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.