Publications using the System 3

Age-related hearing loss (AHL) is a progressive sensorineural hearing loss in elderly people. Although no prevention or treatments have been established for AHL, recent studies have demonstrated that oxidative stress is closely related to pathogenesis of AHL, suggesting that suppression of oxidative stress leads to inhibition of AHL progression. NRF2 is a master transcription factor that regulates various antioxidant proteins and cytoprotection factors. To examine whether NRF2 pathway activation prevents AHL, we used Keap1 -knockdown ( Keap1 FA/FA ) mice, in which KEAP1, a negative regulator of NRF2, is decreased, resulting in the elevation of NRF2 activity. We compared 12-month-old Keap1 FA/FA mice with age-matched wild-type (WT) mice in the same breeding colony. In the Keap1 FA/FA mice, the expression levels of multiple NRF2 target genes were verified to be significantly higher than the expression levels of these genes in the WT mice. Histological analysis showed that cochlear degeneration at the apical and middle turns was ameliorated in the Keap1 FA/FA mice. Auditory brainstem response (ABR) thresholds in the Keap1 FA/FA mice were significantly lower than those in the WT mice, in particular at low–mid frequencies. Immunohistochemical detection of oxidative stress markers suggested that oxidative stress accumulation was attenuated in the Keap1 FA/FA cochlea. Thus, we concluded that NRF2 pathway activation protects the cochlea from oxidative damage during aging, in particular at the apical and middle turns. KEAP1-inhibiting drugs and phytochemicals are expected to be effective in the prevention of AHL.

Usher syndrome is a syndromic form of hereditary hearing impairment that includes sensorineural hearing loss and delayed-onset retinitis pigmentosa (RP). Type 1 Usher syndrome (USH1) is characterized by congenital profound sensorineural hearing impairment and vestibular areflexia, with adolescent-onset RP. Systemic treatment with antisense oligonucleotides (ASOs) targeting the human USH1C c.216G>A splicing mutation in a knockin mouse model of USH1 restores hearing and balance. Herein, we explore the effect of delivering ASOs locally to the ear to treat hearing and vestibular dysfunction associated with Usher syndrome. Three localized delivery strategies were investigated in USH1C mice: inner ear injection, trans-tympanic membrane injection, and topical tympanic membrane application. We demonstrate, for the first time, that ASOs delivered directly to the ear correct Ush1c expression in inner ear tissue, improve cochlear hair cell transduction currents, restore vestibular afferent irregularity, spontaneous firing rate, and sensitivity to head rotation, and successfully recover hearing thresholds and balance behaviors in USH1C mice. We conclude that local delivery of ASOs to the middle and inner ear reach hair cells and can rescue both hearing and balance. These results also demonstrate the therapeutic potential of ASOs to treat hearing and balance deficits associated with Usher syndrome and other ear diseases.

Reduced tolerance to sound stimuli (hyperacusis) is commonly seen in tinnitus patients. Dysfunction of limbic systems, such as the nucleus accumbens (NAc), may be involved in emotional reactions to the sound stimuli in tinnitus patients. To study the functional changes in the NAc in hyperacusis, we have examined the neural activity changes of the NAc using c-Fos staining in an animal model of hyperacusis. The c-Fos staining was also examined in the medial geniculate nucleus (MGN), a central auditory pathway which has neural projections to the NAc. Postnatal rats (14 days) were exposed to loud noise (115 dB SPL, 4 hours for two consecutive days) to induce hyperacusis ( n = 4 ). Rats without noise exposure were used as the controls ( n = 4 ). After P35, rats in both groups were put in a behavioral training for sound detection. After they were trained to detect sound stimuli, their reaction time to noise bursts centered at 2 kHz (40-110 dB SPL) was measured. Rats in the noise group showed a significantly shorter reaction time than those in the control group to the noise bursts at high intensities, suggesting the noise exposure induced hyperacusis behavior. The c-Fos expressions in the NAc and the MGNs of the noise group were significantly higher than those of the control group. Our results suggested that early-age noise exposure caused hyperactivity in the NAc and the MGNs which may induce the loudness increase in these rats.

Noise induces oxidative stress in the cochlea followed by sensory cell death and hearing loss. The proof of principle that injections of antioxidant vitamins and Mg2+ prevent noise-induced hearing loss (NIHL) has been established. However, effectiveness of oral administration remains controversial and otoprotection mechanisms are unclear. Using auditory evoked potentials, quantitative PCR, and immunocytochemistry, we explored effects of oral administration of vitamins A, C, E, and Mg2+ (ACEMg) on auditory function and sensory cell survival following NIHL in rats. Oral ACEMg reduced auditory thresholds shifts after NIHL. Improved auditory function correlated with increased survival of sensory outer hair cells. In parallel, oral ACEMg modulated the expression timeline of antioxidant enzymes in the cochlea after NIHL. There was increased expression of glutathione peroxidase-1 and catalase at 1 and 10 days, respectively. Also, pro-apoptotic caspase-3 and Bax levels were diminished in ACEMg-treated rats, at 10 and 30 days, respectively, following noise overstimulation, whereas, at day 10 after noise exposure, the levels of anti-apoptotic Bcl-2, were significantly increased. Therefore, oral ACEMg improves auditory function by limiting sensory hair cell death in the auditory receptor following NIHL. Regulation of the expression of antioxidant enzymes and apoptosis-related proteins in cochlear structures is involved in such an otoprotective mechanism.

Regeneration of auditory hair cells in adult mammals is challenging. It is also difficult to track the sources of regenerated hair cells, especially in vivo. Previous paper found newly generated hair cells in deafened mouse by injecting a DNA methyltransferase inhibitor 5-azacytidine into the inner ear. This paper aims to investigate the cell sources of new hair cells. Transgenic mice with enhanced green fluorescent protein (EGFP) expression controlled by the Sox2 gene were used in the study. A combination of kanamycin and furosemide was applied to deafen adult mice, which received 4 mM 5-azacytidine injection into the inner ear three days later. Mice were followed for 3, 5, 7 and 14 days after surgery to track hair cell regeneration. Immunostaining of Myosin VIIa and EGFP signals were used to track the fate of Sox2-expressing supporting cells. The results show that (i) expression of EGFP in the transgenic mice colocalized the supporting cells in the organ of Corti, and (ii) the cell source of regenerated hair cells following 5-azacytidine treatment may be supporting cells during 5–7 days post 5-azacytidine injection. In conclusion, 5-azacytidine may promote the conversion of supporting cells to hair cells in chemically deafened adult mice.

Oscillations in the granule cell layer (GCL) of the cerebellar cortex have been related to behavior and could facilitate communication with the cerebral cortex. These local field potential (LFP) oscillations, strong at 4-12 Hz in the rodent cerebellar cortex during awake immobility, should also be an indicator of an underlying influence on the patterns of the cerebellar cortex neuronal firing during rest. To address this hypothesis, cerebellar cortex LFPs and simultaneous single-neuron activity were collected during LFP oscillatory periods in the GCL of awake resting rats. During these oscillatory episodes, different types of units across the GCL and Purkinje cell layers showed variable phase-relation with the oscillatory cycles. Overall, 74% of the Golgi cell firing and 54% of the Purkinje cell simple spike (SS) firing were phase-locked with the oscillations, displaying a clear phase relationship. Despite this tendency, fewer Golgi cells (50%) and Purkinje cell's SSs (25%) showed an oscillatory firing pattern. Oscillatory phase-locked spikes for the Golgi and Purkinje cells occurred towards the peak of the LFP cycle. GCL LFP oscillations had a strong capacity to predict the timing of Golgi cell spiking activity, indicating a strong influence of this oscillatory phenomenon over the GCL. Phase-locking was not as prominent for the Purkinje cell SS firing, indicating a weaker influence over the Purkinje cell layer, yet a similar phase relation. Overall, synaptic activity underlying GCL LFP oscillations likely exert an influence on neuronal population firing patterns in the cerebellar cortex in the awake resting state and could have a preparatory neural network shaping capacity serving as a neural baseline for upcoming cerebellar operations.

Background and objectives: Animal results have suggested that auditory brainstem responses (ABRs) to transient sounds presented at supra-threshold levels may be useful for measuring hearing damage that is hidden to current audiometric tests. Evaluating such ABRs requires extracting the latencies and amplitudes of relevant deflections, or "waves". Currently, this is mostly done by human observers manually picking the waves' peaks and troughs in each individual response - a process that is both time-consuming and requiring of expert experience. Here, we propose a highly automated procedure for extracting individual ABR wave latencies and amplitudes based on the well-established methodology of non-linear curve registration. Methods: First, the to-be-analysed individual ABRs are temporally aligned - either with one another or, if available, with a pre-existing template - by locally compressing or stretching their time axes with smooth and invertible time warping functions. Then, the individual latencies and amplitudes of relevant ABR waves are obtained by picking the latencies of the waves' peaks and troughs on the common (aligned) time axis and combining these with the individual aligned responses and inverse time warping functions. Results: Using an example ABR data set with a wide range of response latencies and signal-to-noise ratios (SNRs), we test different choices for fitting the time warping functions. We cross-validate the warping results using independent response replicates and compare automatically and manually extracted latencies and amplitudes for ABR waves I and V. Using a Bayesian approach, we show that, for the best registration condition, automatic and manual data were statistically similar. Conclusions: Non-linear curve registration can be used to temporally align individual ABRs and extract their wave latencies and amplitudes in a way that closely matches results from manual picking.

Exposure to excessive sound causes noise-induced hearing loss through complex mechanisms and represents a common and unmet neurological condition. We investigate how noise insults affect the cochlea with proteomics and functional assays. Quantitative proteomics reveals that exposure to loud noise causes proteotoxicity. We identify and confirm hundreds of proteins that accumulate, including cytoskeletal proteins, and several nodes of the proteostasis network. Transcriptomic analysis reveals that a subset of the genes encoding these proteins also increases acutely after noise exposure, including numerous proteasome subunits. Global cochlear protein ubiquitylation levels build up after exposure to excess noise, and we map numerous posttranslationally modified lysines residues. Several collagen proteins decrease in abundance, and Col9a1 specifically localizes to pillar cell heads. After two weeks of recovery, the cochlea selectively elevates the abundance of the protein synthesis machinery. We report that overstimulation of the auditory system drives a robust cochlear proteotoxic stress response.

In deaf subjects using a cochlear implant (CI) for hearing restoration, the auditory nerve is subject to degeneration, which may negatively impact CI effectiveness. This nerve degeneration can be reduced by neurotrophic treatment. Here, we compare the preservative effects of the naturally occurring tyrosine receptor kinase B (TrkB) agonist brain-derived neurotrophic factor (BDNF) and the small-molecule TrkB agonist 7,8,3′-trihydroxyflavone (THF) on the auditory nerve in deafened guinea pigs. THF may be more effective than BDNF throughout the cochlea because of better pharmacokinetic properties. The neurotrophic compounds were delivered by placement of a gelatin sponge on the perforated round window membrane. To complement the histology of spiral ganglion cells (SGCs), electrically evoked compound action potential (eCAP) recordings were performed four weeks after treatment initiation. We analyzed the eCAP inter-phase gap (IPG) effect and measures derived from pulse-train evoked eCAPs, both indicative of SGC healthiness. BDNF but not THF yielded a significantly higher survival of SGCs in the basal cochlear turn than untreated controls. Regarding IPG effect and pulse-train responses, the BDNF-treated animals exhibited more normal responses than both untreated and THF-treated animals. We have thus confirmed the protective effect of BDNF, but we have not confirmed previously reported protective effects of THF with our clinically applicable delivery method.

Atg7 is an indispensable factor that plays a role in canonical nonselective autophagy. Here we show that genetic ablation of Atg7 in outer hair cells (OHCs) in mice caused stereocilium damage, somatic electromotility disturbances, and presynaptic ribbon degeneration over time, which led to the gradual wholesale loss of OHCs and subsequent early-onset profound hearing loss. Impaired autophagy disrupted OHC mitochondrial function and triggered the accumulation of dysfunctional mitochondria that would otherwise be eliminated in a timely manner. Atg7-independent autophagy/mitophagy processes could not compensate for Atg7 deficiency and failed to rescue the terminally differentiated, non-proliferating OHCs. Our results show that OHCs orchestrate intricate nonselective and selective autophagic/mitophagy pathways working in concert to maintain cellular homeostasis. Overall, our results demonstrate that Atg7-dependent autophagy plays a pivotal cytoprotective role in preserving OHCs and maintaining hearing function.

Fear conditioning is a form of associative learning that is known to involve different brain areas, notably the amygdala, the prefrontal cortex and the periaqueductal grey (PAG). Here, we describe the functional role of pathways that link the cerebellum with the fear network. We found that the cerebellar fastigial nucleus (FN) sends glutamatergic projections to vlPAG that synapse onto glutamatergic and GABAergic vlPAG neurons. Chemogenetic and optogenetic manipulations revealed that the FN-vlPAG pathway controls bi-directionally the strength of the fear memories, indicating an important role in the association of the conditioned and unconditioned stimuli, a function consistent with vlPAG encoding of fear prediction error. Moreover, FN-vlPAG projections also modulate extinction learning. We also found a FN-parafascicular thalamus pathway, which may relay cerebellar influence to the amygdala and modulates anxiety behaviors. Overall, our results reveal multiple contributions of the cerebellum to the emotional system.

Large-scale epidemiological surveys suggest that hearing loss (HL) is a significant risk factor for dementia. We previously showed that noise-induced HL (NIHL) impairs hippocampal cognitive function and decreases hippocampal neurogenesis and neuronal complexity, suggesting a causal role of HL in dementia. To further investigate the influence of acquired peripheral HL on hippocampal neurogenesis with the aging process as well as the underlying mechanism, we produced NIHL in male CBA/J mice and assessed hippocampal neurogenesis and microglial morphology in the auditory brain and hippocampus at 4 days post-noise exposure (DPN) or 1, 3, 6, or 12 months post-noise exposure (MPN) by immunofluorescence labeling. We found that the age-related decline in hippocampal neurogenesis was accelerated in mice with NIHL. Furthermore, in mice with NIHL, prolonged microglial activation occurred from 1 MPN to 12 MPN across multiple auditory nuclei, while aggravated microglial deterioration occurred in the hippocampus and correlated with the age-related decline in hippocampal neurogenesis. These results suggest that acquired peripheral HL accelerates the age-related decline in hippocampal neurogenesis and that hippocampal microglial degeneration may contribute to the development of neurodegeneration following acquired peripheral HL.

Currently, there are limited reports available regarding the treatment and prevention of progressive age-related hearing loss. This is because age-related hearing loss is not a critical disease with direct fatalities and has several well-established countermeasures such as hearing aids and cochlear implants. This study evaluated the efficacy of Ninjinyoeito (NYT) in the treatment of age-related hearing loss. C57BL/6J mice were divided into three groups: baseline group, untreated group, and NYT-treated group, with the latter receiving NYT treatment for 2 months. The mice were fed with NYT extract mixed with 4% mouse normal chow. Hearing loss was confirmed by a reduction in intact cell density of the auditory nerve from the age of 5-7 months. The suppression of hearing loss with aging and decrease in the intact cell density of the auditory nerve were significant in mice fed with NYT for 2 months. NYT has been reported to improve blood flow and enhance mitochondrial activity and may exert its protective effects on spiral neurons through these mechanisms. There was no decrease in the size of the stria vascularis from the age of 5-7 months in C57BL/6J mice. The present model failed to reveal the effect of NYT on atrophy of the stria vascularis of the cochlear duct. In conclusion, NYT appears to have a protective effect on the auditory nerve and suppress the progression of age-related hearing loss by reducing age-related auditory nerve degeneration.

The round window of the cochlea provides an ideal route for delivering medicines and gene therapy reagents that can cross the round window membrane (RWM) into the inner ear. Recombinant adeno‐associated viruses (rAAVs) have several advantages and are recommended as viral vectors for gene transfection. However, rAAVs cannot cross an intact RWM. Consequently, ultrasound‐mediated microbubble (USMB) cavitation is potentially useful, because it can sonoporate the cell membranes, and increase their permeability to large molecules. The use of USMB cavitation for drug delivery across the RWM has been tested in a few animal studies but has not been used in the context of AAV‐mediated gene transfection. The currently available large size of the ultrasound probe appears to be a limiting factor in the application of this method to the RWM. In this study, we used home‐made ultrasound probe with a decreased diameter to 1.5 mm, which enabled the easy positioning of the probe close to the RWM. In guinea pigs, we used this probe to determine that (1) USMB cavitation caused limited damage to the outer surface layer or the RWM, (2) an eGFP‐gene carrying rAAV could effectively pass the USMB‐treated RWM and reliably transfect cochlear cells, and (3) the hearing function of the cochlea remained unchanged. Our results suggest that USMB cavitation of the RWM is a good method for rAAV‐mediated cochlear gene transfection with clear potential for clinical translation. We additionally discuss several advantages of the small probe size.

The auditory efferent system comprises descending projections from the cerebral cortex to subcortical nuclei, reaching the cochlear receptor through olivocochlear fibres. One of the functions attributed to this corticofugal system is to suppress irrelevant sounds during selective attention to visual stimuli. Medial olivocochlear neurons can also be activated by sounds through a brainstem reflex circuit. Whether the individual variability of this reflex is related to the cognitive capacity to suppress auditory stimuli is still controversial. Here we propose that the individual strength per animal of the olivocochlear reflex is correlated with the ability to suppress auditory distractors during visual attention in awake chinchillas. The olivocochlear reflex was elicited with a contralateral broad-band noise at ~ 60 dB and ipsilateral distortion product otoacoustic emissions were obtained at different frequencies (1–8 kHz). Fourteen chinchillas were evaluated in a behavioural protocol of visual attention with broad-band noise and chinchilla vocalizations as auditory distractors. Results show that the behavioural performance was affected by both distractors and that the magnitudes of the olivocochlear reflex evaluated at multiple frequencies were relevant for behavioural performance during visual discrimination with auditory distractors. These results stress the ecological relevance of the olivocochlear system for suppressing natural distractors.

The contribution of cerebellum to motor learning is often considered to be limited to adaptation, a short-timescale tuning of reflexes and previous learned skills. Yet, the cerebellum is reciprocally connected to two main players of motor learning, the motor cortex and the basal ganglia, via the ventral and midline thalamus respectively. Here, we evaluated the contribution of cerebellar neurons projecting to these thalamic nuclei in a skilled locomotion task in mice. In the cerebellar nuclei, we found task-specific neuronal activities during the task, and lasting changes after the task suggesting an offline processing of task-related information. Using pathway-specific inhibition, we found that dentate neurons projecting to the midline thalamus contribute to learning and retrieval, while interposed neurons projecting to the ventral thalamus contribute to the offline consolidation of savings. Our results thus show that two parallel cerebello-thalamic pathways perform distinct computations operating on distinct timescales in motor learning.

Congenital sensorineural hearing loss (SNHL) affects thousands of infants each year and results in significant delays in speech and language development. Previous studies have shown that early exposure to a simple augmented acoustic environment (AAE) can limit the effects of progressive SNHL on hearing sensitivity. However, SNHL is also accompanied by “hidden hearing loss” that is not assessed on standard audiological exams, such as reduced temporal processing acuity. To assess whether sound therapy may improve these hidden deficits, a mouse model of congenital SNHL was exposed to simple or temporally complex AAE. Peripheral function and sound sensitivity in auditory midbrain neurons improved following exposure to both types of AAE. However, only exposure to a novel, temporally complex AAE significantly improved a measure of temporal processing acuity, neural gap-in-noise detection in the auditory midbrain. These experiments suggest that targeted sound therapy may improve hearing outcomes for children suffering from congenital SNHL.

A noninvasive sleep screen uncovers a Vamp2 mouse mutation with synaptic deficits underlying substantial sleep disturbances.

Deafness, the most frequent sensory deficit in humans, is extremely heterogenous with hundreds of genes probably involved. Clinical and genetic analyses of an extended consanguineous family with pre-lingual, moderate-to-profound autosomal recessive sensorineural hearing loss, allowed us to identify CLRN2 , encoding a tetraspan protein as a new deafness gene. Homozygosity mapping followed by exome sequencing identified a 15.2 Mb locus on chromosome 4p15.32p15.1 containing a missense pathogenic variant in CLRN2 (c.494C>A, NM_001079827.2) segregating with the disease. Using in vitro RNA splicing analysis, we show that the CLRN2 c.494C>A mutation leads to two events: 1) the substitution of a highly conserved threonine (uncharged amino acid) to lysine (charged amino acid) at position 165, p.(Thr165Lys), and 2) aberrant splicing, with the retention of intron 2 resulting in a stop codon after 26 additional amino acids, p.(Gly146Lysfs*26). Expression studies and phenotyping of newly produced zebrafish and mouse models deficient for clarin 2 further confirm that clarin 2, expressed in the inner ear hair cells, is essential for normal organization and maintenance of the auditory hair bundles, and for hearing function. Together, our findings identify CLRN2 as a new deafness gene, which will impact future diagnosis and treatment for deaf patients.

Tinnitus is a common auditory disease worldwide; it is estimated that more than 10% of all individuals experience this hearing disorder during their lifetime. Tinnitus is sometimes accompanied by hearing loss. However, hearing loss is not acquired in some other tinnitus generations. In this study, we injected adult rats with salicylate sodium (SS) (200 mg/kg/day for 10 days) and found no significant hearing threshold changes at 2, 4, 8, 12, 14, 16, 20, or 24 kHz (all p > 0.05 ). Tinnitus was confirmed in the treated rats via Behaviour Testing of Acoustic Startle Response (ASR) and Gap Prepulse Inhibition Test of Acoustic Startle Reflex (GPIAS). A immunostaining study showed that there is significant loss of anti-CtBP2 puncta (a marker of cochlear inner hair cell (HC) ribbon synapses) in treated animals in apical, middle, and basal turns (all p < 0.05 ). The ABR wave I amplitudes were significantly reduced at 4, 8, 12, 14, 16, and 20 kHz (all p < 0.05 ). No significant losses of outer HCs, inner HCs, or HC cilia were observed (all p > 0.05 ). Thus, our study suggests that loss of cochlear inner HC ribbon synapse after SS exposure is a contributor to the development of tinnitus without changing hearing threshold.