recordings during CI stimulation, it is unclear to what extent the apparent deficits are caused by the binaural comparator neurons or arise already on the input level. Here, we use a bottom-up approach to compare response features between electric and acoustic stimulation in an animal model of CI hearing. Conducting extracellular single neuron recordings in gerbils, we find severe hyper-precision and moderate hyper-entrainment of both the excitatory and inhibitory brainstem inputs to the binaural comparator neurons during electrical pulse-train stimulation. This finding establishes conclusively that the binaural processing stage must cope with highly altered input statistics during CI stimulation. To estimate the consequences of these effects on ITD sensitivity, we used a computational model of the auditory brainstem. After tuning the model parameters to match its response properties to our physiological data during either stimulation type, the model predicted that ITD sensitivity to electrical pulses is maintained even for the hyper-precise inputs. However, the model exhibits severely altered spatial sensitivity during electrical stimulation compared to acoustic: while resolution of ITDs near midline was increased, more lateralized adjacent source locations became inseparable. These results directly resemble recent findings in rodent and human CI listeners. Notably, decreasing the phase-locking precision of inputs during electrical stimulation recovered a wider range of separable ITDs. Together, our findings suggest that a central problem underlying the diminished ITD sensitivity in CI users might be the temporal hyper-precision of inputs to the binaural comparator stage induced by electrical stimulation.

We examined the effect of neonatal deafening on frequency-specific pathways for processing of interaural time differences (ITDs) in cochlear-implant stimuli. Animal studies have demonstrated differences in neural ITD sensitivity in the inferior colliculus (IC) depending on the intracochlear location of intracochlear stimulating electrodes. We used neonatally deafened (ND) rats of both sexes and recorded the responses of single neurons in the IC to electrical stimuli with ITDs delivered to the apical or basal cochlea and compared them with acutely deafened (AD) rats of both sexes with normal hearing (NH) during development. We found that neonatal deafness significantly impacted the ITD sensitivity and the ITD tuning patterns restricted to apically driven IC neurons. In ND rats, the ITD sensitivity of apically driven neurons is reduced to values similar to basally driven neurons. The prevalence of ITD-sensitive apical neurons with a peak-shaped ITD tuning curve, which may reflect predominant input from the medial superior olivary (MSO) complex, in ND rats was diminished compared with that in AD rats (67%, AD vs 40%, ND). Conversely, monotonic-type responses rarely occurred in AD rats (14%) but were approximately equally as prevalent as peak-type tuning curves in ND rats (42%). Nevertheless, in ND rats, the ITD at the maximum slope of the ITD tuning curve was still more concentrated within the physiological ITD range in apically driven than in basally driven neurons. These results indicate that the development of high ITD sensitivity processed by low-frequency pathways depends on normal auditory experience and associated biases in ITD tuning strategies.

Cisplatin is a widely used and efficacious chemotherapeutic agent for treating solid tumors, yet it causes systemic end-organ damage that is often irreversible and detrimental to quality of life. This includes severe sensorineural hearing loss, hepatotoxicity, and renal injury. Based on the hard-soft acid-base theory, we recently developed two acetophenone-derived, enol-based compounds that directly interfere with the side effects of cisplatin. We investigated organ-specific and generalized toxicity in order to define dose-dependent responses in rodents injected with cisplatin with or without the protective compounds. All metrics that were used as indicators of toxicity showed retention of baseline or control measurements when animals were pre-treated with acetophenones prior to cisplatin administration, while animals injected with no protective compounds showed expected elevations in toxicity measurements or depressions in measurements of organ function. These data support the further investigation of novel acetophenone compounds for the prevention of cisplatin-induced end-organ toxicity.

The cochlea’s ability to discriminate sound frequencies is facilitated by a special topography along its longitudinal axis known as tonotopy. Auditory hair cells located at the base of the cochlea respond to high-frequency sounds, whereas hair cells at the apex respond to lower frequencies. Gradual changes in morphological and physiological features along the length of the cochlea determine each region’s frequency selectivity, but it remains unclear how tonotopy is established during cochlear development. Recently, sonic hedgehog (SHH) was proposed to initiate the establishment of tonotopy by conferring regional identity to the primordial cochlea. Here, using mouse genetics, we provide in vivo evidence that regional identity in the embryonic cochlea acts as a framework upon which tonotopy-specific properties essential for frequency selectivity in the mature cochlea develop. We found that follistatin (FST) is required for the maintenance of apical cochlear identity, but dispensable for its initial induction. In a fate-mapping analysis, we found that FST promotes expansion of apical cochlear cells, contributing to the formation of the apical cochlear domain. SHH, in contrast, is required both for the induction and maintenance of apical identity. In the absence of FST or SHH, mice produce a short cochlea lacking its apical domain. This results in the loss of apex-specific anatomical and molecular properties and low-frequency-specific hearing loss.

Inner ear gene therapy using adeno-associated viral vectors (AAVs) in neonatal mice can alleviate hearing loss in mouse models of deafness. However, efficient and safe transgene delivery to the adult mouse cochlea is critical for the effectiveness of AAV-mediated therapy. Here, we examined three gene delivery approaches including posterior semicircular canal (PSCC) canalostomy, round window membrane (RWM) injection, and tubing-RWM+PSCC (t-RP) in adult mice. Transduction rates and survival rates of cochlear hair cells were analyzed, hearing function was recorded, AAV distribution in the sagittal brain sections was evaluated, and cochlear histopathologic images were appraised. We found that an injection volume of 1 μL AAV through the PSCC is safe and highly efficient and does not impair hearing function in adult mice, but local injection allows AAV vectors to spread slightly into the brain. We then tested five AAV serotypes (PHP.eB, IE, Anc80L65, AAV2, and PHP.s) in parallel and observed the most robust eGFP expression in inner hair cells, outer hair cells, and spiral ganglion neurons throughout the cochlea after AAV-Anc80L65 injection. Thus, PSCC-injected Anc80L65 provides a foundation for gene therapy in the adult cochlea and will facilitate the development of inner ear gene therapy.

Neurons that process sensory information exhibit bursts of electrical activity during development, providing early training to circuits that will later encode similar features of the external world. In the mammalian auditory system, this intrinsically generated activity emerges from the cochlea prior to hearing onset, but its role in maturation of auditory circuitry remains poorly understood. We show that selective suppression of cochlear supporting cell spontaneous activity disrupts patterned burst firing of central auditory neurons without affecting cell survival or acoustic thresholds. However, neurons in the inferior colliculus of these mice exhibit enhanced acoustic sensitivity and broader frequency tuning, resulting in wider isofrequency laminae. Despite this enhanced neural responsiveness, total tone-responsive regions in the auditory cortex are substantially smaller. Thus, disruption of pre-hearing cochlear activity causes profound changes in neural encoding of sound, with important implications for restoration of hearing in individuals who experience reduced activity during this critical developmental period.

Neurexins are presynaptic transmembrane proteins crucial for synapse development and organization. Deletion and missense mutations in all three Neurexin genes have been identified in psychiatric disorders, with mutations in the NRXN1 gene most strongly linked to schizophrenia (SZ) and autism spectrum disorder (ASD). While the consequences of NRXN1 deletion have been extensively studied on the synaptic and behavioral levels, circuit endophenotypes that translate to the human condition have not been characterized yet. Therefore, we investigated the electrophysiology of cortico-striatal-thalamic circuits in Nrxn1α −/− rats and wildtype littermates focusing on a set of translational readouts, including spontaneous oscillatory activity, auditory-evoked oscillations and potentials, as well as mismatch negativity-like (MMN) responses and responses to social stimuli. On the behavioral level Nrxn1α −/− rats showed locomotor hyperactivity. In vivo freely moving electrophysiology revealed pronounced increases of spontaneous oscillatory power within the gamma band in all studied brain areas and elevation of gamma coherence in cortico-striatal and thalamocortical circuits of Nrxn1α −/− rats. In contrast, auditory-evoked oscillations driven by chirp-modulated tones showed reduced power in cortical areas confined to slower oscillations. Finally, Nrxn1α −/− rats exhibited altered auditory evoked-potentials and profound deficits in MMN-like responses, explained by reduced prediction error. Despite deficits for auditory stimuli, responses to social stimuli appeared intact. A central hypothesis for psychiatric and neurodevelopmental disorders is that a disbalance of excitation-to-inhibition is underlying oscillatory and sensory deficits. In a first attempt to explore the impact of inhibitory circuit modulation, we assessed the effects of enhancing tonic inhibition via δ-containing GABA A receptors (using Gaboxadol) on endophenotypes possibly associated with network hyperexcitability. Pharmacological experiments applying Gaboxadol showed genotype-specific differences, but failed to normalize oscillatory or sensory processing abnormalities. In conclusion, our study revealed endophenotypes in Nrxn1α −/− rats that could be used as translational biomarkers for drug development in psychiatric disorders.

Inhibitory G proteins (GNAI/Gα i ) bind to the scaffold G protein signaling modulator 2 (GPSM2) to form a conserved polarity complex that regulates cytoskeleton organization. GPSM2 keeps GNAI in a guanosine diphosphate (GDP)-bound state, but how GPSM2-GNAI is generated or relates to heterotrimeric G protein signaling remains unclear. We find that RGS12, a GTPase-activating protein (GAP), is required to polarize GPSM2-GNAI at the hair cell apical membrane and to organize mechanosensory stereocilia in rows of graded heights. Accordingly, RGS12 and the guanine nucleotide exchange factor (GEF) DAPLE are asymmetrically co-enriched at the hair cell apical junction, and Rgs12 mouse mutants are deaf. GPSM2 and RGS12 share GoLoco motifs that stabilize GNAI(GDP), and GPSM2 outcompetes RGS12 to bind GNAI. Our results suggest that polarized GEF/GAP junctional activity might dissociate heterotrimeric G proteins, generating free GNAI(GDP) for GPSM2 at the adjacent apical membrane. GPSM2-GNAI(GDP), in turn, imparts asymmetry to the forming stereocilia to enable sensory function in hair cells.

The lateral superior olive (LSO) is a key structure in the central auditory system of mammals that exerts efferent control on cochlear sensitivity and is involved in the processing of binaural level differences for sound localization. Understanding how the LSO contributes to these processes requires knowledge about the resident cells and their connections with other auditory structures. We used standard histological stains and retrograde tracer injections into the inferior colliculus (IC) and cochlea in order to characterize two basic groups of neurons: (1) Principal and periolivary (PO) neurons have projections to the IC as part of the ascending auditory pathway; and (2) lateral olivocochlear (LOC) intrinsic and shell efferents have descending projections to the cochlea. Principal and intrinsic neurons are intermixed within the LSO, exhibit fusiform somata, and have disk-shaped dendritic arborizations. The principal neurons have bilateral, symmetric, and tonotopic projections to the IC. The intrinsic efferents have strictly ipsilateral projections, known to be tonotopic from previous publications. PO and shell neurons represent much smaller populations (<10% of principal and intrinsic neurons, respectively), have multipolar somata, reside outside the LSO, and have non-topographic, bilateral projections. PO and shell neurons appear to have widespread projections to their targets that imply a more diffuse modulatory function. The somata and dendrites of principal and intrinsic neurons form a laminar matrix within the LSO and share quantifiably similar alignment to the tonotopic axis. Their restricted projections emphasize the importance of frequency in binaural processing and efferent control for auditory perception. This study addressed and expanded on previous findings of cell types, circuit laterality, and projection tonotopy in the LSO of the mouse.

Hearing loss is a chronic health condition that affects millions of people worldwide. In addition to age-related hearing impairment, excessive noise exposure is a leading cause of hearing loss. Beyond the devastating effects of hearing impairment itself, epidemiological studies have identified hearing loss as a major risk factor for age-related cognitive decline, including dementia. At present, we currently lack a full understanding of the brain regions and underlying molecular changes that are responsible for mediating the link between hearing loss and cognitive impairment across aging. In the present study, we exposed 6-month-old rats to an occupational-like noise (100 dB SPL, 4 h/day × 30 days) or sham exposure and investigated both hippocampal-dependent (i.e., spatial learning and memory, assessed using the Morris water maze) and striatal-dependent (i.e., visuomotor associative learning, assessed using an operant-conditioning task) cognitive function across aging at 7, 10, and 13 months of age. We also investigated brain region-specific changes in microglial expression following noise/sham exposure in order to assess the potential contribution of this cell type to noise-induced cognitive impairments. Consistent with human studies, the occupational-like noise exposure resulted in high-frequency hearing loss, evidenced by a significant increase in hearing thresholds at 20 kHz. Ultimately, our results suggest that not all higher-level cognitive tasks or their associated brain regions appear to be equally susceptible to noise-induced deficits during aging, as the occupational-like noise exposure caused an age-dependent deficit in spatial but not visuomotor associative learning, as well as altered microglial expression in the hippocampus but not the striatum. Interestingly, we found no significant relationships between spatial learning ability and the level of hearing loss or altered microglial density in the hippocampus following noise exposure, suggesting that other changes in the brain likely contribute to hippocampal-dependent cognitive dysfunction following noise exposure. Lastly, we found that a subset of younger animals also showed noise-induced deficits in spatial learning; findings which suggest that noise exposure may represent an increased risk for cognitive impairment in vulnerable subjects. Overall, our findings highlight that even a mild occupational-like noise exposure earlier in adulthood can have long lasting implications for cognitive function later in life.

Animal research focused on chronic tinnitus associated with noise-induced hearing loss can be expensive and time-consuming as a result of the behavioral training required. Although there exist a number of behavioral tests for tinnitus; there have been few formal direct comparisons of these tests. Here, we evaluated animals in two different tinnitus assessment methods. CBA/CaJ mice were trained in an operant conditioning, active avoidance (AA) test, and a reflexive, gap-induced pre-pulse inhibition of acoustic startle (GPIAS) test, or both. Tinnitus was induced in awake mice by unilateral continuous sound exposure using a 2-kHz- or 1 2 octave-wide noise centered at 16 kHz and presented at 113- or 116-dB SPL. Tinnitus was assessed 8 weeks after sound overexposure. Most mice had evidence of tinnitus behavior in at least one of the two behaviors. Of the mice evaluated in AA, over half (55%) had tinnitus positive behavior. In GPIAS, fewer animals (13%) were positive than were identified using the AA test. Few mice were positive in both tests (10%), and only one was positive for tinnitus behavior at the same spectral frequency in both tests. When the association between tinnitus behavior and spontaneous activity recorded in the inferior colliculus was compared, animals with tinnitus behavior in AA exhibited increased spontaneous activity, while those positive in GPIAS did not. Thus, it appears that operant conditioning tests, like AA, maybe more reliable and accurate tests for tinnitus than reflexive tests.

Centrosomal protein dysfunction might cause ciliopathies. However, the role of centrosomal proteins in male infertility remains poorly defined. Here, we identified a pathogenic splicing mutation in CEP78 in male infertile patients with severely reduced sperm number and motility, and the typical multiple morphological abnormalities of the sperm flagella phenotype. We further created Cep78 knockout mice, which showed an extremely low sperm count, completely aberrant sperm morphology, and approximately null sperm motility. The infertility of the patients and knockout mice could not be rescued by an intracytoplasmic sperm injection treatment. Mechanistically, CEP78 might regulate USP16 expression, which further stabilizes Tektin levels via the ubiquitination pathway. Cep78 knockout mice also exhibited impairments in retina and outer hair cells of the cochlea. Collectively, our findings identified nonfunctional CEP78 as an indispensable factor contributing to male infertility and revealed a role for this gene in regulating retinal and outer hair cell function in mice.

Severe hearing loss or deafness is often caused by cochlear hair cell loss and can be mitigated by a cochlear implant (CI). CIs target the auditory nerve, consisting of spiral ganglion cells (SGCs), which degenerate gradually, following hair cell loss. In animal models, it has been established that treatment with the neurotrophins brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) reduce SGC degeneration. In this study, we aimed to investigate whether treatment with both BDNF and NT-3 (Cocktail) is superior to treatment with each neurotrophin separately regarding cell preservation and neural responsiveness to electrical stimulation. To this end, deafened guinea pigs received neurotrophic treatment in their right ear via a gelatin sponge on the perforated round window membrane, followed by cochlear implantation 4 weeks later in the same ear for electrophysiological recordings to various stimulation paradigms. Normal-hearing and deafened untreated guinea pigs were included as positive and negative controls, respectively. Substantial SGC loss occurred in all deafened animals. Each of the neurotrophic treatments led to enhanced SGC survival mainly in the basal turn of the cochlea, gradually decreasing toward the apex. The Cocktail treatment resulted in the highest SGC survival in the treated ear, followed by BDNF, with the least protection of SGCs following NT-3 treatment. Survival of the SGC’s peripheral processes (PPs) followed the same trend in response to the treatment. However, survival of SGCs and PPs in the contralateral untreated ears was also highest in the Cocktail group. Consequently, analysis of the ratio between the treated and untreated ears showed that the BDNF group, which showed low SGC survival in the untreated ear, had the highest relative SGC survival of the three neurotrophin-treated groups. Neurotrophic treatment had positive effects in part of the electrically evoked compound action-potential recording paradigms. These effects were only observed for the BDNF or Cocktail treatment. We conclude that treatment with either BDNF or a cocktail of BDNF and NT-3 is preferred to NT-3 alone. Furthermore, since the Cocktail treatment resulted in better electrophysiological responsiveness and overall higher SGC survival than BDNF alone, we are inclined to recommend the Cocktail treatment rather than BDNF alone.

Nuclear positioning is important for the functionality of many cell types and is mediated by interactions of cytoskeletal elements and nucleoskeleton proteins. Nesprin proteins, part of the linker of nucleoskeleton and cytoskeleton (LINC) complex, have been shown to participate in nuclear positioning in multiple cell types. Outer hair cells (OHCs) in the inner ear are specialized sensory epithelial cells that utilize somatic electromotility to amplify auditory signals in the cochlea. Recently, Nesprin-4 (encoded by Syne4 ) was shown to play a crucial role in nuclear positioning in OHCs. Syne4 deficiency in humans and mice leads to mislocalization of the OHC nuclei and cell death resulting in deafness. However, it is unknown how Nesprin-4 mediates the position of the nucleus, and which other molecular components are involved in this process. Here, we show that the interaction of Nesprin-4 and the microtubule motor kinesin-1 is mediated by a conserved 4 amino-acid motif. Using in vivo AAV gene delivery, we show that this interaction is critical for nuclear positioning and hearing in mice. Nuclear mislocalization and cell death of OHCs coincide with the onset of hearing and electromotility and are solely restricted to outer, but not inner, hair cells. Likewise, the C. elegans functional homolog of Nesprin-4, UNC-83, uses a similar motif to mediate interactions between migrating nuclei and kinesin-1. Overall, our results suggest that OHCs require unique cellular machinery for proper nuclear positioning at the onset of electromotility. This machinery relies on the interaction between Nesprin-4 and kinesin-1 motors supporting a microtubule cargo model for nuclear positioning.

Vertigo is a debilitating disease affecting 15–20% of adults worldwide. Vestibular peripheral vertigo is the most common cause of vertigo, often due to Meniere's disease and benign paroxysmal positional vertigo. Although some vertigo symptoms can be controlled by conservative treatment and/or vestibular rehabilitation therapy, these treatments do not work for some patients. Semicircular canal occlusion surgery has proven to be very effective for these patients with intractable vertigo. However, its application is limited due to concern that the procedure will disrupt normal hearing. In this study, we investigated if occlusion of two semicircular canals would jeopardize auditory function by comparing auditory function and hair cell morphology between the surgical and contralateral ears before and after the surgery in a mouse model. By measuring the auditory brainstem response and distortion product otoacoustic emission 4 weeks post-surgery, we show that auditory function does not significantly change between the surgical and contralateral ears. In addition, confocal imaging has shown no hair cell loss in the cochlear and vestibular sensory epithelia, and scanning electron microscopy also indicates normal stereocilia morphology in the surgical ear. More importantly, the endocochlear potential measured from the surgical ear is not significantly different than that seen in the contralateral ear. Our study suggests that occlusion of two semicircular canals does not disrupt normal hearing in the mouse model, providing a basis to extend the procedure to patients, even those with normal hearing, benefitting more patients with intractable vertigo attacks.