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Supplementary Figure: Additional data from single preparations illustrating basilar membrane responses to acoustic stimulation of the mouse cochlea. Threshold frequency tuning curves (open symbols), referred to the malleus, measured from the basal turn BM in the cochleae of (a) a Tecta+/+ and (b) a Tecta∆ENT/∆ENT mouse. The phase of BM displacement relative to the motion of the malleus (solid symbols) measured 15 dB above threshold are also shown, relative to the right vertical axis. (c, d). Basilar membrane displacement divided by malleus displacement as a function of stimulus frequency measured from (c) the Tecta+/+ mouse used for (a) for tones at levels between 20 – 60 dB SPL in 5 dB steps and (d) the Tecta∆ENT/∆ENT mouse used for (b) for tones at levels between 48 – 80 dB SPL in 2 dB steps. Black traces in (c and d) are for the highest levels. Vertical dashed lines indicate characteristic frequency (CF), estimated CF (CFe), R2, and R1 frequencies.

SUPPLEMENTARY MATERIAL OUTER HAIR CELL SOMATIC NOT HAIR BUNDLE MOTILITY IS THE BASIS OF THE COCHLEAR AMPLIFIER Marcia M. Mellado Lagarde, Markus Drexl, Victoria A. Lukashkina, Andrei N. Lukashkin and Ian J. Russell School of Life Sciences, University of Sussex, Falmer, Brighton, BN1 9QG

METHODS Animal preparation and maintenance Experiments were performed on Tecta+/+ and Tecta∆ENT/∆ENT mice1, 4–6 weeks old, on a C57B6/J or CBA /J background that were bred and maintained at the University of Sussex. Animals were initially anaesthetized (i.p.) with Fentanyl (0.05 mg/ kg bodyweight,), Midazolam (5mg/ kg bodyweight) and Medetomidine (0.5 mg/ kg bodyweight). Anaesthesia was maintained throughout the experiment. At the end of the experiments the mice were overdosed with pentobarbital sodium (150 mg/ kg bodyweight i.p.). Carbogen (95% O2, 5% CO2, flow rate ~ 0.3 l/ min) was provided via a head mask. Heartbeat was monitored with skin electrodes and core temperature maintained at 38°C. The right auditory bulla was opened with a ventrolateral approach to gain access to the round window. The pinna was removed and the sound system was placed into the meatus ~1mm from the eardrum1.

Electrophysiological recording and stimulation We adopted Nuttall and Ren’s2 technique to deliver extracochlear electrical stimulation and to record cochlear microphonic and compound action potentials using either a silver, or a tungsten electrode placed on the round window and a Ag/ AgCl reference electrode in the neck tissue.

Sodium salicylate application Sodium salicylate was applied as a crystal on the round window membrane for 5 minutes. This technique, and not cochlear perfusion, was employed to avoid cochlear sensitivity reduction when the round window is breached. We were therefore unable to quantify the concentration of salicylate in the perilymph. Enhancement of the CM due to increases in the basolateral and not the mechanoelectrical conductance of the OHCs, which occur when the level of salicylate in the perilymph exceeds ~ 2mM3, were not however observed.

Sound system and electrical stimulation The sound system, its calibration in dB SPL re 2 x 10-5 Pa, and the method of generating computer-controlled command voltages, have been described1, 4, but here we used a custom-built condenser loudspeaker5. For electrical stimulation the loudspeaker was unplugged and the output signal from the GPIB-controlled attenuator was used as the command voltage for a custom-built current-pump with a sensitivity of 100 µA/V. The

current pump delivered sinusoidal current of constant amplitude through the round window electrode corresponding to the applied sinusoidal command voltage.

Basilar membrane measurements BM displacements were measured by focusing the beam of a self-mixing, laserdiode interferometer6 through the round window membrane to form an ~5 µm spot on the centre of the BM in the 60–65 kHz region of the cochlea as previously described1, 6, 7. Experimental control, data acquisition, and data analysis were performed using programs written in TestPoint (CEC). All procedures involving animals were performed in accordance with UK Home Office regulations with approval from the local ethics committee.

REFERENCES 1. Legan, P.K. et al. A targeted deletion in α-tectorin reveals the tectorial membrane is required for the gain and timing of cochlear feedback. Neuron 28, 273–285 (2000). 2. Nuttall, A. L. & Ren, T. Electromotile hearing: evidence from basilar membrane motion and otoacoustic emissions. Hear. Res. 92, 170–177 (1995). 3. Fitzgerald, J. J., Robertson, D. & Johnstone, B.M. Effects of intra-cochlear perfusion of salicylates on cochlear microphonic and other auditory responses in the guinea pig. Hear. Res. 67, 147-56 (1993). 4. Russell, I. J., et al. Sharpened cochlear tuning in a mouse with a genetically modified tectorial membrane. Nat. Neurosci. 10, 215-23 (2007). 5. Schuller, G. A cheap earphone for small animals with good frequency response in the ultrasonic frequency range. J. Neurosci. Methods. 71, 187-90 (1997).

6. Murugasu, E. & Russell, I. J. Salicylate ototoxicity: the effects on basilar membrane displacement, cochlear microphonics, and neural responses in the basal turn of the guinea pig cochlea. Audit. Neurosci. 1, 139-150 (1995). 7. Lukashkin, A. N., Bashtanov, M. E. & Russell, I. J. A self-mixing laser-diode interferometer for measuring basilar membrane vibrations without opening the cochlea. J. Neurosci. Methods 148, 122–129 (2005).