2.5 Auditory brainstem response (ABR) test
Since the ABR test provides auditory information of nuclei in the
central pathway to sound stimulation, a TDT (Tucker Davis Technologies)
RZ6 system was used to collect evoked auditory signals of mice. The mice
were anesthetized with pentobarbital sodium (60 mg/kg, IP injection) and
positioned 10 cm from the free-field speaker (monaural) on a
non-electric heating pad (~37 °C) with three platinum
coated electrodes placed in the scalp, within the sound-proof chamber.
The impedance was adjusted below 5 Ohm before recording, and sound
stimuli is produced through the speaker. For click stimulus, the mouse
is presented with a wide spectrum click (0.1 ms) in a gradient descent
of 10 dB SPL from 90 to 10 dB SPL. Each data point acquisition was
repeated for512 times and the integral signals were averaged for
display. For frequency-specific tone burst stimulus, five frequencies
including 4, 8, 16, 24 and 32 kHz (0.1 ms) were presented in decreasing
levels between 90~20 dB SPL, and each new stimulus was
recorded 5 dB SPL down from the previous. Each point of measurement was
recorded 512 times to be averaged. ABR threshold was considered as the
lowest intensity of recognizable response for the given set of
variables, and all the hearing threshold, latency, waveform, and
amplitude of ABR data were determined using BioSigRZ software. When
finished recording, the mice had electrode removed and were carefully
sent to emergence from anesthesia. The ages and ambient noise levels in
the vivarium were precisely matched for all the C57BL/6J mice used in
ABR test, and the experimental condition was strictly controlled at each
frequency to make the data valid and reliable.