Resonant tectorial membrane motion in the inner ear: Its crucial

Transcription

1 Proc. Natl. Acad. Sci. USA Vol. 93, pp , August 1996 Neurobiology Resonant tectorial membrane motion in the inner ear: Its crucial role in frequency tuning (cochlear amplifier/outer hair cell/basilar membrane/two-dimensional motion) ANTHONY W. GUMMR*, WRNR HMMRT, AND HANS-PTR ZNNR Section of Physiological Acoustics and Communication, Department of Otolaryngology, University of Tubingen, Silcherstrasse 5, Tubingen, Germany Communicated by Jozef J. Zwislocki, Syracuse University, Syracuse, NY, May 22, 1996 (received for review February 26, 1996) ABSTRACT The tectorial membrane has long been postulated as playing a role in the exquisite sensitivity of the cochlea. In particular, it has been proposed that the tectorial membrane provides a second resonant system, in addition to that of the basilar membrane, which contributes to the amplification of the motion of the cochlear partition. Until now, technical difficulties had prevented vibration measurements of the tectorial membrane and, therefore, precluded direct evidence of a mechanical resonance. In the study reported here, the vibration of the tectorial membrane was measured in two orthogonal directions by using a novel method of combining laser interferometry with a photodiode technique. It is shown experimentally that the motion of the tectorial membrane is resonant at a frequency of octave (oct) below the resonant frequency of the basilar membrane and polarized parallel to the reticular lamina. It is concluded that the resonant motion of the tectorial membrane is due to a parallel resonance between the mass of the tectorial membrane and the compliance of the stereocilia of the outer hair cells. Moreover, in combination with the contractile force of outer hair cells, it is proposed that inertial motion of the tectorial membrane provides the necessary conditions to allow positive feedback of mechanical energy into the cochlear partition, thereby amplifying and tuning the cochlear response. Understanding the micromechanical mechanisms underlying the extraordinary sensitivity of the cochlea is a cardinal goal of auditory physiology. It is generally agreed that motion of the tectorial membrane (TM) relative to the cuticular plate of a sensory hair cell stimulates transduction channels in its stereocilia-directly through physical contact to the TM of the longest stereocilia of the outer hair cells (OHCs) and indirectly by fluid motion around the stereocilia of the inner hair cells (1-5). Moreover, because OHCs undergo somatic length changes in response to electrical (6-8) and chemical (9) stimuli, OHCs and their stereocilia are supposed to feed mechanical energy back into the cochlear partition, thereby reducing its impedance (10-12). Therefore, the TM is expected to be functionally connected not only to the input of mechanoelectrical transducers in hair-cell stereocilia, but also to the output of electromechanical transducers in the OHC membrane. Technical difficulties have prevented measurements of TM vibration. Therefore, functional information has been inferred from morphological investigations (13-15), stiffness measurements post mortem (16) and in vivo (17), a physical model (18), mathematical models (5, 10, 11, 19-24), together with the frequency tuning properties of evoked otoacoustic emissions (25, 26) and cochlear microphonic potentials (17). In general, the latter models (5, 10, 18-26) require that the TM be mechanically resonant. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C solely to indicate this fact. The aim of the present study was to experimentally characterize the vibration response of the TM. This was achieved by developing an optical system that detected vibrations in two orthogonal directions: one perpendicular to the basilar membrane (BM), in the transverse direction, and the other parallel to the BM, in the radial direction (Fig. 1). Recordings were made in the apical turn of the cochlea where the upper surface of the TM is optically accessible (27-29). An isolated temporal bone preparation of the guinea pig cochlea was developed for this purpose. Provided appropriate precautions are taken, this region of the cochlea has the advantage that the frequency selectivity of the in vitro vibration responses of Hensen's cells, Reissner's membrane, and the cuticular plate of hair cells, measured in the transverse direction (30), is similar to that of tuning curves for primary auditory nerve fibers (31, 32) and receptor potentials (1, 33) measured at a sound pressure level (SPL) of db above threshold. Two experimental protocols were employed. First, the transverse component in response to intracochlear current injection (34) was used to uncover resonant motion of the TM and also to locate the resonant frequency of the BM. Second, responses to sound were measured in both the transverse and the radial directions to describe the dynamics of TM motion. In our experiments, the rationale for the current-injection experiments was based on the premise that sinusoidal current injected locally into the organ of Corti causes the OHCs to exert synchronous, sinusoidal forces of equal magnitude on the TM-stereocilia complex and on the BM-Deiters cell complex. If these structures can be considered to be in series mechanically, so that each experiences the same force delivered by the OHC, then measurement of the velocity of the TM and BM yields the TM impedance relative to the BM impedance. According to experiments of Mammano and Ashmore (35), for positive current injection in scala media the OHCs hyperpolarize and elongate, causing the TM to move toward scala vestibuli and the BM toward scala tympani. Moreover, the electromotors in the OHC wall function independent of frequency, at least up to 22 khz (36). Therefore, instead of the pressure transducer (the loudspeaker) being located at the external ear canal, as it normally is for acoustical stimulation, it is located within the cochlear partition for electrical stimulation; the OHCs become the frequency-independent, electromechanical transducers. MATRIALS AND MTHODS Temporal bones were removed from guinea pigs ( g), which were decapitated after cervical dislocation. The temporal bone was cemented (Harvard dental cement) to a delrin sound delivery cone and the ventral bulla region opened to expose the apical end of the cochlea. A sheet of Parafilm was Abbreviations: TM, tectorial membrane; OHC, outer hair cell; BM, basilar membrane; BF, best frequency; oct, octave; SPL, sound pressure level. *To whom reprint requests should be addressed. 8727

2 8728 Neurobiology: Gummer et al. FIG. 1. Optical measurement of the vibration of the organ of Corti. Velocity in the transverse direction (T) was measured with a laser Doppler velocimeter (LDV) coupled into the side-arm of an epifluorescence microscope (M) and displacement in the radial direction (R) with a double photodiode (PD) mounted on the microscope. The drawing of the cross-section of the organ of Corti was made from light-microscopic observations of histological sections of the basal part of the fourth cochlear turn. The orientation of the "radial" fibers in the TM is derived from ref. 15; they represent collagenous, type A protofibrils. Note that (i) the stereocilia of the OHCs are orthogonal to the reticular lamina and the lower margin of the TM; (ii) the long axis of the OHCs are inclined at about 550 to the reticular lamina; (iii) the reticular lamina is inclined at about 350 to the BM or 145 to the positive radial direction; and (iv) at the upper and lower margins of the TM, the radial fibers run approximately parallel to the margins and are extremely dense at the lower margin near the stereocilia. glued (Histoacryl) to the bony wall of the cochlear apex to mechanically support a fluid droplet, thereby ensuring that the middle-ear cavities remained free of fluid. Maintenance of air-filled middle-ear cavities is the main difference to the temporal bone preparation described in ref. 27, where the bone was totally submerged in life-support medium. A fluid-filled middle ear has the disadvantage of an overall attenuation of 35 db and artificial sharpening of the tuning curves (28). A small hole was made in the cochlear wall over scala vestibuli; this operation was performed under the droplet to ensure that stria vascularis, Reissner's membrane, and the helicotrema remained intact. Recordings were usually made in the fourth turn, at about 1/4 turn distant from the third turn, or about 2.3 mm from the apical extremity of the cochlea. Depending on the experimental protocol, Reissner's membrane was either left intact or intentionally opened; for the former the fluid droplet was Hanks' solution, and for the latter it was artificial endolymph composed of 150 mm K, 1 mm Na, 2 mm Ca, 130 mm Cl, 25 mm HCO3, 5 mm Hepes (308 mosm, ph 7.4), according to the recipe in ref. 14. A normal in vivo value for endolymphatic calcium, 20,tM (37), was not used because the TM did not remain stable under that condition; it tended to retract radially. The relative stability of the TM for the higher calcium concentration is qualitatively consistent with the results of a detailed study (38) describing the osmotic responses of the mouse TM to isosmotic changes of sodium, potassium and calcium. The fluid droplet served not only to prevent drying of the cochlea and hydrodynamic imbalance between Proc. Natl. Acad. Sci. USA 93 (1996) the scalae, but also to avoid light reflections and refractions at an air interface by placing the microscope objective directly on the fluid (16). Vibration measurements began about 20 min post mortem, lasted h, and were done in different order on different animals to exclude the possibility of time artifacts. Velocity in the transverse direction was measured with a laser Doppler velocimeter (model OFV-302; Polytec, Waldbronn, Germany) coupled into the side-arm of an epifluorescence microscope (Leitz Aristomet), in a manner similar to that described in refs. 39 and 40; the transverse direction coincides with the optical axis of the microscope (Fig. 1). Displacement in the radial direction was measured with a double photodiode (model BPX 48; Siemens, Munich, Germany) mounted on the microscope parallel to the focal plane. The need for a small hole in the cochlear wall meant that it was possible to adjust the focal plane of the microscope parallel to the BM but rarely possible to adjust it parallel to the reticular lamina. Therefore, the preparation was placed such that the transverse direction was approximately orthogonal to the BM. The focal plane was no more than ± 100 from the plane of the BM; it was determined from the transverse and radial distances between a focal point on a border cell of the internal spiral sulcus and one on a Claudius cell, with distances calculated from turns of the micrometers on the microscope. Data were not corrected to place the radial direction parallel to the reticular lamina because the possibility of more than one degree of vibrational freedom would require data interpretation before presentation. The microscope lens was a Zeiss x 40, 0.75 NA water immersion with working distance of 1.92 mm and focal depth of 1.4,um. For the laser Doppler velocimeter, the wavelength was 633 nm, the output power was 1 mw, and the diameter of the scattered laser beam was about 5,tm, which is less than that of an OHC (7-10 Am). For the photodiode, the object was illuminated from above with the microscope light and the magnified image (X203) of an edge of the object projected onto the double photodiode. Cochlear structures in the focal plane were discerned on a video display using a Hamamatsu camera (C3077) with contrast enhancement (C2400). For each recording configuration, an in situ calibration of the photodiode measurement system was made with the aid of a piezoelectric bimorph on which the photodiode was mounted. Stimulation of the bimorph with a sinusoidal calibration voltage (72.5 Hz) produced a spectral peak in the photodiode output, the magnitude of which (176 nm) served as the in situ calibration signal. This signal was used to position the image of an edge of the microsphere in the region of maximum photodiode sensitivity. The harmonic content of the photodiode output for sinusoidal bimorph stimulation was used to ensure that acoustic vibration responses were measured in the linear range of the photodiode (harmonic distortion components 40 db below the fundamental). The frequency response of the measurement system was calibrated by measuring the frequency response of a microsphere glued to a calibrated piezoelectric crystal; the crystal was calibrated with the laser Doppler velocimeter (resonant frequency, 25 khz). Sound was presented closed field from a Beyer DT48 loudspeaker and sound pressure was measured with a Bruel & Kjaer (Narrum, Denmark) 1/4-inch condensor microphone inserted axially into a conical sound coupler. Sound pressure was flat (±5 db) from -4 khz, with measured second and third harmonics more than 40 and 50 db, respectively, below the fundamental component at 110 db SPL. For current injection, an insulated Ag-AgCl electrode of diameter 300,um was placed at the apical end of the cochlea in scala vestibuli, just above the measurement location. The ground electrode was in the fossa of the paraflocculus, which was filled with Hanks' balanced salt solution. The amplitude spectrum of the injected current was flat (+ 1 db) up to 2 khz;

3 Neurobiology: Gummer et al. measured harmonic distortion components were less than -60 db for 300,uA. For velocity measurements with the laser Doppler velocimeter, acoustical or electrical stimuli consisted of a multi-tone complex, derived from a FFT analyzer (model AD-3525; AND, San Diego), with 740 frequencies equally spaced (2.5 Hz) from khz and with equal amplitude but random phase. This signal simulates a band-limited white noise process. The noise process was sampled at 2048 instants in a frame of duration 400 ms. The number of frames used to form an average frequency response was 20 for a Hensen's cell recording and for the TM and BM. Although the commercially available laser Doppler velocimeter was sensitive enough to measure acoustically induced motion of the cuticular plate and Hensen's cells (30), as well as Claudius cells on the BM, microreflectors were required for all TM measurements. Moreover, for electrical stimulation experiments the maximal allowable injected current, to avoid tissue damage, was such that the velocities were smaller than the acoustically induced velocities (typically 40 db), meaning that a microreflector was also required for detection of electrically induced BM motion. Therefore, after having opened Reissner's membrane under artificial endolymph, polystyrene (Polysciences) microspheres, diameter 10,tm and specific gravity 1.05, were introduced onto the upper surface of the TM and onto the Claudius cells overlying the BM. The relatively small focal depth of the microscope objective (1.4,um) and the large reflectance of a microsphere, at least 10 db greater than that of the Hensen's cells, meant that the reflected signal was derived primarily from the microsphere. This assertion was supported by the loss of the detector signal when the laser beam was focused nearby but not on the microsphere. vidence that the microspheres did not vibrate relative to the TM [an extremely sticky structure (29)] comes from vibration measurements of the lipid droplets in Hensen's cells: their phase responses were identical to those of the TM and their amplitude responses were of the same form (but larger by about 6 db). The coupling of the TM and Hensen's cell responses was expected because of the physical coupling between these structures (Fig. 1). Similarly, there was no detectable difference between the responses of a BM microsphere and a reflecting point within a Claudius cell located a few tens of,tms radially from it. For displacement measurements with the double photodiode, the stimulus was a multi-tone complex, derived from the FFT analyzer (model AD-3525; AND), with 44 frequencies from 2.5 Hz to khz, which were multiples of 2.5 Hz but with a minimum spacing of 1/5.3 octave (oct); the amplitudes were equal but the phases were random. This signal simulates a colored noise process in which the power spectral density decreases with frequency. The noise process was sampled at 2048 instants in a frame of duration 400 ms. With 20 frames to form an average frequency response, the noise floor was less than 1 nm and the reproducibility ±3 db. The colored noise has the advantage that the total spectral energy is less than for the band-limited white noise (12 db); moreover, for the same signal-to-noise ratio, the acquisition time is shorter (44-fold) than for sinusoidal stimulus paradigms. Acoustic responses are referred to sound pressure near the tympanic membrane and are not corrected for the middle-ear response because the motion of the stapedial crux was measured to be independent of frequency up to about 2 khz. lectrical responses are relative to positive current injected into scala vestibuli. Phase in the transverse direction is positive for motion toward scala vestibuli and in the radial direction for motion toward the spiral limbus. RSULTS lectromotility. Current injection experiments (n = 24) uncovered major differences between the micromechanical Proc. Natl. Acad. Sci. USA 93 (1996) 8729 properties of the TM and BM, which were not detectable in the acoustic responses. Although the acoustically induced motion was larger for the TM than for the BM, the amplitude curves had similar forms up to 1 khz (Fig. 24). The larger overall amplitude for the TM was due to the geometry of the cochlear partition, the TM being recorded closer to the center of the partition than the BM. The frequency of largest acoustic response, called the best frequency (BF), was 660 Hz (full arrow in Fig. 2A). [In accordance with others (41), when the amplitude response exhibited a flat maximum rather than a well-defined peak, the BF was estimated as the frequency above which the acoustic amplitude response began to "rapidly" decay.] In contrast to the situation for acoustic stimulation, for electrical stimulation, there was a maximum in the amplitude response at 450 Hz (broken arrow in Fig. 2C), suggesting that the TM was resonant-not at the BF of the cochlear partition, but at oct'below BF. This TM resonance was coincident with a minimum in the acoustic amplitude responses of the TM an'd BM (Fig. 2A). In addition, there was an electrically induced antiresonance in the BM response coincident with the TM resonance (Fig. 2C), with minimum 14 db below the TM maximum. Since the TM resonance and BM antiresonance were effectively mirror images, one can be sure that they were not due to resonances in the electromechanical parameters of the OHCs. Moreover, at low and high frequencies there was a phase difference of cycles between TM and BM motion (below 250 Hz and above 900 Hz in Fig. 2D), meaning that bulk fluid flow and electrophonics can be excluded as the source of the electrically induced motion. Instead, electrically induced tuning of the TM must be due to a mechanical resonance associated with the TM-stereocilia complex, the BM exhibiting the antiresonance because in this frequency range the TM-stereocilia complex shunts the greater part of the mechanical energy from the OHCs. The resonant frequency of the BM was readily obtained from the TM and BM phase responses for electrical stimulation. By definition, at resonance the force acting on a body is resistive whereas sufficiently above resonance the force is inertial, meaning that motion above resonance must lag motion at resonance by 5 cycle. Therefore, the resonant frequency of the BM was given as the frequency above TM resonance at which electrically induced BM motion lagged TM motion by 5 cycle (600 Hz in Fig. 2D). In general, the BM resonant frequency was about oct above the TM resonant frequency and coincided with the acoustic BF of the TM and BM Ṫhe acoustic responses in Fig. 2A exhibit two amplitude minima or antiresonances-one at 770 Hz and the other at 1 khz. The first of these antiresonances might be associated with the two maxima predicted by Zwislocki (5) for the two resonator system formed by the TM-stereocilia complex and the BM-Deiters cell complex; the first maximum is located at the BF and the other at 890 Hz. However, an internal antiresonance in the organ of Corti cannot be discounted. The second acoustic antiresonance (at 1 khz in Fig. 2A) marks the end of traveling wave motion because the acoustic phase curves (Fig. 2C) exhibited a plateau beginning at this frequency (41). The TM resonance was found to be vulnerable, vanishing within 30 min after beginning the current injection experiments. Then, the electrically induced responses were that of a first-order high-pass filter with corner frequency coincident with the resonant frequency of the cochlear partition (not illustrated). Nevertheless, there was still antiphase motion, implying that the OHCs were electromotive. Two-Dimensional Motion. In the second series of experiments, measurement of the acoustic vibration response of the TM in both the transverse and radial directions enabled the dynamics of the TM to be spatially characterized (n = 23). Velocity in the transverse direction was measured, as usual,

4 8730 Neurobiology: Gummer et al. Proc. Natl. Acad. Sci. USA 93 (1996) 2 Velocity Velocity 'Icn :L "a a) 0.05 U) a) 0C.) U) (0) 0:02 * 0.01_ ) U) I- U) -C 0.0 L -.5 D I 4.6 pa ;~~~~~~ ' ~~~~~T 1 2 Frequency (khz) Frequency (khz) FIG. 2. Comparison of acoustically and electrically induced motion of the tectorial membrane (v) and the basilar membrane (0) in the fourth cochlear turn. Amplitude (A) and phase (B) of velocity for acoustic stimulation; amplitude (C) and phase (D) of velocity for 4.6 pa (per spectral point) injected into scala vestibuli. Symbols are used as a visual aid. The velocity amplitudes were linearly dependent on current amplitude up to 300,uA, implying that OHC electromotility was linear for the currents used in determining the frequency responses. Likewise, acoustic amplitude responses were linearly dependent on SPL up to 100 db SPL. Acoustic responses were usually measured at db SPL and have been linearly corrected to the same SPL; 40 db was chosen as the reference SPL to give amplitudes comparable with the electrically induced values. The full and broken arrows indicate the best frequencies of the cochlear partition and the TM, respectively. Distance from apex: 2.3 mm. Note that the electrically induced TM response has a resonance at 450 Hz, but that the acoustically induced TM and BM amplitude responses have a local minimum in this region. with the laser Doppler velocimeter, and displacement in the radial direction was measured with an in situ calibrated photodiode. The transverse component exhibited an antiresonance below BF, at 0.4 oct in the example given in Fig. 3 (BF of 735 Hz). The radial component, however, exhibited a resonance at the antiresonant frequencies of the transverse component. The radial component was 30 db larger than the transverse component at the antiresonant frequency. The amplitude and phase data demonstrate the existence of at least two degrees of vibrational freedom in the TM. However, BM motion showed only one degree of vibrational freedomorthogonal to the BM surface (data not illustrated)- consistent with the classical beam-bending model of BM vibration (16). For two degrees of freedom at a single frequency, a point in the TM must undergo an elliptical trajectory, which can degenerate to rectilinear motion at some frequencies. As shown in the Inset of Fig. 3A, the velocity trajectories depend on the relative amplitudes and phases of the two components. From Hz ( oct below BF) the transverse component is larger, consistent with the classical model that this component is principally derived from rotation of the organ of Corti about the spiral limbus. This rotational motion provides the first degree of freedom. Motion below 215 Hz is presumed to be influenced by the helicotrema (42), so that this very low-frequency range will not be discussed further. For Hz (1.0- oct below BF), the radial component dominates, the difference (30 db) being greatest close to the -oct frequency. Taken together with the presence of only one degree of freedom in the BM motion and the results of the electrostimulation experiments, this implies that the radial component in this frequency range is due to a mechanical resonance associated with the TM-stereocilia complex, which is tuned to oct below BF of the cochlear partition. This resonant, translational motion provides the second degree of freedom. The major axis of the elliptical trajectory (1700) at the -oct frequency is approximately parallel to the reticular lamina. At 908 Hz, or 0.3 oct above BF, there was a "mild" antiresonance in the transverse component and a corresponding peak in the radial component, such that the major axis of the elliptical trajectory (164 ) was also aligned approximately parallel to the reticular lamina. The depths of the antiresonances were dependent on recording angle. This is illustrated by the dashed curves in Fig. 3 which, based on the data measured in the two orthogonal directions, are the amplitudes and phases of the velocity that would be recorded in the transverse direction if the preparation were rotated anticlockwise by 30. Two important features inherent to two-dimensional motion become obvious from such an axis rotation. First, the antiresonance below the BF is now shallow and resembles that for the amplitude response for sound stimulation in Fig. 2A. Second, the phase response in the transverse direction is no longer shallow and becomes consistent with traveling-wave motion (41).

5 I L 0.0 X - a) o -1.0 `- a) X) -1.5.C il -in -Z.U -2.5 Neurobiology: Gummer et al _ Velo city Proc. Natl. Acad. Sci. USA 93 (1996) 8731 derives from a parallel resonance of the TM mass with the stereocilia compliance, as predicted by Zwislocki (5, 18). The I ;00 db SPL two conditions are (i) the axial mechanical impedance of the I G+ OHCs is large compared with the radial mechanical impedance of the stereocilia bundle of the OHCs, as model calculations from in vitro data suggest (22, 43, 44); and (ii) the BM l t. impedance is large compared with the impedances of the TM I and g OHC stereocilia, as in vivo (17, 45) and in vitro (16, 46, 47) experiments indicate. Then for both acoustic (5, 18) and electrical stimulation, resonant TM motion must occur when l. the impedance of the OHC stereocilia bundle cancels the TM impedance; namely, at f = 1/27TmTMCp where Cp = CTMCS/ (CTM + Cs), or Cp Cs, for the TM compliance much greater than the stereocilia compliance. Thus, the experimentally determined resonant frequency of 450 Hz for TM motion 1 2 means that the product MTMCP is 1.3 x 10-7.s2 at the 16 mm point of the guinea-pig cochlea. Moreover, the current injection experiments showed that the BM resonant frequency is located oct above the frequency at which the TM mass is resonant with the stereocilia compliance, in agreement with T the value of 4 oct predicted by Zwislocki (5). The major problem with the isolated temporal bone preparation is that the endocochlear potential was -0 mv, meaning that the electrical drive to the hair cells was about half of its normal in vivo value (48, 49). The absence of an endocochlear potential might explain the linearity of the responses (50). Nevertheless, the amplitude responses measured in the trans- IR Qx ~ verse direction were similar to those for neural (31, 32) and hair-cell (1, 33) responses at db above threshold for this region of the cochlea. This similarity is consistent with the 1 2 observation that endocochlear potential in the apical region of Frequen cy (khz) the cochlea is not as important to cochlear tuning as in the base FIG. 3. Transverse (T; ) and radial (R; 0) components of acoustically induced tectorial-membrane motion. Amplitude (A) and phase (B) of velocity. Dashed curves are the amplitudes and phases that would be recorded by the laser Doppler velocimeter if the preparation were rotated anticlockwise by 300; they are based on the amplitudes and phases measured in the two orthogonal directions (T and R). Displacement was measured in the radial direction and converted to velocity. Vibration measurements are from a microsphere placed on the upper surface of the TM over the OHCs. All responses were linear and are scaled to 100 db SPL. The elliptical, velocity trajectories at the designated frequencies were calculated from the amplitudes and phases of the two measured components. The beginning of a trajectory is indicated by a circle and a quarter-cycle later by a triangle. (Inset) Linear scale for the radial (R) and transverse (T) components of the velocity trajectories (±3 mm.s-1). For orientation purposes, a spatial angle of 1500 is approximately parallel to the reticular lamina (Fig. 1), indicated by the line RL in the Inset. Notice that the major axes of the trajectories at the antiresonances (558 Hz and 908 Hz) are polarized approximately parallel to the reticular lamina. Arrow indicates the BF. Distance from apex, 2.3 mm. DISCUSSION The data provide direct confirmation of the predicted (5, 10, 11, 18-24) and experimentally inferred TM resonances (17, 25, 26). The current injection experiments uncovered resonant TM motion and located the resonant frequency of the BM; the two-dimensional experiments showed that the resonant motion of the TM is polarized parallel to the reticular lamina. Theoretically, resonant motion of the TM is governed primarily by elastic coupling of its mass to two structures: (i) to the spiral limbus through a rich network of highly organized collagenous and non-collagenous protofibrils and (ii) to the organ of Corti through the OHC stereocilia (13-15). Clearly, therefore, it is not possible to determine the absolute mechanical parameters of TM mass, MTM, TM compliance, CTm, and stereocilia compliance, Cs, from these measurements. However, provided two important mechanical conditions are satisfied, one may conclude that resonant motion of the TM TM compliant i~~~~~~~~~~sv XCIT.&, - STin INHIBIT DPOL CONTRACT HYP.POL TM inertial INHIBIT dl~~~~a HYRPOL LONGAT DPOL LONGAT XCIT CONTRACT A B sv -Time ST FIG. 4. The action of the electromechanical force of an OHC for compliant (A) or inertial (B) motion of the TM. The sinusoid represents the displacement of a point on the BM as a function of time. For compliant motion, excitation occurs for BM motion in direction of scala vestibuli (SV), whereas for inertial motion it occurs for BM motion in scala tympani (ST). Because of the membrane time constant of the OHC, the depolarizing receptor potential lags stereociliary displacement by 900. Consequently, for compliant TM motion the OHC electromechanical force produces attenuation, whereas for inertial TM motion it produces amplification of BM motion. Relative dimensions have been exaggerated for illustrative purposes.

6 8732 Neurobiology: Gummer et al. of the cochlea (51). Finally, current injection evoked acoustic emissions in the external ear canal (spectrum with maximum of 7 db SPL at 600 Hz for 4,xA), providing further evidence for electromechanical action of the OHCs and its coupling into the organ of Corti. If these results in the apical region of the cochlea can be extended to the basal region, then the finding that the TMstereocilia complex is tuned to about oct below the BM resonant frequency and the BF of the cochlear partition has important consequences for the function of the healthy and pathological cochlea. It is proposed that inertial TM motion in the BF region produces the necessary phase conditions to allow OHC contraction to reduce the impedance of the cochlear partition. With the aid of the cartoons in Fig. 4, we describe the resultant action of OHC electromechanical forces on the BM when the motion of the TM is either compliant (Fig. 4A) or inertial (Fig. 4B). When the motion of the TM is governed by its compliance, BM displacement toward scala vestibuli (Fig. 4A) causes deflection of the stereocilia tips in the direction of the longest stereocilia (12, 16, 24), the excitatory direction, producing a depolarizing receptor potential (52, 53) and contraction of the OHC (6-8). The resulting contractile force appears to be in phase with the receptor potential (36). However, the receptor potential lags stereocilia displacement by 5 cycle, beginning several octaves below the BF (52). Consequently, the OHC contractile force, which is directed toward scala vestibuli, is maximal at the instant when the BM crosses its baseline in the opposite direction, in the direction of scala tympani (Fig. 4A). As a result, the electromechanical force of the OHC attenuates the motion of the BM. Clearly, this mechanism cannot produce cochlear tuning; the OHCs act as an active "brake." However, when the motion of the TM is inertial, the phase of the stereociliary displacement is rotated cycle relative to its value for compliant TM motion. Then, the stereocilia are displaced in the excitatory direction for BM displacement in the direction of scala tympani rather than scala vestibuli (Fig. 4B). Consequently, the BM crosses its baseline in the same direction and at the same instant as the maximal electromechanical force (Fig. 4B). The electromechanical force of the OHC thereby amplifies the motion of the BM. Indeed, since frictional forces on the BM are expected to be proportional to BM velocity, which in turn is maximal when the BM crosses its baseline, the OHC electromechanical forces act to reduce the effective resistance of the cochlear partition. In other words, active amplification occurs through the synergistic action of the forces of TM inertia and OHC electromotility. We thank our colleagues S. Preyer and J. P. 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