Cochlear outer hair cell electromotility enhances organ of Corti motion on a cycle-by-cycle basis at high frequencies in vivo.

Mammalian hearing depends on an amplification process involving prestin, a voltage-sensitive motor protein that enables cochlear outer hair cells (OHCs) to change length and generate force. However, it has been questioned whether this prestin-based somatic electromotility can operate fast enough in vivo to amplify cochlear vibrations at the high frequencies that mammals hear. In this study, we measured sound-evoked vibrations from within the living mouse cochlea and found that the top and bottom of the OHCs move in opposite directions at frequencies exceeding 20 kHz, consistent with fast somatic length changes. These motions are physiologically vulnerable, depend on prestin, and dominate the cochlea's vibratory response to high-frequency sound. This dominance was observed despite mechanisms that clearly low-pass filter the in vivo electromotile response. Low-pass filtering therefore does not critically limit the OHC's ability to move the organ of Corti on a cycle-by-cycle basis. Our data argue that electromotility serves as the primary high-frequency amplifying mechanism within the mammalian cochlea.

Overturning the mechanisms of cochlear amplification via area deformations of the organ of Corti.

The mammalian ear embeds a cellular amplifier that boosts sound-induced hydromechanical waves as they propagate along the cochlea. The operation of this amplifier is not fully understood and is difficult to disentangle experimentally. In the prevailing view, cochlear waves are amplified by the piezo-electric action of the outer hair cells (OHCs), whose cycle-by-cycle elongations and contractions inject power into the local motion of the basilar membrane (BM). Concomitant deformations of the opposing (or "top") side of the organ of Corti are assumed to play a minor role and are generally neglected. However, analysis of intracochlear motions obtained using optical coherence tomography calls this prevailing view into question. In particular, the analysis suggests that (i) the net local power transfer from the OHCs to the BM is either negative or highly inefficient; and (ii) vibration of the top side of the organ of Corti plays a primary role in traveling-wave amplification. A phenomenological model derived from these observations manifests realistic cochlear responses and suggests that amplification arises almost entirely from OHC-induced deformations of the top side of the organ of Corti. In effect, the model turns classic assumptions about spatial impedance relations and power-flow direction within the sensory epithelium upside down.

Amplification and Suppression of Traveling Waves along the Mouse Organ of Corti: Evidence for Spatial Variation in the Longitudinal Coupling of Outer Hair Cell-Generated Forces.

Mammalian hearing sensitivity and frequency selectivity depend on a mechanical amplification process mediated by outer hair cells (OHCs). OHCs are situated within the organ of Corti atop the basilar membrane (BM), which supports sound-evoked traveling waves. It is well established that OHCs generate force to selectively amplify BM traveling waves where they peak, and that amplification accumulates from one location to the next over this narrow cochlear region. However, recent measurements demonstrate that traveling waves along the apical surface of the organ of Corti, the reticular lamina (RL), are amplified over a much broader region. Whether OHC forces accumulate along the length of the RL traveling wave to provide a form of "global" cochlear amplification is unclear. Here we examined the spatial accumulation of RL amplification. In mice of either sex, we used tones to suppress amplification from different cochlear regions and examined the effect on RL vibrations near and far from the traveling-wave peak. We found that although OHC forces amplify the entire RL traveling wave, amplification only accumulates near the peak, over the same region where BM motion is amplified. This contradicts the notion that RL motion is involved in a global amplification mechanism and reveals that the mechanical properties of the BM and organ of Corti tune how OHC forces accumulate spatially. Restricting the spatial buildup of amplification enhances frequency selectivity by sharpening the peaks of cochlear traveling waves and constrains the number of OHCs responsible for mechanical sensitivity at each location.SIGNIFICANCE STATEMENT Outer hair cells generate force to amplify traveling waves within the mammalian cochlea. This force generation is critical to the ability to detect and discriminate sounds. Nevertheless, how these forces couple to the motions of the surrounding structures and integrate along the cochlear length remains poorly understood. Here we demonstrate that outer hair cell-generated forces amplify traveling-wave motion on the organ of Corti throughout the wave's extent, but that these forces only accumulate longitudinally over a region near the wave's peak. The longitudinal coupling of outer hair cell-generated forces is therefore spatially tuned, likely by the mechanical properties of the basilar membrane and organ of Corti. Our findings provide new insight into the mechanical processes that underlie sensitive hearing.

Neuroplastin Isoform Np55 Is Expressed in the Stereocilia of Outer Hair Cells and Required for Normal Outer Hair Cell Function.

UNLABELLED: Neuroplastin (Nptn) is a member of the Ig superfamily and is expressed in two isoforms, Np55 and Np65. Np65 regulates synaptic transmission but the function of Np55 is unknown. In an N-ethyl-N-nitrosaurea mutagenesis screen, we have now generated a mouse line with an Nptn mutation that causes deafness. We show that Np55 is expressed in stereocilia of outer hair cells (OHCs) but not inner hair cells and affects interactions of stereocilia with the tectorial membrane. In vivo vibrometry demonstrates that cochlear amplification is absent in Nptn mutant mice, which is consistent with the failure of OHC stereocilia to maintain stable interactions with the tectorial membrane. Hair bundles show morphological defects as the mutant mice age and while mechanotransduction currents can be evoked in early postnatal hair cells, cochlea microphonics recordings indicate that mechanontransduction is affected as the mutant mice age. We thus conclude that differential splicing leads to functional diversification of Nptn, where Np55 is essential for OHC function, while Np65 is implicated in the regulation of synaptic function. SIGNIFICANCE STATEMENT: Amplification of input sound signals, which is needed for the auditory sense organ to detect sounds over a wide intensity range, depends on mechanical coupling of outer hair cells to the tectorial membrane. The current study shows that neuroplastin, a member of the Ig superfamily, which has previously been linked to the regulation of synaptic plasticity, is critical to maintain a stable mechanical link of outer hair cells with the tectorial membrane. In vivo recordings demonstrate that neuroplastin is essential for sound amplification and that mutation in neuroplastin leads to auditory impairment in mice.

A common microstructure in behavioral hearing thresholds and stimulus-frequency otoacoustic emissions.

Behavioral hearing thresholds and otoacoustic emission (OAE) spectra often exhibit quasiperiodic fluctuations with frequency. For behavioral and OAE responses to single tones-the latter referred to as stimulus-frequency otoacoustic emissions (SFOAEs)-this microstructure has been attributed to intracochlear reflections of SFOAE energy between its region of generation and the middle ear boundary. However, the relationship between behavioral and SFOAE microstructures, as well as their presumed dependence on the properties of the SFOAE-generation mechanism, have yet to be adequately examined. To address this, behavioral thresholds and SFOAEs evoked by near-threshold tones were compared in 12 normal-hearing female subjects. The microstructures observed in thresholds and both SFOAE amplitudes and delays were found to be strikingly similar. SFOAE phase accumulated an integer number of cycles between the frequencies of microstructure maxima, consistent with a dependence of microstructure periodicity on SFOAE propagation delays. Additionally, microstructure depth was correlated with SFOAE magnitude in a manner resembling that predicted by the intracochlear reflection framework, after assuming reasonable values of parameters related to middle ear transmission. Further exploration of this framework may yield more precise estimates of such parameters and provide insight into their frequency dependence.

Similar Tuning of Distortion-Product Otoacoustic Emission Ratio Functions and Cochlear Vibrations in Mice.

When elicited by two stimulus tones (at frequencies f1 and f2, f2 > f1), the amplitudes of specific distortion-product otoacoustic emission (DPOAE) components exhibit a characteristic bandpass shape as the ratio between f2 and f1 is varied. This bandpass shape has been attributed to various mechanisms including intracochlear resonance, suppression, and wave interference, and has been proposed to be related to cochlear frequency tuning. While human studies suggest modest correlations between psychophysical tuning and the tuning of DPOAE amplitude vs. f2/f1 ratio functions, a relationship between the latter and the tuning of cochlear mechanical responses has yet to be established. This was addressed here through direct comparisons of DPOAEs and cochlear vibrations in wild-type CBA/CaJ mice. DPOAEs were elicited using a fixed-f2, swept-f1 paradigm, and optical coherence tomography was used to measure displacements from cochlear locations with characteristic frequencies near f2. The tuning sharpness of 2f1-f2 DPOAE ratio functions was found to be remarkably similar to that of basilar membrane and/or tectorial membrane responses to single tones, with the tuning sharpness of all responses increasing similarly with decreasing stimulus level. This relationship was observed for f2 frequencies ranging from ~8 to 22 kHz. Intracochlear distortion products did not exhibit a bandpass shape as the f2/f1 ratio was varied, indicating that interference between distortion products traveling to the stapes may be responsible for the tuning of the DPOAE ratio function. While these findings suggest that DPOAE ratio functions could be used to noninvasively infer cochlear tuning, it remains to be determined whether this relationship holds in other species and for lower frequency regions.

Bandpass Shape of Distortion-Product Otoacoustic Emission Ratio Functions Reflects Cochlear Frequency Tuning in Normal-Hearing Mice.

The frequency selectivity of the mammalian auditory system is critical for discriminating complex sounds like speech. This selectivity derives from the sharp tuning of the cochlea's mechanical response to sound, which is largely attributed to the amplification of cochlear vibrations by outer hair cells (OHCs). Due to its nonlinearity, the amplification process also leads to the generation of distortion products (DPs), some of which propagate out to the ear canal as DP otoacoustic emissions (DPOAEs). However, the insight that these signals provide about the tuned micro- and macro-mechanics underlying their generation remains unclear. Using optical coherence tomography to measure cochlear vibrations in mice, we show that the cochlea's frequency tuning is reflected in the bandpass shape that is observed in DPOAE amplitudes when the ratio of the two evoking stimulus frequencies is varied (here termed DPOAE "ratio functions"). The tuning sharpness of DPOAE ratio functions and cochlear vibrations co-varied with stimulus level, with a similar quantitative agreement in tuning sharpness observed for both apical and mid-cochlear locations. Measurement of intracochlear DPs revealed that the tuning of the DPOAE ratio functions was not caused by mechanisms that shape DPs locally near where they are generated. Instead, simple model simulations indicate that the bandpass shape is due to a more global wave interference phenomenon. It appears that the filtering of DPOAEs by wave interactions over an extended spatial region allows them to provide a window onto the frequency tuning of single cochlear locations.

The Remarkable Outer Hair Cell: Proceedings of a Symposium in Honour of W. E. Brownell.

In 1985, Bill Brownell and colleagues published the remarkable observation that cochlear outer hair cells (OHCs) express voltage-driven mechanical motion: electromotility. They proposed OHC electromotility as the mechanism for the elusive "cochlear amplifier" required to explain the sensitivity of mammalian hearing. The finding and hypothesis stimulated an explosion of experiments that have transformed our understanding of cochlear mechanics and physiology, the evolution of hair cell structure and function, and audiology. Here, we bring together examples of current research that illustrate the continuing impact of the discovery of OHC electromotility.

Cubic and quadratic distortion products in vibrations of the mouse cochlear apex.

When the ear is stimulated by two tones presented at frequencies f1 and f2, nonlinearity in the cochlea's vibratory response leads to the generation of distortion products (DPs), with the cubic 2f1-f2 DP commonly viewed as the most prominent. While the quadratic f2-f1 DP is also evident in numerous physiological and perceptual studies, its presence in the cochlea's mechanical response has been less well documented. Here, examination of vibratory DPs within the mouse cochlea confirmed that f2-f1 was a significant and sometimes dominant component, whether DPs were measured near their generation site, or after having propagated from more basal locations.

The Elusive Cochlear Filter: Wave Origin of Cochlear Cross-Frequency Masking.

The mammalian cochlea achieves its remarkable sensitivity, frequency selectivity, and dynamic range by spatially segregating the different frequency components of sound via nonlinear processes that remain only partially understood. As a consequence of the wave-based nature of cochlear processing, the different frequency components of complex sounds interact spatially and nonlinearly, mutually suppressing one another as they propagate. Because understanding nonlinear wave interactions and their effects on hearing appears to require mathematically complex or computationally intensive models, theories of hearing that do not deal specifically with cochlear mechanics have often neglected the spatial nature of suppression phenomena. Here we describe a simple framework consisting of a nonlinear traveling-wave model whose spatial response properties can be estimated from basilar-membrane (BM) transfer functions. Without invoking jazzy details of organ-of-Corti mechanics, the model accounts well for the peculiar frequency-dependence of suppression found in two-tone suppression experiments. In particular, our analysis shows that near the peak of the traveling wave, the amplitude of the BM response depends primarily on the nonlinear properties of the traveling wave in more basal (high-frequency) regions. The proposed framework provides perhaps the simplest representation of cochlear signal processing that accounts for the spatially distributed effects of nonlinear wave propagation. Shifting the perspective from local filters to non-local, spatially distributed processes not only elucidates the character of cochlear signal processing, but also has important consequences for interpreting psychophysical experiments.

In vivo functional imaging of the human middle ear with a hand-held optical coherence tomography device.

We describe an optical coherence tomography and vibrometry system designed for portable hand-held usage in the otology clinic on awake patients. The system provides clinically relevant point-of-care morphological imaging with 14-44 µm resolution and functional vibratory measures with sub-nanometer sensitivity. We evaluated various new approaches for extracting functional information including a multi-tone stimulus, a continuous chirp stimulus, and alternating air and bone stimulus. We also explored the vibratory response over an area of the tympanic membrane (TM) and generated TM thickness maps. Our results suggest that the system can provide real-time in vivo imaging and vibrometry of the ear and could prove useful for investigating otologic pathology in the clinic setting.

LMO7 deficiency reveals the significance of the cuticular plate for hearing function.

Sensory hair cells, the mechanoreceptors of the auditory and vestibular systems, harbor two specialized elaborations of the apical surface, the hair bundle and the cuticular plate. In contrast to the extensively studied mechanosensory hair bundle, the cuticular plate is not as well understood. It is believed to provide a rigid foundation for stereocilia motion, but specifics about its function, especially the significance of its integrity for long-term maintenance of hair cell mechanotransduction, are not known. We discovered that a hair cell protein called LIM only protein 7 (LMO7) is specifically localized in the cuticular plate and the cell junction. Lmo7 KO mice suffer multiple cuticular plate deficiencies, including reduced filamentous actin density and abnormal stereociliar rootlets. In addition to the cuticular plate defects, older Lmo7 KO mice develop abnormalities in inner hair cell stereocilia. Together, these defects affect cochlear tuning and sensitivity and give rise to late-onset progressive hearing loss.

Mammalian Auditory Hair Cell Bundle Stiffness Affects Frequency Tuning by Increasing Coupling along the Length of the Cochlea.

The stereociliary bundles of cochlear hair cells convert mechanical vibrations into the electrical signals required for auditory sensation. While the stiffness of the bundles strongly influences mechanotransduction, its influence on the vibratory response of the cochlear partition is unclear. To assess this, we measured cochlear vibrations in mutant mice with reduced bundle stiffness or with a tectorial membrane (TM) that is detached from the sensory epithelium. We found that reducing bundle stiffness decreased the high-frequency extent and sharpened the tuning of vibratory responses obtained postmortem. Detaching the TM further reduced the high-frequency extent of the vibrations but also lowered the partition's resonant frequency. Together, these results demonstrate that the bundle's stiffness and attachment to the TM contribute to passive longitudinal coupling in the cochlea. We conclude that the stereociliary bundles and TM interact to facilitate passive-wave propagation to more apical locations, possibly enhancing active-wave amplification in vivo.

Profiles of Stimulus-Frequency Otoacoustic Emissions from 0.5 to 20 kHz in Humans.

The characteristics of human otoacoustic emissions (OAEs) have not been thoroughly examined above the standard audiometric frequency range (>8 kHz). This is despite the fact that deterioration of cochlear function often starts at the basal, high-frequency end of the cochlea before progressing apically. Here, stimulus-frequency OAEs (SFOAEs) were obtained from 0.5 to 20 kHz in 23 young, audiometrically normal female adults and three individuals with abnormal audiograms, using a low-to-moderate probe level of 36 dB forward pressure level (FPL). In audiometrically normal ears, SFOAEs were measurable at frequencies approaching the start of the steeply sloping high-frequency portion of the audiogram (∼12-15 kHz), though their amplitudes often declined substantially above ∼7 kHz, rarely exceeding 0 dB SPL above 8 kHz. This amplitude decline was typically abrupt and occurred at a frequency that was variable across subjects and not strongly related to the audiogram. In contrast, certain ears with elevated mid-frequency thresholds but regions of normal high-frequency sensitivity could possess surprisingly large SFOAEs (>10 dB SPL) above 7 kHz. When also measured, distortion-product OAEs (DPOAEs) usually remained stronger at higher stimulus frequencies and mirrored the audiogram more closely than SFOAEs. However, the high-frequency extent of SFOAE and DPOAE responses was similar when compared as a function of the response frequency, suggesting that middle ear transmission may be a common limiting factor at high frequencies. Nevertheless, cochlear factors are more likely responsible for complexities observed in high-frequency SFOAE spectra, such as abrupt amplitude changes and narrowly defined response peaks above 10 kHz, as well as the large responses in abnormal ears. These factors may include altered cochlear reflectivity due to subtle damage or the reduced spatial extent of the SFOAE generation region at the cochlear base. The use of higher probe levels is necessary to further evaluate the characteristics and potential utility of high-frequency SFOAE measurements.

Efferent Modulation of Stimulus Frequency Otoacoustic Emission Fine Structure.

Otoacoustic emissions, sounds generated in the inner ear, have become a convenient non-invasive tool to examine the efferent modulation of cochlear mechanics. Activation of the medial olivocochlear (MOC) efferents has been shown to alter the magnitude of these emissions. When the effects of efferent activation on the detailed spectral structures of these emissions have been examined, a shift of the spectral patterns toward higher frequencies has been reported for distortion product and spontaneous otoacoustic emissions. Stimulus frequency otoacoustic emissions (SFOAEs) have been proposed as the preferred emission type in the study of efferent modulation due to the simplicity of their production leading to the possibility of clearer interpretation of results. The effects of efferent activation on the complex spectral patterns of SFOAEs have not been examined to the best of our knowledge. We have examined the effects of activating the MOC efferents using broadband noise in normal-hearing humans. The detailed spectral structure of SFOAEs, known as fine structure, was recorded with and without contralateral acoustic stimulation. Results indicate that SFOAEs are reduced in magnitude and their fine structure pushed to higher frequencies by contralateral acoustic stimulation. These changes are similar to those observed in distortion product or spontaneous otoacoustic emissions and behavioral hearing thresholds. Taken together with observations made about magnitude and phase changes in otoacoustic emissions and hearing thresholds upon contralateral acoustic stimulation, all changes in otoacoustic emission and hearing threshold fine structure appear to be driven by a common set of mechanisms. Specifically, frequency shifts in fine structure patterns appear to be linked to changes in SFOAE phase due to contralateral acoustic stimulation.

Effects of contralateral acoustic stimulation on spontaneous otoacoustic emissions and hearing threshold fine structure.

Medial olivocochlear (MOC) influence on cochlear mechanics can be noninvasively, albeit indirectly, explored via the effects of contralateral acoustic stimulation (CAS) on otoacoustic emissions. CAS-mediated effects are particularly pronounced for spontaneous otoacoustic emissions (SOAEs), which are typically reduced in amplitude and shifted upward in frequency by CAS. We investigated whether similar frequency shifts and magnitude reductions were observed behaviorally in the fine structure of pure-tone hearing thresholds, a phenomenon thought to share a common underlying mechanism with SOAEs. In normal-hearing listeners, fine-resolution thresholds were obtained over a narrow frequency range centered on the frequency of an SOAE, both in the absence and presence of 60-dB SPL broadband CAS. While CAS shifted threshold fine structure patterns and SOAEs upward in frequency by a comparable amount, little reduction in the presence or depth of fine structure was observed at frequencies near those of SOAEs. In fact, CAS typically improved thresholds, particularly at threshold minima, and increased fine structure depth when reductions in the amplitude of the associated SOAE were less than 10 dB. Additional measurements made at frequencies distant from SOAEs, or near SOAEs that were more dramatically reduced in amplitude by the CAS, revealed that CAS tended to elevate thresholds and reduce threshold fine structure depth. The results suggest that threshold fine structure is sensitive to MOC-mediated changes in cochlear gain, but that SOAEs complicate the interpretation of threshold measurements at nearby frequencies, perhaps due to masking or other interference effects. Both threshold fine structure and SOAEs may be significant sources of intersubject and intrasubject variability in psychoacoustic investigations of MOC function.