Control of hearing sensitivity by tectorial membrane calcium.

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.

Static length changes of cochlear outer hair cells can tune low-frequency hearing.

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 organ's 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.

Minimal basilar membrane motion in low-frequency hearing.

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.

Outer Hair Cell Lateral Wall Structure Constrains the Mobility of Plasma Membrane Proteins.

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.

Filtering of acoustic signals within the hearing organ.

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.

Effects of salicylate on sound-evoked outer hair cell stereocilia deflections.

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.

Perivascular-resident macrophage-like melanocytes in the inner ear are essential for the integrity of the intrastrial fluid-blood barrier.

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.

Perivascular macrophage-like melanocyte responsiveness to acoustic trauma--a salient feature of strial barrier associated hearing loss.

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.

Noise-induced alterations in cochlear mechanics, electromotility, and cochlear amplification.

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.

Soft Electromagnetic Vibrotactile Actuators with Integrated Vibration Amplitude Sensing.

Soft vibrotactile devices have the potential to expand the functionality of emerging electronic skin technologies. However, those devices often lack the necessary overall performance, sensing-actuation feedback and control, and mechanical compliance for seamless integration on the skin. Here, we present soft haptic electromagnetic actuators that consist of intrinsically stretchable conductors, pressure-sensitive conductive foams, and soft magnetic composites. To minimize joule heating, high-performance stretchable composite conductors are developed based on in situ-grown silver nanoparticles formed within the silver flake framework. The conductors are laser-patterned to form soft and densely packed coils to further minimize heating. Soft pressure-sensitive conducting polymer-cellulose foams are developed and integrated to tune the resonance frequency and to provide internal resonator amplitude sensing in the resonators. The above components together with a soft magnet are assembled into soft vibrotactile devices providing high-performance actuation combined with amplitude sensing. We believe that soft haptic devices will be an essential component in future developments of multifunctional electronic skin for future human-computer and human-robotic interfaces.

Sound rhythms are encoded by postinhibitory rebound spiking in the superior paraolivary nucleus.

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.

Self-reported hearing difficulties, main income sources, and socio-economic status; a cross-sectional population-based study in Sweden.

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.

Best frequencies and temporal delays are similar across the low-frequency regions of the guinea pig cochlea.

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.

An outer hair cell-powered global hydromechanical mechanism for cochlear amplification.

It is a common belief that the mammalian cochlea achieves its exquisite sensitivity, frequency selectivity, 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 generate 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 frequencies 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 amplification. This article is part of the Special Issue Outer hair cell Edited by Joseph Santos-Sacchi and Kumar Navaratnam.

The reticular lamina and basilar membrane vibrations in the transverse direction in the basal turn of the living gerbil cochlea.

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 ~ 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.

The endocochlear potential alters cochlear micromechanics.

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.

Persistence of past stimulations: storing sounds within the inner ear.

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.

Membrane cholesterol modulates cochlear electromechanics.

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-ß-cyclodextrin (MßCD) 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 MßCD 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 MßCD in some preparations. Imaging was used to assess cellular integrity which remained unchanged after several hours of exposure to MßCD 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.

Reverse wave propagation in the cochlea.

Otoacoustic emissions, sounds generated by the inner ear, are widely used for diagnosing hearing disorders and studying cochlear mechanics. However, it remains unclear how emissions travel from their generation sites to the cochlear base. The prevailing view is that emissions reach the cochlear base via a backward-traveling wave, a slow-propagating transverse wave, along the cochlear partition. A different view is that emissions propagate to the cochlear base via the cochlear fluids as a compressional wave, a fast longitudinal wave. These theories were experimentally tested in this study by measuring basilar membrane (BM) vibrations at the cubic distortion product (DP) frequency from two longitudinal locations with a laser interferometer. Generation sites of DPs were varied by changing frequencies of primary tones while keeping the frequency ratio constant. Here, we show that BM vibration amplitude and phase at the DP frequency are very similar to responses evoked by external tones. Importantly, the BM vibration phase at a basal location leads that at a more apical location, indicating a traveling wave that propagates in the forward direction. These data are in conflict with the backward- traveling-wave theory but are consistent with the idea that the emission comes out of the cochlea predominantly through compressional waves in the cochlear fluids.

Inner hair cell stereocilia are embedded in the tectorial membrane.

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.