Soft vibrotactile devices have the potential to expandthe functionalityof emerging electronic skin technologies. However, those devices oftenlack the necessary overall performance, sensing-actuation feedbackand control, and mechanical compliance for seamless integration onthe skin. Here, we present soft haptic electromagnetic actuators thatconsist of intrinsically stretchable conductors, pressure-sensitiveconductive foams, and soft magnetic composites. To minimize jouleheating, high-performance stretchable composite conductors are developedbased on in situ-grown silver nanoparticles formed within the silverflake framework. The conductors are laser-patterned to form soft anddensely packed coils to further minimize heating. Soft pressure-sensitiveconducting polymer-cellulose foams are developed and integrated totune the resonance frequency and to provide internal resonator amplitudesensing in the resonators. The above components together with a softmagnet are assembled into soft vibrotactile devices providing high-performanceactuation combined with amplitude sensing. We believe that soft hapticdevices will be an essential component in future developments of multifunctionalelectronic skin for future human-computer and human-roboticinterfaces.

It is a common belief that the mammalian cochlea achieves its exquisite sensitivity, frequency selectiv-ity, and dynamic range through an outer hair cell-based active process, or cochlear amplification. As a sound-induced traveling wave propagates from the cochlear base toward the apex, outer hair cells at a narrow region amplify the low level sound-induced vibration through a local feedback mechanism. This widely accepted theory has been tested by measuring sound-induced sub-nanometer vibrations within the organ of Corti in the sensitive living cochleae using heterodyne low-coherence interferometry and optical coherence tomography. The aim of this short review is to summarize experimental findings on the cochlear active process by the authors group. Our data show that outer hair cells are able to gener-ate substantial forces for driving the cochlear partition at all audible frequencies in vivo. The acoustically induced reticular lamina vibration is larger and more broadly tuned than the basilar membrane vibration. The reticular lamina and basilar membrane vibrate approximately in opposite directions at low frequen-cies and in the same direction at the best frequency. The group delay of the reticular lamina is larger than that of the basilar membrane. The magnitude and phase differences between the reticular lamina and basilar membrane vibration are physiologically vulnerable. These results contradict predictions based on the local feedback mechanism but suggest a global hydromechanical mechanism for cochlear amplifi-cation. This article is part of the Special Issue Outer hair cell Edited by Joseph Santos-Sacchi and Kumar Navaratnam. (c) 2021 Elsevier B.V. All rights reserved.

The cochlea maps tones with different frequencies to distinct anatomical locations. For instance, a faint 5000-hertz tone produces brisk responses at a place approximately 8 millimeters into the 18-millimeter-long guinea pig cochlea, but little response elsewhere. This place code pervades the auditory pathways, where neurons have "best frequencies" determined by their connections to the sensory cells in the hearing organ. However, frequency selectivity in cochlear regions encoding low-frequency sounds has not been systematically studied. Here, we show that low-frequency hearing works according to a unique principle that does not involve a place code. Instead, sound-evoked responses and temporal delays are similar across the low-frequency regions of the cochlea. These findings are a break from theories considered proven for 100 years and have broad implications for understanding information processing in the brainstem and cortex and for optimizing the stimulus delivery in auditory implants.

Cochlear distortions afford researchers and clinicians a glimpse into the conditions and properties of inner ear signal processing mechanisms. Until recently, our examination of these distortions has been limited to measuring the vibration of the basilar membrane or recording acoustic distortion output in the ear canal. Despite its importance, the generation mechanism of cochlear distortion remains a substantial task to understand. The ability to measure the vibration of the reticular lamina in rodent models is a recent experimental advance. Surprising mechanical properties have been revealed. These properties merit both discussion in context with our current understanding of distortion, and appraisal of the significance of new interpretations of cochlear mechanics. This review focusses on some of the recent data from our research groups and discusses the implications of these data on our understanding of vocalization processing in the periphery, and their influence upon future experimental directions. (C) 2021 Elsevier B.V. All rights reserved.

The prevailing theory of cochlear function states that outer hair cells amplify sound-induced vibration to improve hearing sensitivity and frequency specificity. Recent micromechanical measurements in the basal turn of gerbil cochleae through the round window have demonstrated that the reticular lamina vibration lags the basilar membrane vibration, and it is physiologically vulnerable not only at the best frequency but also at the low frequencies. These results suggest that outer hair cells from a broad cochlear region enhance hearing sensitivity through a global hydromechanical mechanism. However, the time difference between the reticular lamina and basilar membrane vibration has been thought to result from a systematic measurement error caused by the optical axis non-perpendicular to the cochlear partition. To address this concern, we measured the reticular lamina and basilar membrane vibrations in the transverse direction through an opening in the cochlear lateral wall in this study. Present results show that the phase difference between the reticular lamina and basilar membrane vibration decreases with frequency by similar to 180 degrees from low frequencies to the best frequency, consistent with those measured through the round window. Together with the round-window measurement, the low-coherence interferometry through the cochlear lateral wall demonstrates that the time difference between the reticular lamina and basilar membrane vibration results from the cochlear active processing rather than a measurement error.

Mammalian hearing depends on sound-evoked displacements of the stereocilia of inner hair cells (IHCs), which cause the endogenous mechanoelectrical transducer channels to conduct inward currents of cations including Ca2+. Due to their presumed lack of contacts with the overlaying tectorial membrane (TM), the putative stimulation mechanism for these stereocilia is by means of the viscous drag of the surrounding endolymph. However, despite numerous efforts to characterize the TM by electron microscopy and other techniques, the exact IHC stereocilia-TM relationship remains elusive. Here we show that Ca2+-rich filamentous structures, that we call Ca2+ ducts, connect the TM to the IHC stereocilia to enable mechanical stimulation by the TM while also ensuring the stereocilia access to TM Ca2+. Our results call for a reassessment of the stimulation mechanism for the IHC stereocilia and the TM role in hearing.

Millions of people are affected by hearing loss. Hearing loss is frequently caused by noise or aging and often associated with loss of pericytes. Pericytes populate the small vessels in the adult cochlea. However, their role in different types of hearing loss is largely unknown. Using an inducible and conditional pericyte depletion mouse model and noise-exposed mouse model, we show that loss of pericytes leads to marked changes in vascular structure, in turn leading to vascular degeneration and hearing loss. In vitro, using advanced tissue explants from pericyte fluorescence reporter models combined with exogenous donor pericytes, we show that pericytes, signaled by VEGF isoform A165 (VEGFA165), vigorously drive new vessel growth in both adult and neonatal mouse inner ear tissue. In vivo, the delivery of an adeno-associated virus serotype 1-mediated (AAV1-mediated) VEGFA165 viral vector to pericyte-depleted or noise-exposed animals prevented and regenerated lost pericytes, improved blood supply, and attenuated hearing loss. These studies provide the first clear-cut evidence that pericytes are critical for vascular regeneration, vascular stability, and hearing in adults. The restoration of vascular function in the damaged cochlea, including in noise-exposed animals, suggests that VEGFA165 gene therapy could be a new strategy for ameliorating vascular associated hearing disorders.

Sound and vibrations that cause the skull bone to vibrate can be heard as ordinary sounds and this is termed hearing by bone conduction (BC). Not all mechanisms that causes a skull vibration to result in BC hearing are known, and one such unknown is how the direction of the vibration influences BC hearing. This direction sensitivity was investigated by providing BC stimulation in five different directions at the vertex of the guinea pig skull. The hearing thresholds for BC stimulation was obtained in the frequency range of 2 to 20 kHz by measurements of compound action potential. During the stimulation by BC, the vibration of the cochlear promontory was measured with a three-dimensional laser Doppler vibrometer resulting in a set of unique three-dimensional velocity magnitude combinations for each threshold estimation. The sets of three-dimensional velocity magnitude at threshold were used to investigate nine different predictors of BC hearing based on cochlear promontory velocity magnitudes, six single direction (x, y and z directions in isolation, the normal to the stapes footplate, the oval to round window direction, and the cochlear base to apex direction), one linear combination of the three dimension velocity magnitudes, one square-rooted sum of the squared velocity magnitudes, and one sum of the weighted three dimensional velocity magnitudes based on a restricted minimum square error (MSE) estimation. The MSE gave the best predictions of the hearing threshold based on the cochlear promontory velocity magnitudes while using only a single direction gave the worst predictions of the hearing thresholds overall. According to the MSE estimation, at frequencies up to 8 kHz the vibration direction between the right and left side gave the greatest contribution to BC hearing in the guinea pig while at the highest frequencies measured, 16 and 20 kHz, the anteroposterior direction of the guinea pig head gave the greatest contribution.

The stereocilia of the inner ear sensory cells contain the actin-binding protein radixin, encoded by RDX. Radixin is important for hearing but remains functionally obscure. To determine how radixin influences hearing sensitivity, we used a custom rapid imaging technique to visualize stereocilia motion while measuring electrical potential amplitudes during acoustic stimulation. Radixin inhibition decreased sound-evoked electrical potentials. Other functional measures, including electrically induced sensory cell motility and sound-evoked stereocilia deflections, showed a minor amplitude increase. These unique functional alterations demonstrate radixin as necessary for conversion of sound into electrical signals at acoustic rates. We identified patients with RDX variants with normal hearing at birth who showed rapidly deteriorating hearing during the first months of life. This may be overlooked by newborn hearing screening and explained by multiple disturbances in postnatal sensory cells. We conclude radixin is necessary for ensuring normal conversion of sound to electrical signals in the inner ear.

Although human bone conduction (BC) hearing is well investigated, there is a lack of information about BC hearing in most other species. In humans, the amount of conductive loss is estimated as the difference between the air conduction (AC) and BC thresholds. Similar estimations for animals are difficult since in most species, the normal BC hearing thresholds have not been established. In the current study, the normal BC thresholds in the frequency range between 2 kHz and 20 kHz are investigated for the Guinea pig. Also, the effect of a middle ear lesion, here modelled by severing the ossicles (ossicular discontinuity) and gluing the ossicles to the bone (otosclerosis), is investigated for both AC and BC. The hearing thresholds in the Guinea pigs were estimated by a regression of the amplitude of the compound action potential (CAP) with stimulation level and was found robust and gave a high resolution of the threshold level. The reference for the BC thresholds was the cochlear promontory bone velocity. This reference enables comparison of BC hearing in animals, both intra and inter species, which is independent on the vibrator and stimulation position. The vibration was measured in three orthogonal directions where the dominating vibration directions was in line with the stimulation direction, here the ventral direction. The BC thresholds lay between -10 and 3 dB re 1 mu m/s. The slopes of CAP growth function were similar for AC and BC at low and high frequencies, but slightly lower for BC than AC at frequencies between 8 and 16 kHz. This was attributed to differences in the stimulus levels used for the slope estimation and not a real difference in CAP slopes between the stimulation modalities. Two kinds of middle ear lesions, ossicular discontinuity and stapes glued to the surrounding bone, gave threshold shifts of between 23 and 53 dB for AC while it was below 16 dB when the stimulation was by BC. Statistically different threshold shifts between the two types of lesions were found where the AC threshold shifts for a glued stapes at 2 and 4 kHz were 9-18 dB greater than for a severed ossicular chain, and the BC threshold shifts for a glued stapes at 4 and 12 kHz were 8-9 dB greater than fora severed ossicular chain. (C) 2019 Elsevier B.V. All rights reserved.

When sound stimulates the stereocilia on the sensory cells in the hearing organ, Ca2+ ions flow through mechanically gated ion channels. This Ca2+ influx is thought to be important for ensuring that the mechanically gated channels operate within their most sensitive response region, setting the fraction of channels open at rest, and possibly for the continued maintenance of stereocilia. Since the extracellular Ca2+ concentration will affect the amount of Ca2+ entering during stimulation, it is important to determine the level of the ion close to the sensory cells. Using fluorescence imaging and fluorescence correlation spectroscopy, we measured the Ca2+ concentration near guinea pig stereocilia in situ. Surprisingly, we found that an acellular accessory structure close to the stereocilia, the tectorial membrane, had much higher Ca2+ than the surrounding fluid. Loud sounds depleted Ca2+ from the tectorial membrane, and Ca2+ manipulations had large effects on hair cell function. Hence, the tectorial membrane contributes to control of hearing sensitivity by influencing the ionic environment around the stereocilia.

Optical coherence tomography (OCT) has revolutionized physiological studies of the hearing organ, the vibration and morphology of which can now be measured without opening the surrounding bone. In this review, we provide an overview of OCT as used in the otological research, describing advances and different techniques in vibrometry, angiography, and structural imaging.

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.

Hair cell mechanoelectrical transduction and adaptation are believed to be regulated by extracellular calcium. However, the majority of experiments addressing calciums role have been performed on reduced preparations in conditions that do not mimic those present in vivo. We used confocal microscopy and a low affinity (k(d) similar to 11 mu M) ratiometric fluorescent indicator to measure the extracellular calcium concentration in scala media in an in vitro preparation of the guinea pig cochlea. Microelectrodes were used to measure the cochlear microphonic potential during acoustic stimulation. The mean calcium concentration is significantly higher in the tectorial membrane (TM) than the surrounding endolymph, suggesting that the membrane acts as a calcium sink. We also observe calcium hot spots along the underside of the TM, near the outer hair cell bundles and near Hensens stripe close to the inner hair cell bundle. This suggests that the local calcium concentration near the hair bundles exceeds 100 mu M, significantly higher than the bulk endolymph. These results were corroborated with fluorescence correlation spectroscopy using a second calcium sensitive dye, Oregon Green 488-BAPTA. Following a brief exposure to loud sound, TM calcium drops dramatically and shows recovery on a similar timescale as the microphonic potential. Our results suggest that the extracellular calcium concentration near the hair bundles is much higher than previously believed and may also serve as a partial control parameter for temporary threshold shifts.

Mammalian hearing depends on deflection of stereocilia on the sensory outer hair cells of the inner ear. Previous data indicate that the stiffness of outer hair cell stereocilia are actively regulated. The molecular mechanism that regulate the deflection of stereocilia are presently less known. The aim of the study is to investigate the mechanistic pathway that underlie the stiffness modulation of outer hair cell stereocilia. Our hypothesis is that the membrane-cytoskeleton linker protein radixin, which is present at high concentration in stereocilia, could contribute to stiffness regulation. To test this hypothesis, we use the radixin blocker DX-52-1 which binds strongly and specifically to radixin. Time-resolved confocal imaging was used to visualize the sound-evoked motion of stereocilia in a semi-intact preparation of the guinea pig temporal bone. Cochlear microphonic potentials were also measured, using electrodes positioned in scala media. We found that the DX-52-1 inhibitor leads to an increase in stereocilia movements and decline in the amplitude of the cochlear microphonic potential. However, DX-52-1 caused a paradoxical increase in electromotility. These results suggest that radixin has a functionally important regulatory role in the mature inner ear.

The cochlea not only transduces sound-induced vibration into neural spikes, it also amplifies weak sound to boost its detection. Actuators of this active process are sensory outer hair cells in the organ of Corti, whereas the inner hair cells transduce the resulting motion into electric signals that propagate via the auditory nerve to the brain. However, how the outer hair cells modulate the stimulus to the inner hair cells remains unclear. Here, we combine theoretical modeling and experimental measurements near the cochlear apex to study the way in which length changes of the outer hair cells deform the organ of Corti. We develop a geometry-based kinematic model of the apical organ of Corti that reproduces salient, yet counter-intuitive features of the organs motion. Our analysis further uncovers a mechanism by which a static length change of the outer hair cells can sensitively tune the signal transmitted to the sensory inner hair cells. When the outer hair cells are in an elongated state, stimulation of inner hair cells is largely inhibited, whereas outer hair cell contraction leads to a substantial enhancement of sound-evoked motion near the hair bundles. This novel mechanism for regulating the sensitivity of the hearing organ applies to the low frequencies that are most important for the perception of speech and music. We suggest that the proposed mechanism might underlie frequency discrimination at low auditory frequencies, as well as our ability to selectively attend auditory signals in noisy surroundings.

Low-frequency hearing is critically important for speech and music perception, but no mechanical measurements have previously been available from inner ears with intact low-frequency parts. These regions of the cochlea may function in ways different from the extensively studied high-frequency regions, where the sensory outer hair cells produce force that greatly increases the sound-evoked vibrations of the basilar membrane. We used laser interferometry in vitro and optical coherence tomography in vivo to study the low-frequency part of the guinea pig cochlea, and found that sound stimulation caused motion of a minimal portion of the basilar membrane. Outside the region of peak movement, an exponential decline in motion amplitude occurred across the basilar membrane. The moving region had different dependence on stimulus frequency than the vibrations measured near the mechanosensitive stereocilia. This behavior differs substantially from the behavior found in the extensively studied high-frequency regions of the cochlea.

Sound processing in the inner ear involves separation of the constituent frequencies along the length of the cochlea. Frequencies relevant to human speech (100 to 500 Hz) are processed in the apex region. Among mammals, the guinea pig cochlear apex processes similar frequencies and is thus relevant for the study of speech processing in the cochlea. However, the requirement for extensive surgery has challenged the optical accessibility of this area to investigate cochlear processing of signals without significant intrusion. A simple method is developed to provide optical access to the guinea pig cochlear apex in two directions with minimal surgery. Furthermore, all prior vibration measurements in the guinea pig apex involved opening an observation hole in the otic capsule, which has been questioned on the basis of the resulting changes to cochlear hydrodynamics. Here, this limitation is overcome by measuring the vibrations through the unopened otic capsule using phase-sensitive Fourier domain optical coherence tomography. The optically and surgically advanced method described here lays the foundation to perform minimally invasive investigation of speech-related signal processing in the cochlea. (C) The Authors. Published by SPIE under a Creative Commons Attribution 3.0 Unported License.

Background Otosclerosis is a disorder that impairs middle ear function, leading to conductive hearing loss. Surgical treatment results in large improvement of hearing at low sound frequencies, but high-frequency hearing often suffers. A likely reason for this is that inner ear sensory cells are damaged by surgical trauma and loud sounds generated during the operation. Animal studies have shown that antioxidants such as N-Acetylcysteine can protect the inner ear from noise, surgical trauma, and some ototoxic substances, but it is not known if this works in humans. This trial was performed to determine whether antioxidants improve surgical results at high frequencies. Methods We performed a randomized, double-blind and placebo-controlled parallel group clinical trial at three Swedish university clinics. Using block-stratified randomization, 156 adult patients undergoing stapedotomy were assigned to intravenous N-Acetylcysteine (150 mg/kg body weight) or matching placebo (1:1 ratio), starting one hour before surgery. The primary outcome was the hearing threshold at 6 and 8 kHz; secondary outcomes included the severity of tinnitus and vertigo. Findings One year after surgery, high-frequency hearing had improved 2.7 +/- 3.8 dB in the placebo group (67 patients analysed) and 2.4 +/- 3.7 dB in the treated group (72 patients; means +/- 95% confidence interval, p = 0.54; linear mixed model). Surgery improved tinnitus, but there was no significant intergroup difference. Post-operative balance disturbance was common but improved during the first year, without significant difference between groups. Four patients receiving N-Acetylcysteine experienced mild side effects such as nausea and vomiting. Conclusions N-Acetylcysteine has no effect on hearing thresholds, tinnitus, or balance disturbance after stapedotomy.

Hearing depends on sound-evoked deflections of the stereocilia that protrude from the sensory hair cells in the inner ear. Although sound provides an important force driving stereocilia, forces generated through mechanically sensitive ion channels and through the motor protein prestin have been shown to influence stereocilia motion in solitary hair cells. While a possible influence of prestin on mechanically sensitive ion channels has not been systematically investigated, a decrease in transducer currents is evident in solitary hair cells when prestin is blocked with salicylate, raising the question of whether a reduced prestin activity or salicylate itself affected the mechanotransduction apparatus. We used two- and three-dimensional time-resolved confocal imaging to visualize outer hair cell stereocilia during sound stimulation in the apical turn of cochlear explant preparations from the guinea pig. Surprisingly, following application of salicylate, outer hair cell stereocilia deflections increased, while cochlear microphonic potentials decreased. However, when prestin activity was altered with the chloride ionophore tributyltin, both the cochlear microphonic potential and the stereocilia deflection amplitude decreased. Neither positive nor negative current stimulation abolished the bundle movements in the presence of salicylate, indicating that the observed effects did not depend on the endocochlear potential. These data suggest that salicylate may alter the mechanical properties of stereocilia, decreasing their bending stiffness.

Otosclerosis is a common disorder that leads to conductive hearing loss. Most patients with otosclerosis also have tinnitus, and surgical treatment is known to improve hearing as well as tinnitus. Some patients however experience worsening of tinnitus after the operation, but there are no known factors that allow surgeons to predict who will be at risk. In this prospective observational study on 133 patients undergoing stapedotomy, we show that postoperative air conduction thresholds at very high stimulus frequencies predict improvement of tinnitus, as assessed with proportional odds logistic regression models. Young patients were significantly more likely to experience reduction of tinnitus and patients whose tinnitus became better were also more satisfied with the outcome of the operation. These findings have practical importance for patients and their surgeons. Young patients can be advised that surgery is likely to be beneficial for their tinnitus, but a less positive message should be conveyed to older patients.

The exceptional sensitivity of mammalian hearing organ is attributed to an outer hair cell-mediated active process, where forces produced by sensory cells boost sound-induced vibrations, making soft sounds audible. This process is thought to be local, with each section of the hearing organ capable of amplifying sound-evoked movement, and nearly instantaneous, since amplification can work for sounds at frequencies up to 100 kHz in some species. To test these precepts, we developed a method for focally stimulating the living hearing organ with light. Light pulses caused intense and highly damped mechanical responses followed by traveling waves that developed with considerable delay. The delayed response was identical to movements evoked by click-like sounds. A physiologically based mathematical model shows that such waves engage the active process, enhancing hearing sensitivity. The experiments and the theoretical analysis show that the active process is neither local nor instantaneous, but requires mechanical waves traveling from the cochlear base toward its apex.

Exposure to loud sounds can lead to both permanent and short term changes in auditory sensitivity. Permanent hearing loss is often associated with gross changes in cochlear morphology including the loss of hair cells and auditory nerve fibers while the mechanisms of short term threshold shifts are much less well understood and may vary at different locations across the cochlea. Previous reports suggest that exposure to loud sounds leads to a decrease in the cochlear microphonic potential and in the stiffness of the organ of Corti. Because the cochlear microphonic reflects changes in the membrane potential of the hair cells, this suggests that hair-bundle motion should be reversibly altered following exposure to loud sounds. Using an in vitro preparation of the guinea pig temporal bone we investigate changes in the micro-mechanical response near the cochlear apex following a brief (up to 10 - 20 minutes) exposure to loud (similar to 120 dB) tones near the best frequency at this location. We use time-resolved confocal imaging to record the motion of outer hair cell bundles before and after acoustic overstimulation. We have also recorded larger-scale structural views of the organ of Corti before and after exposure to the loud sound. Conventional electrophysiological techniques are used measure the cochlear microphonic potential. As has been previously reported, following acoustic overexposure the cochlear microphonic declines in value and typically recovers on the order of 30 - 60 minutes. Hair-bundle trajectories are affected following the loud sound and typically recover on a somewhat faster time scale than the microphonic potential, although the results vary considerably across preparations. Preliminary results also suggest reversible changes in the hair cells resting potential following the loud sound.

Nature's fastest motors are the cochlear outer hair cells (OHCs). These sensory cells use a membrane protein, Slc26a5 (prestin), to generate mechanical force at high frequencies, which is essential for explaining the exquisite hearing sensitivity of mammalian ears. Previous studies suggest that Slc26a5 continuously diffuses within the membrane, but how can a freely moving motor protein effectively convey forces critical for hearing? To provide direct evidence in OHCs for freely moving Slc26a5 molecules, we created a knockin mouse where Slc26a5 is fused with YFP. These mice and four other strains expressing fluorescently labeled membrane proteins were used to examine their lateral diffusion in the OHC lateral wall. All five proteins showed minimal diffusion, but did move after pharmacological disruption of membrane-associated structures with a cholesterol-depleting agent and salicylate. Thus, our results demonstrate that OHC lateral wall structure constrains the mobility of plasma membrane proteins and that the integrity of such membrane-associated structures are critical for Slc26a5's active and structural roles. The structural constraint of membrane proteins may exemplify convergent evolution of cellular motors across species. Our findings also suggest a possible mechanism for disorders of cholesterol metabolism with hearing loss such as Niemann-Pick Type C diseases.

Objectives Questionnaire studies suggest that hearing is declining among young adults. However, few studies have examined the reliability of hearing questionnaires among young adult subjects. This study examined the associations between pure tone audiometrically assessed (PTA) hearing loss and questionnaire responses in young to middle aged adults. Materials and Methods A cross-sectional study using questionnaire and screening PTA (500 through 6000 Hz) data from 15322 Swedish subjects (62% women) aged 18 through 50 years. PTA hearing loss was defined as a hearing threshold above 20 dB in both ears at one or more frequencies. Data were analysed with chi-square tests, nonlinear regression, binary logistic regression, and the generalized estimating equation (GEE) approach. Results The prevalence of PTA hearing loss was 6.0% in men and 2.9% in women (p less than 0.001). Slight hearing impairment was reported by 18.5% of the men and 14.8% of the women (p less than 0.001), whereas 0.5% of men and women reported very impaired hearing. Using multivariate GEE modelling, the odds ratio of PTA hearing loss was 30.4 (95% CI, 12.7-72.9) in men and 36.5 (17.2-77.3) in women reporting very impaired hearing. The corresponding figures in those reporting slightly impaired hearing were 7.06 (5.25-9.49) in men and 8.99 (6.38-12.7) in women. These values depended on the sound stimulus frequency (p = 0.001). The area under the ROC curve was 0.904 (0.892-0.915) in men and 0.886 (0.872-0.900) in women. Conclusions Subjective hearing impairment predicted clinically assessed hearing loss, suggesting that there is cause for concern as regards the future development of hearing in young to middle-aged people.

The precise mechanical behavior of the basilar membrane (BM) at low frequencies is still unknown. To address this issue we use an in vitro preparation of the guinea pig temporal bone to investigate the mechanical behaviour of the organ of Corti at the apex of the cochlea. Confocal laser interferometry is used to record the nanometre displacements of both Hensens cells (HeC) and the BM in response to sound and electrical stimulation. We show that at low frequencies, the BM exhibits greatly reduced sound-evoked movement (similar to 35dB less) and no current-evoked movement, when compared to the HeC at the same position along the spiral. The BM best frequency is found to be an average of 52Hz (0.35 octave) higher than the HeC best frequency. In addition, we demonstrate that BM motion is not affected by inhibition of somatic electromotility or by blocking the mechanoelectrical transduction channels. We therefore propose that the BM primarily acts as a passive support structure at the cochlear apex. We suggest that the micromechanics of the cochlea that are vital to low-frequency amplification and frequency selectivity take place predominantly at the surface of the organ of Corti.

We measured sound-evoked vibrations at the stereociliary side of inner and outer hair cells and their surrounding supporting cells, using optical coherence tomography interferometry in living anesthetized guinea pigs. Our measurements demonstrate a gradient in frequency tuning among different cell types, going from a high best frequency at the inner hair cells to a lower one at the Hensen cells. This causes the locus of maximum inner hair cell activation to be shifted toward the apex of the cochlea as compared to the outer hair cells. These observations show that additional processing and filtering of acoustic signals occurs within the organ of Corti prior to inner hair cell excitation, thus reinstating a transformed second filter as a mechanism contributing to cochlear frequency tuning.

In this study, we measure the in vivo apical-turn vibrations of the guinea pig organ of Corti in both axial and radial directions using phase-sensitive Fourier domain optical coherence tomography. The apical turn in guinea pig cochlea has best frequencies around 100 - 500 Hz which are relevant for human speech. Prior measurements of vibrations in the guinea pig apex involved opening the otic capsule, which has been questioned on the basis of the resulting changes to cochlear hydrodynamics. Here this limitation is overcome by measuring the vibrations through bone without opening the otic capsule. Furthermore, we have significantly reduced the surgery needed to access the guinea pig apex in the axial direction by introducing a miniature mirror inside the bulla. The method and preliminary data are discussed in this article.

The following is an edited transcript of a recorded discussion session on the topic of "What Shapes the Stimulus to the Inner Hair Cell?". The discussion, moderated by the authors, took place at the 12th International Workshop on the Mechanics of Hearing held at Cape Sounio, Greece, in June 2014. All participants knew that the session was being recorded. In view of both the spontaneous nature of the discussion and the editing, however, this transcript may not represent the considered or final views of the participants, and may not represent a consensus of experts in the field. The reader is advised to consult additional independent publications.

The detection of sound by the mammalian hearing organ involves a complex mechanical interplay among different cell types. The inner hair cells, which are the primary sensory receptors, are stimulated by the structural vibrations of the entire organ of Corti. The outer hair cells are thought to modulate these sound-evoked vibrations to enhance hearing sensitivity and frequency resolution, but it remains unclear whether other structures also contribute to frequency tuning. In the current study, sound-evoked vibrations were measured at the stereociliary side of inner and outer hair cells and their surrounding supporting cells, using optical coherence tomography interferometry in living anesthetized guinea pigs. Our measurements demonstrate the presence of multiple vibration modes as well as significant differences in frequency tuning and response phase among different cell types. In particular, the frequency tuning at the inner hair cells differs from other cell types, causing the locus of maximum inner hair cell activation to be shifted toward the apex of the cochlea compared with the outer hair cells. These observations show that additional processing and filtering of acoustic signals occur within the organ of Corti before inner hair cell excitation, representing a departure from established theories.

The exceptional sensitivity of mammalian hearing organs is attributed to an active process, where force produced by sensory cells boost sound-induced vibrations, making soft sounds audible. This process is thought to be local, with each section of the hearing organ capable of amplifying sound-evoked movement, and nearly instantaneous, since amplification can work for sounds at frequencies up to 100 kHz in some species. To test these fundamental precepts, we developed a method for focally stimulating the living hearing organ with light. Light pulses caused intense and highly damped mechanical responses followed by traveling waves that developed with considerable delay. The delayed response was identical to movements evoked by click-like sounds. This shows that the active process is neither local nor instantaneous, but requires mechanical waves traveling from the cochlear base toward its apex. A physiologically-based mathematical model shows that such waves engage the active process, enhancing hearing sensitivity.

This protocol describes a growth medium-based approach for obtaining cochlear endothelial cells (ECs), pericytes (PCs) and perivascular resident macrophage-like melanocytes (PVM/Ms) from the stria vascularis of mice aged between P10 and P15 (P, postnatal day). The procedure does not involve mechanical or enzymatic digestion of the sample tissue. Explants of stria vascularis, 'mini-chips', are selectively cultured in growth medium, and primary cell lines are obtained in 7-10 d. The method is simple and reliable, and it provides high-quality ECs, PVM/Ms and PCs with a purity >90% after two passages. This protocol is suitable for producing primary culture cells from organs and tissues of small volume and high anatomical complexity, such as the inner ear capillaries. The highly purified primary cell lines enable cell culture-based in vitro modeling of cell-cell interactions, barrier control function and drug action.

Loud sounds are a common cause of hearing loss. Very intense sounds may result in permanent hearing loss, but lower levels typically cause a transient decrease in auditory sensitivity. Studies have arrived at different conclusions as regards the physiological mechanisms underlying such temporary threshold shifts. Here, we investigated the effect of acoustic overstimulation on the mechanics of the low-frequency areas of the guinea pig cochlea. We demonstrate that brief loud sound exposure results in an increased phase lag and a paradoxical frequency-specific increase of sound-evoked displacement. Despite the increased displacement, electrically evoked motion is reduced. Because electromotility is important for amplifying low-level sounds, this change was associated with a decrease in measures of cochlear amplification. These changes recovered over the course of 30-40 min. Overstimulation also caused an increase in cytoplasmic calcium levels of both hair cells and supporting cells. These data suggest that reduced organ of Corti stiffness contributes to temporary threshold shifts.

Tissue perivascular resident macrophages (PVM/Ms), a hybrid cell type with characteristics of both macrophages and melanocytes, are critical for establishing and maintaining the endocochlear potential (EP) required for hearing. The PVM/Ms modulate expression of tight- and adherens-junction proteins in the endothelial barrier of the stria vascularis (intrastrial fluid-blood barrier) through secretion of a signaling molecule, pigment epithelium growth factor (PEDF). Here, we identify a significant link between abnormalities in PVM/Ms and endothelial barrier breakdown from acoustic trauma to the mouse ear. We find that acoustic trauma causes activation of PVM/Ms and physical detachment from capillary walls. Concurrent with the detachment, we find loosened tight junctions between endothelial cells and decreased production of tight- and adherens-junction protein, resulting in leakage of serum proteins from the damaged barrier. A key factor in the intrastrial fluid-blood barrier hyperpermeability exhibited in the mice is down-regulation of PVM/M modulated PEDF production. We demonstrate that delivery of PEDF to the damaged ear ameliorates hearing loss by restoring intrastrial fluid-blood barrier integrity. PEDF up-regulates expression of tight junction-associated proteins (ZO-1 and VE-cadherin) and PVM/M stabilizing neural cell adhesion molecule (NCAM-120). These studies point to the critical role PVM/Ms play in regulating intrastrial fluid-blood barrier integrity in healthy and noise-damaged ears.

BACKGROUND: Mammalian hearing is refined by amplification of the sound-evoked vibration of the cochlear partition. This amplification is at least partly due to forces produced by protein motors residing in the cylindrical body of the outer hair cell. To transmit power to the cochlear partition, it is required that the outer hair cells dynamically change their length, in addition to generating force. These length changes, which have not previously been measured in vivo, must be correctly timed with the acoustic stimulus to produce amplification.

METHODOLOGY/PRINCIPAL FINDINGS: Using in vivo optical coherence tomography, we demonstrate that outer hair cells in living guinea pigs have length changes with unexpected timing and magnitudes that depend on the stimulus level in the sensitive cochlea.

CONCLUSIONS/SIGNIFICANCE: The level-dependent length change is a necessary condition for directly validating that power is expended by the active process presumed to underlie normal hearing.

Georg von Békésy designed the instruments needed for his research. He also created physical models of the cochlea allowing him to manipulate the parameters (such as volume elasticity) that could be involved in controlling traveling waves. This review is about the specific devices that he used to study the motion of the basilar membrane thus allowing the analysis that lead to his Nobel Prize Award. The review moves forward in time mentioning the subsequent use of von Békésy's methods and later technologies important for motion studies of the organ of Corti. Some of the seminal findings and the controversies of cochlear mechanics are mentioned in relation to the technical developments.

RNA interference (RNAi) using short interfering RNA (siRNA) is an attractive therapeutic approach for treatment of dominant-negative mutations. Some rare missense dominant-negative mutations lead to congenital-hearing impairments. A variety of viral vectors have been tested with variable efficacy for modulating gene expression in inner ear. However, there is concern regarding their safety for clinical use. Here, we report a novel cell-penetrating peptide (CPP)-based nonviral approach for delivering siRNA into inner ear tissue using organotypic cultures as model system. PepFect6 (PF6), a variant of stearyl-TP10, was specially designed for improved delivery of siRNA by facilitating endosomal release. We show that PF6 was internalized by all cells without inducing cytotoxicity in cochlear cultures. PF6/siRNA nanoparticles lead to knockdown of target genes, a housekeeping gene and supporting cell-specific connexin 26. Interestingly, application of PF6/connexin 26 siRNA exhibited knockdown of both connexin 26 and 30 mRNA and their absence led to impaired intercellular communication as demonstrated by reduced transfer of calcein among the PF6/connexin 26-siRNA-treated cells. Thus, we conclude that PF6 is an efficient nonviral vector for delivery of siRNA, which can be applied as a tool for the development of siRNA-based therapeutic applications for hearing impairments.Molecular Therapy - Nucleic Acids (2012) 1, e61; doi:10.1038/mtna.2012.50; published online 11 December 2012.

The microenvironment of the cochlea is maintained by the barrier between the systemic circulation and the fluids inside the stria vascularis. However, the mechanisms that control the permeability of the intrastrial fluid-blood barrier remain largely unknown. The barrier comprises endothelial cells connected to each other by tight junctions and an underlying basement membrane. In a recent study, we found that the intrastrial fluid-blood barrier also includes a large number of perivascular cells with both macrophage and melanocyte characteristics. The perivascular-resident macrophage-like melanocytes (PVM/Ms) are in close contact with vessels through cytoplasmic processes. Here we demonstrate that PVM/Ms have an important role in maintaining the integrity of the intrastrial fluid-blood barrier and hearing function. Using a cell culture-based in vitro model and a genetically induced PVM/M-depleted animal model, we show that absence of PVM/Ms increases the permeability of the intrastrial fluid-blood barrier to both low- and high-molecular-weight tracers. The increased permeability is caused by decreased expression of pigment epithelial-derived factor, which regulates expression of several tight junction-associated proteins instrumental to barrier integrity. When tested for endocochlear potential and auditory brainstem response, PVM/M-depleted animals show substantial drop in endocochlear potential with accompanying hearing loss. Our results demonstrate a critical role for PVM/Ms in regulating the permeability of the intrastrial fluid-blood barrier for establishing a normal endocochlear potential hearing threshold.

BACKGROUND: Hearing difficulties constitute the most common cause of disability globally. Yet, studies on people with hearing difficulties regarding socio-economic status (SES), work, long-term unemployment, sickness absence, and disability pension are scarce. The aim of the present study was to investigate the main income sources of men and women of working ages with and without self-reported hearing difficulties and associations with gender, age, SES, type of living area, and country of birth.

METHODS: A cross-sectional population-based study, using information on self-reported hearing difficulties and SES of 19 045 subjects aged 20-64 years participating in Statistics Sweden's annual Living Conditions Surveys in any of the years 2004 through 2008. The information was linked to a nationwide database containing data on demographics and income sources. Odds ratios (ORs) and their 95% confidence intervals (CIs) were calculated, using binary logistic regression analysis.

RESULTS: Hearing difficulties increased with age and were more common in men (age-adjusted OR: 1.42 (95% CI: 1.30-1.56)) with an overall prevalence of 13.1% in men and 9.8% in women. Using working men as reference, the OR of having hearing difficulties was 1.23 (0.94-1.60) in men with unemployment benefits and 1.36 (1.13-1.65) in men with sickness benefits or disability pension, when adjusting for age and SES. The corresponding figures in women were 1.59 (1.17-2.16) and 1.73 (1.46-2.06). The OR of having sickness benefits or disability pension in subjects with hearing difficulties was 1.36 (1.12-1.64) in men and 1.70 (1.43-2.01) in women, when adjusting for age and SES and using men and women with no hearing difficulties as reference.

CONCLUSIONS: Hearing difficulties were more prevalent in men. After adjustment with age and SES as well as with type of living area and country of birth, a significant association with unemployment benefits was found only in women, and the associations with long-term sickness absence and disability pension tended to be stronger in women.

Hearing relies on mechanical stimulation of stereocilia bundles on the sensory cells of the inner ear. When sound hits the ear, each stereocilium pivots about a neck-like taper near their base. More than three decades of research have established that sideways deflection of stereocilia is essential for converting mechanical stimuli into electrical signals. Here we show that mammalian outer hair cell stereocilia not only move sideways but also change length during sound stimulation. Currents that enter stereocilia through mechanically sensitive ion channels control the magnitude of both length changes and bundle deflections in a reciprocal manner: the smaller the length change, the larger is the bundle deflection. Thus, the transduction current is important for maintaining the resting mechanical properties of stereocilia. Hair cell stimulation is most effective when bundles are in a state that ensures minimal length change.

The ear is a remarkably sensitive pressure fluctuation detector. In guinea pigs, behavioral measurements indicate a minimum detectable sound pressure of ∼20 μPa at 16 kHz. Such faint sounds produce 0.1-nm basilar membrane displacements, a distance smaller than conformational transitions in ion channels. It seems that noise within the auditory system would swamp such tiny motions, making weak sounds imperceptible. Here we propose a new mechanism contributing to a resolution of this problem and validate it through direct measurement. We hypothesized that vibration at the apical side of hair cells is enhanced compared with that at the commonly measured basilar membrane side. Using in vivo optical coherence tomography, we demonstrated that apical-side vibrations peaked at a higher frequency, had different timing and were enhanced compared with those at the basilar membrane. These effects depend nonlinearly on the stimulus sound pressure level. The timing difference and enhancement of vibrations are important for explaining how the noise problem is circumvented.

Changing the concentration of cholesterol in the plasma membrane of isolated outer hair cells modulates electromotility and prestin-associated charge movement, suggesting that a similar manipulation would alter cochlear mechanics. We examined cochlear function before and after depletion of membrane cholesterol with methyl-β-cyclodextrin (MβCD) in an excised guinea pig temporal bone preparation. The mechanical response of the cochlear partition to acoustic and/or electrical stimulation was monitored using laser interferometry and time-resolved confocal microscopy. The electromechanical response in untreated preparations was asymmetric with greater displacements in response to positive currents. Exposure to MβCD increased the magnitude and asymmetry of the response, without changing the frequency tuning of sound-evoked mechanical responses or cochlear microphonic potentials. Sodium salicylate reversibly blocked the enhanced electromechanical response in cholesterol depleted preparations. The increase of sound-evoked vibrations during positive current injection was enhanced following MβCD in some preparations. Imaging was used to assess cellular integrity which remained unchanged after several hours of exposure to MβCD in several preparations. The enhanced electromechanical response reflects an increase in outer hair cell electromotility and may reveal features of cholesterol distribution and trafficking in outer hair cells.

Tones cause vibrations within the hearing organ. Conventionally, these vibrations are thought to reflect the input and therefore end with the stimulus. However, previous recordings of otoacoustic emissions and cochlear microphonic potentials suggest that the organ of Corti does continue to move after the end of a tone. These after-vibrations are characterized here through recordings of basilar membrane motion and hair cell extracellular receptor potentials in living anesthetized guinea pigs. We show that after-vibrations depend on the level and frequency of the stimulus, as well as on the sensitivity of the ear. Even a minor loss of hearing sensitivity caused a sharp reduction in after-vibration amplitude and duration. Mathematical models suggest that after-vibrations are driven by energy added into organ of Corti motion after the end of an acoustic stimulus. The possible importance of after-vibrations for psychophysical phenomena such as forward masking and gap detection are discussed.

The superior paraolivary nucleus (SPON) is a prominent structure in the auditory brainstem. In contrast to the principal superior olivary nuclei with identified roles in processing binaural sound localization cues, the role of the SPON in hearing is not well understood. A combined in vitro and in vivo approach was used to investigate the cellular properties of SPON neurons in the mouse. Patch-clamp recordings in brain slices revealed that brief and well timed postinhibitory rebound spiking, generated by the interaction of two subthreshold-activated ion currents, is a hallmark of SPON neurons. The I(h) current determines the timing of the rebound, whereas the T-type Ca(2+) current boosts the rebound to spike threshold. This precisely timed rebound spiking provides a physiological explanation for the sensitivity of SPON neurons to sinusoidally amplitude-modulated (SAM) tones in vivo, where peaks in the sound envelope drive inhibitory inputs and SPON neurons fire action potentials during the waveform troughs. Consistent with this notion, SPON neurons display intrinsic tuning to frequency-modulated sinusoidal currents (1-15Hz) in vitro and discharge with strong synchrony to SAMs with modulation frequencies between 1 and 20 Hz in vivo. The results of this study suggest that the SPON is particularly well suited to encode rhythmic sound patterns. Such temporal periodicity information is likely important for detection of communication cues, such as the acoustic envelopes of animal vocalizations and speech signals.

Acoustic stimulation gates mechanically sensitive ion channels in cochlear sensory hair cells. Even in the absence of sound, a fraction of these channels remains open, forming a conductance between hair cells and the adjacent fluid space, scala media. Restoring the lost endogenous polarization of scala media in an in vitro preparation of the whole cochlea depolarizes the hair cell soma. Using both digital laser interferometry and time-resolved confocal imaging, we show that this causes a structural refinement within the organ of Corti that is dependent on the somatic electromotility of the outer hair cells (OHCs). Specifically, the inner part of the reticular lamina up to the second row of OHCs is pulled toward the basilar membrane, whereas the outer part (third row of OHCs and the Hensen's cells) unexpectedly moves in the opposite direction. A similar differentiated response pattern is observed for sound-evoked vibrations: restoration of the endogenous polarization decreases vibrations of the inner part of the reticular lamina and results in up to a 10-fold increase of vibrations of the outer part. We conclude that the endogenous polarization of scala media affects the function of the hearing organ by altering its geometry, mechanical and electrical properties.

A method for three-dimensional motion analysis designed for live cell imaging by fluorescence confocal microscopy is described. The approach is based on optical flow computation and takes into account brightness variations in the image scene that are not due to motion, such as photobleaching or fluorescence variations that may reflect changes in cellular physiology. The 3-D optical flow algorithm allowed almost perfect motion estimation on noise-free artificial sequences, and performed with a relative error of <10% on noisy images typical of real experiments. The method was applied to a series of 3-D confocal image stacks from an in vitro preparation of the guinea pig cochlea. The complex motions caused by slow pressure changes in the cochlear compartments were quantified. At the surface of the hearing organ, the largest motion component was the transverse one (normal to the surface), but significant radial and longitudinal displacements were also present. The outer hair cell displayed larger radial motion at their basolateral membrane than at their apical surface. These movements reflect mechanical interactions between different cellular structures, which may be important for communicating sound-evoked vibrations to the sensory cells. A better understanding of these interactions is important for testing realistic models of cochlear mechanics.

The auditory sensory organ, the cochlea, not only detects but also generates sounds. Such sounds, otoacoustic emissions, are widely used for diagnosis of hearing disorders and to estimate cochlear nonlinearity. However, the fundamental question of how the otoacoustic emission exits the cochlea remains unanswered. In this study, emissions were provoked by two tones with a constant frequency ratio, and measured as vibrations at the basilar membrane and at the stapes, and as sound pressure in the ear canal. The propagation direction and delay of the emission were determined by measuring the phase difference between basilar membrane and stapes vibrations. These measurements show that cochlea-generated sound arrives at the stapes earlier than at the measured basilar membrane location. Data also show that basilar membrane vibration at the emission frequency is similar to that evoked by external tones. These results conflict with the backward-traveling-wave theory and suggest that at low and intermediate sound levels, the emission exits the cochlea predominantly through the cochlear fluids.

Noise, ototoxic substances and various genetic factors are common causes of profound hearing loss. Cochlear implants can often restore hearing in these cases, but only if a sufficient number of responsive auditory nerve fibers remain. Over time, these nerve fibers degenerate in the damaged ear, and it is therefore important to establish factors that control neuronal survival and maintain neural excitability. Recent studies show that neuregulins and their receptors are important for survival and proper targeting of neurons in the developing inner ear. A role for neuregulins as maintainers of the neuronal population in the mature inner ear was therefore hypothesized. Here, this hypothesis was directly tested by chronic local application of substances that block neuregulin receptors. Using auditory brainstem response measurements, we demonstrate that such receptor block leads to a progressive hearing impairment that develops over the course of weeks. This impairment occurs despite a normal number of auditory neurons and preserved outer hair cell function. Real-time quantitative reverse transcriptase-polymerase chain reaction shows alterations in neurotrophin-3 expression, suggesting that this growth factor participates in regulating cochlear sensitivity. The present work demonstrates the critical importance of neuregulin/erbB signaling in long-term functional regulation in the mature guinea pig hearing organ.

Laser interferometry is the technique of choice for studying the smallest displacements of the hearing organ. For low intensity sound stimulation, these displacements may be below 1 nm. This cannot be reliably measured with other presently available techniques in an intact organ of Corti. In a heterodyne interferometer, light is projected against an object of study and motion of the target along the optical axis causes phase and frequency modulations of the back-reflected light. To recover object motion, the reflected light is made to interfere with a reference beam of artificially altered frequency, producing a beating signal. In conventional interferometers, this carrier signal is demodulated with analog electronics. In this paper, we describe a digital implementation of the technique, using direct carrier sampling. In order to obtain the necessary reference signal for demodulation we introduce an additional third light path. Together, this results in lower noise and reduces the cost of the system.

Within the hearing organ, different structures may move in different directions. It is therefore necessary to precisely measure the angle of incidence of the laser light, and to precisely localize the anatomical structure where the measurement is performed. Therefore, the interferometer is integrated with a laser scanning confocal microscope that permits us to map crucial morphometric parameters in each experiment. We provide key construction parameters and a detailed performance characterization. We also show that the system accurately measures the diminutive vibrations present in the apical turn of the cochlea during low-level sound stimulation.

There are well known differences between males and females in hearing. In the present study, the role of estrogen receptor-beta (ER-beta; listed as ESR2 in the MGI Database) in hearing was investigated by comparing hearing and morphology of the inner ear in ER-beta knock-out mice (ER-beta(-/-)) with that of wild-type (WT) littermates. Hearing was analyzed with auditory brainstem response audiometry at 3 and 12 months. The ER-beta(-/-) mice were deaf at 1 year of age, and the morphological analysis showed absence of hair cells and loss of the whole organ of Corti initiated in the basal turn of the cochlea. Furthermore, in ER-beta(-/-), but not in WT mice, the spiral ganglion was lacking many of its neurons. Immunostaining showed the presence of both ER-alpha (listed as ESR1 in the MGI Database) and ER-beta in the nuclei of some neurons in the inner ear in WT mice, but no ER-beta was found in the ER-beta(-/-) mice as expected. ER-alpha staining was predominant in the nuclei of large neurons and ER-beta in nuclei of small neurons and fibroblasts. These results reveal that both ERs are present in the inner ear at specific localizations suggesting subtype-specific functions. It is concluded that ER-beta is important for the prevention of age-related hearing loss. These findings strengthen the hypothesis that estrogen has a direct effect on hearing functions.