Radial Flow Field of Spiral Cochlea and Its Effect on Stereocilia.

The opening of the ion channels ultimately depends on the movement and energy conversion of the microstructural organization. But the role was not yet clear how the active sound amplification function is generated by the microstructure in the cochlear characteristic spiral shape. In this paper, an analytical model of the spiral cochlea is developed to investigate the radial flow field generated by the spiral shape of the cochlea and its effect on the outer hair cell stereocilia, and to analyze the effect of the spiral shape on the micromechanics of the cochlea. The results show that the spiral shape of the cochlea exerts a radial shear force on the hair cell stereocilia by generating a radial flow field, causing the stereocilia to deflect in the radial flow field, with the maximum deflection occurring at the apex of the cochlea. This finding explains from the microscopic mechanism that cochlear spiral shape can enhance low-frequency hearing in humans, which provides a basis for further studies on the contribution of the movement of stereocilia applied by the radial flow field of lymphatic fluid to activate ion channels for auditory production.

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.

Stereocilia Bundle Imaging with Nanoscale Resolution in Live Mammalian Auditory Hair Cells.

Inner ear hair cells detect sound-induced displacements and transduce these stimuli into electrical signals in a hair bundle that consists of stereocilia that are arranged in rows of increasing height. When stereocilia are deflected, they tug on tiny (~5 nm in diameter) extracellular tip links interconnecting stereocilia, which convey forces to the mechanosensitive transduction channels. Although mechanotransduction has been studied in live hair cells for decades, the functionally important ultrastructural details of the mechanotransduction machinery at the tips of stereocilia (such as tip link dynamics or transduction-dependent stereocilia remodeling) can still be studied only in dead cells with electron microscopy. Theoretically, scanning probe techniques, such as atomic force microscopy, have enough resolution to visualize the surface of stereocilia. However, independent of imaging mode, even the slightest contact of the atomic force microscopy probe with the stereocilia bundle usually damages the bundle. Here we present a detailed protocol for the hopping probe ion conductance microscopy (HPICM) imaging of live rodent auditory hair cells. This non-contact scanning probe technique allows time lapse imaging of the surface of live cells with a complex topography, like hair cells, with single nanometers resolution and without making physical contact with the sample. The HPICM uses an electrical current passing through the glass nanopipette to detect the cell surface in close vicinity to the pipette, while a 3D-positioning piezoelectric system scans the surface and generates its image. With HPICM, we were able to image stereocilia bundles and the links interconnecting stereocilia in live auditory hair cells for several hours without noticeable damage. We anticipate that the use of HPICM will allow direct exploration of ultrastructural changes in the stereocilia of live hair cells for better understanding of their function.

A binding protein regulates myosin-7a dimerization and actin bundle assembly.

Myosin-7a, despite being monomeric in isolation, plays roles in organizing actin-based cell protrusions such as filopodia, microvilli and stereocilia, as well as transporting cargoes within them. Here, we identify a binding protein for Drosophila myosin-7a termed M7BP, and describe how M7BP assembles myosin-7a into a motile complex that enables cargo translocation and actin cytoskeletal remodeling. M7BP binds to the autoinhibitory tail of myosin-7a, extending the molecule and activating its ATPase activity. Single-molecule reconstitution show that M7BP enables robust motility by complexing with myosin-7a as 2:2 translocation dimers in an actin-regulated manner. Meanwhile, M7BP tethers actin, enhancing complex's processivity and driving actin-filament alignment during processive runs. Finally, we show that myosin-7a-M7BP complex assembles actin bundles and filopodia-like protrusions while migrating along them in living cells. Together, these findings provide insights into the mechanisms by which myosin-7a functions in actin protrusions.

Serial scanning electron microscopy of anti-PKHD1L1 immuno-gold labeled mouse hair cell stereocilia bundles.

Serial electron microscopy techniques have proven to be a powerful tool in biology. Unfortunately, the data sets they generate lack robust and accurate automated segmentation algorithms. In this data descriptor publication, we introduce a serial focused ion beam scanning electron microscopy (FIB-SEM) dataset consisting of six outer hair cell (OHC) stereocilia bundles, and the supranuclear part of the hair cell bodies. Also presented are the manual segmentations of stereocilia bundles and the gold bead labeling of PKHD1L1, a coat protein of hair cell stereocilia important for hearing in mice. This depository includes all original data and several intermediate steps of the manual analysis, as well as the MATLAB algorithm used to generate a three-dimensional distribution map of gold labels. They serve as a reference dataset, and they enable reproduction of our analysis, evaluation and improvement of current methods of protein localization, and training of algorithms for accurate automated segmentation.

Distinct roles of stereociliary links in the nonlinear sound processing and noise resistance of cochlear outer hair cells.

Outer hair cells (OHCs) play an essential role in hearing by acting as a nonlinear amplifier which helps the cochlea detect sounds with high sensitivity and accuracy. This nonlinear sound processing generates distortion products, which can be measured as distortion-product otoacoustic emissions (DPOAEs). The OHC stereocilia that respond to sound vibrations are connected by three kinds of extracellular links: tip links that connect the taller stereocilia to shorter ones and convey force to the mechanoelectrical transduction channels, tectorial membrane-attachment crowns (TM-ACs) that connect the tallest stereocilia to one another and to the overlying TM, and horizontal top connectors (HTCs) that link adjacent stereocilia. While the tip links have been extensively studied, the roles that the other two types of links play in hearing are much less clear, largely because of a lack of suitable animal models. Here, while analyzing genetic combinations of tubby mice, we encountered models missing both HTCs and TM-ACs or HTCs alone. We found that the tubby mutation causes loss of both HTCs and TM-ACs due to a mislocalization of stereocilin, which results in OHC dysfunction leading to severe hearing loss. Intriguingly, the addition of the modifier allele modifier of tubby hearing 1 in tubby mice selectively rescues the TM-ACs but not the HTCs. Hearing is significantly rescued in these mice with robust DPOAE production, indicating an essential role of the TM-ACs but not the HTCs in normal OHC function. In contrast, the HTCs are required for the resistance of hearing to damage caused by noise stress.

Myosin-XVa Controls Both Staircase Architecture and Diameter Gradation of Stereocilia Rows in the Auditory Hair Cell Bundles.

Mammalian hair cells develop their mechanosensory bundles through consecutive phases of stereocilia elongation, thickening, and retraction of supernumerary stereocilia. Many molecules involved in stereocilia elongation have been identified, including myosin-XVa. Significantly less is known about molecular mechanisms of stereocilia thickening and retraction. Here, we used scanning electron microscopy (SEM) to quantify postnatal changes in number and diameters of the auditory hair cell stereocilia in shaker-2 mice (Myo15sh2) that lack both "long" and "short" isoforms of myosin-XVa, and in mice lacking only the "long" myosin-XVa isoform (Myo15∆N). Previously, we observed large mechanotransduction current in young postnatal inner (IHC) and outer (OHC) hair cells of both these strains. Stereocilia counts showed nearly identical developmental retraction of supernumerary stereocilia in control heterozygous, Myo15sh2/sh2, and Myo15∆N/∆N mice, suggesting that this retraction is largely unaffected by myosin-XVa deficiency. However, myosin-XVa deficiency does affect stereocilia diameters. In control, the first (tallest) and second row stereocilia grow in diameter simultaneously. However, the third row stereocilia in IHCs grow only until postnatal day 1-2 and then become thinner. In OHCs, they also grow slower than taller stereocilia, forming a stereocilia diameter gradation within a hair bundle. The sh2 mutation disrupts this gradation and makes all stereocilia nearly identical in thickness in both IHCs and OHCs, with only subtle residual diameter differences. All Myo15sh2/sh2 stereocilia grow postnatally including the third row, which is not a part of normal development. Serial sections with focused ion beam (FIB)-SEM confirmed that diameter changes of Myo15sh2/sh2 IHC and OHC stereocilia resulted from corresponding changes of their actin cores. In contrast to Myo15sh2/sh2, Myo15∆N/∆N hair cells develop prominent stereocilia diameter gradation. Thus, besides building the staircase, the short isoform of myosin-XVa is essential for controlling the diameter of the third row stereocilia and formation of the stereocilia diameter gradation in a hair bundle.

Mechanotransduction-Dependent Control of Stereocilia Dimensions and Row Identity in Inner Hair Cells.

Actin-rich structures, like stereocilia and microvilli, are assembled with precise control of length, diameter, and relative spacing. By quantifying actin-core dimensions of stereocilia from phalloidin-labeled mouse cochleas, we demonstrated that inner hair cell stereocilia developed in specific stages, where a widening phase is sandwiched between two lengthening phases. Moreover, widening of the second-tallest stereocilia rank (row 2) occurred simultaneously with the appearance of mechanotransduction. Correspondingly, Tmc1KO/KO;Tmc2KO/KO or TmieKO/KO hair cells, which lack transduction, have significantly altered stereocilia lengths and diameters, including a narrowed row 2. EPS8 and the short splice isoform of MYO15A, identity markers for mature row 1 (the tallest row), lost their row exclusivity in transduction mutants. GNAI3, another member of the mature row 1 complex, accumulated at mutant row 1 tips at considerably lower levels than in wild-type bundles. Alterations in stereocilia dimensions and in EPS8 distribution seen in transduction mutants were mimicked by block of transduction channels of cochlear explants in culture. In addition, proteins normally concentrated at mature row 2 tips were also distributed differently in transduction mutants; the heterodimeric capping protein subunit CAPZB and its partner TWF2 never concentrated at row 2 tips like they do in wild-type bundles. The altered distribution of marker proteins in transduction mutants was accompanied by increased variability in stereocilia length. Transduction channels thus specify and maintain row identity, control addition of new actin filaments to increase stereocilia diameter, and coordinate stereocilia height within rows.

Hair Bundle Stimulation Mode Modifies Manifestations of Mechanotransduction Adaptation.

Sound detection in auditory sensory hair cells depends on the deflection of the stereocilia hair bundle which opens mechano-electric transduction (MET) channels. Adaptation is hypothesized to be a critical property of MET that contributes to the auditory system's wide dynamic range and sharp frequency selectivity. Our recent work using a stiff probe to displace hair bundles showed that the fastest adaptation mechanism (fast adaptation) does not require calcium entry. Using fluid-jet stimuli, others obtained data showing only a calcium-dependent fast adaptation response. Because cochlear stereocilia do not move coherently and the hair cell response depends critically on the magnitude and time course of the hair bundle deflection, we developed a high-speed imaging technique to quantify this deflection in rat cochlear hair cells. The fluid jet delivers a force stimulus, and force steps lead to a complex time course of hair bundle displacement (mechanical creep), which affects the hair cell's macroscopic MET current response by masking the time course of the fast adaptation response. Modifying the fluid-jet stimulus to generate a hair bundle displacement step produced rapidly adapting currents that did not depend on membrane potential, confirming that fast adaptation does not depend on calcium entry. MET current responses differ with stimulus modality and will shape receptor potentials of different hair cell types based on their in vivo stimulus mode. These transformations will directly affect how stimuli are encoded.SIGNIFICANCE STATEMENT Mechanotransduction by sensory hair cells represents a key first step for the sound sensing ability in vertebrates. The sharp frequency tuning and wide dynamic range of sound sensation are hypothesized to require a mechanotransduction adaptation mechanism. Recent work indicated that the apparent calcium dependence of the fastest adaptation differs with the method of cochlear hair cell stimulation. Here, we reconcile existing data and show that calcium entry does not drive the fastest adaptation process, independent of the stimulation method. With force stimulation of the hair bundle, adaptation manifests differently than with displacement stimulation, indicating that the stimulation mode of the hair bundle will affect the hair cell receptor current and stimulus coding.

Nanomechanics of tip-link cadherins.

Hearing and balance rely on the transduction of mechanical stimuli arising from sound waves or head movements into electrochemical signals. This archetypal mechanoelectrical transduction process occurs in the hair-cell stereocilia of the inner ear, which experience continuous oscillations driven by undulations in the endolymph in which they are immersed. The filamentous structures called tip links, formed by an intertwined thread composed of an heterotypic complex of cadherin 23 and protocadherin 15 ectodomain dimers, connect each stereocilium to the tip of the lower sterocilium, and must maintain their integrity against continuous stimulatory deflections. By using single molecule force spectroscopy, here we demonstrate that in contrast to the case of classical cadherins, tip-link cadherins are mechanoresilient structures even at the exceptionally low Ca2+ concentration of the endolymph. We also show that the D101G deafness point mutation in cadherin 23, which affects a Ca2+ coordination site, exhibits an altered mechanical phenotype at the physiological Ca2+ concentration. Our results show a remarkable case of functional adaptation of a protein's nanomechanics to extremely low Ca2+ concentrations and pave the way to a full understanding of the mechanotransduction mechanism mediated by auditory cadherins.

New insights into regulation and function of planar polarity in the inner ear.

Acquisition of cell polarity generates signaling and cytoskeletal asymmetry and thus underpins polarized cell behaviors during tissue morphogenesis. In epithelial tissues, both apical-basal polarity and planar polarity, which refers to cell polarization along an axis orthogonal to the apical-basal axis, are essential for epithelial morphogenesis and function. A prime example of epithelial planar polarity can be found in the auditory sensory epithelium (or organ of Corti, OC). Sensory hair cells, the sound receptors, acquire a planar polarized apical cytoskeleton which is uniformely oriented along an axis orthogonal to the longitudinal axis of the cochlear duct. Both cell-intrinsic and tissue-level planar polarity are necessary for proper perception of sound. Here we review recent insights into the novel roles and mechanisms of planar polarity signaling gained from genetic analysis in mice, focusing mainly on the OC but also with some discussions on the vestibular sensory epithelia.

TRIOBP-5 sculpts stereocilia rootlets and stiffens supporting cells enabling hearing.

TRIOBP remodels the cytoskeleton by forming unusually dense F-actin bundles and is implicated in human cancer, schizophrenia, and deafness. Mutations ablating human and mouse TRIOBP-4 and TRIOBP-5 isoforms are associated with profound deafness, as inner ear mechanosensory hair cells degenerate after stereocilia rootlets fail to develop. However, the mechanisms regulating formation of stereocilia rootlets by each TRIOBP isoform remain unknown. Using 3 new Triobp mouse models, we report that TRIOBP-5 is essential for thickening bundles of F-actin in rootlets, establishing their mature dimensions and for stiffening supporting cells of the auditory sensory epithelium. The coiled-coil domains of this isoform are required for reinforcement and maintenance of stereocilia rootlets. A loss of TRIOBP-5 in mouse results in dysmorphic rootlets that are abnormally thin in the cuticular plate but have increased widths and lengths within stereocilia cores, and causes progressive deafness recapitulating the human phenotype. Our study extends the current understanding of TRIOBP isoform-specific functions necessary for life-long hearing, with implications for insight into other TRIOBPopathies.

GPSM2-GNAI Specifies the Tallest Stereocilia and Defines Hair Bundle Row Identity.

The transduction compartment of inner ear hair cells, the hair bundle, is composed of stereocilia rows of graded height, a property essential for sensory function that remains poorly understood at the molecular level. We previously showed that GPSM2-GNAI is enriched at stereocilia distal tips and required for their postnatal elongation and bundle morphogenesis-two characteristics shared with MYO15A (short isoform), WHRN, and EPS8 proteins. Here we first performed a comprehensive genetic analysis of the mouse auditory epithelium to show that GPSM2, GNAI, MYO15A, and WHRN operate in series within the same pathway. To understand how these functionally disparate proteins act as an obligate complex, we then systematically analyzed their distribution in normal and mutant bundles over time. We discovered that WHRN-GPSM2-GNAI is an extra module recruited by and added to a pre-existing MYO15A-EPS8 stereocilia tip complex. This extended complex is only present in the first, tallest row, and is required to stabilize larger amounts of MYO15A-EPS8 than in shorter rows, which at tips harbor only MYO15A-EPS8. In the absence of GPSM2 or GNAI function, including in the epistatic Myo15a and Whrn mutants, bundles retain an embryonic-like organization that coincides with generic stereocilia at the molecular level. We propose that GPSM2-GNAI confers on the first row its unique tallest identity and participates in generating differential row identity across the hair bundle.

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.

Building and repairing the stereocilia cytoskeleton in mammalian auditory hair cells.

Despite all recent achievements in identification of the molecules that are essential for the structure and mechanosensory function of stereocilia bundles in the auditory hair cells of mammalian species, we still have only a rudimentary understanding of the mechanisms of stereocilia formation, maintenance, and repair. Important molecular differences distinguishing mammalian auditory hair cells from hair cells of other types and species have been recently revealed. In addition, we are beginning to solve the puzzle of the apparent life-long stability of the stereocilia bundles in these cells. New data link the stability of the cytoskeleton in the mammalian auditory stereocilia with the normal activity of mechanotransduction channels. These data suggest new ideas on how a terminally-differentiated non-regenerating hair cell in the mammalian cochlea may repair and tune its stereocilia bundle throughout the life span of the organism.

Variable number of TMC1-dependent mechanotransducer channels underlie tonotopic conductance gradients in the cochlea.

Functional mechanoelectrical transduction (MET) channels of cochlear hair cells require the presence of transmembrane channel-like protein isoforms TMC1 or TMC2. We show that TMCs are required for normal stereociliary bundle development and distinctively influence channel properties. TMC1-dependent channels have larger single-channel conductance and in outer hair cells (OHCs) support a tonotopic apex-to-base conductance gradient. Each MET channel complex exhibits multiple conductance states in ~50 pS increments, basal MET channels having more large-conductance levels. Using mice expressing fluorescently tagged TMCs, we show a three-fold increase in number of TMC1 molecules per stereocilium tip from cochlear apex to base, mirroring the channel conductance gradient in OHCs. Single-molecule photobleaching indicates the number of TMC1 molecules per MET complex changes from ~8 at the apex to ~20 at base. The results suggest there are varying numbers of channels per MET complex, each requiring multiple TMC1 molecules, and together operating in a coordinated or cooperative manner.

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.

Non-tip auditory-nerve responses that are suppressed by low-frequency bias tones originate from reticular lamina motion.

Recent cochlear mechanical measurements show that active processes increase the motion response of the reticular lamina (RL) at frequencies more than an octave below the local characteristic frequency (CF) for CFs above 5 kHz. A possible correlate is that in high-CF (>5 kHz) auditory-nerve (AN) fibers, responses to frequencies 1-3 octaves below CF ("tail" frequencies) can be inhibited by medial olivocochlear (MOC) efferents. These results indicate that active processes enhance the sensitivity of tail-frequency RL and AN responses. Perhaps related is that some apical low-CF AN fibers have tuning-curve (TC) "side-lobe" response areas at frequencies above and below the TC-tip that are MOC inhibited. We hypothesized that the tail and side-lobe responses are enhanced by the same active mechanisms as CF cochlear amplification. If responses to CF, tail-frequency, and TC-side-lobe tones are all enhanced by prestin motility controlled by outer-hair-cell (OHC) transmembrane voltage, then they should depend on OHC stereocilia position in the same way. To test this, we cyclically changed the OHC-stereocilia mechano-electric-transduction (MET) operating point with low-frequency "bias" tones (BTs) and increased the BT level until the BT caused quasi-static OHC MET saturation that reduced or "suppressed" the gain of OHC active processes. While measuring cat AN-fiber responses, 50 Hz BT level series, 70-120 dB SPL, were run alone and with CF tones, or 2.5 kHz tail-frequency tones, or side-lobe tones. BT-tone-alone responses were used to exclude BT sound levels that produced AN responses that might obscure BT suppression. Data were analyzed to show the BT phase that suppressed the tone responses at the lowest sound level. We found that AN responses to CF, tail-frequency, and side-lobe tones were suppressed at the same BT phase in almost all cases. The data are consistent with the enhancement of responses to CF, tail-frequency, and side-lobe tones all being due to the same OHC-stereocilia MET-dependent active process. Thus, OHC active processes enhance AN responses at frequencies outside of the cochlear-amplified TC-tip region in both high- and low-frequency cochlear regions. The data are consistent with the AN response enhancements being due to enhanced RL motion that drives IHC-stereocilia deflection by traditional RL-TM shear and/or by changing the RL-TM gap. Since tail-frequency basilar membrane (BM) motion is not actively enhanced, the tail-frequency IHC drive is from a vibrational mode little present on the BM, not a "second filter" of BM motion.

A Model for Link Pruning to Establish Correctly Polarized and Oriented Tip Links in Hair Bundles.

Tip links are thought to gate the mechanically sensitive transduction channels of hair cells, but how they form during development and regeneration remains mysterious. In particular, it is unclear how tip links are strung between stereocilia so that they are oriented parallel to a single axis; why their polarity is uniform despite their constituent molecules' intrinsic asymmetry; and why only a single tip link is present at each tip-link position. We present here a series of simple rules that reasonably explain why these phenomena occur. In particular, our model relies on each of the two ends of the tip link having distinct Ca2+-dependent stability and being connected to different motor complexes. A simulation employing these rules allowed us to explore the parameter space for the model, demonstrating the importance of the feedback between transduction channels and angled links, links that are 60° off-axis with respect to mature tip links. We tested this key aspect of the model by examining angled links in chick cochlea hair cells. As implied by the assumptions used to generate the model, we found that angled links were stabilized if there was no tip link at the tip of the upper stereocilium, and appeared when transduction channels were blocked. The model thus plausibly explains how tip-link formation and pruning can occur.

Model-based estimation of the frequency tuning of the inner-hair-cell stereocilia from neural tuning curves.

This study proposes that the frequency tuning of the inner-hair-cell (IHC) stereocilia in the intact organ of Corti can be derived from the responses of the auditory fibers (AFs) using computational tools. The frequency-dependent relationship between the AF threshold and the amplitude of the stereocilia vibration is estimated using a model of the IHC-mediated mechanical to neural transduction. Depending on the response properties of the considered AF, the amplitude of stereocilia deflection required to drive the simulated AF above threshold is 1.4 to 9.2 dB smaller at low frequencies (≤500 Hz) than at high frequencies (≥4 kHz). The estimated frequency-dependent relationship between ciliary deflection and neural threshold is employed to derive constant-stereocilia-deflection contours from previously published AF recordings from the chinchilla cochlea. This analysis shows that the transduction process partially accounts for the observed differences between the tuning of the basilar membrane and that of the AFs.