Procedure: Auditory brainstem response (ABR)

1. Mice are anesthetized and placed on heating blanket in the sound chamber.
2. Sub-dermal needle electrodes are inserted (active electrode on vertex; reference electrode overlying left bulla; ground electrode overlying right bulla).
3. Mouse is placed unrestrained in a prone position, nose forward, at the calibrated distance from the leading edge of the speaker to the mouse's interaural axis, on a thermostatically controlled blanket, inside a sound-attenuating booth.
4. A click-evoked ABR (70 dB sound pressure level (SPL)) is recorded to ensure a good ABR is present (optional).
5. ABRs are recorded to clicks (10 µs duration, positive transient) presented from 0-85 dB SPL in 5 dB steps, presented 256 times at 42.6/s.
6. ABRs are recorded to the following frequencies at 0-85 dB SPL, if not otherwise indicated (stimulus extension used for some to 90 dB SPL (see download file)): 6kHz, 12kHz, 18 kHz, 24 kHz and 30 kHz, presented in 5 dB intervals. Tone pips are 5 ms in duration, with a 1 ms rise-fall time, presented 256 times at 42.6/s. Tone stimuli are presented in decreasing frequency order for a particular sound level and from low to high stimulus level.
7. A final click-evoked ABR is recorded (70 dB SPL) to check for any deterioration of the click-evoked response during recordings (optional).
8. Data are analyzed using Sanger Bespoke Averager software for ABR.
9. For statistical analysis values above measurement limit (80 or 90 kHz) were set to 100.