Reticular lamina and basilar membrane vibrations in living mouse cochleae

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1 Reticular lamina and basilar membrane vibrations in living mouse cochleae Tianying Ren a,1, Wenxuan He a, and David Kemp b a Oregon Hearing Research Center, Department of Otolaryngology, Oregon Health & Science University, Portland, OR 97239; and b University College London Ear Institute, University College London, London WC1E 6BT, United Kingdom Edited by Mario A. Ruggero, Northwestern University, Evanston, IL, and accepted by Editorial Board Member Peter L. Strick July 1, 2016 (received for review May 9, 2016) It is commonly believed that the exceptional sensitivity of mammalian hearing depends on outer hair cells which generate forces for amplifying sound-induced basilar membrane vibrations, yet how cellular forces amplify vibrations is poorly understood. In this study, by measuring subnanometer vibrations directly from the reticular lamina at the apical ends of outer hair cells and from the basilar membrane using a custom-built heterodyne low-coherence interferometer, we demonstrate in living mouse cochleae that the soundinduced reticular lamina vibration is substantially larger than the basilar membrane vibration not only at the best frequency but surprisingly also at low frequencies. The phase relation of reticular lamina to basilar membrane vibration changes with frequency by up to 180 degrees from 135 degrees at low frequencies to -45 degrees at the best frequency. The magnitude and phase differences between reticular lamina and basilar membrane vibrations are absent in postmortem cochleae. These results indicate that outer hair cells do not amplify the basilar membrane vibration directly through a local feedback as commonly expected; instead, they actively vibrate the reticular lamina over a broad frequency range. The outer hair cell-driven reticular lamina vibration collaboratively interacts with the basilar membrane traveling wave primarily through the cochlear fluid, which boosts peak responses at the best-frequency location and consequently enhances hearing sensitivity and frequency selectivity. cochlea outer hair cells hearing cochlear amplifier interferometry As the auditory sensory organ, the mammalian cochlea is able to detect soft sounds at levels as low as 20 μpa, equivalent to eardrum vibrations of <1-pm displacement (1), which is >100-fold smaller than the diameter of a hydrogen atom. The cochlea s remarkable sensitivity is commonly attributed to a micromechanical feedback system, which amplifies soft sounds using forces generated by outer hair cells (2 10). In addition to active bundle movements (2), mammalian outer hair cells are capable of changing their lengths in response to electrical stimulation in vitro (11 14). This electromechanical force production is termed outer hair cell electromotility or somatic motility, which is produced by the membrane protein, prestin (15). Transgenic mice without prestin or with altered prestin suffer from severe hearing loss (4, 16). Targeted inactivation of prestin over well-defined cochlear segments dramatically reduces the traveling wave s peak response (17). Recent micromechanical measurements in sensitive living mouse cochleae showed (18) that electrical stimulation of outer hair cells induces vigorous vibrations of the reticular lamina at the apical surfaces of the outer hair cells. Whereas this experiment demonstrates that outer hair cells are capable of generating substantial forces to vibrate cochlear microstructures in vivo, the lack of frequency selectivity of the electrically induced reticular lamina vibration, however, is inconsistent with the sharp tuning of the basilar membrane vibration. Because electrical stimulation is not a natural stimulus of the auditory system, we here investigate how acoustically induced reticular lamina vibrations interact with basilar membrane vibrations in sensitive living mouse cochleae, using a custom-built scanning heterodyne low-coherence interferometer (Fig. 1 A and B and Materials and Methods). The results from this study do not demonstrate the commonly expected cochlear local feedback but instead indicate a global hydromechanical mechanism for outer hair cells to enhance hearing sensitivity. Results Vibrations of the Reticular Lamina and Basilar Membrane in Sensitive Cochleae. Vibrations of the reticular lamina and basilar membrane measured from a sensitive cochlea are presented in Fig. 1 C J. Below 50 db sound pressure level (SPL) (0 db SPL = 20 μpa), the basilar membrane vibration increased with frequency and reached the maximum at 48 khz (best frequency) and then decreased, forming a sharp peak (Fig. 1C). The peak became broader and shifted toward low frequencies as the sound level increased. The ratios of the basilar membrane displacement to the malleus displacement at low sound levels confirm the best frequency of 48 khz (Fig. 1E). The peak ratio decreased from >100 to 3 as the sound level increased from 20 to 80 db SPL, indicating 30-dB nonlinear compression. The overlapping curves at frequencies below 35 khz (Fig. 1E) indicate the linear growth at low frequencies. The basilar membrane vibration phase (referred to as the malleus phase) decreased with frequency (Fig. 1G). Data in Fig. 1 C and E demonstrate the high sensitivity, sharp tuning, and nonlinearity of basilar membrane responses to ultrasonic sounds in the living mouse cochlea, which are similar to previous measurements in squirrel monkeys (19), gerbils (20 23), chinchillas (17, 24, 25), guinea pigs (26 28), and mice (7, 29, 30). By contrast, the displacement of the reticular lamina vibration (Fig. 1D) was substantially larger than that of the basilar membrane vibration. At 20 db SPL, reticular lamina vibrations were the greatest approximately at the same frequency as the best frequency of the basilar membrane ( 48 khz). Notably, the peak Significance The remarkable sensitivity of mammalian hearing depends on auditory sensory outer hair cells, yet how these cells enhance the hearing sensitivity remains unclear. By measuring subnanometer vibrations directly from motile outer hair cells in living inner ear, we demonstrate that outer hair cells do not directly amplify the basilar membrane vibration by local feedback as commonly expected; instead, they actively vibrate the reticular lamina over a broad frequency range. The outer hair cell-driven reticular lamina vibration interacts with the basilar membrane traveling wave through the cochlear fluid, resulting in maximal vibrations at the best-frequency location, consequently enhancing hearing sensitivity. This finding advances our knowledge of mammalian hearing and may lead to strategies for restoring hearing in patients. Author contributions: T.R. designed research; T.R. and W.H. performed research; T.R., W.H., and D.K. analyzed data; and T.R., W.H., and D.K. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. M.A.R. is a Guest Editor invited by the Editorial Board. 1 To whom correspondence should be addressed. rent@ohsu.edu PNAS August 30, 2016 vol. 113 no. 35

2 Fig. 1. Diagram of the organ of Corti and vibrations of the reticular lamina and basilar membrane in a sensitive mouse cochlea. (A) Intracochlear image through the intact round window membrane in a living mouse. BM, basilar membrane; OSL, osseous spiral lamina. (B) Diagram of the organ of Corti. RL, reticular lamina; IHC, inner hair cell; OHCs, outer hair cells; TM, tectorial membrane; DCs, Deiters cells. (C and D) Displacements of the BM (red) and RL (blue) as a function of frequency. The noise floor (black dotted line in C) is <0.01 nm. BF, best frequency. (E and F) Displacement ratios of the BM and RL to the malleus. (G and H) BM and RL phase referred to the malleus. (I)Theratio of the RL to BM displacement. (J) Phase difference between the RL and BM. displacement of the reticular lamina was 10-fold greater than that of the basilar membrane. At sound levels above 40 db SPL, overlapping curves near the best frequency indicate strong nonlinear compression. Surprisingly, displacements of the reticular lamina at frequencies far below the best frequency were also 10- fold greater than those of the basilar membrane (Fig. 1D). Displacement ratios of the reticular lamina to the malleus in Fig. 1F show a peak magnitude of 1,000 at low sound levels, which is larger than that of the basilar membrane (Fig. 1E). With an increase in sound level from 20 to 80 db SPL, the peak magnitude decreased by 45 db for the reticular lamina vibration (Fig. 1F) and by 30 db for the basilar membrane vibration (Fig. 1E). The phase responses of the reticular lamina (Fig. 1H) were similar to those of the basilar membrane (Fig. 1G). Ratios of the reticular lamina to basilar membrane displacement greater than one (Fig. 1I) confirm that the reticular lamina vibrated more than the basilar membrane at all frequencies and sound levels. Near 48 khz, 30-dB SPL tone-induced reticular lamina vibration was approximately sevenfold greater than that of the basilar membrane. This difference decreased with sound level and approached one at 80 db SPL. At a given sound level, the magnitude ratio decreased with frequency up to 48 khz. The magnitude ratio curves also shifted toward low frequencies with the sound level increase. Surprisingly, the largest-magnitude ratios occurred at frequencies far below the best frequency and at high sound levels, which likely resulted from the saturation near the best frequency. The phase difference of >90 degrees in Fig. 1J indicates that the reticular lamina and basilar membrane vibrated approximately in opposite directions at frequencies below 15 khz. The lack of substantial phase difference near 48 khz, however, demonstrates that the reticular lamina and the basilar membrane moved in the same direction at the best frequency. Vibrations of the Reticular Lamina and Basilar Membrane in Insensitive Cochleae. Under postmortem conditions, the basilar membrane and reticular lamina vibrations (blue lines in Fig. 2 A and B) showed decreased magnitude near the best frequency, peak shift toward low frequencies, and linear growth with the sound level. In contrast with the lack of substantial postmortem change in basilar membrane vibration at frequencies below 30 khz in Fig. 2A, the reticular lamina displacements decreased by 10-fold (or 20 db) over the same frequency range (Fig. 2B). These postmortem magnitude changes were confirmed by the displacement ratios of the basilar membrane and reticular lamina to the malleus in Fig. 2 C and D. In contrast to the basilar membrane phase responses (Fig. 2E), the reticular lamina vibration showed a substantial postmortem phase decrease at low frequencies (Fig. 2F). Compared with sensitive responses, the ratio of the reticular lamina to basilar membrane displacement decreased dramatically and approached one (blue lines in Fig. 2G) in the postmortem cochlea, and the phase difference approached zero (blue lines in Fig. 2H). Thus, under postmortem conditions, the reticular lamina and basilar membrane vibrated synchronously in the same direction and with similar magnitudes at all observed frequencies and sound levels. The Relationship Between the Reticular Lamina and Basilar Membrane Vibrations. Average displacements at the best frequency as a function of the sound level from eight cochleae (Fig. 3A) showed that reticular lamina displacements were significantly greater than those of the basilar membrane at 20, 30, 40, and 50 db SPL (t > 1.89, P < 0.05, n = 8). The displacement difference decreased with the sound level and became insignificant at 60, 70, and 80 db SPL (t < 0.73, P > 0.35, n = 8). The phase of the reticular lamina increased slightly with the sound level up to 70 db SPL (Fig. 3B). In postmortem cochleae, the reticular lamina and basilar membrane vibrations increased proportionally with the sound level (Fig. 3C), whereas the corresponding phase changed insignificantly (Fig. 3D). The average displacement ratio of the reticular lamina to the basilar membrane and the corresponding phase difference from five sensitive and six insensitive cochleae are presented as a NEUROSCIENCE Ren et al. PNAS August 30, 2016 vol. 113 no

3 Discussion In this study, the reticular lamina and basilar membrane vibrations were measured at a longitudinal location 0.85 mm from the cochlear base (red arrows in Fig. 4 A and B) asafunctionof frequency. Because the peak location of the cochlear partition vibration is determined by frequency (31), the change in stimulus frequency from low to high caused the peak of the traveling wave to move from apex to base. Whereas the vibration at 48 khz was Fig. 2. Postmortem changes in reticular lamina and basilar membrane vibrations. Postmortem data were collected immediately after death (blue) and are compared with those measured under sensitive conditions (red) in the same cochlea. (A) Displacements of basilar membrane (BM) as a function of frequency at different sound levels. (B) Reticular lamina (RL) displacements. (C) Displacement ratios of the BM to malleus. (D) Displacement ratios of the RL to malleus. (E and F) BM and RL phase referred to the malleus. (G) Magnitude ratios of the RL to BM. (H) Phase difference between the BM and RL. In addition to poor sensitivity, broad tuning, and linear growth, the magnitude and phase differences between the RL and BM were absent under postmortem conditions. function of frequency in Fig. 3 E H. Because the basilar membrane vibrations at low frequencies can be recorded only at relatively high sound levels due to sharp tuning in sensitive cochleae, the data in Fig. 3 E H were collected at 80 db SPL. Fig. 3 E and F shows that reticular lamina vibration at 10 khz was >fivefold larger than the basilar membrane vibration, with a phase lead of >90 degrees in sensitive cochleae. Near the best frequency, reticular lamina and basilar membrane vibrations had a similar magnitude and phase. The magnitude and phase differences were absent in postmortem cochleae (Fig. 3 G and H). Fig. 3. Differences between the reticular lamina and basilar membrane vibrations. (A) Reticular lamina (RL) and basilar membrane (BM) displacements at different sound levels in sensitive cochleae. (B) RL and BM phase. Phase was normalized to that at 80 db SPL. (C) RL and BM displacements in postmortem cochleae. (D) Insensitive RL and BM phase. (E) Displacement ratios of the RL to BM show that the RL vibrated >fivefold more than the BM at low frequencies. This difference decreased with frequency and approached one near the best frequency. (F) RL phase led the BM phase by 135 degree at low frequencies and lagged the BM phase by -45 degrees at the best frequency (48.08 ± 0.25 khz, n = 5) in sensitive cochleae. (G and H) The magnitude and phase differences were absent in postmortem cochleae Ren et al.

4 Fig. 4. Longitudinal patterns and interaction of the cochlear partition vibrations. The active component of the reticular lamina vibration (green lines in panels C F) was obtained by vector subtraction of the basilar membrane vibration from the measured reticular lamina vibration. (A) Diagram of the conventional traveling wave at 48 khz shows that the vibration was measured at the peak of the traveling wave (red arrow). (B) At a frequency lower than the BF, the vibration was measured at a basal part of the traveling wave. (C) At 40 db SPL, the reticular lamina (RL) and basilar membrane (BM) vibrations were at a small region centered at the BF location. (E) At 70 db SPL, both RL and BM displacements extended from the BF location to the base. (D and F) Near the base, RL phase led BM phase by >120 degrees; they were approximately in-phase at the BF location. (G) Diagram of a single tone-induced cochlear traveling wave. D OHC, the outer hair cell force-induced displacement; D TW, the traveling wave-induced displacement. (H) At the base of the traveling wave, RL and BM move in approximately opposite directions. The active RL movement creates a negative fluid pressure in the scala vestibuli and a positive fluid pressure inside the organ of Corti. (I) At the peak of the measured at the response peak location (Fig. 4A), the vibration at a lower frequency was measured at the basal part of the traveling wave because the response peak moved toward an apical location (Fig. 4B). Therefore, the frequency responses measured in this study can reflect the relationship between the reticular lamina and basilar membrane vibrations at different parts of traveling waves. The longitudinal patterns of the reticular lamina and basilar membrane vibrations at 48 khz were obtained from measured frequency responses (Fig. 1 C, D, G,andH)(Materials and Methods) and are presented by blue and red lines in Fig. 4 C F.Whereas40-dB SPL responses occurred within a 200-μm region(fig. 4C and D), a 70-dB SPL tone-induced vibration extended over the entire region from the response peak to the base (Fig. 4 E and F). The reticular lamina vibration was substantially larger than the basilar membrane vibration not only at the peak (Fig. 4C) but also at the basal part of the traveling wave (Fig. 4E). Phase difference between the reticular lamina and basilar membrane vibrations was >120 degrees at the base (Fig. 4F) but insignificant at the response peak (Fig. 4 D and F). Because outer hair cells sit on the basilar membrane via Deiters cells, they can move passively, following the basilar membrane traveling wave. Outer hair cells can also change their length (11) in response to the membrane potential change caused by sound stimulation (32). Because the reticular lamina vibration is a vector sum of the basilar membrane vibration and the outer hair celldriven movement, the active component of the reticular lamina vibration was obtained by vector subtraction of the basilar membrane vibration from the measured reticular lamina vibration and is shown by green lines in Fig. 4 C F. The overlapping blue and green lines in Fig. 4 C and D indicate that reticular lamina vibration was dominated by the outer hair cell-driven component at 40 db SPL. In Fig. 4E,the green line diverges from the blue line and almost overlaps the red line near the best frequency, reflecting saturation of the outer hair celldriven component at high sound levels near the response peak. These data indicate that acoustically induced reticular lamina vibration is dominated by outer hair cell-driven vibration over the entire traveling wave region. This is confirmed by postmortem changes, which show no substantial difference between the reticular lamina and basilar membrane vibrations (blue lines in Figs. 2 G and H and 3 G and H). The data also show that neither destructive interference between the basilar membrane and reticular lamina vibrations at the base nor constructive interference near the peak is significant (indicated by the negligible gap between blue and green lines in Fig. 4 C F) due to the large-magnitude difference between the reticular lamina and basilar membrane vibrations. Because force production of outer hair cells depends on metabolic energy in vivo and the generated forces act equally on both ends of the cells but in opposite directions, it is expected that the outer hair cell-driven reticular lamina and basilar membrane vibrations are physiologically vulnerable and in opposite directions (13, 33, 34). The lack of postmortem changes in the basilar membrane vibration at low frequencies (Fig. 2 A and C) and approximately in-phase vibrations of the reticular lamina and basilar membrane near the best frequency (Figs. 1J and 3F) indicate that, under physiological conditions, locally generated outer hair cell forces do not drive the basilar membrane effectively, likely because outer hair cell-generated forces could not be coupled back to the basilar membrane (35). When sound causes the stapes to move out toward the middle ear, displacing fluid out of the scala vestibuli, the basilar membrane at the cochlear base reacts by moving up (Fig. 4H). This upward movement of the basilar membrane depolarizes outer hair cells by deflecting hair bundles toward the excitatory direction (35). Due to traveling wave, the RL and BM move in the same direction, which results in the maximal vibration at the apical ends of outer hair cells through constructive interference. Dotted lines show resting positions. NEUROSCIENCE Ren et al. PNAS August 30, 2016 vol. 113 no

5 configuration change in the motor protein, prestin, outer hair cells contract, pulling the reticular lamina toward the basilar membrane (Fig. 4H). Unexpectedly, the present results indicate a 10-fold greater displacement of the reticular lamina than the basilar membrane. Furthermore, the observed phase difference of up to 135 degrees between the two vibrations (Fig. 4F) indicates that they move in approximately opposite directions at the base. The fluid displaced by this opposing reticular lamina motion may have a significant influence on the developing traveling wave. Indeed, electrically evoked reticular lamina vibration has already been shown to initiate a conventional traveling wave (18). The unexpectedly large opposing motion of the reticular lamina must also result in substantial fluid displacements inside the organ of Corti. When the reticular lamina moves toward the basilar membrane (Fig. 4H) at a basal location, the reticular lamina at a more apical location 0.5 wavelength (or 180 degrees) from the basal location moves away from the basilar membrane at the same time. Active differential movements of the reticular lamina could therefore result in longitudinal fluid movements. As the traveling waves propagate forward, these longitudinal fluid movements help transfer energy toward best-frequency locations (6, 36) (Fig. 4G), boosting the peak response. Our interpretation of the present results is also consistent with the following previous studies. Out-of-phase vibrations of the reticular lamina and basilar membrane at low frequencies (Figs. 1J and 3F) are compatible with electrically evoked opposite movements at both ends of outer hair cells (11, 12) and at the reticular lamina (or tectorial membrane) and the basilar membrane in excised cochleae (13, 33, 34). The acoustically induced in-phase vibrations near the best frequency are similar to electrically evoked reticularlaminaandbasilarmembranevibrations(18)andlowfrequency tone-induced vibrations of Hensen s cells and basilar membrane (37). The proposed outer hair cell-driven fluid movement inside the organ of Corti is in agreement with the electrically induced fluid movement inside the tunnel of Corti in vitro (38). Because the outer hair cell-generated pressure inside the organ of Corti acts in all directions, the proposed mechanism can interpret complex vibrations of the organ of Corti (18, 27, 29, 30). Phase inconsistency between our results and others likely resulted from differences in phase detection techniques, animal species, and measurement locations. Whereas the proposed cochlear micromechanical mechanism is well supported by experimental data, it does not exclude other mechanisms (33, 39 49). In summary, heterodyne low-coherence interferometry in living mouse cochleae demonstrates that the sound-induced reticular lamina vibration is substantially larger than basilar membrane vibration and is physiologically vulnerable not only at the best frequency but also at low frequencies and that the reticular lamina and basilar membrane vibrate approximately in the same direction near the best frequency. These results indicate that, contrary to expectations, outer hair cells do not amplify the basilar membrane vibration directly by local feedback. Rather, they actively vibrate the reticular lamina over a broad frequency range. The interaction of the outer hair cell-driven reticular lamina vibration with the basilar membrane traveling wave through the cochlear fluid results in maximum responses at the best-frequency location, which consequently enhances hearing sensitivity and frequency selectivity. Materials and Methods Eighteen male and female CBA/CaJ mice at 3 5 wk of age were used in this study. The animal use protocol was approved by the Oregon Health & Science University Institutional Animal Care and Use Committee. The Scanning Heterodyne Low-Coherence Interferometer. Based on a scanning laser interferometer (50, 51), a scanning heterodyne low-coherence interferometer was built by replacing the light source and related optical and electronic components (18). Taking advantage of the small coherence length, low-coherence interferometers can measure vibration with a high axial resolution (27, 29, 52). Due to the use of the 40-MHz carrier frequency for heterodyne detection, the current low-coherence interferometer has unprecedented sensitivity, wide dynamic range, no 180-degree phase ambiguity, and high temporal resolution. Although the measurement noise floor is below 0.01 nm (black dotted line in Fig. 1C) and heterodyne interferometry does not require a stable optical path length, occasional animal movement disturbed low-level measurements. Thus, data points showing irregular patterns, particularly with <10-dB signal-to-noise ratio, were not presented. The use of low-coherence light and an objective lens with relatively large numerical aperture (NA = 0.42) provides adequate spatial selectivity for measuring the reticular lamina and basilar membrane vibrations through the intact round window membrane in mice. Measurement of the Reticular Lamina and Basilar Membrane Vibrations. Animal anesthesia and surgical procedures were the same as described previously (18). Under direct visualization, low-coherence light from the interferometer was focused on the center of the outer hair cell region of the cochlear partition through the intact round window membrane (Fig. 1A). The distance from the cochlear base to the measurement location was 0.85 mm. The transverse locations of the basilar membrane and reticular lamina were determined by measuring the backscattered light level and the vibration magnitude and phase as a function of the transverse location. When the object beam of the interferometer was focused on the basilar membrane, or reticular lamina, acoustical tones at different frequencies and levels were delivered to the ear canal. The tone frequency was changed from 0.75 to 67.5 khz by <0.75 khz per step. Data Analysis and Statistical Methods. Igor Pro (Version 6.35A5, WaveMetrics) was used for processing and analyzing data. Frequency responses of the reticular lamina and basilar membrane were presented by plotting displacement and phase as a function of frequency. The transfer functions were estimated by the displacement ratio of the reticular lamina or basilar membrane to the malleus at different frequencies. The relationship between the reticular lamina and basilar membrane vibrations was presented by the ratio of the reticular lamina displacement to the basilar membrane displacement and the phase difference obtained by subtracting the basilar membrane phase from the reticular lamina phase. The longitudinal patterns of the reticular lamina and basilar membrane vibrations at the best frequency of the measured location were presented by plotting displacement and phase as a function of the longitudinal location. The longitudinal location was derived from the stimulus frequency according to the frequency location function in mouse cochleae (31) [d = ð logðfþþ= , where d is distance from the base in millimeters and f is frequency in khz]. The grouped results were presented by mean and SE calculated across animals at given frequencies and stimulus levels. Displacement difference between the reticular lamina and basilar membrane vibration at the best frequency was determined using a two-tailed t test, and P value <0.05 was considered statistically significant. ACKNOWLEDGMENTS. We thank Peter Barr-Gillespie and John V. Brigande for valuable comments on the manuscript, Alfred L. Nuttall and other colleagues at Oregon Hearing Research Center for helpful discussion of the data, and Edward Porsov for technical help. This study was funded by NIH Grant R01 DC (to T.R.). 1. Dalhoff E, Turcanu D, Zenner HP, Gummer AW (2007) Distortion product otoacoustic emissions measured as vibration on the eardrum of human subjects. Proc Natl Acad Sci USA 104(5): Hudspeth AJ (2014) Integrating the active process of hair cells with cochlear function. Nat Rev Neurosci 15(9): Kemp DT (1986) Otoacoustic emissions, travelling waves and cochlear mechanisms. Hear Res 22: Dallos P (2008) Cochlear amplification, outer hair cells and prestin. Curr Opin Neurobiol 18(4): Fettiplace R, Hackney CM (2006) The sensory and motor roles of auditory hair cells. Nat Rev Neurosci 7(1): Reichenbach T, Hudspeth AJ (2014) The physics of hearing: Fluid mechanics and the active process of the inner ear. Rep Prog Phys 77(7): Russell IJ, et al. (2007) Sharpened cochlear tuning in a mouse with a genetically modified tectorial membrane. Nat Neurosci 10(2): Peng AW, Ricci AJ (2011) Somatic motility and hair bundle mechanics, are both necessary for cochlear amplification? Hear Res 273(1-2): Dong W, Olson ES (2013) Detection of cochlear amplification and its activation. Biophys J 105(4): Robles L, RuggeroMA (2001) Mechanics ofthe mammalian cochlea. Physiol Rev 81(3): Brownell WE, Bader CR, Bertrand D, de Ribaupierre Y (1985) Evoked mechanical responses of isolated cochlear outer hair cells. Science 227(4683): Ren et al.

6 12. Santos-Sacchi J (1989) Asymmetry in voltage-dependent movements of isolated outer hair cells from the organ of Corti. J Neurosci 9(8): Mammano F, Ashmore JF (1993) Reverse transduction measured in the isolated cochlea by laser Michelson interferometry. Nature 365(6449): Frank G, Hemmert W, Gummer AW (1999) Limiting dynamics of high-frequency electromechanical transduction of outer hair cells. Proc Natl Acad Sci USA 96(8): Zheng J, et al. (2000) Prestin is the motor protein of cochlear outer hair cells. Nature 405(6783): Liberman MC, et al. (2002) Prestin is required for electromotility of the outer hair cell and for the cochlear amplifier. Nature 419(6904): Fisher JA, Nin F, Reichenbach T, Uthaiah RC, Hudspeth AJ (2012) The spatial pattern of cochlear amplification. Neuron 76(5): Ren T, He W, Barr-Gillespie PG (2016) Reverse transduction measured in the living cochlea by low-coherence heterodyne interferometry. Nat Commun 7: Rhode WS (1971) Observations of the vibration of the basilar membrane in squirrel monkeys using the Mössbauer technique. J Acoust Soc Am 49(4):2, Overstreet EH, 3rd, Temchin AN, Ruggero MA (2002) Basilar membrane vibrations near the round window of the gerbil cochlea. J Assoc Res Otolaryngol 3(3): Versteegh CP, van der Heijden M (2012) Basilar membrane responses to tones and tone complexes: Nonlinear effects of stimulus intensity. J Assoc Res Otolaryngol 13(6): van der Heijden M, Versteegh CP (2015) Energy flux in the cochlea: Evidence against power amplification of the traveling wave. J Assoc Res Otolaryngol 16(5): Ren T, Nuttall AL (2001) Basilar membrane vibration in the basal turn of the sensitive gerbil cochlea. Hear Res 151(1-2): Ruggero MA, Narayan SS, Temchin AN, Recio A (2000) Mechanical bases of frequency tuning and neural excitation at the base of the cochlea: Comparison of basilarmembrane vibrations and auditory-nerve-fiber responses in chinchilla. Proc Natl Acad Sci USA 97(22): Recio A, Rich NC, Narayan SS, Ruggero MA (1998) Basilar-membrane responses to clicks at the base of the chinchilla cochlea. J Acoust Soc Am 103(4): Cooper NP, Rhode WS (1992) Basilar membrane mechanics in the hook region of cat and guinea-pig cochleae: Sharp tuning and nonlinearity in the absence of baseline position shifts. Hear Res 63(1-2): Chen F, et al. (2011) A differentially amplified motion in the ear for near-threshold sound detection. Nat Neurosci 14(6): Nilsen KE, Russell IJ (2000) The spatial and temporal representation of a tone on the guinea pig basilar membrane. Proc Natl Acad Sci USA 97(22): Lee HY, et al. (2015) Noninvasive in vivo imaging reveals differences between tectorial membrane and basilar membrane traveling waves in the mouse cochlea. Proc Natl Acad Sci USA 112(10): Gao SS, et al. (2014) Vibration of the organ of Corti within the cochlear apex in mice. J Neurophysiol 112(5): Müller M, von Hünerbein K, Hoidis S, Smolders JW (2005) A physiological placefrequency map of the cochlea in the CBA/J mouse. Hear Res 202(1-2): Corey DP, Hudspeth AJ (1979) Ionic basis of the receptor potential in a vertebrate hair cell. Nature 281(5733): Gummer AW, Hemmert W, Zenner HP (1996) Resonant tectorial membrane motion in the inner ear: Its crucial role in frequency tuning. Proc Natl Acad Sci USA 93(16): Nowotny M, Gummer AW (2011) Vibration responses of the organ of Corti and the tectorial membrane to electrical stimulation. J Acoust Soc Am 130(6): Reichenbach T, Hudspeth AJ (2010) A ratchet mechanism for amplification in lowfrequency mammalian hearing. Proc Natl Acad Sci USA 107(11): Lighthill J (1981) Energy flow in the cochlea. J Fluid Mech 106: Khanna SM, Hao LF (2000) Amplification in the apical turn of the cochlea with negative feedback. Hear Res 149(1-2): Karavitaki KD, Mountain DC (2007) Evidence for outer hair cell driven oscillatory fluid flow in the tunnel of Corti. Biophys J 92(9): Markin VS, Hudspeth AJ (1995) Modeling the active process of the cochlea: Phase relations, amplification, and spontaneous oscillation. Biophys J 69(1): Zwislocki JJ, Kletsky EJ (1979) Tectorial membrane: A possible effect on frequency analysis in the cochlea. Science 204(4393): Neely ST, Kim DO (1983) An active cochlear model showing sharp tuning and high sensitivity. Hear Res 9(2): Allen JB (1980) Cochlear micromechanics a physical model of transduction. J Acoust Soc Am 68(6): Hubbard A (1993) A traveling-wave amplifier model of the cochlea. Science 259(5091): Ghaffari R, Aranyosi AJ, Richardson GP, Freeman DM (2010) Tectorial membrane travelling waves underlie abnormal hearing in Tectb mutant mice. Nat Commun 1: van der Heijden M (2014) Frequency selectivity without resonance in a fluid waveguide. Proc Natl Acad Sci USA 111(40): Wang Y, Steele CR, Puria S (2016) Cochlear outer-hair-cell power generation and viscous fluid loss. Sci Rep 6: Ramamoorthy S, Deo NV, Grosh K (2007) A mechano-electro-acoustical model for the cochlea: Response to acoustic stimuli. J Acoust Soc Am 121(5 Pt1): Liu Y, Gracewski SM, Nam JH (2015) Consequences of location-dependent organ of Corti micro-mechanics. PLoS One 10(8):e Ó Maoiléidigh D, Hudspeth AJ (2013) Effects of cochlear loading on the motility of active outer hair cells. Proc Natl Acad Sci USA 110(14): Ren T (2002) Longitudinal pattern of basilar membrane vibration in the sensitive cochlea. Proc Natl Acad Sci USA 99(26): Ren T (2004) Reverse propagation of sound in the gerbil cochlea. Nat Neurosci 7(4): Hong SS, Freeman DM (2006) Doppler optical coherence microscopy for studies of cochlear mechanics. J Biomed Opt 11(5): NEUROSCIENCE Ren et al. PNAS August 30, 2016 vol. 113 no