Normal hearing in mammals depends on sound amplification by outer hair cells (OHCs) presumably by their somatic motility and force production. However, the role of OHC force production in cochlear amplification and frequency tuning are not yet fully understood. Currently, available OHC manipulation techniques for physiological or clinical studies are limited by their invasive nature, lack of precision, and poor temporal-spatial resolution. To overcome these limitations, we explored an optogenetic approach based on channelrhodopsin 2 (ChR-2), a direct light-activated nonselective cation channel originally discovered in Chlamydomonas reinhardtii. Three approaches were compared: 1) adeno-associated virus-mediated in utero transfer of the ChR-2 gene into the developing murine otocyst, 2) expression of ChR-2(H134R) in an auditory cell line (HEI-OC1), and 3) expression of ChR-2 in the OHCs of a mouse line carrying a ChR-2 conditional allele. Whole cell recording showed that blue light (470 nm) elicited the typical nonselective cation current of ChR-2 with reversal potential around zero in both mouse OHCs and HEI-OC1 cells and generated depolarization in both cell types. In addition, pulsed light stimulation (10 Hz) elicited a 1:1 repetitive depolarization and ChR-2 currents in mouse OHCs and HEI-OC1 cells, respectively. The time constant of depolarization in OHCs, 1.45 ms, is 10 times faster than HEI-OC1 cells, which allowed light stimulation up to rates of 10/s to elicit corresponding membrane potential changes. Our study demonstrates that ChR-2 can successfully be expressed in mouse OHCs and HEI-OC1 cells and that these present a typical light-sensitive current and depolarization. However, the amount of ChR-2 current induced in our in vivo experiments was insufficient to result in measurable cochlear effects.

The phospholipid- and Ca(2+)-binding protein annexin A5 (ANXA5) is the most abundant membrane-associated protein of ~P23 mouse vestibular hair bundles, the inner ear's sensory organelle. Using quantitative mass spectrometry, we estimated that ANXA5 accounts for ~15,000 copies per stereocilium, or ~2% of the total protein there. Although seven other annexin genes are expressed in mouse utricles, mass spectrometry showed that none were present at levels near ANXA5 in bundles and none were upregulated in stereocilia of Anxa5(-/-) mice. Annexins have been proposed to mediate Ca(2+)-dependent repair of membrane lesions, which could be part of the repair mechanism in hair cells after noise damage. Nevertheless, mature Anxa5(-/-) mice not only have normal hearing and balance function, but following noise exposure, they are identical to wild-type mice in their temporary or permanent changes in hearing sensitivity. We suggest that despite the unusually high levels of ANXA5 in bundles, it does not play a role in the bundle's key function, mechanotransduction, at least until after two months of age in the cochlea and six months of age in the vestibular system. These results reinforce the lack of correlation between abundance of a protein in a specific compartment or cellular structure and its functional significance.

The endothelial-blood/tissue barrier is critical for maintaining tissue homeostasis. The ear harbors a unique endothelial-blood/tissue barrier which we term "blood-labyrinth-barrier". This barrier is critical for maintaining inner ear homeostasis. Disruption of the blood-labyrinth-barrier is closely associated with a number of hearing disorders. Many proteins of the blood-brain-barrier and blood-retinal-barrier have been identified, leading to significant advances in understanding their tissue specific functions. In contrast, capillaries in the ear are small in volume and anatomically complex. This presents a challenge for protein analysis studies, which has resulted in limited knowledge of the molecular and functional components of the blood-labyrinth-barrier. In this study, we developed a novel method for isolation of the stria vascularis capillary from CBA/CaJ mouse cochlea and provided the first database of protein components in the blood-labyrinth barrier as well as evidence that the interaction of Na(+)/K(+)-ATPase α1 (ATP1A1) with protein kinase C eta (PKCη) and occludin is one of the mechanisms of loud sound-induced vascular permeability increase.

The DFNB31 gene plays an indispensable role in the cochlea and retina. Mutations in this gene disrupt its various isoforms and lead to non-syndromic deafness, blindness and deaf-blindness. However, the known expression of Dfnb31, the mouse ortholog of DFNB31, in vestibular organs and the potential vestibular-deficient phenotype observed in one Dfnb31 mutant mouse (Dfnb31(wi/wi)) suggest that DFNB31 may also be important for vestibular function. In this study, we find that full-length (FL-) and C-terminal (C-) whirlin isoforms are expressed in the vestibular organs, where their stereociliary localizations are similar to those of developing cochlear inner hair cells. No whirlin is detected in Dfnb31(wi/wi) vestibular organs, while only C-whirlin is expressed in Dfnb31(neo/neo) vestibular organs. Both FL- and C-whirlin isoforms are required for normal vestibular stereociliary growth, although they may play slightly different roles in the central and peripheral zones of the crista ampullaris. Vestibular sensory-evoked potentials demonstrate severe to profound vestibular deficits in Dfnb31(neo/neo) and Dfnb31(wi/wi) mice. Swimming and rotarod tests demonstrate that the two Dfnb31 mutants have balance problems, with Dfnb31(wi/wi) mice being more affected than Dfnb31(neo/neo) mice. Because Dfnb31(wi/wi) and Dfnb31(neo/neo) mice faithfully recapitulate hearing and vision symptoms in patients, our findings of vestibular dysfunction in these Dfnb31 mutants raise the question of whether DFNB31-deficient patients may acquire vestibular as well as hearing and vision loss.

Signal transducers and activators of transcription 3 (STAT3) is a stress responsive transcription factor that plays a key role in oxidative stress-mediated tissue injury. As reactive oxygen species (ROS) are a known source of damage to tissues of the inner ear following loud sound exposure, we examined the role of the Janus kinase 2 (JAK2)/STAT3 signaling pathway in noise induce hearing loss using the pathway specific inhibitor, JSI-124. Mice were exposed to a moderately damaging level of loud sound revealing the phosphorylation of STAT3 tyrosine 705 residues and nuclear localization in many cell types in the inner ear including the marginal cells of the stria vascularis, type II, III, and IV fibrocytes, spiral ganglion cells, and in the inner hair cells. Treatment of the mice with the JAK2/STAT3 inhibitor before noise exposure reduced levels of phosphorylated STAT3 Y705. We performed auditory brain stem response and distortion product otoacoustic emission measurements and found increased recovery of hearing sensitivity at two weeks after noise exposure with JAK2/STAT3 inhibition. Performance of cytocochleograms revealed improved outer hair cell survival in JSI-124 treated mice relative to control. Finally, JAK2/STAT3 inhibition reduced levels of ROS detected in outer hair cells at two hours post noise exposure. Together, these findings demonstrate that inhibiting the JAK2/STAT3 signaling pathway is protective against noise-induced cochlear tissue damage and loss of hearing sensitivity.

A synchronized dual-wavelength laser speckle contrast imaging (DWLSCI) system and a Doppler optical microangiography (DOMAG) system was developed to determine several ischemic parameters in the cochlea due to a systemic hypoxic challenge. DWLSCI can obtain two-dimensional data, and was used to determine the relative changes in cochlear blood flow, and change in the concentrations of oxyhemoglobin (HbO), deoxyhemoglobin (Hb) and total hemoglobin (HbT) in mice. DOMAG can obtain three-dimensional data, and was used to determine the changes in cochlear blood flow with single vessel resolution. It was demonstrated that during a hypoxic challenge there was an increase in the concentrations of Hb, a decrease in the concentrations of HbO and cochlear blood flow, and a slight decrease in the concentration of HbT. Also, the rate of change in the concentrations of Hb and HbO was quantified during and after the hypoxic challenge. The ability to simultaneously measure these ischemic parameters with high spatio-temporal resolution will allow the detailed quantitative analysis of several hearing disorders, and will be useful for diagnosing and developing treatments.

With their essential role in inner ear function, stereocilia of sensory hair cells demonstrate the importance of cellular actin protrusions. Actin packing in stereocilia is mediated by cross-linkers of the plastin, fascin, and espin families. Although mice lacking espin (ESPN) have no vestibular or auditory function, we found that mice that either lacked plastin 1 (PLS1) or had nonfunctional fascin 2 (FSCN2) had reduced inner ear function, with double-mutant mice most strongly affected. Targeted mass spectrometry indicated that PLS1 was the most abundant cross-linker in vestibular stereocilia and the second most abundant protein overall; ESPN only accounted for ∼15% of the total cross-linkers in bundles. Mouse utricle stereocilia lacking PLS1 were shorter and thinner than wild-type stereocilia. Surprisingly, although wild-type stereocilia had random liquid packing of their actin filaments, stereocilia lacking PLS1 had orderly hexagonal packing. Although all three cross-linkers are required for stereocilia structure and function, PLS1 biases actin toward liquid packing, which allows stereocilia to grow to a greater diameter.

Calcitonin gene-related peptide (CGRP) is a neuroactive peptide that is thought to play a role at efferent synapses in hair cell organs including the cochlea, lateral line, and semicircular canal. The deletion of CGRP in transgenic mice is associated with a significant reduction in suprathreshold cochlear nerve activity and vestibulo-ocular reflex (VOR) gain efficacy when compared to littermate controls. Here we asked whether the loss of CGRP also influences otolithic end organ function and contributes to balance impairments. Immunostaining for CGRP was absent in the otolithic end organs of αCGRP null (-/-) mice while choline acetyltransferase (ChAT) immunolabeling appeared unchanged suggesting the overall gross development of efferent innervation in otolithic organs was unaltered. Otolithic function was assessed by quantifying the thresholds, suprathreshold amplitudes, and latencies of vestibular sensory-evoked potentials (VsEPs) while general balance function was assessed using a modified rotarod assay. The loss of αCGRP in null (-/-) mice was associated with: (1) shorter VsEP latencies without a concomitant change in amplitude or thresholds, and (2) deficits in the rotarod balance assay. Our findings show that CGRP loss results in faster otolith afferent activation timing, suggesting that the CGRP component of the efferent vestibular system (EVS) also plays a role in otolithic organ dynamics, which when coupled with reduced VOR gain efficacy, impairs balance.

The mammalian cochlea possesses unique acoustic sensitivity due to a mechanoelectrical 'amplifier', which requires the metabolic support of the cochlear lateral wall. Loud sound exposure sufficient to induce permanent hearing damage causes cochlear blood flow reduction, which may contribute to hearing loss. However, sensory epithelium involvement in the cochlear blood flow regulation pathway is not fully described. We hypothesize that genetic manipulation of the mechanoelectrical transducer complex will abolish sound induced cochlear blood flow regulation. We used salsa mice, a Chd23 mutant with no mechanoelectrical transduction, and deafness before p56. Using optical coherence tomography angiography, we measured the cochlear blood flow of salsa and wild-type mice in response to loud sound (120 dB SPL, 30 minutes low-pass filtered noise). An expected sound induced decrease in cochlear blood flow occurred in CBA/CaJ mice, but surprisingly the same sound protocol induced cochlear blood flow increases in salsa mice. Blood flow did not change in the contralateral ear. Disruption of the sympathetic nervous system partially abolished the observed wild-type blood flow decrease but not the salsa increase. Therefore sympathetic activation contributes to sound induced reduction of cochlear blood flow. Additionally a local, non-sensory pathway, potentially therapeutically targetable, must exist for cochlear blood flow regulation.

Cyclin-dependent kinase 2 (CDK2) is a potential therapeutic target for the treatment of hearing loss and cancer. Previously, we identified AZD5438 and AT7519-7 as potent inhibitors of CDK2, however, they also targeted additional kinases, leading to unwanted toxicities. Proteolysis Targeting Chimeras (PROTACs) are a new promising class of small molecules that can effectively direct specific proteins to proteasomal degradation. Herein we report the design, synthesis, and characterization of PROTACs of AT7519-7 and AZD5438 and the identification of PROTAC-8, an AZD5438-PROTAC, that exhibits selective, partial CDK2 degradation. Furthermore, PROTAC-8 protects against cisplatin ototoxicity and kainic acid excitotoxicity in zebrafish. Molecular dynamics simulations reveal the structural requirements for CDK2 degradation. Together, PROTAC-8 is among the first-in-class PROTACs with in vivo therapeutic activities and represents a new lead compound that can be further developed for better efficacy and selectivity for CDK2 degradation against hearing loss and cancer.

To understand speech, the slowly varying outline, or envelope, of the acoustic stimulus is used to distinguish words. A small amount of information about the envelope is sufficient for speech recognition, but the mechanism used by the auditory system to extract the envelope is not known. Several different theories have been proposed, including envelope detection by auditory nerve dendrites as well as various mechanisms involving the sensory hair cells. We used recordings from human and animal inner ears to show that the dominant mechanism for envelope detection is distortion introduced by mechanoelectrical transduction channels. This electrical distortion, which is not apparent in the sound-evoked vibrations of the basilar membrane, tracks the envelope, excites the auditory nerve, and transmits information about the shape of the envelope to the brain.

Shank proteins (1-3) are considered the master organizers of glutamatergic postsynaptic densities in the central nervous system, and the genetic deletion of either Shank1, 2, or 3 results in altered composition, form, and strength of glutamatergic postsynapses. To investigate the contribution of Shank proteins to glutamatergic afferent synapses of the inner ear and especially cochlea, we used immunofluorescence and quantitative real time PCR to determine the expression of Shank1, 2, and 3 in the cochlea. Because we found evidence for expression of Shank1 but not 2 and 3, we investigated the morphology, composition, and function of afferent postsynaptic densities from defined tonotopic regions in the cochlea of Shank1(-/-) mice. Using immunofluorescence, we identified subtle changes in the morphology and composition (but not number and localization) of cochlear afferent postsynaptic densities at the lower frequency region (8 kHz) in Shank1(-/-) mice compared to Shank1(+/+) littermates. However, we detected no differences in auditory brainstem responses at matching or higher frequencies. We also identified Shank1 in the vestibular afferent postsynaptic densities, but detected no differences in vestibular sensory evoked potentials in Shank1(-/-) mice compared to Shank1(+/+) littermates. This work suggests that Shank proteins play a different role in the development and maintenance of glutamatergic afferent synapses in the inner ear compared to the central nervous system.

Studies of developmental and functional biology largely rely on conditional expression of genes in a cell type-specific manner. Therefore, the importance of specificity and lack of inherent phenotypes for Cre-driver animals cannot be overemphasized. The Gfi1Cre mouse is commonly used for conditional hair cell-specific gene deletion/reporter gene activation in the inner ear. Here, using immunofluorescence and flow cytometry, we show that the Gfi1Cre mice produce a pattern of recombination that is not strictly limited to hair cells within the inner ear. We observe a broad expression of Cre recombinase in the Gfi1Cre mouse neonatal inner ear, primarily in inner ear resident macrophages, which outnumber the hair cells. We further show that heterozygous Gfi1Cre mice exhibit an early onset progressive hearing loss as compared with their wild-type littermates. Importantly, vestibular function remains intact in heterozygotes up to 10 months, the latest time point tested. Finally, we detect minor, but statistically significant, changes in expression of hair cell-enriched transcripts in the Gfi1Cre heterozygous mice cochleae compared with their wild-type littermate controls. Given the broad use of the Gfi1Cre mice, both for gene deletion and reporter gene activation, these data are significant and necessary for proper planning and interpretation of experiments.

Dizziness and hearing loss are among the most common disabilities. Many forms of hereditary balance and hearing disorders are caused by abnormal development of stereocilia, mechanosensory organelles on the apical surface of hair cells in the inner ear. The deaf whirler mouse, a model of human Usher syndrome (manifested by hearing loss, dizziness, and blindness), has a recessive mutation in the whirlin gene, which renders hair cell stereocilia short and dysfunctional. In this study, wild-type whirlin cDNA was delivered to the inner ears of neonatal whirler mice using adeno-associated virus serotype 2/8 (AAV8-whirlin) by injection into the posterior semicircular canal. Unilateral whirlin gene therapy injection was able to restore balance function as well as improve hearing in whirler mice for at least 4 months. Our data indicate that gene therapy is likely to become a treatment option for hereditary disorders of balance and hearing.

Sensorineural hearing loss is a common and currently irreversible disorder, because mammalian hair cells (HCs) do not regenerate and current stem cell and gene delivery protocols result only in immature HC-like cells. Importantly, although the transcriptional regulators of embryonic HC development have been described, little is known about the postnatal regulators of maturating HCs. Here we apply a cell type-specific functional genomic analysis to the transcriptomes of auditory and vestibular sensory epithelia from early postnatal mice. We identify RFX transcription factors as essential and evolutionarily conserved regulators of the HC-specific transcriptomes, and detect Rfx1,2,3,5 and 7 in the developing HCs. To understand the role of RFX in hearing, we generate Rfx1/3 conditional knockout mice. We show that these mice are deaf secondary to rapid loss of initially well-formed outer HCs. These data identify an essential role for RFX in hearing and survival of the terminally differentiating outer HCs.

Control of the dimensions of actin-rich processes like filopodia, lamellipodia, microvilli, and stereocilia requires the coordinated activity of many proteins. Each of these actin structures relies on heterodimeric capping protein (CAPZ), which blocks actin polymerization at barbed ends. Because dimension control of the inner ear's stereocilia is particularly precise, we studied the CAPZB subunit in hair cells. CAPZB, present at ∼100 copies per stereocilium, concentrated at stereocilia tips as hair cell development progressed, similar to the CAPZB-interacting protein TWF2. We deleted Capzb specifically in hair cells using Atoh1-Cre, which eliminated auditory and vestibular function. Capzb-null stereocilia initially developed normally but later shortened and disappeared; surprisingly, stereocilia width decreased concomitantly with length. CAPZB2 expressed by in utero electroporation prevented normal elongation of vestibular stereocilia and irregularly widened them. Together, these results suggest that capping protein participates in stereocilia widening by preventing newly elongating actin filaments from depolymerizing.

Each vestibular sensory epithelium in the inner ear is divided morphologically and physiologically into two zones, called the striola and extrastriola in otolith organ maculae, and the central and peripheral zones in semicircular canal cristae. We found that formation of striolar/central zones during embryogenesis requires Cytochrome P450 26b1 (Cyp26b1)-mediated degradation of retinoic acid (RA). In Cyp26b1 conditional knockout mice, formation of striolar/central zones is compromised, such that they resemble extrastriolar/peripheral zones in multiple features. Mutants have deficient vestibular evoked potential (VsEP) responses to jerk stimuli, head tremor and deficits in balance beam tests that are consistent with abnormal vestibular input, but normal vestibulo-ocular reflexes and apparently normal motor performance during swimming. Thus, degradation of RA during embryogenesis is required for formation of highly specialized regions of the vestibular sensory epithelia with specific functions in detecting head motions.

Age-related loss of vestibular function and hearing are common disorders that arise from the loss of function of the inner ear and significantly decrease quality of life. The underlying pathophysiological mechanisms are poorly understood and difficult to investigate in humans. Therefore, our study examined young (1.5-month-old) and old (24-month-old) C57BL/6 mice, utilizing physiological, histological, and transcriptomic methods. Vestibular sensory-evoked potentials revealed that older mice had reduced wave I amplitudes and delayed wave I latencies, indicating reduced vestibular function. Immunofluorescence and image analysis revealed that older mice exhibited a significant decline in type I sensory hair cell density, particularly in hair cells connected to dimorphic vestibular afferents. An analysis of gene expression in the isolated vestibule revealed the upregulation of immune-related genes and the downregulation of genes associated with ossification and nervous system development. A comparison with the isolated cochlear sensorineural structures showed similar changes in genes related to immune response, chondrocyte differentiation, and myelin formation. These findings suggest that age-related vestibular hypofunction is linked to diminished peripheral vestibular responses, likely due to the loss of a specific subpopulation of hair cells and calyceal afferents. The upregulation of immune- and inflammation-related genes implies that inflammation contributes to these functional and structural changes. Furthermore, the comparison of gene expression between the vestibule and cochlea indicates both shared and distinct mechanisms contributing to age-related vestibular and hearing impairments. Further research is necessary to understand the mechanistic connection between inflammation and age-related balance and hearing disorders and to translate these findings into clinical treatment strategies.

Little is known about the function of the cholinergic efferents innervating peripheral vestibular hair cells. We measured vestibular sensory evoked potentials (VsEPs) in α9 knockout (KO) mice, α10 KO mice, α7 KO mice, α9/10 and α7/9 double KO mice, and wild-type (WT) controls. We also studied the morphology and ultrastructure of efferent terminals on vestibular hair cells in α9, α10, and α9/10 KOs. Both type I and type ll vestibular hair cells express the α9 and α10 subunits. The efferent boutons on vestibular cells in α9, α10, and α9/10 KOs appeared normal, but a quantitative analysis was not performed. Mean VsEP thresholds were significantly elevated in α9 and α9/10 KO animals. Some α9 and α9/10 KO animals, however, had normal or near-normal thresholds, whereas others were greatly affected. Despite individual variability in threshold responses, latencies were consistently shortened. The double α7/9 KO resulted in decreased variance by normalizing waveforms and latencies. The phenotypes of the α7 and α10 single KOs were identical. Both α7 and α10 KO mice evidenced normal thresholds, decreased activation latencies, and larger amplitudes compared with WT mice. The data suggest a complex interaction of nicotinic acetylcholine receptors (nAChRs) in regulating vestibular afferent gain and activation timing. Although the α9/10 heteromeric nAChR is an important component of vestibular efferent activity, other peripheral or central nAChRs involving the α7 subunit or α10 subunit and α9 homomeric receptors are also important. J. Comp. Neurol. 525:1216-1233, 2017. © 2016 Wiley Periodicals, Inc.

Potassium (K+) channels shape the response properties of neurons. Although enormous progress has been made to characterize K+ channels in the primary auditory neurons, the molecular identities of many of these channels and their contributions to hearing in vivo remain unknown. Using a combination of RNA sequencing and single molecule fluorescent in situ hybridization, we localized expression of transcripts encoding the sodium-activated potassium channels KNa1.1 (SLO2.2/Slack) and KNa1.2 (SLO2.1/Slick) to the primary auditory neurons (spiral ganglion neurons, SGNs). To examine the contribution of these channels to function of the SGNs in vivo, we measured auditory brainstem responses in KNa1.1/1.2 double knockout (DKO) mice. Although auditory brainstem response (wave I) thresholds were not altered, the amplitudes of suprathreshold responses were reduced in DKO mice. This reduction in amplitude occurred despite normal numbers and molecular architecture of the SGNs and their synapses with the inner hair cells. Patch clamp electrophysiology of SGNs isolated from DKO mice displayed altered membrane properties, including reduced action potential thresholds and amplitudes. These findings show that KNa1 channel activity is essential for normal cochlear function and suggest that early forms of hearing loss may result from physiological changes in the activity of the primary auditory neurons.