Mice were anesthetized with intraperitoneal injection of a mixture of zolazepam-tiletamine (5 mg/kg) and xylazine (5 mg/kg). All tests were conducted in a custom-made sound-attenuated chamber for mice. For hearing screening, auditory brainstem responses (ABRs) to click (0.1 msec, 19.3/sec) and tone bursts (8/16/32 kHz, 1.5 msec, 21.1/sec) were recorded using SmartEP fitted with high-frequency transducers and high-frequency software ver. 2.33 (Intelligent Hearing Systems, Miami, FL, USA). Test stimuli were delivered to the ear canal through a miniature insert earphone from 90 dB to 10 dB sound pressure level (SPL) in 5 dB steps. Electrical potentials in the first 12 msec after the stimuli were sampled via subdermal stainless steel needle electrodes at the vertex (active) and pinna (reference) with 0.1–3 kHz bandpass filtering, and averaged for 256 sweeps. Thresholds were defined as the lowest stimulus intensity where the typical wave pattern was still identified. SmartOAE ver. 4.26 (Intelligent Hearing Systems) was used to measure the 2f₁-f₂ DPOAE in the ear canal and to obtain DP-gram that displays DPOAE amplitude plotted as a function of f₂ frequency. DPOAE recordings were made for f₂ frequencies from 6.5 to 35 kHz at 8 points per octave using primary tone paradigm set as follows: L₁=65 dB, L₂=55 dB SPL, and f₁/f₂=1.22. Stimulus signals were passed through ER-10B+ probe microphone inserted into the ear canal in conjunction with two types of transducers: ER-2 transducer (Etymotic Research, Elk Grove Village, IL, USA) for frequencies from 6 to 16 kHz and high-frequency transducer (Intelligent Hearing Systems) for frequencies from 16 to 32 kHz. The output was sampled at 128 kHz using a 16-bit A/D converter. Four blocks were acquired in total and each block consisted of 32 sweeps. Tests were performed under two stimulus conditions of the opposite ear with and without CAS. Broadband noise MOC elicitor was presented to the contralateral ear at 55 dB SPL that does not evoke stapedial reflex in mammals [13].

Mice were anesthetized and killed to harvest the cochleas, which were processed for whole mount immunofluorescent staining and confocal microscopy. The cochlea was perfused and fixed with 2% paraformaldehyde, and then decalcified in ethylenediaminetetraacetic acid (EDTA) for 2–4 days. The lateral wall, Reissner’s membrane, and tectorial membrane were removed in order after the otic capsule was opened. In whole mount cochleas, the ETs were made visible by immunofluorescent staining for α-synuclein, an efferent synaptic vesicular protein. Specimens were blocked in 5% normal goat serum and 0.3% Triton X-100 for 1 hour, and incubated with 1:100 anti-α-synuclein antibody (purified mouse anti-α-synuclein, BD Biosciences, San Jose, CA, USA) at 4°C overnight in a humid chamber. They were rinsed and incubated with 1:4,000 fluorescent secondary antibody (Alexa Fluor 555 goat anti-mouse IgG, Thermo Fisher Scientific, Waltham, MA, USA) for 2 hours. After the secondary antibodies had been removed, specimens were exposed to Vectashield with DAPI (Vector Laboratories, Burlingame, CA, USA) which allows visualization of the OHC nuclei. The frequency regions of interest were localized along the cochlear spiral at a low magnification according to a place-frequency map of mouse cochlea published previously [14]. Eighteen percent distance from the apex designated 8 kHz region, 43% distance designated 16 kHz, and 68% distance designated 32 kHz. Stained whole mounts were examined by confocal laser scanning microscopy (LSM 510 Meta, Carl Zeiss, Jena, Germany). As the ET-OHC synapses are located at different heights, a confocal z-stack was obtained from each region of the cochlea. Approximately 30 series of images were acquired per a region by focusing from top to bottom with a 0.6 μm step in the z-axis through the whole mount specimen under a magnification of 1,000. The acquired z-plane images were reconstructed using IPLab software (BioVision Technologies Inc., Exton, PA, USA) to create a single z-stack image. The captured images were imported into Image J software (National Institutes of Health, Bethesda, MD, USA). For quantitative morphometric analysis, two indices were used: the ET diameter and the number of ETs per OHC. The diameters of all visible ETs in a single image were measured and averaged. The number of all ETs (range, 44 to 91) was counted and divided by the number of OHCs (range, 31 to 39) shown in that image.

DPOAE amplitude was measured from the noise floor at each test frequency. Data points of an amplitude less than 6 dB were rejected. A DPOAE change by the MOC effect was defined as CAS-on amplitude minus baseline amplitude. Negative changes indicated suppression and positive changes, enhancement. The DPOAE change was normalized to the baseline amplitude and termed suppression ratio as the measure of MOCR strength at a valid data point. F₂ frequencies were grouped into three ranges based on the same mouse place-frequency map: the low frequencies (LF) included 6,573–10,779 Hz, which corresponds approximately to a <30 percent distance region from the apex; the middle frequencies (MF), 11,904–23,592 Hz, a 30–60 percent distance region; the high frequencies (HF), 26,048–35,056 kHz, a >60 percent distance region. Within each frequency range, the suppression ratios at all data points from 16 ears were averaged. Statistical analysis was done using SPSS ver. 18.0 (SPSS Inc., Chicago, IL, USA) at a two-tailed significance level of 0.05. Two way ANOVA was performed in advance of every statistical analysis to rule out any interaction between the group and the side of the tested ears. The suppression ratios were compared across the three frequency ranges. The morphometric indices of the ETs were compared across the three regions of the cochlea. One way ANOVA and post hoc Tukey’s test were performed after Shapiro-Wilk test for normal distribution and Levene’s test for the equality of variances. Otherwise, Kruskal-Wallis test and post hoc Mann-Whitney U-test were applied with Bonferroni’s correction.

ABR thresholds at click, 8, 16, and 32 kHz tone bursts were 16.3±0.6 (mean±standard error of mean [SE]), 16.3±0.6, 18.4±0.9, and 19.4±0.8 dB SPL respectively. All experimental mice had good hearing with ABR thresholds less than 20 dB SPL at the test stimuli on both ears. DP-grams obtained from 16 ears displayed a consistent pattern of amplitude variations with alternating peaks and dips in quiet, and in the presence of CAS (Fig. 1). There were no statistical differences in DPOAE levels between the right and left ears: 12.4±7.7 dB SPL for the right and 11.8±7.5 dB SPL for the left ears (grand mean of the mean DPOAEs at test frequencies±standard deviation). DPOAE amplitudes were in the range of –12 to 39 dB SPL without CAS, and –14 to 38 dB SPL with CAS. Valid data points for CAS were 204 (71%) out of 288 (18 frequencies in 16 ears): 78, 84, and 42 from LF, MF, and HF respectively. CAS-evoked DPOAE changes exhibited both suppressions and enhancements ranged from –16 to 11 dB SPL (Fig. 2A). Mean (SE) suppression ratio in LF/MF/HF was 0.19 (0.02)/0.39 (0.06)/0.42 (0.09) from 39/49/24 suppression points. Significant differences were shown between LF and MF (P=0.012), and between LF and HF (P=0.005) (Fig. 2B).

Whole mount organ of Corti labeled with anti-α-synuclein showed clustered MOC ETs in the OHC area (Fig. 3A). Mean (SE) diameters of MOC terminals were 1.70 (0.04), 1.84 (0.04), and 1.53 (0.06) μm in the 8, 16, and 32 kHz regions respectively with significant differences between the 16 and 32 kHz (P<0.001), and between the 8 and 32 kHz regions (P=0.048) (Fig. 3B). Mean (SE) numbers of MOC terminals per OHC were 1.7 (0.06), 2.3 (0.04), and 1.8 (0.03) in the 8, 16, and 32 kHz regions respectively with significant differences between the 8 and 16 kHz (P<0.001), and between the 16 and 32 kHz regions (P<0.001) (Fig. 3C).