Table1. Primers for qRT-PCR.
Figure 1.
Noise exposure causes hair cell loss in the cochlear base.
(A) One representative whole-mount cytocochleogram from a mouse cochlea harvested seven days after noise exposure is shown. Z-stacks were collected and summed using confocal microscopy. Prestin (red) labels OHCs. Myosin VIIa (green) labels both IHCs and OHCs, although we only summed the z-sections containing the IHCs for this image to distinguish them from OHCs. There was partial loss of hair cells in the basal region of the cochlea, whereas there was no hair cell loss in the apical region. The arrowhead highlights the 11 kHz region and arrow highlights the 32 kHz region. (B) The cytocochleogram counts revealed that more OHCs were lost than IHCs, and there was a gradient, with the most loss at the extreme cochlear base. (C)Total OHCs numbers progressively declined at 7 days and 1 month after noise exposure, whereas IHC numbers stabilized 7 days after noise exposure.
Figure 2.
Functional prestin increases after noise exposure.
(A,B) Current tracings in response to voltage steps from −100 mV to +80 mV in 20 mV steps from representative OHCs from control mice and mice 7 days after noise exposure. (C) Average capacitance as a function of voltage from control and noise-exposed mice. The non-linear capacitance was significantly higher in OHCs 7 days after noise exposure. (D) The linear capacitance of OHCs from control and noise-exposed mice were not significantly different. (E) The total charge moved (Qmax) was greater in OHCs from noise-exposed mice. (F) The charge density was greater in OHCs from noise-exposed mice.
Figure 3.
Prestin mRNA increases after noise exposure.
(A) The total amount of myosin VIIa mRNA was not significantly different after noise exposure. (B) After normalizing myosin VIIa to the number of residual hair cells at 7 days and one month after noise exposure, there still was no difference in the amount of myosin VIIa per hair cell. (C) The total amount of prestin mRNA was not significantly different after noise exposure. (D) After normalizing prestin to the number of residual OHCs at 7 days and one month after noise exposure, there were statistically significant increases in the amount of prestin mRNA per OHC. (E) The prestin/myosin VIIa ratio increased after noise exposure. (F) After normalizing the prestin/myosin VIIa ratio to the number of residual OHCs, the increase after noise exposure persisted.
Figure 4.
Prestin protein level increases after noise exposure.
(A) Representative Western blots of prestin and myosin VIIa from mice seven days after noise exposure and from control mice. These images come from the same gel. (B) Quantification of the band density revealed that the prestin/myosin VIIa ratio increased after noise exposure. (C) After normalizing the prestin/myosin VIIa ratio to the number of residual OHCs, the increase after noise exposure persisted.
Figure 5.
The measured prestin increases are greater than modeling predictions.
(A) Representative whole-mount preparation immunolabeled for prestin imaged by two-photon microscopy. We measured the diameter of the OHC at the top (t), the diameter of the OHC at the bottom (b), and the length of the OHC (l) to calculate the amount of prestin-containing membrane (PCM) per OHC. (B) The PCM/OHC measured at six different cochlear locations demonstrated a linear relationship. The region where the small OHCs from the base that were lost after noise exposure is shown. (C) The data from the cytocochleograms were used to predict what would happen, assuming the amount of prestin per OHC does not change and only the smaller OHCs from the base are removed from the average. According to this model, a statistically significant increase in the PCM/OHC should be expected. (D) However, the fold-increases in prestin we measured by qPCR and Western blot were larger than that predicted by the model. For reference, the fold-increase in the functional prestin density measured with the patch clamping experiments is also shown.
Figure 6.
ABR, DPOAE, and cochlear microphonic measurements.
(A, B) ABR and DPOAE thresholds were dramatically elevated 0.5 days after noise exposure. There was complete recovery in the low frequencies and partial recovery in the high frequencies. (C) DPOAE growth curves demonstrate similar magnitude emissions between noise-exposed and control mice at 11.3 kHz, the area where we performed the patch-clamp studies. (D) At 32 kHz, a region where there was partial OHC loss, noise-exposed mice had lower DPOAE magnitudes. (E) The cochlear microphonic magnitude measured at the round window using a 6 kHz stimulus was lower in noise-exposed mice compared to controls.
Figure 7.
(A, B) Basilar membrane vibratory magnitude and phase measured in the 8–9 kHz region using a 60 dB SPL stimulus are shown. In order to allow averaging, the frequency was normalized so that the resonance frequencies were 1.0, and the magnitudes was normalized to the middle ear response. (C) There were no differences between in the peak magnitudes of tuning curves measured in noise-exposed and control mice. (D) There were no differences between in the sharpness of the tuning curves measured in noise-exposed and control mice. (E) CAP tuning curves collected serially in a cohort of mice using a 12 kHz probe tone. (F) There were no significant difference in the sharpness of the tuning curves. (G) CAP tuning curves were also collected using a 32 kHz probe tone. (H) The tuning curve sharpness dropped after noise exposure in this region where some OHCs were lost.