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Home , Antiresonance, Basilar membrane, Claudius cell, Cochlear amplifier, Endolymph, Tectorial membrane

Proc. Natl. Acad. Sci. USA Vol. 93, pp. 8727-8732, August 1996 Neurobiology

Resonant motion in the inner : Its crucial role in tuning (/outer //two-dimensional motion) ANTHONY W. GUMMER*, WERNER HEMMERT, AND HANS-PETER ZENNER Section of Physiological and Communication, Department of Otolaryngology, University of Tubingen, Silcherstrasse 5, 72076 Tubingen, Germany Communicated by JozefJ. Zwislocki, Syracuse University, Syracuse, NY, May 22, 1996 (received for review February 26, 1996)

ABSTRACT The tectorial membrane has long been pos- The aim of the present study was to experimentally charac- tulated as playing a role in the exquisite sensitivity of the terize the vibration response of the TM. This was achieved by . In particular, it has been proposed that the tectorial developing an optical system that detected vibrations in two membrane provides a second resonant system, in addition to orthogonal directions: one perpendicular to the basilar mem- that of the basilar membrane, which contributes to the brane (BM), in the transverse direction, and the other parallel amplification of the motion of the cochlear partition. Until to the BM, in the radial direction (Fig. 1). Recordings were now, technical difficulties had prevented vibration measure- made in the apical turn of the cochlea where the upper surface ments of the tectorial membrane and, therefore, precluded of the TM is optically accessible (27-29). An isolated temporal direct evidence of a mechanical . In the study bone preparation of the guinea pig cochlea was developed for reported here, the vibration of the tectorial membrane was this purpose. Provided appropriate precautions are taken, this measured in two orthogonal directions by using a novel region of the cochlea has the advantage that the frequency method of combining laser interferometry with a photodiode selectivity of the in vitro vibration responses of Hensen's cells, technique. It is shown experimentally that the motion of the Reissner's membrane, and the cuticular plate of hair cells, tectorial membrane is resonant at a frequency of 0.5 octave measured in the transverse direction (30), is similar to that of (oct) below the resonant frequency of the basilar membrane tuning curves for primary auditory nerve fibers (31, 32) and and polarized parallel to the reticular lamina. It is concluded receptor potentials (1, 33) measured at a sound pressure level that the resonant motion of the tectorial membrane is due to (SPL) of 20-50 dB above threshold. a parallel resonance between the mass of the tectorial mem- Two experimental protocols were employed. First, the trans- brane and the compliance of the of the outer hair verse component in response to intracochlear current injection cells. Moreover, in combination with the contractile force of (34) was used to uncover resonant motion of the TM and also outer hair cells, it is proposed that inertial motion of the to locate the resonant frequency of the BM. Second, responses tectorial membrane provides the necessary conditions to allow to sound were measured in both the transverse and the radial positive feedback of mechanical energy into the cochlear directions to describe the dynamics of TM motion. partition, thereby amplifying and tuning the cochlear re- In our experiments, the rationale for the current-injection sponse. experiments was based on the premise that sinusoidal current injected locally into the causes the OHCs to Understanding the micromechanical mechanisms underlying exert synchronous, sinusoidal forces of equal magnitude on the the extraordinary sensitivity of the cochlea is a cardinal goal of TM-stereocilia complex and on the BM-Deiters cell complex. auditory physiology. It is generally agreed that motion of the If these structures can be considered to be in series mechan- tectorial membrane relative to the cuticular of a ically, so that each experiences the same force delivered by the (TM) plate OHC, then measurement of the velocity of the TM and BM sensory hair cell stimulates transduction channels in its stereo- yields the TM impedance relative to the BM impedance. cilia-directly through physical contact to the TM of the According to experiments of Mammano and Ashmore (35), for longest stereocilia of the outer hair cells (OHCs) and indirectly positive current injection in scala media the OHCs hyperpo- by fluid motion around the stereocilia of the inner hair cells larize and elongate, causing the TM to move toward scala (1-5). Moreover, because OHCs undergo somatic length vestibuli and the BM toward scala tympani. Moreover, the changes in response to electrical (6-8) and chemical (9) electromotors in the OHC wall function independent of fre- stimuli, OHCs and their stereocilia are supposed to feed quency, at least up to 22 kHz (36). Therefore, instead of the mechanical energy back into the cochlear partition, thereby pressure transducer (the loudspeaker) being located at the reducing its impedance (10-12). Therefore, the TM is ex- external , as it normally is for acoustical stimulation, pected to be functionally connected not only to the input of it is located within the cochlear partition for electrical stimu- mechanoelectrical transducers in hair-cell stereocilia, but also lation; the OHCs become the frequency-independent, elec- to the output of electromechanical transducers in the OHC tromechanical transducers. membrane. Technical difficulties have prevented measure- ments of TM vibration. Therefore, functional information has been inferred from morphological investigations (13-15), stiff- MATERIALS AND METHODS ness measurements post mortem (16) and in vivo (17), a Temporal bones were removed from guinea pigs (250-400 g), physical model (18), mathematical models (5, 10, 11, 19-24), which were decapitated after cervical dislocation. The tem- together with the frequency tuning properties of evoked poral bone was cemented (Harvard dental cement) to a delrin otoacoustic emissions (25, 26) and cochlear microphonic po- sound delivery cone and the ventral bulla region opened to tentials (17). In general, the latter models (5, 10, 18-26) expose the apical end of the cochlea. A sheet of Parafilm was require that the TM be mechanically resonant. Abbreviations: TM, tectorial membrane; OHC, outer hair cell; BM, The publication costs of this article were defrayed in part by page charge basilar membrane; BF, best frequency; oct, octave; SPL, sound payment. This article must therefore be hereby marked "advertisement" in pressure level. accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed.

8727 Downloaded by guest on October 2, 2021 8728 Neurobiology: Gummer et al. 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 1-1.5 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, Wald- bronn, Germany) coupled into the side-arm of an epifluores- cence 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, Ger- many) 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 , 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 interpreta- FIG. 1. Optical measurement of the vibration of the organ of Corti. tion before presentation. The microscope lens was a Zeiss x40, Velocity in the transverse direction (T) was measured with a laser 0.75 NA water immersion with working distance of 1.92 mm Doppler velocimeter (LDV) coupled into the side-arm of an epiflu- orescence microscope (M) and displacement in the radial direction (R) and focal depth of 1.4 ,um. For the laser Doppler velocimeter, with a double photodiode (PD) mounted on the microscope. The the wavelength was 633 nm, the output power was 1 mW, and drawing of the cross-section of the organ of Corti was made from the diameter of the scattered laser beam was about 5 ,tm, which light-microscopic observations of histological sections of the basal part is less than that of an OHC (7-10 Am). For the photodiode, the of the fourth cochlear turn. The orientation of the "radial" fibers in object was illuminated from above with the microscope light the TM is derived from ref. 15; they represent collagenous, type A and the magnified image (X203) of an edge of the object protofibrils. Note that (i) the stereocilia of the OHCs are orthogonal projected onto the double photodiode. Cochlear structures in to the reticular lamina and the lower margin of the TM; (ii) the long the focal plane were discerned on a video display using a axis of the OHCs are inclined at about 550 to the reticular lamina; (iii) Hamamatsu camera (C3077) with contrast enhancement 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 (C2400). TM, the radial fibers run approximately parallel to the margins and are For each recording configuration, an in situ calibration of extremely dense at the lower margin near the stereocilia. the photodiode measurement system was made with the aid of a piezoelectric bimorph on which the photodiode was glued (Histoacryl) to the bony wall of the cochlear apex to mounted. Stimulation of the bimorph with a sinusoidal cali- mechanically support a fluid droplet, thereby ensuring that the bration voltage (72.5 Hz) produced a spectral peak in the middle-ear cavities remained free of fluid. Maintenance of photodiode output, the magnitude of which (176 nm) served air-filled middle-ear cavities is the main difference to the as the in situ calibration signal. This signal was used to position temporal bone preparation described in ref. 27, where the bone the image of an edge of the microsphere in the region of was totally submerged in life-support medium. A fluid-filled maximum photodiode sensitivity. The harmonic content of the has the disadvantage of an overall attenuation of 35 photodiode output for sinusoidal bimorph stimulation was dB and artificial sharpening of the tuning curves (28). A small used to ensure that acoustic vibration responses were mea- hole was made in the cochlear wall over scala vestibuli; this sured in the linear range of the photodiode (harmonic distor- operation was performed under the droplet to ensure that stria tion components 40 dB below the fundamental). The fre- vascularis, Reissner's membrane, and the re- quency response of the measurement system was calibrated by mained intact. Recordings were usually made in the fourth measuring the of a microsphere glued to turn, at about 1/4 turn distant from the third turn, or about 2.3 a calibrated piezoelectric crystal; the crystal was calibrated mm from the apical extremity of the cochlea. Depending on with the laser Doppler velocimeter (resonant frequency, 25 the experimental protocol, Reissner's membrane was either kHz). left intact or intentionally opened; for the former the fluid Sound was presented closed field from a Beyer DT48 droplet was Hanks' solution, and for the latter it was artificial loudspeaker and sound pressure was measured with a Bruel & composed of 150 mM K, 1 mM Na, 2 mM Ca, 130 Kjaer (Narrum, Denmark) 1/4-inch condensor microphone mM Cl, 25 mM HCO3, 5 mM Hepes (308 mOsm, pH 7.4), inserted axially into a conical sound coupler. Sound pressure according to the recipe in ref. 14. A normal in vivo value for was flat (±5 dB) from 0.1-4 kHz, with measured second and endolymphatic calcium, 20 ,tM (37), was not used because the third harmonics more than 40 and 50 dB, respectively, below TM did not remain stable under that condition; it tended to the fundamental component at 110 dB SPL. retract radially. The relative stability of the TM for the higher For current injection, an insulated Ag-AgCl electrode of calcium concentration is qualitatively consistent with the re- diameter 300 ,um was placed at the apical end of the cochlea sults of a detailed study (38) describing the osmotic responses in scala vestibuli, just above the measurement location. The of the mouse TM to isosmotic changes of sodium, potassium ground electrode was in the fossa of the paraflocculus, which and calcium. The fluid droplet served not only to prevent was filled with Hanks' balanced salt solution. The drying of the cochlea and hydrodynamic imbalance between spectrum of the injected current was flat (+ 1 dB) up to 2 kHz; Downloaded by guest on October 2, 2021 Neurobiology: Gummer et al. Proc. Natl. Acad. Sci. USA 93 (1996) 8729

measured harmonic distortion components were less than -60 properties of the TM and BM, which were not detectable in the dB for 300 ,uA. acoustic responses. Although the acoustically induced motion For velocity measurements with the laser Doppler veloci- was larger for the TM than for the BM, the amplitude curves meter, acoustical or electrical stimuli consisted of a multi-tone had similar forms up to 1 kHz (Fig. 24). The larger overall complex, derived from a FFT analyzer (model AD-3525; AND, amplitude for the TM was due to the geometry of the cochlear San Diego), with 740 equally spaced (2.5 Hz) from partition, the TM being recorded closer to the center of the 0.05-1.9 kHz and with equal amplitude but random . This partition than the BM. The frequency of largest acoustic signal simulates a band-limited white noise process. The noise response, called the best frequency (BF), was 660 Hz (full process was sampled at 2048 instants in a frame of duration 400 arrow in Fig. 2A). [In accordance with others (41), when the ms. The number of frames used to form an average frequency amplitude response exhibited a flat maximum rather than a response was 20 for a Hensen's cell recording and 20-100 for well-defined peak, the BF was estimated as the frequency the TM and BM. Although the commercially available laser above which the acoustic amplitude response began to "rap- Doppler velocimeter was sensitive enough to measure acous- idly" decay.] In contrast to the situation for acoustic stimula- tically induced motion of the cuticular plate and Hensen's cells tion, for electrical stimulation, there was a maximum in the (30), as well as Claudius cells on the BM, microreflectors were amplitude response at 450 Hz (broken arrow in Fig. 2C), required for all TM measurements. Moreover, for electrical suggesting that the TM was resonant-not at the BF of the stimulation experiments the maximal allowable injected cur- cochlear partition, but at 0.5 oct'below BF. This TM resonance rent, to avoid tissue damage, was such that the velocities were was coincident with a minimum in the acoustic amplitude smaller than the acoustically induced velocities (typically 40 responses of the TM an'd BM (Fig. 2A). In addition, there was dB), meaning that a microreflector was also required for an electrically induced antiresonance in the BM response detection of electrically induced BM motion. Therefore, after coincident with the TM resonance (Fig. 2C), with minimum 14 having opened Reissner's membrane under artificial en- dB below the TM maximum. Since the TM resonance and BM dolymph, polystyrene (Polysciences) microspheres, diameter antiresonance were effectively mirror images, one can be sure 10 ,tm and specific gravity 1.05, were introduced onto the that they were not due to in the electromechanical upper surface of the TM and onto the Claudius cells overlying parameters of the OHCs. Moreover, at low and high frequen- the BM. The relatively small focal depth of the microscope cies there was a phase difference of 0.5 cycles between TM and objective (1.4 ,um) and the large reflectance of a microsphere, BM motion (below 250 Hz and above 900 Hz in Fig. 2D), at least 10 dB greater than that of the Hensen's cells, meant meaning that bulk fluid flow and electrophonics can be that the reflected signal was derived primarily from the excluded as the source of the electrically induced motion. microsphere. This assertion was supported by the loss of the Instead, electrically induced tuning of the TM must be due to detector signal when the laser beam was focused nearby but not a mechanical resonance associated with the TM-stereocilia on the microsphere. Evidence that the microspheres did not complex, the BM exhibiting the antiresonance because in this vibrate relative to the TM [an extremely sticky structure (29)] frequency range the TM-stereocilia complex shunts the comes from vibration measurements of the lipid droplets in greater part of the mechanical energy from the OHCs. Hensen's cells: their phase responses were identical to those of The resonant frequency of the BM was readily obtained the TM and their amplitude responses were of the same form from the TM and BM phase responses for electrical stimula- (but larger by about 6 dB). The coupling of the TM and tion. By definition, at resonance the force acting on a body is Hensen's cell responses was expected because of the physical resistive whereas sufficiently above resonance the force is coupling between these structures (Fig. 1). Similarly, there was inertial, meaning that motion above resonance must lag mo- no detectable difference between the responses of a BM tion at resonance by 0.25 cycle. Therefore, the resonant microsphere and a reflecting point within a Claudius cell frequency of the BM was given as the frequency above TM located a few tens of ,tms radially from it. resonance at which electrically induced BM motion lagged TM For displacement measurements with the double photo- motion by 0.25 cycle (600 Hz in Fig. 2D). In general, the BM diode, the stimulus was a multi-tone complex, derived from the resonant frequency was about 0.5 oct above the TM resonant FFT analyzer (model AD-3525; AND), with 44 frequencies frequency and coincided with the acoustic BF of the TM and from 2.5 Hz to 1.9725 kHz, which were multiples of 2.5 Hz but BM. with a minimum spacing of 1/5.3 octave (oct); the The acoustic responses in Fig. 2A exhibit two amplitude were equal but the phases were random. This signal simulates minima or antiresonances-one at 770 Hz and the other at 1 a colored noise process in which the power kHz. The first of these antiresonances might be associated with decreases with frequency. The noise process was sampled at the two maxima predicted by Zwislocki (5) for the two 2048 instants in a frame of duration 400 ms. With 20 frames to resonator system formed by the TM-stereocilia complex and form an average frequency response, the noise floor was less the BM-Deiters cell complex; the first maximum is located at than 1 nm and the reproducibility ±3 dB. The colored noise the BF and the other at 890 Hz. However, an internal has the advantage that the total spectral energy is less than for antiresonance in the organ of Corti cannot be discounted. The the band-limited white noise (12 dB); moreover, for the same second acoustic antiresonance (at 1 kHz in Fig. 2A) marks the signal-to-noise ratio, the acquisition time is shorter (44-fold) end of traveling wave motion because the acoustic phase than for sinusoidal stimulus paradigms. curves (Fig. 2C) exhibited a plateau beginning at this fre- Acoustic responses are referred to sound pressure near the quency (41). tympanic membrane and are not corrected for the middle-ear The TM resonance was found to be vulnerable, vanishing response because the motion of the stapedial crux was mea- within 30 min after beginning the current injection experi- sured to be independent of frequency up to about 2 kHz. ments. Then, the electrically induced responses were that of a Electrical responses are relative to positive current injected first-order high-pass filter with corner frequency coincident into scala vestibuli. Phase in the transverse direction is positive with the resonant frequency of the cochlear partition (not for motion toward scala vestibuli and in the radial direction for illustrated). Nevertheless, there was still antiphase motion, motion toward the . implying that the OHCs were electromotive. Two-Dimensional Motion. In the second series of experi- ments, measurement of the acoustic vibration response of the RESULTS TM in both the transverse and radial directions enabled the Electromotility. Current injection experiments (n = 24) dynamics of the TM to be spatially characterized (n = 23). uncovered major differences between the micromechanical Velocity in the transverse direction was measured, as usual, Downloaded by guest on October 2, 2021 8730 Neurobiology: Gummer et al. Proc. Natl. Acad. Sci. USA 93 (1996) Velocity Velocity 2

cn'I 0.5 U) E a) :L "a 0.2 a) 0C.) 0.1 U) E 0.05 (0)

0:02 * 0.01_ 0.1 0.2 0.5 1 2 0.1 0.2 0.5 1 2 10 - 0.5 D I 4.6pA

U) 0.0 I-E ;~~~~~~ 0) U) -C E ' ~~~~~T L -.5

-1.0 0.1 0.2 0.5 1 2 0.1 0.2 0.5 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 .60-70 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 dominates, the difference (30 dB) being greatest close to the radial direction was measured with an in situ calibrated 0.5-oct frequency. Taken together with the presence of only photodiode. The transverse component exhibited an antireso- one degree of freedom in the BM motion and the results of the nance below BF, at 0.4 oct in the example given in Fig. 3 (BF electrostimulation experiments, this implies that the radial of 735 Hz). The radial component, however, exhibited a component in this frequency range is due to a mechanical resonance at the antiresonant frequencies of the transverse resonance associated with the TM-stereocilia complex, which component. The radial component was 30 dB larger than the is tuned to 0.5 oct below BF of the cochlear partition. This transverse component at the antiresonant frequency. The resonant, translational motion provides the second degree of amplitude and phase data demonstrate the existence of at least freedom. The major axis of the elliptical trajectory (1700) at two degrees of vibrational freedom in the TM. However, BM the 0.5-oct frequency is approximately parallel to the reticular motion showed only one degree of vibrational freedom- lamina. At 908 Hz, or 0.3 oct above BF, there was a "mild" orthogonal to the BM surface (data not illustrated)- antiresonance in the transverse component and a correspond- consistent with the classical beam-bending model of BM ing peak in the radial component, such that the major axis of vibration (16). the elliptical trajectory (164°) was also aligned approximately For two degrees of freedom at a single frequency, a point in parallel to the reticular lamina. the TM must undergo an elliptical trajectory, which can The depths of the antiresonances were dependent on re- degenerate to rectilinear motion at some frequencies. As cording angle. This is illustrated by the dashed curves in Fig. shown in the Inset of Fig. 3A, the velocity trajectories depend 3 which, based on the data measured in the two orthogonal on the relative amplitudes and phases of the two components. directions, are the amplitudes and phases of the velocity that From 215-360 Hz (1.8-1.0 oct below BF) the transverse would be recorded in the transverse direction if the prepara- component is larger, consistent with the classical model that tion were rotated anticlockwise by 30°. Two important features this component is principally derived from rotation of the inherent to two-dimensional motion become obvious from organ of Corti about the spiral limbus. This rotational motion such an axis rotation. First, the antiresonance below the BF is provides the first degree of freedom. Motion below 215 Hz is now shallow and resembles that for the amplitude response for presumed to be influenced by the helicotrema (42), so that this sound stimulation in Fig. 2A. Second, the phase response in the very low-frequency range will not be discussed further. For transverse direction is no longer shallow and becomes consis- 360-670 Hz (1.0-0.1 oct below BF), the radial component tent with traveling-wave motion (41). Downloaded by guest on October 2, 2021 Neurobiology: Gummer et al. Proc. Natl. Acad. Sci. USA 93 (1996) 8731 Velocity derives from a parallel resonance of the TM mass with the stereocilia compliance, as predicted by Zwislocki (5, 18). The 2 I ;00 dB SPL two conditions are (i) the axial mechanical impedance of the I+G OHCs is large compared with the radial mechanical imped- I- 1 ance of the stereocilia bundle of the OHCs, as model calcu- data suggest (22, 43, 44); and (ii) the BM E 0.5 lations from in vitro E l t. | impedance is large compared with the impedances of the TM 0 0.2 I andg OHC stereocilia, as in vivo (17, 45) and in vitro (16, 46, 47) experiments indicate. Then for both acoustic (5, 18) and 0.1 electrical stimulation, resonant TM motion must occur when E l §§ . the impedance of the OHC stereocilia bundle cancels the TM 0.05 impedance; namely, at f = 1/27TmTMCp where Cp = CTMCS/ (CTM + Cs), or Cp Cs, for the TM compliance much greater 0.02 than the stereocilia compliance. Thus, the experimentally L determined resonant frequency of 450 Hz for TM motion 0.1 0.2 0.5 1 2 means that the product MTMCP is 1.3 x 10-7.s2 at the 16 mm 0.5 point of the guinea-pig cochlea. Moreover, the current injec- tion experiments showed that the BM resonant frequency is 0.0 located 0.5 oct above the frequency at which the TM mass is resonant with the stereocilia compliance, in agreement with T Xa) -0.5 the value of 0.54 oct predicted by Zwislocki (5). The major problem with the isolated temporal bone prep- o -1.0 `- aration is that the endocochlear potential was -0 mV, mean- X)a) -1.5 ing that the electrical drive to the hair cells was about half of .C its normal in vivo value (48, 49). The absence of an endococh- iL -Z.U-in lear potential might explain the linearity of the responses (50). Nevertheless, the amplitude responses measured in the trans- -2.5 IR Qx ~ verse direction were similar to those for neural (31, 32) and hair-cell (1, 33) responses at 20-50 dB above threshold for this -3.0 _ region of the cochlea. This similarity is consistent with the 0.1 0.2 0.5 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 TM compliant A 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 micro- i~~~~~~~~~~sv sphere placed on the upper surface of the TM over the OHCs. All , .& - responses were linear and are scaled to 100 dB SPL. The elliptical, EXCITE STin velocity trajectories at the designated frequencies were calculated from the amplitudes and phases of the two measured components. The DEPOL HYERPOL beginning of a trajectory is indicated by a circle and a quarter-cycle CONTRACT ELONGATE later by a triangle. (Inset) Linear scale for the radial (R) and transverse INHIBIT (T) components of the velocity trajectories (±3 mm.s-1). For orien- tation purposes, a spatial angle of 1500 is approximately parallel to the TM inertial B 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. dL~~~~A DISCUSSION sv The data provide direct confirmation of the predicted (5, 10, -Time 11, 18-24) and experimentally inferred TM resonances (17, 25, INHIBIT ST 26). The current injection experiments uncovered resonant TM motion and located the resonant frequency of the BM; the HYPE.POL DEPOL two-dimensional experiments showed that the resonant mo- ELONGATE EXCITE CONTRACT tion of the TM is polarized parallel to the reticular lamina. Theoretically, resonant motion of the TM is governed primar- FIG. 4. The action of the electromechanical force of an OHC for ily by elastic coupling of its mass to two structures: (i) to the compliant (A) or inertial (B) motion of the TM. The sinusoid spiral limbus through a rich network of highly organized represents the displacement of a point on the BM as a function of time. collagenous and non-collagenous protofibrils and (ii) to the For compliant motion, excitation occurs for BM motion in direction OHC of scala vestibuli (SV), whereas for inertial motion it occurs for BM organ of Corti through the stereocilia (13-15). Clearly, motion in scala tympani (ST). Because of the membrane time constant therefore, it is not possible to determine the absolute mechan- of the OHC, the depolarizing receptor potential lags stereociliary ical parameters of TM mass, MTM, TM compliance, CTm, and displacement by 900. Consequently, for compliant TM motion the stereocilia compliance, Cs, from these measurements. How- OHC electromechanical force produces attenuation, whereas for ever, provided two important mechanical conditions are sat- inertial TM motion it produces amplification of BM motion. Relative isfied, one may conclude that resonant motion of the TM dimensions have been exaggerated for illustrative purposes. Downloaded by guest on October 2, 2021 8732 Neurobiology: Gummer et al. Proc. Natl. Acad. Sci. USA 93 (1996) of the cochlea (51). Finally, current injection evoked acoustic 10. Allen, J. B. & Neely, S. T. (1992) Phys. Today 7, 40-47. emissions in the external ear canal (spectrum with maximum 11. Mammano, F. & Nobili, R. (1993) J. Acoust. Soc. Am. 93, of 7 dB SPL at 600 Hz for 4 ,xA), providing further evidence 3320-3332. for electromechanical action of the OHCs and its coupling into 12. Zwislocki, J. J. (1990) in The and Biophysics ofHear- the organ of Corti. ing, eds. Dallos, P., Geisler, C. D., Mathews, J. W., Ruggero, If these results in the apical region of the cochlea can be M. A. & Steele, C. R. (Springer, Heidelberg), pp. 114-120. extended to the 13. Lim, D. J. (1972) Arch. Otolaryngol. 93, 199-215. basal region, then the finding that the TM- 14. Kronester-Frei, A. (1979) Hear. Res. 1, 81-94. stereocilia complex is tuned to about 0.5 oct below the BM 15. Hasko, J. A. & Richardson, G. P. (1988) Hear. Res. 35, 21-38. resonant frequency and the BF of the cochlear partition has 16. von Bekesy, G. (1964) Experiments in (McGraw-Hill, important consequences for the function of the healthy and New York). pathological cochlea. It is proposed that inertial TM motion in 17. Zwislocki, J. J. & Cefaratti, L. K. (1989) Hear. Res. 42, 211-227. the BF region produces the necessary phase conditions to 18. Zwislocki, J. J. (1980) J. Acoust. Soc. Am. 67, 1679-1685. allow OHC contraction to reduce the impedance of the 19. Allen, J. B. (1980) J. Acoust. Soc. Am. 68, 1660-1670. cochlear partition. With the aid of the cartoons in Fig. 4, we 20. de Boer, E. (1991) Phys. Rep. 203, 125-231. describe the resultant action of OHC electromechanical forces 21. Neely, S. T. & Kim, D. 0. (1986) J. Acoust. Soc. Am. 79, on the BM when the motion of the TM is either compliant (Fig. 1472-1480. 22. Mountain, D. C. & Hubbard, A. E. (1994) J. Acoust. Soc. Am. 95, 4A) or inertial (Fig. 4B). When the motion of the TM is 350-354. governed by its compliance, BM displacement toward scala 23. Zwislocki, J. J. & Kletsky, E. J. (1979) Science 204, 639-641. vestibuli (Fig. 4A) causes deflection of the stereocilia tips in 24. Rhode, W. S. & Geisler, C. D. (1967) J. Acoust. Soc. Am. 42, the direction of the longest stereocilia (12, 16, 24), the exci- 185-190. tatory direction, producing a depolarizing receptor potential 25. Allen, J. B. & Fahey, P. F. (1993) J. Acoust. Soc. Am. 94,809-816. (52, 53) and contraction of the OHC (6-8). The resulting 26. Brown, A. M., Gaskill, S. A. & Williams, D. M. (1992) Proc. R contractile force appears to be in phase with the receptor Soc. London B 250, 29-34. potential (36). However, the receptor potential lags stereocilia 27. Khanna, S. M. (1989) Acta Oto-Laryngol. Suppl. 467, 1-279. displacement by 0.25 cycle, beginning several octaves below 28. Ulfendahl, M., Khanna, S. M. & Flock, A. (1991) Hear. Res. 57, the BF (52). Consequently, the OHC contractile 31-37. force, which 29. Orman, S. S. & Geisler, C. D. (1986) Am. J. Otolaryngol. 7, is directed toward scala vestibuli, is maximal at the instant 140-146. when the BM crosses its baseline in the opposite direction, in 30. Gummer, A. W., Hemmert, W., Morioka, I., Reis, P., Reuter, G. the direction of scala tympani (Fig. 4A). As a result, the & Zenner, H.-P. (1993) in Biophysics ofHair Cell Sensory Systems, electromechanical force of the OHC attenuates the motion of eds. Duifhuis, H., Horst, J. W., van Dijk, P. & van Netten, S. M. the BM. Clearly, this mechanism cannot produce cochlear (World Scientific, Singapore), pp. 229-236. tuning; the OHCs act as an active "brake." However, when the 31. Rose, J. E., Hind, J. E., Anderson, D. J. & Brugge, J. F. (1971) motion of the TM is inertial, the phase of the stereociliary J. Neurophysiol. 34, 685-699. displacement is rotated 0.5 cycle relative to its value for 32. Evans, E. F. (1972) J. Physiol. (London) 226, 263-287. compliant TM motion. Then, the stereocilia are displaced in 33. Dallos, P. (1986) Hear. Res. 22, 185-198. 34. Xue, S., Mountain, D. C. & Hubbard, A. E. (1993) Hear. Res. 70, the excitatory direction for BM displacement in the direction 121-126. of scala tympani rather than scala vestibuli (Fig. 4B). Conse- 35. Mammano, F. & Ashmore, J. F. (1993) Nature (London) 365, quently, the BM crosses its baseline in the same direction and 838-841. at the same instant as the maximal electromechanical force 36. Dallos, P. & Evans, B. N. (1995) Science 267, 2006-2009. (Fig. 4B). The electromechanical force of the OHC thereby 37. Bosher, S. K. & Warren, R. L. (1978) Nature (London) 273, amplifies the motion of the BM. Indeed, since frictional forces 377-379. on the BM are expected to be proportional to BM velocity, 38. Shah, D. M., Freeman, D. M. & Weiss, T. F. (1995) Hear. Res. 87, which in turn is maximal when the BM crosses its baseline, the 187-207. OHC electromechanical forces act to reduce the effective 39. Nuttall, A. L., Dolan, D. F. & Avinash, G. (1991) Hear. Res. 51, resistance of the cochlear partition. In other 203-213. words, active 40. Ruggero, M. A. & Rich, N. C. (1991) Hear. Res. 51, 215-230. amplification occurs through the synergistic action of the 41. Wilson, J. P. & Johnstone, J. R. (1975) J. Acoust. Soc. Am. 57, forces of TM inertia and OHC electromotility. 705-723. 42. Dallos, P. (1970) J. Acoust. Soc. Am. 48, 489-499. We thank our colleagues S. Preyer and J. P. Ruppersberg for their 43. Iwasa, K H. (1994) J. Acoust. Soc. 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