Differences in Calcium Clearance at Inner Hair Cell Active Zones May Underlie the Difference in Susceptibility to Noise-Induced Cochlea Synaptopathy of C57BL/6J and CBA/CaJ Mice

Abstract

Noise exposure of a short period at a moderate level can produce permanent cochlear synaptopathy without seeing lasting changes in audiometric threshold. However, due to the species differences in inner hair cell (IHC) calcium current that we have recently discovered, the susceptibility to noise exposure may vary, thereby impact outcomes of noise exposure. In this study, we investigate the consequences of noise exposure in the two commonly used animal models in hearing research, CBA/CaJ (CBA) and C57BL/6J (B6) mice, focusing on the functional changes of cochlear IHCs. In the CBA mice, moderate noise exposure resulted in a typical fully recovered audiometric threshold but a reduced wave I amplitude of auditory brainstem responses. In contrast, both auditory brainstem response threshold and wave I amplitude fully recovered in B6 mice at 2 weeks after noise exposure. Confocal microscopy observations found that ribbon synapses of IHCs recovered in B6 mice but not in CBA mice. To further characterize the molecular mechanism underlying these different phenotypes in synaptopathy, we compared the ratio of Bax/Bcl-2 with the expression of cytochrome-C and found increased activity in CBA mice after noise exposure. Under whole-cell patch clamped IHCs, we acquired two-photon calcium imaging around the active zone to evaluate the Ca²⁺ clearance rate and found that CBA mice have a slower calcium clearance rate. Our results indicated that excessive accumulation of calcium due to acoustic overexposure and slow clearance around the presynaptic ribbon might lead to disruption of calcium homeostasis, followed by mitochondrial dysfunction of IHCs that cause susceptibility of noise-induced cochlear synaptopathy in CBA mice.

Keywords: calcium clearance; inner hair cell; mitochondrial; noise-induced cochlear synaptopathy; presynaptic ribbon.

Figure 1

Auditory brainstem response (ABR) threshold and wave I analysis. (A,B) Hearing threshold of ABR before and after noise exposure. (C,D) Representative traces of ABR response to 80 dB SPL at 8.0 kHz. (E,F) ABR wave I amplitudes, evoked by suprathreshold tones at 8.0 kHz. Data are presented as mean ± SEM; statistical significance was assessed by two-way ANOVA followed by the Bonferroni post-hoc test, *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 2

Inner hair cell ribbon synapse counts in B6 and CBA mice before and after noise exposure. (A,C) Representative confocal images of IHCs co-labeled for the presynaptic marker CtBP2 (red) and postsynaptic marker GluA2 (green) of B6 and CBA mice at pre, 1 day, and 14 days after noise exposure in the apical turn of the basilar membrane. (B,D) Puncta of co-labeling are presumably ribbon synapses, and the numbers of synapses were counted (mean ± SEM) per IHC. Data were analyzed by one-way ANOVA followed by Bonferroni post-hoc test. *P < 0.05 and ***P < 0.001. N.S., no significant difference was found.

Figure 3

Comparison of protein expression at 1 day after noise exposure between CBA and B6. (A) Protein levels of cleaved caspase-3, Bax, Bcl 2, cytochrome-C, calpain 1/2, and calpastatin were detected by Western blotting; GAPDH was used as an internal control. (B,C) Relative protein levels are presented as relative ratio of target proteins to GAPDH. Data were analyzed by one-way ANOVA followed by Bonferroni post-hoc test. *P < 0.05. N.S., no significant difference was found.

Figure 4

Two-photon calcium imaging in IHCs from the apical turn of the organ of Corti without noise exposure. (A) IHCs were loaded with Cy3-conjugated peptide binding to the synaptic ribbon to visualize synaptic ribbons at the active zone, and the hotspots of depolarization-evoked Ca²⁺ influx, visualized by increased fluorescence of the Ca²⁺ indicator Fluo-4FF. (B) Example line scan of a fluorescently-labeled ribbon and Fluo-4FF fluorescence change at an individual IHC active zone during 20-ms depolarization. (C) Representative recording show the evoked whole-cell Ca²⁺ current and the depolarization-evoked increase in fluorescence at a single active zone; ΔF from line-scans was normalized to their baseline fluorescence F₀ hence ΔF/F₀. (D–F) Decay of Ca²⁺ fluorescence intensity was fitted by a double exponential. Statistical significance was assessed by the Mann–Whitney test, **P < 0.01. N.S., no significant difference was found.

Figure 5

Immunofluorescence analysis of the presynaptic abundance of mitochondria counts between CBA and B6 mice without noise exposure. (A) Organ of Corti confocal image stacks of B6 and CBA mice, stained for CtBP2 and PNPase, show similar distribution in IHC at the apical turn of basilar membrane. (B) Normalized distributions of volumes of confocal z-sections integrated within a 0.5-μm radius around the center of mass of CtBP2 fluorescence in single confocal sections show near-identical distribution pattern. (C) Number of mitochondria within a 0.5-μm radius around the center of mass of CtBP2. Statistical significance was assessed by two-sample Kolmogorov–Smirnov test and unpaired t-test. N.S., no significant difference was found.

Figure 6

Expression of calretinin in IHCs. (A) Whole-mount preparation of organ of Corti double-stained for calretinin and myosin VIIa. (B) Quantification of calretinin fluorescence at the base of IHCs in apical region without noise exposure. Statistical significance was assessed by unpaired t-test. N.S., no significant difference.