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Hearing and balance depend upon the precise morphogenesis and mechanosensory function of stereocilia, the specialized structures on the apical surface of sensory hair cells in the inner ear. Previous studies of Grxcr1 mutant mice indicated a critical role for this gene in control of stereocilia dimensions during development. In this study, we analyzed expression of the paralog Grxcr2 in the mouse and evaluated auditory and vestibular function of strains carrying targeted mutations of the gene. Peak expression of Grxcr2 occurs during early postnatal development of the inner ear and GRXCR2 is localized to stereocilia in both the cochlea and in vestibular organs. Homozygous Grxcr2 deletion mutants exhibit significant hearing loss by 3 weeks of age that is associated with developmental defects in stereocilia bundle orientation and organization. Despite these bundle defects, the mechanotransduction apparatus assembles in relatively normal fashion as determined by whole cell electrophysiological evaluation and FM1-43 uptake. Although Grxcr2 mutants do not exhibit overt vestibular dysfunction, evaluation of vestibular evoked potentials revealed subtle defects of the mutants in response to linear accelerations. In addition, reduced Grxcr2 expression in a hypomorphic mutant strain is associated with progressive hearing loss and bundle defects. The stereocilia localization of GRXCR2, together with the bundle pathologies observed in the mutants, indicate that GRXCR2 plays an intrinsic role in bundle orientation, organization, and sensory function in the inner ear during development and at maturity., Competing Interests: The authors have declared that no competing interests exist.

In experimental animal models of auditory hair cell (HC) loss, insults such as noise or ototoxic drugs often lead to secondary changes or degeneration in non-sensory cells and neural components, including reduced density of spiral ganglion neurons, demyelination of auditory nerve fibers and altered cell numbers and innervation patterns in the cochlear nucleus (CN). However, it is not clear whether loss of HCs alone leads to secondary degeneration in these neural components of the auditory pathway. To elucidate this issue, we investigated changes of central components after cochlear insults specific to HCs using diphtheria toxin receptor (DTR) mice expressing DTR only in HCs and exhibiting complete HC loss when injected with diphtheria toxin (DT). We showed that DT-induced HC ablation has no significant impacts on the survival of auditory neurons, central synaptic terminals, and myelin, despite complete HC loss and profound deafness. In contrast, noise exposure induced significant changes in synapses, myelin and CN organization even without loss of inner HCs. We observed a decrease of neuronal size in the auditory pathway, including peripheral axons, spiral ganglion neurons, and CN neurons, likely due to loss of input from the cochlea. Taken together, selective HC ablation and noise exposure showed different patterns of pathology in the auditory pathway and the presence of HCs is not essential for the maintenance of central synaptic connectivity and myelination., (Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.)

Neurons of the lateral olivocochlear (LOC) system project from the auditory brainstem to the cochlea, where they synapse on radial dendrites of auditory nerve fibers. Selective LOC disruption depresses sound-evoked auditory nerve activity in the guinea pig, but enhances it in the mouse. Here, LOC disruption depressed spontaneous auditory nerve activity in the guinea pig. Recordings from single auditory nerve fibers revealed a significantly reduced proportion of fibers with the highest spontaneous firing rates (SRs) and an increased proportion of neurons with lower SRs. Ensemble activity, estimated using round window noise, also decreased after LOC disruption. Decreased spontaneous activity after LOC disruption may be a consequence of reduced tonic release of excitatory transmitters from the LOC terminals in guinea pigs., (Copyright © 2014. Published by Elsevier Ireland Ltd.)

The mammalian auditory epithelium (AE) cannot replace supporting cells and hair cells once they are lost. Therefore, sensorineural hearing loss associated with missing cells is permanent. This inability to regenerate critical cell types makes the AE a potential target for cell replacement therapies such as stem cell transplantation. Inserting stem cells into the AE of deaf ears is a complicated task due to the hostile, high potassium environment of the scala media in the cochlea, and the robust junctional complexes between cells in the AE that resist stem cell integration. Here, we evaluate whether temporarily reducing potassium levels in the scala media and disrupting the junctions in the AE make the cochlear environment more receptive and facilitate survival and integration of transplanted cells. We used sodium caprate to transiently disrupt the AE junctions, replaced endolymph with perilymph, and blocked stria vascularis pumps with furosemide. We determined that these three steps facilitated survival of HeLa cells in the scala media for at least 7 days and that some of the implanted cells formed a junctional contact with native AE cells. The data suggest that manipulation of the cochlear environment facilitates survival and integration of exogenously transplanted HeLa cells in the scala media.

Diverse cellular and environmental stresses can activate the heat shock response, an evolutionarily conserved mechanism to protect proteins from denaturation. Stressors activate heat shock transcription factor 1 (HSF1), which binds to heat shock elements in the genes for heat shock proteins, leading to rapid induction of these important molecular chaperones. Both heat and noise stress are known to activate the heat shock response in the cochlea and protect it from subsequent noise trauma. However, the contribution of HSF1 to induction of heat shock proteins following noise trauma has not been investigated at the molecular level. We evaluated the role of HSF1 in the cochlea following noise stress by examining induction of heat shock proteins in Hsf1 ( +/- ) control and Hsf1 ( -/- ) mice. Heat stress rapidly induced expression of Hsp25, Hsp47, Hsp70.1, Hsp70.3, Hsp84, Hsp86, and Hsp110 in the cochleae of wild-type and Hsf1 ( +/- ) mice, but not in Hsf1 ( -/- ) mice, confirming the essential role of HSF1 in mediating the heat shock response. Exposure to broadband noise (2-20 kHz) at 106 dB SPL for 2 h produced partial hearing loss. Maximal induction of heat shock proteins occurred 4 h after the noise. In comparison to heat stress, noise stress resulted in lower induced levels of Hsp25, Hsp70.1, Hsp70.3, Hsp86, and Hsp110 in Hsf1 ( +/- ) mice. Induction of these heat shock proteins was attenuated, but not completely eliminated, in Hsf1 ( -/- ) mice. These same noise exposure conditions induced genes for several immediate early transcription factors and maximum induction occurred earlier than for heat shock proteins. Thus, additional signaling pathways and transcriptional regulators that are activated by noise probably contribute to induction of heat shock proteins in the cochlea.

When sinusoidal electric stimulation is applied to the intact cochlea, a frequency-specific acoustic emission can be recorded in the ear canal. Acoustic emissions are produced by basilar membrane motion, and have been used to suggest a corresponding acoustic sensation termed "electromotile hearing." Electromotile hearing has been specifically attributed to electric stimulation of outer hair cells in the intact organ of Corti. To determine the nature of the auditory perception produced by electric stimulation of a cochlea with intact outer hair cells, guinea pigs were tested in a psychophysical task. First, subjects were trained to report detection of sinusoidal acoustic stimuli and dynamic range was assessed using response latency. Subjects were then implanted with a ball electrode placed into scala tympani. Following the surgical implant procedure, subjects were transferred to a task in which acoustic signals were replaced by sinusoidal electric stimulation, and dynamic range was assessed again. Finally, the ability of acoustic pure-tone stimuli to mask the detection of the electric signals was assessed. Based on the masking effects, it is concluded that sinusoidal electric stimulation of the intact cochlea results in perception of a tonal (rather than a broadband or noisy) sound at a frequency of 8 kHz or above.

The inferior colliculus (IC) is a processing center in both the ascending and descending auditory pathways. It has been demonstrated anatomically to send descending projections to the region of the medial olivocochlear (MOC) neurons in the auditory brainstem. Activation of MOC system produces reductions in cochlear neural activity. Individual MOC fibers innervate relatively restricted regions of the cochlea. Recent studies have shown that selective electrical stimulation within the IC central nucleus (ICC) produces frequency-specific reductions of neural activity in the contralateral cochlea (Ota, Y., Oliver, D.L., Dolan, D.F., 2004. Frequency-specific effects on cochlear responses during activation of the inferior colliculus in the guinea pig. J. Neurophysiol. 91, 2185-2193). This efferent effect is likely mediated through selective activation of MOC cells. In this study, we investigated the effects of selective stimulation of one ICC on cochlear output in both ears in anesthetized and paralyzed guinea pigs to explore possible differences in the effective efferent innervation of the two ears. ICC stimulation had a similar tonotopically tuned effect on the distortion product otoacoustic emission (DPOAE) and the cochlear whole-nerve action potential (CAP) in each cochlea. The bandwidth of the efferent effect in each ear was measured and compared at different stimulation levels. For a given ICC stimulation site, the efferent effects were larger for the CAP response. The effect on each response measure was greater in the contralateral than the ipsilateral ear. The effective bandwidth of the efferent effect on the CAP was current-level-dependent but less so for the DPOAE. The results of transections at various locations within the brainstem suggest that the effects were mediated by the MOC system. From the results presented here, the descending efferent system, which originates in the auditory cortex, has frequency-specific, spatially restricted, bilateral effects. The effects are greater in the contralateral ear.

The shaker2 (sh2) mouse is a murine model for human non-syndromic deafness DFNB3. The mice have abnormal circling behavior suggesting a balanced disorder, and profound deafness. The insertion of a bacterial artificial chromosome (BAC) transgene containing the Myo15a gene into sh2/sh2 zygotes confers hearing capability and abolishes the circling behavior in 1-month-old transgenic animals. In this study, we investigated both the hearing and the morphology of the cochlea in Myo15a mutants carrying this BAC transgene at two, four, or six months of age. The hearing threshold of these mice is normal, with no physiologically significant differences compared to age-matched heterozygous sh2J mice (with or without the BAC transgene). In six-month-old transgenic mice with the BAC, the morphology of hair cells in the apical and upper basal turns of the cochlea is normal. Hair cells of lower basal turn, however, were missing in some mutant animals. This study demonstrates that BAC transgene correction cannot only maintain normal morphology but also confer stable hearing function in Myo15a mutant mice for as long as 6 months. In addition, excess Myo15a expression has no physiologically significant protective or deleterious effects on hearing of normal mice, suggesting that the dosage of Myo15a may not be problematic for gene therapy.

Electrically evoked otoacoustic emissions (EEOAEs) are sounds measured in the ear canal when alternating current (AC) stimulation is passed into the cochlea. These sounds are attributed to the motile responses of outer hair cells (OHCs). The EEOAE has characteristic amplitude, phase, and fine structure. Multicomponent analysis of the EEOAE shows short (SDC) and long delay components (LDC) that are thought to originate from OHCs near the AC stimulating site and from OHCs at more remote locations, respectively. We measured the effects of various loud noise exposures on the EEOAE and the cochlear whole-nerve action potential (CAP) in animals chronically implanted with a scala tympani electrode. Noise exposures that produced permanent (PTS) or temporary threshold shifts (TTS) were associated with frequency-specific changes in CAP thresholds, EEOAE fine structure, and reductions in the amplitude of the LDC. A frequent observation in this study was an increase in the overall EEOAE amplitude after the noise exposure. The increase was correlated with increased SDC amplitude. The SDC was present in animals chemically treated with ototoxic drugs and mechanical damage to the cochlea. The SDC was eliminated after disarticulation of the ossicular chain. The presence of EEOAE fine structure in the postexposure response is an indicator of TTS in advance of CAP recovery. The results suggest that the EEOAE might be used to differentiate the mechanisms associated with TTS and PTS.

Brief cochlear excitotoxicity produces temporary neural swelling and transient deficits in auditory sensitivity; however, the consequences of long-lasting excitotoxic insult have not been tested. Chronic intra-cochlear infusion of the glutamate agonist AMPA (a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) resulted in functional deficits in the sound-evoked auditory brainstem response, as well as in behavioral measures of hearing. The electrophysiological deficits were similar to those observed following acute infusion of AMPA into the cochlea; however, the concentration-response curve was significantly shifted as a consequence of the slower infusion rate used with chronic cochlear administration. As observed following acute excitotoxic insult, complete functional recovery was evident within 7 days of discontinuing the AMPA infusion. Distortion product otoacoustic emissions were not affected by chronic AMPA infusion, suggesting that trauma to outer hair cells did not contribute to AMPA-induced deficits in acoustic sensitivity. Results from the current experiment address the permanence of deficits induced by chronic (14 day) excitotoxic insult as well as deficits in psychophysical detection of longer duration acoustic signals.

The unconventional myosin genes Myo15, Myo6 and Myo7a are essential for hearing in both humans and mice. Despite the expression of each gene in multiple organs, mutations result in identifiable phenotypes only in auditory or ocular sensory organs. The pirouette (pi) mouse also exhibits deafness and an inner ear pathology resembling that of Myo15 mutant mice and thus may be functionally related to Myo15. In order to investigate possible interactions between Myo15 and Myo6, Myo7a, and the gene affected in pirouette, we crossed Myo15(sh2/sh2) mice to the three other mutant mouse strains. Hearing in doubly heterozygous mice was similar to age-matched singly heterozygous animals, indicating that partial deficiency for both Myo15 and one of these other deafness genes does not reduce hearing. Viable double mutants were obtained from each cross, indicating that potential overlapping functions between these genes in other organs are not essential for viability. All critical cell types of the cochlear sensory epithelium were present in double mutant mice and cochlear stereocilia exhibited a superimposition of single mutant phenotypes. These data suggest that the function of Myo15 is distinct from that of Myo6, Myo7a or pi in development and/or maintenance of stereocilia.

Our previous study demonstrated rapamycin added to diet at 4 months of age had significantly less age-related outer hair cell loss in the basal half of the cochlea at 22 months of age compared to mice without rapamycin. The present study tested adding rapamycin to diet later in life, at 14 months of age, and added a longitudinal assessment of auditory brain stem response (ABR). The present study used UMHET4 mice, a 4 way cross in which all grandparental strains lack the Cdh23753A allele that predisposes to early onset, progressive hearing loss. UMHET4 mice typically have normal hearing until 16–17 months, then exhibit threshold shifts at low frequencies/apical cochlea and later in more basal high frequency regions. ABR thresholds at 4, 12, 24, and 48 kHz were assessed at 12, 18, and 24 months of age and compared to baseline ABR thresholds acquired at 5 months of age to determine threshold shifts (TS). There was no TS at 12 months of age at any frequency tested. At 18 months of age mice with rapamycin added to diet at 14 months had a significantly lower mean TS at 4 and 12 kHz compared to mice on control diet with no significant difference at 24 and 48 kHz. At 24 months of age, the mean 4 kHz TS in rapamycin diet group was no longer significantly lower than the control diet group, while the 12 kHz mean remained significantly lower. Mean TS at 24 and 48 kHz in the rapamycin diet group became significantly lower than in the control diet group at 24 months. Hair cell counts at 24 months showed large loss in the apical half of most rapamycin and control diet mice cochleae with no significant difference between groups. There was only mild outer hair cell loss in the basal half of rapamycin and control diet mice cochleae with no significant difference between groups. The results show that a later life addition of rapamycin can decrease age-related hearing loss in the mouse model, however, it also suggests that this decrease is a delay/deceleration rather than a complete prevention. [ABSTRACT FROM AUTHOR]

The human deafness-pigmentation syndromes, Waardenburg syndrome ( WS) type 2a, and Tietz syndrome are characterized by profound deafness but only partial cutaneous pigmentary abnormalities. Both syndromes are caused by mutations in MITF. To illuminate differences between cutaneous and otic melanocytes in these syndromes, their development and survival in heterozygous Microphthalmia- White ( Mitf Mi-wh/+) mice were studied and hearing function of these mice characterized. Mitf Mi-wh/+ mice have a profound hearing deficit, characterized by elevated auditory brainstem response thresholds, reduced distortion product otoacoustic emissions, absent endocochlear potential, loss of outer hair cells, and stria vascularis abnormalities. Mitf Mi-wh/+ embryos have fewer melanoblasts during embryonic development than their wild-type littermates. Although cochlear melanocytes are present at birth, they disappear from the Mitf Mi-wh/+ cochlea between P1 and P7. These findings may provide insight into the mechanism of melanocyte and hearing loss in human deafness-pigmentation syndromes such as WS and Tietz syndrome and illustrate differences between otic and follicular melanocytes. [ABSTRACT FROM AUTHOR]

Electrically evoked otoacoustic emissions (EEOAEs) are sounds measured in the ear canal when alternating current (AC) stimulation is passed into the cochlea. These sounds are attributed to the motile responses of outer hair cells (OHCs). The EEOAE has characteristic amplitude, phase, and fine structure. Multicomponent analysis of the EEOAE shows short (SDC) and long delay components (LDC) that are thought to originate from OHCs near the AC stimulating site and from OHCs at more remote locations, respectively. We measured the effects of various loud noise exposures on the EEOAE and the cochlear whole-nerve action potential (CAP) in animals chronically implanted with a scala tympani electrode. Noise exposures that produced permanent (PTS) or temporary threshold shifts (TTS) were associated with frequency-specific changes in CAP thresholds, EEOAE fine structure, and reductions in the amplitude of the LDC. A frequent observation in this study was an increase in the overall EEOAE amplitude after the noise exposure. The increase was correlated with increased SDC amplitude. The SDC was present in animals chemically treated with ototoxic drugs and mechanical damage to the cochlea. The SDC was eliminated after disarticulation of the ossicular chain. The presence of EEOAE fine structure in the postexposure response is an indicator of TTS in advance of CAP recovery. The results suggest that the EEOAE might be used to differentiate the mechanisms associated with TTS and PTS. [ABSTRACT FROM AUTHOR]

In the mammalian auditory system, sensory cell loss resulting from aging, ototoxic drugs, infections, overstimulation and other causes is irreversible and leads to permanent sensorineural hearing loss. To restore hearing, it is necessary to generate new functional hair cells. One potential way to regenerate hair cells is to induce a phenotypic transdifferentiation of nonsensory cells that remain in the deaf cochlea. Here we report that Atoh1, a gene also known as Math1 encoding a basic helix-loop-helix transcription factor and key regulator of hair cell development, induces regeneration of hair cells and substantially improves hearing thresholds in the mature deaf inner ear after delivery to nonsensory cells through adenovectors. This is the first demonstration of cellular and functional repair in the organ of Corti of a mature deaf mammal. The data suggest a new therapeutic approach based on expressing crucial developmental genes for cellular and functional restoration in the damaged auditory epithelium and other sensory systems. [ABSTRACT FROM AUTHOR]

• Small arms fire-like noise induced loss of inner hair cell synaptic ribbons in rat cochleae. • Small arms fire-like noise also reduced Gap Detection many noise-induced rats, this can be associated with Tinnitus. • Noise-exposed rats with reduced Gap Detection had greater loss of hair cell ribbons than those with normal Gap Detection. • Piribedil, memantine, and ACEMg treatment significantly reduced the noise-induced loss of ribbons. • A treatment induced reduction in incidence of noise-induced reduced Gap Detection did not reach significance. A noise-induced loss of inner hair cell (IHC) – auditory nerve synaptic connections has been suggested as a factor that can trigger the progression of maladaptive plastic changes leading to noise-induced tinnitus. The present study used a military relevant small arms fire (SAF)-like noise (50 biphasic impulses over 2.5 min at 152 dB SPL given unilaterally to the right ear) to induce loss (∼1/3) of IHC synaptic ribbons (associated with synapse loss) in rat cochleae with only minor (less than 10%) loss of outer hair cells. Approximately half of the noise-exposed rats showed poorer Gap Detection post-noise, a behavioral indication suggesting the presence of tinnitus. There was significantly greater loss of IHC ribbons in noise-exposed rats with reduced Gap Detection compared to noise-exposed rats retaining normal Gap Detection. We have previously shown systemic administration of piribedil, memantine, and/or ACEMg significantly reduced loss of IHC ribbons induced by a 3 h 4 kHz octave band 117 dB (SPL) noise. The present study examined if this treatment would also reduce ribbon loss from the SAF-like noise exposure and if this would prevent the reduced Gap Detection. As in the previous study, piribedil, memantine, and ACEMg treatment significantly reduced the noise-induced loss of ribbons, such that it was no longer significantly different from normal. However, it did not prevent development of the reduced Gap Detection indication of tinnitus in all treated noise-exposed rats, reducing the incidence but not reaching significance. [ABSTRACT FROM AUTHOR]

• A single exposure to noise given to young mice that does not cause immediate permanent hearing loss will accelerate and enhance later age-related hearing loss. • The effects of noise in youth on age-related hearing loss is largely an acceleration, with age-related hearing appearing earlier. • Different noise exposure conditions cause different paces and patterns of accelerated age-related hearing loss. Many factors contribute to hearing loss commonly found in older adults. There can be natural aging of cellular elements, hearing loss previously induced by environmental factors such as noise or ototoxic drugs as well as genetic and epigenetic influences. Even when noise overstimulation does not immediately cause permanent hearing loss it has recently been shown to increase later age-related hearing loss (ARHL). The present study further investigated this condition in the UMHET4 mouse model by comparing a small arms fire (SAF)-like impulse noise exposure that has the greatest immediate effect in more apical cochlear regions to a broadband noise (BBN) exposure that has the greatest immediate effect in more basal cochlear regions. Both noise exposures were given at levels that only induced temporary auditory brainstem response (ABR) threshold shifts (TS). Mice were noise exposed at 5 months of age followed by ABR assessment at 6, 12, 18, 21, and 24 months of age. Mice that received the SAF-like impulse noise had accelerated age-related TS at 4 kHz that appeared at 12 months of age (significantly increased compared to no-noise controls). This increased TS at 4 kHz continued at 18 and 21 months but was no longer significantly greater at 24 months of age. The SAF-like impulse noise also induced a significantly greater mean TS at 48 kHz, first appearing at 18 months of age and continuing to be significantly greater than controls at 21 and 24 months. The BBN induced a different pace and pattern of enhanced age-related ABR TS. The mean TS for the BBN group first became significantly greater than controls at 18 months of age and only at 48 kHz. It remained significantly greater than controls at 21 months but was no longer significantly greater at 24 months of age. Results, therefore, show different influences on ARHL for the two different noise exposure conditions. Noise-induced enhancement appears to provide more an acceleration than overall total increase in ARHL. [ABSTRACT FROM AUTHOR]

Thyroid hormone has pleiotropic effects on cochlear development, and genomic variation influences the severity of associated hearing deficits. DW/J-Pou1f1dw/dw mutant mice lack pituitary thyrotropin, which causes severe thyroid hormone deficiency and profound hearing impairment. To assess the genetic complexity of protective effects on hypothyroidism-induced hearing impairment, an F1 intercross was generated between DW/J-Pou1f1dw/+ carriers and an inbred strain with excellent hearing derived from Mus castaneus, CAST/EiJ. Approximately 24% of the (DW/J x CAST/EiJ) Pou1f1dw/dw F2 progeny had normal hearing. A genome scan revealed a locus on chromosome 2, named modifier of dw hearing, or Mdwh, that rescues hearing despite persistent hypothyroidism. This chromosomal region contains the modifier of tubby hearing 1 (Moth1) locus that encodes a protective allele of the microtubuleassociated protein MTAP1A. DW/J-Pou1f1dw/+carriers were crossed with the AKR strain, which also carries a protective allele of Mtap1a, and we found that AKR is not protective for hearing in the (DW/J x AKR) Pou1f1dw/dw F2 progeny. Thus, protective alleles of Mtap1a are not sufficient to rescue DW/J-Pou1f1dw/dw hearing. We expect that identification of protective modifiers will enhance our understanding of the mechanisms of hypothyroidism-induced hearing impairment. [ABSTRACT FROM AUTHOR]