Research ArticleCELLULAR NEUROSCIENCE

Decades-old model of slow adaptation in sensory hair cells is not supported in mammals

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Science Advances  14 Aug 2020:
Vol. 6, no. 33, eabb4922
DOI: 10.1126/sciadv.abb4922
  • Fig. 1 Intracellular Ca2+ buffering modulates slow adaptation magnitude.

    (A) With a step-like force stimulus (M), the hair-bundle displacement (X) exhibits a creep. The creep appears to correlate with the slow adaptation receptor current decay (I). This correlation was presumed to be causal and led to the motor model of adaptation (schematized in the yellow stereocilia), with myosin motors indicated in red. The motor model predicts that lower adaptation magnitudes would lead to lower creep magnitudes (gray and light blue traces). (B) Top and side view schematic representations of the experimental setup for recording MET currents in OHCs using fluid-jet stimulation showing the patch pipette (P), the fluid-jet stimulator (FJ), and perfusion pipette (Perf). (C) Bundle displacements (X) and currents (I) recorded with stimulus waveform timing (M) at −80 mV (darker) and +80 mV (lighter) with different intracellular BAPTA concentrations. (D) Activation curves generated from cells shown in (C). (E) Summary plots of the percentage of adaptation and resting open probability (resting Popen). Data (+80 mV) have the gray shaded background. Cells shown in (C) are marked with filled symbols. Mean ± SD shown. Paired Student’s t tests were performed for the data in each internal solution between −80 and +80 holding potentials. Unpaired, unequal variance Student’s t tests were used to compare among all intracellular solutions at −80 mV and separately at +80 mV. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Number of cells (animals): 0.1 mM BAPTA, 7 (7); 1 mM BAPTA, 17 (17); 10 mM BAPTA, 10 (10); 1.4 mM Ca2+, 4 (4).

  • Fig. 2 Hair-bundle creep does not correlate with adaptation magnitude for different intracellular Ca2+ buffering and holding potentials.

    (A) Mean (solid) ± SD (shaded) of the normalized displacements eliciting a current with an amplitude of ~50% Imax from 0.1 mM BAPTA recorded at −80 mV and +80 mV and 10 mM BAPTA at −80 mV. (B) Summary plots of the bundle creep properties for all conditions in Fig. 1. From the top, the plots show the fast (τ1) and slow (τ2) time constants of the creep, the relative magnitude of the slow component of the creep [A2/(A1 + A2)], the magnitude of creep normalized to the total displacement [(A1 + A2)/y0], and the magnitude of the slow creep component normalized to the total displacement (A2/y0) for a given step. (C) No correlation (Pearson’s r = 9.2 × 10−4, P = 0.99) was found between A2/y0 and the adaptation magnitude for cells recorded with an intracellular concentration of 0.1 mM BAPTA at negative (black) and positive (gray) potentials. (D) A slight correlation (Pearson’s r = 0.24, P = 8.7 × 10−4) was found between τ2 and the slow adaptation τ in all cells recorded with an intracellular concentration of 0.1 mM BAPTA at negative (black) and positive (gray) potentials. Dashed line represents the trend for an ideal linear correlation. Correlation analysis includes all positive deflections in all cells except for the smallest deflection, where the signal-to-noise ratio was low. (E) Correlation between the inferred shift and the slow component of the creep (A2) in all cells recorded with an intracellular concentration of 0.1 mM BAPTA at negative (black) and positive (gray) potentials. The cells held at positive potentials show a decreased slope in the inferred shift versus creep magnitude, indicating that these two variables are not causally related. Same statistical details and cell (animal) numbers are shown as in Fig. 1.

  • Fig. 3 Inhibition of myosin ATPase activity and PMCA pumps modulates slow adaptation, but not the hair-bundle creep.

    (A) Hair-bundle displacements (X) and currents (I) recorded with stimulus waveform (M) using 0.1 mM BAPTA (control, black) with 60 mM cesium sulfate (Cs2SO4, orange) or 4 mM sodium orthovanadate (Na3VO4, purple) intracellularly. Using 1 mM BAPTA intracellularly, data were taken in normal extracellular solution (pH 7.4, red) and after 10-min PMCA inhibition with pH 9 (blue) in the same cell. Inhibition of ATPase activity and PMCA pumps has opposite effects on the adaptation magnitude. (B to D) Summary plots of adaptation magnitude and (E to G) resting open probability. (B and E) Using 0.1 mM BAPTA intracellular buffer with SO42− or VO43−. (C and F) Using 1 mM BAPTA intracellular buffer with SO42− or VO43−. (D and G) Before (pH 7.4) and after PMCA inhibition (pH 9). Gray shading indicates data at +80 mV. Additional control data plots show the adaptation magnitude and resting open probability at 5 min (pH 7.4) and 20 to 25 min in the normal pH 7.4 extracellular solution (Control). The cells shown in (A) are shaded. (H and I) Mean (solid) ± SD (shaded) of normalized displacements eliciting ~50% Imax with a color legend and an onset motion expansion (gray box). Summary plots of the bundle creep properties recorded during (J) ATPase inhibition or (K) PMCA inhibition. Dashed lines connect data points for a given cell. Mean ± SD shown. *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001. Number of cells (animals): 0.1 mM BAPTA, 15 (13); 0.1 mM BAPTA-Cs2SO4, 8 (7); 0.1 mM BAPTA-Na3VO4, 16 (15); 1 mM BAPTA, 8 (8); 1 mM BAPTA-Cs2SO4, 10 (10); 1 mM BAPTA-Na3VO4, 14 (13); pH 9 to 10 (10); Control, 5 (5).

  • Fig. 4 The hair-bundle creep is uncorrelated with slow adaptation in vestibular hair cells when stimulated with flexible glass fibers.

    (A) Schematic representation of the experimental setup during the recording of MET currents in vestibular hair cells when stimulated with a flexible fiber. (B) Image of the vestibular hair bundle used for the recording in (C) with a black line to show where the bundle displacement was analyzed. Scale bar, 2 μm. (C) Currents and bundle displacements from the same cell in a P21 gerbil utricle when stimulated with a flexible glass fiber (0.139 mN/m stiffness) at +80 mV (red) and −80 mV (black). Marked traces (violet and blue) highlight a similar step size and are directly compared below. (D) Summary plots of the current properties for the stimulation eliciting ~50% Imax fit with a double exponential decay equation. The left plot summarizes the percentage of adaptation at −80 mV and +80 mV. Fast (τ1) and slow (τ2) adaptation time constants at −80 mV, as well as the Imax at −80 mV and +80 mV, are shown. (E) Summary plots of the bundle motion properties. Gray lines connect data points for a given cell, with cells shown in (A) marked. Bars represent mean ± SD. **P ≤ 0.01, ****P ≤ 0.0001. Number of cells (animals): 13 (13).

  • Fig. 5 In vestibular hair cells, Myo1c inhibition modulates the adaptation magnitude but not the hair-bundle creep; however, in the cochlea, Myo1c inhibition has no effect on slow adaptation.

    In the utricle, (A) bundle displacements (X) and currents (I) recorded with stimulus waveform (M) using 0.1 mM BAPTA intracellularly with and without 250 μM NMB-ADP in WT and Myo1cY61G/Y61G type II utricular hair cells. (B) Summary plots of Imax and current decay properties for steps eliciting ~90% Imax because slow adaptation was more prominent for larger step sizes in vestibular hair cells with fluid-jet stimulation. (C) Mean (solid) ± SD (shaded) of the normalized displacements eliciting ~90% Imax from each experimental condition. (D) Summary plots for properties of the hair-bundle creep show no effect on the mechanical properties of the bundle. In the cochlea, (E) bundle displacements (X) and currents (I) recorded with stimulus waveform (M) using 0.1 mM BAPTA intracellularly with 250 μM NMB-ADP in WT (gray) and Myo1cY61G/Y61G (light blue) OHCs. (F) Summary plots show no difference in the percentage of adaptation (P = 0.65), resting open probability (P = 0.64), and peak current (P = 0.76). Percentages of adaptation were calculated for the stimulus eliciting ~50% Imax. Cell examples are marked as diamonds. Black error bars represent mean ± SD. Unpaired, unequal variance Student’s t tests were used. *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001. For utricle, numbers of cells (animals): Myo1cY61G/Y61G, 10 (6); Myo1cY61G/Y61G + NMB-ADP, 8 (6); WT, 10 (5); and WT + NMB-ADP, 8 (5). For cochlea, numbers of cells (animals): WT + NMB-ADP, 9 (9); Myo1cY61G/Y61G + NMB-ADP, 6 (5).

  • Fig. 6

    Schematic representation of the MET machinery between two adjacent rows of stereocilia comparing the old motor model and revised model of adaptation. (A) In the old model, MET channels are located at both ends of the tip link. Myosin motors are located at the upper tip-link insertion, generate resting tension, and mediate adaptation by climbing and slipping along the side of the stereocilium to regulate tip link tension. (B) In our revised model, different myosin motor populations are responsible for tension generation at the upper tip-link insertion and for mediating slow adaptation. We propose that slow adaptation does not involve the modulation of tip-link tension, requires the function of myosin motors near the MET channels, and involves PIP2.

Supplementary Materials

  • Supplementary Materials

    Decades-old model of slow adaptation in sensory hair cells is not supported in mammals

    Giusy A. Caprara, Andrew A. Mecca, Anthony W. Peng

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