Reliable Long-Term Serial Evaluation of Cochlear Function Using Pulsed Distortion-Product Otoacoustic Emissions: Analyzing Levels and Pressure Time Courses.

OBJECTIVES: To date, there is no international standard on how to use distortion-product otoacoustic emissions (DPOAEs) in serial measurements to accurately detect changes in the function of the cochlear amplifier due, for example, to ototoxic therapies, occupational noise, or the development of regenerative therapies. The use of clinically established standard DPOAE protocols for serial monitoring programs appears to be hampered by multiple factors, including probe placement and calibration effects, signal-processing complexities associated with multiple sites of emission generation as well as suboptimal selection of stimulus parameters. DESIGN: Pulsed DPOAEs were measured seven times within 3 months for f2 = 1 to 14 kHz and L2 = 25 to 80 dB SPL in 20 ears of 10 healthy participants with normal hearing (mean age = 32.1 ± 9.7 years). L1 values were computed from individual optimal-path parameters derived from the corresponding individual DPOAE level map in the first test session. Three different DPOAE metrics for evaluating the functional state of the cochlear amplifier were investigated with respect to their test-retest reliability: (1) the interference-free, nonlinear-distortion component level (LOD), (2) the time course of the DPOAE-envelope levels, LDP(t), and (3) the squared, zero-lag correlation coefficient () between the time courses of the DPOAE-envelope pressures, pDP(t), measured in two sessions. The latter two metrics include the two main DPOAE components and their state of interference. RESULTS: Collated over all sessions and frequencies, the median absolute difference for LOD was 1.93 dB and for LDP(t) was 2.52 dB; the median of was 0.988. For the low (f2 = 1 to 3 kHz), mid (f2 = 4 to 9 kHz), and high (f2 = 10 to 14 kHz) frequency ranges, the test-retest reliability of LOD increased with increasing signal to noise ratio (SNR). CONCLUSIONS: On the basis of the knowledge gained from this study on the test-retest reliability of pulsed DPOAE signals and the current literature, we propose a DPOAE protocol for future serial monitoring applications that takes into account the following factors: (1) separation of DPOAE components, (2) use of individually optimal stimulus parameters, (3) SNR of at least 15 dB, (4) accurate pressure calibration, (5) consideration of frequency- and level-dependent test-retest reliabilities and corresponding reference ranges, and (6) stimulus levels L2 that are as low as possible with sufficient SNR to capture the nonlinear functional state of the cochlear amplifier operating at its highest gain.

[Pulsed DPOAEs in serial measurements : Combined analysis paradigm of simultaneously occurring changes in hearing thresholds and DPOAEs. German version]. / Gepulste DPOAE in Verlaufsmessungen : Kombiniertes Analyseparadigma von gleichzeitig auftretenden Veränderungen in Hörschwellen und DPOAE.

BACKGROUND: To date, there is no consensus on how to standardize the assessment of ototoxicity in serial measurements. For the diagnosis of damage to the cochlear amplifier, measurement methods are required that have the highest possible test-retest reliability and validity for detecting persistent damage. Estimated distortion-product thresholds (LEDPT) based on short-pulse distortion-product otoacoustic emission (DPOAE) level maps use individually optimal DPOAE stimulus levels and allow reliable quantitative estimation of cochlea-related hearing loss. MATERIALS AND METHODS: Hearing thresholds were estimated objectively using LEDPT and subjectively using modified Békésy tracking audiometry (LTA). Recordings were performed seven times within three months at 14 frequencies (f2 = 1-14 kHz) in 20 ears (PTA4 (0.5-4 kHz) < 20 dB HL). Reconstruction of the DPOAE growth behavior as a function of the stimulus levels L1, L2 was performed on the basis of 21 DPOAE amplitudes. A numerical fit of a nonlinear mathematical function to the three-dimensional DPOAE growth function yielded LEDPT for each stimulus frequency. For the combined analysis, probability distributions of hearing thresholds (LTA, LEDPT), DPOAE levels (LDP), and combinations thereof were determined. RESULTS: LTA and LEDPT each exhibited a test-retest reliability with a median of absolute differences (AD) of 3.2 dB and 3.3 dB, respectively. Combining LEDPT, LDP, and LTA into a single parameter yielded a significantly smaller median AD of 2.0 dB. CONCLUSION: It is expected that an analysis paradigm based on a combination of LEDPT, suprathreshold LDP, and fine-structure-reduced LTA would achieve higher test performance (sensitivity and specificity), allowing reliable detection of pathological or regenerative changes in the outer hair cells.

Understanding the impact of modiolus porosity on stimulation of spiral ganglion neurons by cochlear implants.

Moderate-to-profound sensorineural hearing loss in humans is treatable by electrically stimulating the auditory nerve (AN) with a cochlear implant (CI). In the cochlea, the modiolus presents a porous bony interface between the CI electrode and the AN. New bone growth caused by the presence of the CI electrode or neural degeneration inflicted by ageing or otological diseases might change the effective porosity of the modiolus and, thereby, alter its electrical material properties. Using a volume conductor description of the cochlea, with the aid of a 'mapped conductivity' method and an ad-hoc 'regionally kinetic' equation system, we show that even a slight variation in modiolus porosity or pore distribution can disproportionately affect AN stimulation. Hence, because of porosity changes, an inconsistent CI performance might occur if neural degeneration or new bone growth progress after implantation. Appropriate electrical material properties in accordance with modiolar morphology and pathology should be considered in patient-specific studies. The present first-of-its-kind in-silico study advocates for contextual experimental studies to further explore the utility of modiolus porous morphology in optimising the CI outcome.

An additional source of distortion-product otoacoustic emissions from perturbation of nonlinear force by reflection from inhomogeneities.

The basilar membrane in the cochlea can be modeled as an array of fluid coupled segments driven by stapes vibration and by the undamping nonlinear force simulating cochlear amplification. If stimulated with two tones, the model generates additional tones due to nonlinear distortion. These distortion products (DPs) can be transmitted into the ear canal and produce distortion-product otoacoustic emissions (DPOAEs) known to be generated in the healthy ear of various vertebrates. This study presents a solution for DPs in a two-dimensional nonlinear cochlear model with cochlear roughness-small irregularities in the impedance along the basilar membrane, which may produce additional DPs due to coherent reflection. The solution allows for decomposition of various sources of DPs in the model. In addition to the already described nonlinear-distortion and coherent-reflection mechanisms of DP generation, this study identifies a long-latency DPOAE component due to perturbation of nonlinear force. DP wavelets that are coherently reflected due to impedance irregularities travel toward the stapes across the primary generation region of DPs and there evoke perturbation of the nonlinear undamping force. The ensuing DP wavelets have opposite phase to the wavelets arising from coherent reflection, which results in partial cancellation of the coherent-reflection DP wavelets.

Vesicle traffic in the outer hair cell.

The plasma-membrane marker FM1-43 was employed to reveal the relative significance of different types of endocytic and transcytic mechanisms in outer hair cells (OHCs) of the guinea-pig cochlea. A double-barrel local perfusion system was used to label independently the apical or synaptic pole of the isolated OHC to study mechanisms of vesicle uptake at the poles and of vesicle trafficking along and across the cell. Treatment with an inhibitor of macropino- and phagocytosis, phenylarsine oxide, or of clathrin-mediated endocytic activity, concanavalin A, significantly reduced the dye uptake at both the apical and the synaptic poles, indicating the presence of both clathrin-independent and clathrin-mediated processes at both poles. However, measurement of uptake speed in the presence of the inhibitors suggested that clathrin-independent processes contribute more extensively to endocytosis at the basal pole than the apical pole. Treatment with an inhibitor of myosin VI, 2,4,6-triiodophenol, significantly delayed both the apicobasal and the basoapical fluorescence signals. However, treatment with an inhibitor of kinesin, monastrol, or of dynein, ciliobrevin D, significantly delayed the signals only in the basoapical direction. The myosinVI inhibitor, but neither the kinesin nor dynein inhibitors, significantly delayed the signals to the subsurface cisternae. That is, myosin VI carries vesicles in both longitudinal directions as well as radially to the subsurface cisternae, whereas kinesin and dynein participate primarily in basoapical trafficking. This fundamental information is essential for elucidating recycling mechanisms of specific proteins involved in establishing, controlling and maintaining the electromechanical action of OHCs and, therefore, is vital for understanding auditory perception.

Test-retest reliability of distortion-product thresholds compared to behavioral auditory thresholds.

When referred to baseline measures, serial monitoring of pure-tone behavioral thresholds and distortion-product otoacoustic emissions (DPOAEs) can be used to detect the progression of cochlear damage. Semi-logarithmic DPOAE input-output (I/O) functions enable the computation of estimated distortion-product thresholds (EDPTs) by means of linear regression, a metric that provides a quantitative estimate of hearing loss due to cochlear-amplifier degradation. DPOAE wave interference and a suboptimal choice of stimulus levels limit the accuracy of EDPTs. This work identifies the test-retest reliability of EDPTs derived from short-pulse DPOAE level maps (EDPTLM), a method that circumvents limitations associated with both wave interference and suboptimal choice of stimulus levels. The test-retest reliability was compared to that of EDPTs derived from semi-logarithmic I/O functions (EDPTI/O) and that of behavioral thresholds acquired with pure-tone audiometry (PTA) and modified Békésy tracking audiometry (TA) to provide a foundation for identifying and interpreting significant threshold shifts. The DPOAE-based auditory thresholds (EDPTLM and EDPTI/O) and behavioral thresholds (PTA and TA) were recorded seven times within three months at 14 frequencies with f2 = 1-14 kHz in 20 ears from ten subjects with normal hearing (4PTA0.5-4kHz < 20 dB HL). To obtain EDPTLM, short-pulse DPOAEs were recorded using 21 L1,L2 pairs. Reconstruction of DPOAE growth behavior as a function of L1 and L2 using nonlinear curve fitting enabled the derivation of EDPTLM for each frequency. Test-retest reliability was determined using three different approaches: 1) centered thresholds, 2) average threshold differences, and 3) average absolute threshold differences, between each possible test session (N = 21). Test-retest reliability based on centered thresholds and average threshold differences showed no statistically significant difference between EDPTLM, EDPTI/O, PTA, and TA for the pooled analysis incorporating all stimulus frequencies. Average absolute threshold differences presented small but significant differences in test-retest reliability with median values of 3.00 dB for PTA, 3.20 dB for TA, 3.34 dB for EDPTLM, and 3.51 dB for EDPTI/O. A considerable frequency dependence of test-retest reliability was found; namely, the highest test-retest reliability was for EDPTLM at f2 = 11 - 14 kHz. Otherwise, at lower frequencies, the highest test-retest reliability was for TA at f2 =1 - 2 kHz. Overall, the test-retest reliability of EDPTLM was better than that of EDPTI/O and was similar to that for behavioral thresholds. Hence, deriving EDPTLM from individual level maps is a promising and sensitive method for objectively monitoring the state of the cochlea. Furthermore, the detection of an equidirectional threshold change at a single frequency in both EDPTLM and TA might allow reducing the threshold shift as indication of a follow-up examination from the clinical standard of 10 dB down to 5 dB. This stricter indicator might be beneficial when monitoring cochlear damage, for example ototoxicity, in the presence of (remnant) cochlear amplification at baseline.

Neural Tissue Degeneration in Rosenthal's Canal and Its Impact on Electrical Stimulation of the Auditory Nerve by Cochlear Implants: An Image-Based Modeling Study.

Sensorineural deafness is caused by the loss of peripheral neural input to the auditory nerve, which may result from peripheral neural degeneration and/or a loss of inner hair cells. Provided spiral ganglion cells and their central processes are patent, cochlear implants can be used to electrically stimulate the auditory nerve to facilitate hearing in the deaf or severely hard-of-hearing. Neural degeneration is a crucial impediment to the functional success of a cochlear implant. The present, first-of-its-kind two-dimensional finite-element model investigates how the depletion of neural tissues might alter the electrically induced transmembrane potential of spiral ganglion neurons. The study suggests that even as little as 10% of neural tissue degeneration could lead to a disproportionate change in the stimulation profile of the auditory nerve. This result implies that apart from encapsulation layer formation around the cochlear implant electrode, tissue degeneration could also be an essential reason for the apparent inconsistencies in the functionality of cochlear implants.

Nonlinear reflection as a cause of the short-latency component in stimulus-frequency otoacoustic emissions simulated by the methods of compression and suppression.

Stimulus-frequency otoacoustic emissions (SFOAEs) are generated by coherent reflection of forward traveling waves by perturbations along the basilar membrane. The strongest wavelets are backscattered near the place where the traveling wave reaches its maximal amplitude (tonotopic place). Therefore, the SFOAE group delay might be expected to be twice the group delay estimated in the cochlear filters. However, experimental data have yielded steady-state SFOAE components with near-zero latency. A cochlear model is used to show that short-latency SFOAE components can be generated due to nonlinear reflection of the compressor or suppressor tones used in SFOAE measurements. The simulations indicate that suppressors produce more pronounced short-latency components than compressors. The existence of nonlinear reflection components due to suppressors can also explain why SFOAEs can still be detected when suppressors are presented more than half an octave above the probe-tone frequency. Simulations of the SFOAE suppression tuning curves showed that phase changes in the SFOAE residual as the suppressor frequency increases are mostly determined by phase changes of the nonlinear reflection component.

Derivation of input-output functions from distortion-product otoacoustic emission level maps.

Distortion-product otoacoustic emissions (DPOAEs) emerge from the cochlea when elicited with two tones of frequencies f1 and f2. DPOAEs mainly consist of two components, a nonlinear-distortion and a coherent-reflection component. Input-output (I/O) functions of DPOAE pressure at the cubic difference frequency, fDP=2f1-f2, enable the computation of estimated distortion-product thresholds (EDPTs), offering a noninvasive approach to estimate auditory thresholds. However, wave interference between the DPOAE components and suboptimal stimulus-level pairs reduces the accuracy of EDPTs. Here, the amplitude P of the nonlinear-distortion component is extracted from short-pulse DPOAE time signals. DPOAE level maps representing the growth behavior of P in L1,L2 space are recorded for 21 stimulus-level pairs and 14 frequencies with f2=1 to 14 kHz (f2/f1=1.2) from 20 ears. Reproducing DPOAE growth behavior using a least-squares fit approach enables the derivation of ridge-based I/O functions from model level maps. Objective evaluation criteria assess the fit results and provide EDPTs, which correlate significantly with auditory thresholds (p < 0.001). In conclusion, I/O functions derived from model level maps provide EDPTs with high precision but without the need of predefined optimal stimulus-level pairs.

Nanomechanical mapping reveals localized stiffening of the basilar membrane after cochlear implantation.

Cochlear implantation leads to many structural changes within the cochlea which can impair residual hearing. In patients with preserved low-frequency hearing, a delayed hearing loss can occur weeks-to-years post-implantation. We explore whether stiffening of the basilar membrane (BM) may be a contributory factor in an animal model. Our objective is to map changes in morphology and Young's modulus of basal and apical areas of the BM after cochlear implantation, using quantitative nanomechanical atomic force microscopy (QNM-AFM) after cochlear implant surgery. Cochlear implantation was undertaken in the guinea pig, and the BM was harvested at four time-points: 1 day, 14 days, 28 days and 84 days post-implantation for QNM-AFM analysis. Auditory brainstem response thresholds were determined prior to implantation and termination. BM tissue showed altered morphology and a progressive increase in Young's modulus, mainly in the apex, over time after implantation. BM tissue from the cochlear base demonstrated areas of extreme stiffness which are likely due to micro-calcification on the BM. In conclusion, stiffening of the BM after cochlear implantation occurs over time, even at sites far apical to a cochlear implant.

Distributed sources as a cause of abrupt amplitude decrease in cubic distortion-product otoacoustic emissions at high stimulus intensities.

The amplitudes of distortion-product otoacoustic emissions (DPOAEs) may abruptly decrease even though the stimulus level is relatively high. These notches observed in the DPOAE input/output functions or distortion-product grams have been hypothesized to be due to destructive interference between wavelets generated by distributed sources of the nonlinear-distortion component of DPOAEs. In this paper, simulations with a smooth cochlear model and its analytical solution support the hypothesis that destructive interference between individual wavelets may lead to the amplitude notches and explain the cause for onset and offset amplitude overshoots in the DPOAE signal measured for intensity pairs in the notches.

The influence of distributed source regions in the formation of the nonlinear distortion component of cubic distortion-product otoacoustic emissions.

Distortion product otoacoustic emissions (DPOAEs) are evoked by two stimulus tones with frequency f1 and f2 of ratio f2/f1 in the range between approximately 1.05 and 1.4. This study theoretically and experimentally analyzes the cubic 2f1-f2 DPOAE for different stimulus levels of one of the tones while the other is constant. Simulations for f2/f1 of 1.2 and moderate stimulus levels (30-70 dB sound pressure level) indicate that cubic distortion products are generated along a relatively large length of the basilar membrane, the extent of which increases with stimulus level. However, apical from the place of maximum nonlinear force, the wavelets generated by these distributed sources mutually cancel. Therefore, although the spatial extent of the primary DPOAE sources broadens with increasing stimulus level (up to 1.5 oct), the basilar-membrane region contributing to the DPOAE signal is relatively narrow (0.6 oct) and level independent. The observed dependence of DPOAE amplitude on stimulus level can be well-approximated by a point source at the basilar-membrane place where the largest distortion product (maximum of the nonlinear force) is generated. Onset and offset of the DPOAE signal may contain amplitude overshoots (complexities), which are in most cases asymmetrical. Two-tone suppression was identified as the main cause of these onset and offset complexities. DPOAE measurements in two normal-hearing subjects support the level dependence of the steady-state DPOAE amplitude and the asymmetry in the onset and offset responses predicted by the theoretical analysis.

Double fluorescent labelling of a bipolar epithelial cell in vitro: The outer hair cell.

BACKGROUND: Fluorescence membrane markers are efficient tools for visualizing the dynamics of membrane recycling processes in living cells. The outer hair cell (OHC) - a bipolar epithelial cell in the cochlea - possesses endocytic activity at both its apical and basal poles. The best visual overview of transcytosis in the OHC is achieved when the cell is isolated, so that both the apical and the basal poles are in the same focal plane to allow confocal imaging. Until now, fluorescent markers were applied to the extracellular environment of isolated OHCs without distinguishing the apical and basal poles. The drawback of that configuration is that apicobasal and basoapical vesicle traffic labelled at the opposite poles cannot be visualized independently because the same fluorescent marker has access to both poles. NEW METHOD: A double-barrel, capillary perfusion system was developed to independently stain either one pole or both the apical and the basal poles of isolated OHCs using different types of fluorescence membrane markers. RESULTS: Producing laminar fluid flow, the double-barrel perfusor allows investigation of the dynamics of apicobasal and basoapical vesicle traffic independently and/or simultaneously in the same OHC. COMPARISON WITH EXISTING METHOD: This method offers a unique option for investigating bidirectional vesicle traffic in bipolar epithelial cells, which is superior to other already established labelling techniques. CONCLUSIONS: The double-barrel perfusion system, suitable for selectively staining a longitudinal section of the plasma membrane of an isolated bipolar epithelial cell, opens new possibilities for investigating cell labelling and intracellular vesicle traffic.

Electrophysiological Evidence of the Basilar-Membrane Travelling Wave and Frequency Place Coding of Sound in Cochlear Implant Recipients.

AIM: To obtain direct evidence for the cochlear travelling wave in humans by performing electrocochleography from within the cochlea in subjects implanted with an auditory prosthesis. BACKGROUND: Sound induces a travelling wave that propagates along the basilar membrane, exhibiting cochleotopic tuning with a frequency-dependent phase delay. To date, evoked potentials and psychophysical experiments have supported the presence of the travelling wave in humans, but direct measurements have not been made. METHODS: Electrical potentials in response to rarefaction and condensation acoustic tone bursts were recorded from multiple sites along the human cochlea, directly from a cochlear implant electrode during, and immediately after, its insertion. These recordings were made from individuals with residual hearing. RESULTS: Electrocochleography was recorded from 11 intracochlear electrodes in 7 ears from 6 subjects, with detectable responses on all electrodes in 5 ears. Cochleotopic tuning and frequency-dependent phase delay of the cochlear microphonic were demonstrated. The response latencies were slightly shorter than those anticipated which we attribute to the subjects' hearing loss. CONCLUSIONS: Direct evidence for the travelling wave was observed. Electrocochleography from cochlear implant electrodes provides site-specific information on hair cell and neural function of the cochlea with potential diagnostic value.

Input-output functions of the nonlinear-distortion component of distortion-product otoacoustic emissions in normal and hearing-impaired human ears.

Distortion-product otoacoustic emissions (DPOAEs) arise in the cochlea in response to two tones with frequencies f1 and f2 and mainly consist of two components, a nonlinear-distortion and a coherent-reflection component. Wave interference between these components limits the accuracy of DPOAEs when evaluating the function of the cochlea with conventional continuous stimulus tones. Here, DPOAE components are separated in the time domain from DPOAE signals elicited with short stimulus pulses. The extracted nonlinear-distortion components are used to derive estimated distortion-product thresholds (EDPTs) from semi-logarithmic input-output (I/O) functions for 20 normal-hearing and 21 hearing-impaired subjects. I/O functions were measured with frequency-specific stimulus levels at eight frequencies f2 = 1,…, 8 kHz (f2/f1 = 1.2). For comparison, DPOAEs were also elicited with continuous primary tones. Both acquisition paradigms yielded EDPTs, which significantly correlated with behavioral thresholds (p < 0.001) and enabled derivation of estimated hearing thresholds (EHTs) from EDPTs using a linear regression relationship. DPOAE-component separation in the time domain significantly reduced the standard deviation of EHTs compared to that derived from continuous DPOAEs (p < 0.01). In conclusion, using frequency-specific stimulus levels and DPOAE-component separation increases the reliability of DPOAE I/O functions for assessing cochlear function and estimating behavioral thresholds.

Comparison of time-domain source-separation techniques for short-pulse distortion-product otoacoustic emissions.

Distortion-product otoacoustic emissions (DPOAEs) are presumed to consist mainly of two components, a nonlinear-distortion component and a coherent-reflection component. Wave interference between these two components reduces the accuracy of DPOAEs when used to evaluate cochlear function. Here, short tone pulses are utilized to record DPOAE signals in normal-hearing subjects. DPOAE components are extracted from recordings at discrete frequencies using two different techniques in the time domain. The extracted DPOAE components are compared to recordings obtained with conventional, continuous primary tones.

Coupling of an active middle-ear implant to the long process of the incus using an elastic clip attachment.

The active middle-ear implant Vibrant Soundbridge© (VSB) is used to treat mild-to-severe sensorineural hearing losses. The standard surgical approach for incus vibroplasty is a mastoidectomy and a posterior tympanotomy, crimping the Floating Mass Transducer (FMT) to the long process of the incus (LPI) (standard crimped application). However, tight crimping increases the risk of necrosis of the LPI, resulting in reduction of energy transfer and loss of amplification. The aim of this study was to develop a new coupling device for the LPI, that does not require crimping, and to test its vibrational transfer properties in temporal-bone preparations. An extended antrotomy and a posterior tympanotomy were performed in ten fresh human temporal bones. As a control for normal middle-ear function, the tympanic membrane was stimulated acoustically and the vibration of the stapes footplate was measured by laser Doppler vibrometry (LDV). FMT-induced vibration responses of the stapes were then measured for the standard crimped application at the LPI and for the newly designed elastic long process coupler (LP coupler). For the LP coupler, velocity-amplitude responses in temporal-bone preparations showed increased mean amplitudes at around 1 kHz (∼10 dB) and a reduction between 1.8 and 6 kHz (13 dB on average for 2 ≤ f ≤ 5 kHz). In conclusion, attachment of the FMT to the LPI with the LP coupler leads to generally good mechanical and functional coupling in temporal-bone preparations with a notable disadvantage between 1.8 and 6 kHz. Due to its elastic clip attachment it is expected that the LP coupler will reduce the risk of necrosis of the incus long process, which has to been shown in further studies. Clinical results of the LP coupler are pending.

The chloride-channel blocker 9-anthracenecarboxylic acid reduces the nonlinear capacitance of prestin-associated charge movement.

The basis of the extraordinary sensitivity and frequency selectivity of the cochlea is a chloride-sensitive protein called prestin which can produce an electromechanical response and which resides in the basolateral plasma membrane of outer hair cells (OHCs). The compound 9-anthracenecarboxylic acid (9-AC), an inhibitor of chloride channels, has been found to reduce the electromechanical response of the cochlea and the OHC mechanical impedance. To elucidate these 9-AC effects, the functional electromechanical status of prestin was assayed by measuring the nonlinear capacitance of OHCs from the guinea-pig cochlea and of prestin-transfected human embryonic kidney 293 (HEK 293) cells. Extracellular application of 9-AC caused reversible, dose-dependent and chloride-sensitive reduction in OHC nonlinear charge transfer, Qmax . Prestin-transfected cells also showed reversible reduction in Qmax . For OHCs, intracellular 9-AC application as well as reduced intracellular pH had no detectable effect on the reduction in Qmax by extracellularly applied 9-AC. In the prestin-transfected cells, cytosolic application of 9-AC approximately halved the blocking efficacy of extracellularly applied 9-AC. OHC inside-out patches presented the whole-cell blocking characteristics. Disruption of the cytoskeleton by preventing actin polymerization with latrunculin A or by decoupling of spectrin from actin with diamide did not affect the 9-AC-evoked reduction in Qmax . We conclude that 9-AC acts on the electromechanical transducer principally by interaction with prestin rather than acting via the cytoskeleton, chloride channels or pH. The 9-AC block presents characteristics in common with salicylate, but is almost an order of magnitude faster. 9-AC provides a new tool for elucidating the molecular dynamics of prestin function.