Ohc Somatic Supp

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b

c d

a

20 30 40 50 60 70

Stimulus frequency (kHz)

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100

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10000

B M g a

i n r e m a

l l e u s

CFR1 R2

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-4.0-3.5-3.0-2.5-2.0-1.5-1.0-0.50.00.5

B M p

h a s e r e m a

l l e u s

( c y c

l e s )

10

20

30

40

5060

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B M t h r e s

h o

l d ( d B

S P L )

CFR1 R2

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 (CF e ), R2, and

R1 frequencies.

20 30 40 50 60 70


10

100

1000

10000

B M d i s p

l a c e e n

t /

m a l

l e u s

d i s p

l a c e m e n

tCFeR2

20 30 40 50 60 70


-2.0

-1.5

-1.0

-0.5

0.0

0.5

B M p h a s e r e m a l

l e u s

( c y c

l e s )

20

30

40

50

6070

80

B M t h r e s h o l

d ( d B S P L )

CFeR2


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Electrophysiological recording and stimulation

We adopted Nuttall and Rens 2 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 ~ 2mM 3, 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 described 1, 4, but here we

used a custom-built condenser loudspeaker 5. 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 A/V. The


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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, laser-

diode interferometer 6 through the round window membrane to form an ~5 m spot on the

centre of the BM in the 6065 kHz region of the cochlea as previously described 1, 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 -tectorin reveals the tectorial membrane is

required for the gain and timing of cochlear feedback. Neuron 28 , 273285 (2000).

2. Nuttall, A. L. & Ren, T. Electromotile hearing: evidence from basilar membrane

motion and otoacoustic emissions. Hear. Res. 92 , 170177 (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 theguinea 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).


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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, 122129 (2005).

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