Effect of Sodium Salicylate on Calcium Currents and Exocytosis in Cochlear Inner Hair Cells: Implications for Tinnitus Generation

Abstract

Sodium salicylate is an anti-inflammatory medication with a side-effect of tinnitus. Here, we used mouse cochlear cultures to explore the effects of salicylate treatment on cochlear inner hair cells (IHCs). We found that IHCs showed significant damage after exposure to a high concentration of salicylate. Whole-cell patch clamp recordings showed that 1-5 mmol/L salicylate did not affect the exocytosis of IHCs, indicating that IHCs are not involved in tinnitus generation by enhancing their neuronal input. Instead, salicylate induced a larger peak amplitude, a more negative half-activation voltage, and a steeper slope factor of Ca²⁺ current. Using noise analysis of Ca²⁺ tail currents and qRT-PCR, we further found that salicylate increased the number of Ca²⁺ channels along with Ca_V1.3 expression. All these changes could act synergistically to enhance the Ca²⁺ influx into IHCs. Inhibition of intracellular Ca²⁺ overload significantly attenuated IHC death after 10 mmol/L salicylate treatment. These results implicate a cellular mechanism for tinnitus generation in the peripheral auditory system.

Keywords: CaV1.3 channel; Calcium current; Exocytosis; Inner hair cell; Salicylate; Tinnitus; Whole-cell patch clamp.

Fig. 1

Experimental design. A Timeline of the experimental protocol. P6, postnatal day 6; IF, immunofluorescence; qPCR, reverse transcription quantitative polymerase chain reaction. B Middle turn of a cochlear explant used for further experiments marked ROI (region of interest) (scale bar, 100 μm). C High magnification of a cochlea after 5 days in culture showing three rows of outer hair cells (OHCs), one row of inner hair cells (IHCs), and the approach to an inner hair cell with a recording pipette (40× water-immersion lens) (scale bar, 10 μm).

Fig. 2

Morphological changes of cochlear hair cells induced by different concentrations of salicylate. A Representative images of immunofluorescence staining for prestin (green), myosin 7a (red), and DAPI (blue) in the middle turn of the cochlea in different groups (arrowheads, damaged inner or outer hair cells; scale bars, 10 μm). B Quantification of inner hair cells from different groups (****P <0.0001 vs control, 1, 3, and 5 mmol/L salicylate group, one-way ANOVA). C Numbers of outer hair cells per 100 µm show no significant difference among the six groups. Data are presented as the mean ± SEM; n = 3 cochlear explants per group; statistical significance by one-way ANOVA followed by the Least Significant Difference test.

Fig. 3

Ca²⁺ current in inner hair cells (IHCs) after exposure to different concentrations of salicylate. A Representative Ca²⁺ currents recorded from four IHCs, one from each treatment group. B Peak amplitude (I_Ca) of Ca²⁺ current. C Half-activation voltage (V_half) of Ca²⁺ current. D Slope factor (k_slope) of Ca²⁺ current. Data are presented as the mean ± SEM. ns, P >0.05, *P <0.05, **P <0.01, ***P <0.001, ****P <0.0001, unpaired Student’s t-test; n = 14, 10, 11, and 11 IHCs in control, 1, 3, and 5 mmol/L salicylate groups, respectively.

Fig. 4

Exocytosis from inner hair cells (IHCs) after exposure to different concentrations of salicylate. A Representative Ca²⁺ currents (upper panel) and exocytotic capacitance increase (ΔC_m, lower panel) in response to 100 ms depolarization recorded from four IHCs, one from each treatment group. B Ca²⁺ influx (Q_Ca) of IHCs in response to stimulation from 20 to 200 ms. C Capacitance change (ΔC_m) of IHCs with 2 mmol/L intracellular EGTA. D Capacitance change (ΔC_m) of IHCs with 0.2 mmol/L intracellular EGTA. E–G The efficiency of exocytosis, i.e., the ΔC_m/Q_Ca ratio, in response to stimulation for 20 ms (E), 50 ms (F), and 100 ms (G). Data are presented as the mean ± SEM; ns P >0.05, *P <0.05, **P <0.01, ***P <0.001, ****P <0.0001, one-way ANOVA (B–D) followed by Least Significant Difference test (E–G).

Fig. 5

Noise analysis of Ca²⁺ tail currents: the numbers of Ca²⁺ channels and single-channel current after exposure to different concentrations of salicylate. A The applied voltage clamp protocol (upper trace) and representative evoked Ca²⁺ tail currents (lower traces) recorded from four inner hair cells (IHCs), one from each treatment group. B The mean (I_mean, solid lines) and variance (dashed lines) calculated from the ensembles of Ca²⁺ tail currents as in A. C Variance plotted against the I_mean (solid lines) and fitted to a parabolic function (dashed lines). D The calculated single Ca²⁺ channel current (i_Ca) in IHCs does not significantly differ among the four groups (P = 0.489, one-way ANOVA). E Number of Ca²⁺ channels in single IHCs (N_Ca) (*P <0.05, **P <0.01, unpaired Student’s t-test). F RT-qPCR determination of the mRNA expression of Ca_V1.3 reveals that Ca_V1.3 is up-regulated after salicylate treatment. Each experiment repeated three times. Data are presented as the mean ± SEM; *P <0.05, **P <0.01, Least Significant Difference test.

Fig. 6

Effects of BAPTA-AM on salicylate-exposed inner hair cells (IHCs). A Immunofluorescence staining for prestin (green), myosin 7a (red), and DAPI (blue) in the middle turn of the cochlea in different groups (arrowheads, damaged IHCs; scale bar, 10 μm). B Quantification of IHCs from different groups. C–E Half-activation voltage (V_half, C), slope factor (k_slope, D), and peak amplitude (I_Ca, E) of Ca²⁺ current in IHCs with or without BAPTA-AM pretreatment. F Numbers of Ca²⁺ channels (N_Ca) in IHCs with or without BAPTA-AM pretreatment. G RT-qPCR determination of the mRNA expression of Ca_V1.3 with or without BAPTA-AM pretreatment. Data are presented as the mean ± SEM. NS, P >0.05, *P <0.05, **P <0.01, unpaired Student’s t-test.