Sound-induced length changes in outer hair cell stereocilia

Abstract

Hearing relies on mechanical stimulation of stereocilia bundles on the sensory cells of the inner ear. When sound hits the ear, each stereocilium pivots about a neck-like taper near their base. More than three decades of research have established that sideways deflection of stereocilia is essential for converting mechanical stimuli into electrical signals. Here we show that mammalian outer hair cell stereocilia not only move sideways but also change length during sound stimulation. Currents that enter stereocilia through mechanically sensitive ion channels control the magnitude of both length changes and bundle deflections in a reciprocal manner: the smaller the length change, the larger is the bundle deflection. Thus, the transduction current is important for maintaining the resting mechanical properties of stereocilia. Hair cell stimulation is most effective when bundles are in a state that ensures minimal length change.

Fig. 1. Staining and imaging of stereocilia bundles

a. Schematic representation of an outer hair cell stereocilium of the tallest row depicting its attachment to the tectorial membrane. b. Confocal imaging of the cochlear partition before staining hair cells with the fluorescent dye RH-795. A glass pipette, the tip of which is inserted through Reissner's membrane, is used to iontophoretically inject dye. Reissner's membrane was stained by expelling a small amount of dye from the electrode prior to penetration. c. Hair cells and their stereocilia bundles are visible after dye release. d. Optical section showing RH-795 staining of the membranes of individual stereocilia within the bundle. e. Maximum brightness projection reconstructed from a deconvolved Z-stack acquired at the end of an experiment. f. Cochlear microphonic potential recordings before and after staining of hair cells with RH-795. Neither tuning nor amplitude is affected by the dye. g. Profile view of stereocilia bundle of an inner hair cell. h. Profile view of stereocilia bundle of an outer hair cell. Supplementary Movie 1 shows sound-evoked motion of the bundle.

Fig. 2

Effect of electrical stimulation on sound-evoked motion of outer hair cell stereocilia. a. Effect of +10 μA current injection during sound stimulation at the best frequency. A separate series of measurements showed that +10 μA current generates a potential of 120 ± 8 mV in scala media (n=7). The sound-evoked motion of the base of the bundle follows the blue pattern, whereas the tip of the bundle moves along the red trace. The green trace depicts the deflection of the bundle, which is known to gate the MET channels. Note that the deflection amplitude is maximal in the horizontal direction. b. Effect of negative current on the same cell as in a. The sound evoked movements of the base and the tip of the bundle are shown in blue and red respectively. The bundle deflection, shown in green, reorients itself as a result of the negative current. As in a, the trajectories were computed with a wavelet-based optical flow algorithm. c. Effect of electrical stimulation on the length of the bundle. Length changes are small during positive current. The traces shown are associated with data in a and b respectively. d. Electrical stimulation reveals a reciprocal relationship between sound-evoked length change (red) and deflection (black) magnitude. e. Distribution of the strain ratio in 26 different cells. The small width of the distribution indicates that bending movements do not contribute to the bundle length changes. f. Deflection phases measured at the tip (I) and the center of the bundle (II) are not significantly different (Wilcoxon rank sum test, p=0.6; n=26). Error bars represent the standard error of the mean.

Fig. 3

Bundle length changes persist in three dimensions. a. A reconstruction of the apical part of an outer hair cell, obtained during sound stimulation at 81 dB SPL and 200 Hz. The bundle length is the distance between the two asterisks. Scale bars, 2 μm in the x and y directions and 5 μm for the z axis. b. Change in distance between the two asterisks shown in panel A (representative example of 4 independent experiments).

Fig. 4

Effect of FM1-43, and salicylate on bundle movements at BF and 72 dB SPL. a. Effect of FM1-43 on the cochlear microphonic potential. The amplitude decreases as a result of MET channel block. (n=7 animals). b. Averaged amplitudes for length changes and deflection at negative (blue) and positive (red) current in presence of FM1-43. c. Averaged amplitudes for length changes and deflection at negative (blue) and positive (red) current in the absence of FM1-43. d. Averaged amplitudes for length changes and deflection at negative (blue) and positive (red) current in presence of salicylate. BF, best frequency. Error bars in panel b – d represent the standard error of the mean. One asterisk denotes a P-value less than 0.05; three asterisks a P-value less than 0.001. The Wilcoxon rank sum test was used in all panels of this figure.

Fig. 5

Fluorescence recovery after photobleaching in outer hair cell bundles. In each trace, laser light bleached a 1-micron spot on the bundle at time = 0. The fluorescence within the bleached spot is tracked by confocal microscopy before and after bleaching. As dye molecules wander into the bleached area, fluorescence recovers with an exponential time course. a. During negative current stimulation, complete fluorescence recovery is observed, but the extent of recovery is much smaller during positive current. This signifies a decrease in the fraction of dye molecules that are free to move within the membrane. b. Salicylate increases the mobile fraction and the rate of fluorescence recovery. All data in this panel were acquired without current injection. In both panels, smooth lines represent least-squares fits to a single-exponential function. Asterisks signify a p-value less than 0.001 by the Wilcoxon rank sum test.

Fig. 6

Effect of electrical stimulation on sound-evoked motion of inner hair cell stereocilia. The sound stimulus was delivered at the best frequency and 72dB SPL.Blue traces show motion at the bundle base, red traces motion at the bundle tip, and the deflection of the bundle is shown in green. Effect of positive (a) and negative (b) current. The polarity of the current affects neither the orientation nor the magnitude of the deflection trajectory, which remains horizontal, indicating absence of measurable bundle length changes in these cells.