Figure 2.

Monaural conductive hearing loss decreases vGluT1 expression within the cytoplasm of auditory nerve endings, and vGluT1 expression increases after earplug removal. A, Top row, Examples of auditory nerve endings on bushy cells immunolabeled with vGluT1 (10-nm-diameter gold particles) at low magnification. White boxed areas are magnified on the bottom row. Arrowheads note the edge of the PSD. AN, Auditory nerve; BC, bushy cell. Scale bars: top row, 250 nm; bottom row, 200 nm. B, Summary of the mean area of auditory nerve endings and number and density of gold particles for vGluT1 per auditory nerve ending. Areas of endbulb profiles were the same between the earplugged group and the age-matched P40 shams (p > 0.05, Mann–Whitney U test). vGluT1 gold labeling was significantly decreased in the earplugged animals compared with age-matched shams (number: P40 sham, 197.6 ± 30.57; P40 EP, 54.4 ± 7.91; ****p < 0.0001; density: P40 sham, 54.51 ± 7.10; P40 EP, 15.68 ± 1.7; ****p < 0.0001, Mann–Whitney U test). Density and gold labeling for vGluT1 after earplug removal was higher than age-matched shams (number: P50 sham, 183.6 ± 27.45; P50 EP-removal, 486.2 ± 61.75; ***p < 0.003; density: P50 sham, 57.95 ± 5.93; EP-removal, 150 ± 23.4; ****p < 0.001, Mann–Whitney U test). Number and density of vGluT1 labeling was the same in P40 and P50 shams (p > 0.05, Mann–Whitney U test). C, Average number of vGluT1 gold particles per SV in sham and earplugged animal groups. The number of gold particles per SV was significantly decreased in the earplugged animals compared with age-matched shams (****p < 0.0001, Mann–Whitney U test). No difference was found in the number of vGluT1 gold particles per SV after earplugged removal compared with age-matched shams (p = 0.20, Mann–Whitney U test). The difference between earplugged animals and age-matched controls was not found statistically significant (p > 0.05, Mann–Whitney U test test).