The spatial pattern of cochlear amplification

Abstract

Sensorineural hearing loss, which stems primarily from the failure of mechanosensory hair cells, changes the traveling waves that transmit acoustic signals along the cochlea. However, the connection between cochlear mechanics and the amplificatory function of hair cells remains unclear. Using an optical technique that permits the targeted inactivation of prestin, a protein of outer hair cells that generates forces on the basilar membrane, we demonstrate that these forces interact locally with cochlear traveling waves to achieve enormous mechanical amplification. By perturbing amplification in narrow segments of the basilar membrane, we further show that a cochlear traveling wave accumulates gain as it approaches its peak. Analysis of these results indicates that cochlear amplification produces negative damping that counters the viscous drag impeding traveling waves; targeted photoinactivation locally interrupts this compensation. These results reveal the locus of amplification in cochlear traveling waves and connect the characteristics of normal hearing to molecular forces.

Figure 1. Active Traveling Waves Measured In Vivo

(A) Schematic illustrations depict the shapes of traveling waves in a normal, active cochlea (left) and an anoxic, passive one (right). The black lines show the instantaneous position of the cochlear partition; the shaded areas represent the envelopes of entire cycles of oscillation. In these diagrams and all subsequent plots of basilar-membrane data, the cochlear base lies to the left and the apex to the right. (B) Interferometric measurements indicate the magnitudes of the velocities at which the basilar membrane oscillated up-and-down in traveling waves elicited by pure-tone stimulation at 9 kHz; the abscissa's scale is shown in (C). In this and subsequent plots of basilar-membrane data, the abscissa depicts the distance along the basilar membrane relative to the most basal point of recording. The results are plotted on a logarithmic scale and at 10 dB decrements in sound-pressure level (SPL) descending from 90 dB. As demonstrated by the convergence of the curves toward the peak, active traveling waves (left) are compressive at stimulus levels above 40 dB SPL. In contrast, the traveling waves measured in an anoxic animal (right) scale linearly with sound pressure and the peaks are shifted basally. (C) Dividing the velocities by the stimulus pressures yields sensitivity measurements for the foregoing data. At each cochlear position, the ratio of the maximal to the minimal sensitivity provides a measure of the wave's gain accumulated by an active cochlea (left) up to that point. Because the anoxic preparation (right) lacks gain, the sensitivity curves are superimposed. (D) The velocities measured at the peak of the traveling wave in another control experiment (red) demonstrate compressive nonlinearity above 40 dB SPL. In contrast, the velocities in the same animal following anoxia (black) are smaller and scale linearly. The dashed line represents a linear relation. (E) The maximal cumulative gain of the basilar membrane's active response is the ratio of the control sensitivity at low stimulus levels (red) to that following anoxia (black). See also Figure S1.

Figure 2. Photoinactivation of Somatic Motility

(A) UV irradiation of 4-azidosalicylate yields a highly reactive nitrene moiety that covalently attaches to nearby amino-acid residues. (B) The optical absorbance spectrum of an aqueous solution containing 5 mM 4-azidosalicylate (red) was altered by irradiation with UV light (black). (C) Repeated measurements from a prestin-transfected HEK293T cell showed a gradual decrease in the nonlinear capacitance during a two-minute exposure to 5 mM 4-azidosalicylate (yellow region). In the absence of exposure to UV light, the capacitance gradually returned to its baseline value as the cell was then washed with extracellular saline solution. In this and the subsequent two records, each hump represents the capacitance measured during a linear voltage ramp from a holding potential of –150 to +100 mV. (D) The nonlinear capacitance in a prestin-transfected HEK293T cell was unaffected by a 1.5 s pulse of UV light. (E) Exposure of a prestin-transfected HEK293T cell to 5 mM 4-azidosalicylate for two minutes (yellow region) abolished the nonlinear capacitance. After the cell had been irradiated with a 1 s pulse of UV light, the nonlinear capacitance did not recover following washout of the free 4-azidosalicylate. (F) In an enlarged plot of one measurement of the voltage-dependent capacitance similar to those in (C–E), the abscissa shows the range of holding potentials over which the capacitance was measured. The nonlinear component measured in the absence of 4-azidosalicylate (red points) reflects the gating currents associated with prestin; the superimposed curve portrays a fit of the data with the derivative of a Boltzmann relation, C(V) = C_LIN + (Q_MAXze/kT)e^{–ze(V–V_1/2)/kT} [1 + e^{–ze(V–V_1/2)/kT}]^–2, in which C_LIN is the linear capacitative component, Q_MAX is the maximal charge displacement, z is the valence, k is Boltzmann's constant, T is the absolute temperature, and V_1/2 is the voltage at half-maximal charge movement. The fit values are C_LIN = 5.75 pF, Q_MAX = 0.22 pC, z = 0.28, and V_1/2 = –63 mV. The nonlinear capacitance vanished in the presence of 5 mM 4-azidosalicylate (blue points). (G) The changes in length of an isolated outer hair cell in response to a voltage step delivered through a wide-mouthed pipette were similar before (red) and after (blue) exposure to 5 mM 4-azidosalicylate and subsequent washout. (H) Motility was similar for an outer hair cell isolated from a control preparation (red) and one isolated from an excised organ of Corti after exposure to UV light in the absence of drug (blue). (I) Somatic motility was abolished in an outer hair cell isolated from an organ of Corti that was irradiated with UV light during exposure to 5 mM 4-azidosalicylate (blue). A control cell from the untreated cochlea of the same animal shows normal motility (red). See also Figure S2.

Figure 3. The Effect of 4-Azidosalicylate and UV Light

The most sensitive control responses, posttreatment responses, and anoxic responses measured under the specified conditions are indicated by respectively red, blue, and black lines. (A) The sensitivity profile of an active traveling wave before and after irradiation with UV light demonstrates that the sensitivity was unaffected. The stimulus level was 60 dB SPL. (B) While the scala tympani was perfused with 4-azidosalicylate, the traveling wave's sensitivity at 50 dB SPL fell to the level measured during anoxia. (C) Following exposure to 7.5 mM 4-azidosalicylate and subsequent washout with artificial perilymph, the wave recovered its original sensitivity. The responses were measured at 50 dB SPL. (D) After three bouts of irradiation over the entire exposed region of the basilar membrane during perfusion with 4-azidosalicylate, the basilar membrane's sensitivity remained roughly twice that of an anoxic preparation. The stimulus level was initially 50 dB SPL but increased to 70 dB SPL to account for the higher threshold after repeated photolysis. See also Figure S4.

Figure 4. Targeted Photoinactivation of Somatic Motility

The schematic diagrams of the traveling waves atop each column, which are not drawn to scale, depict qualitatively the locus of photoinactivation. The violet-shaded area in each diagram indicates the region of UV irradiation in one of three animals. In each graph of the upper row, the red line depicts the sensitivity of a traveling wave under control circumstances and the blue line shows that after targeted photoinactivation of the specified segment of the basilar membrane. The black line indicates the sensitivity after the animal had become anoxic. In each graph of the lower row, the cumulative gain is plotted as a function of distance along the basilar membrane under control conditions (red) and after photoinactivation (blue). We define the cumulative gain at any point along the abscissa as the ratio of the point's sensitivity before to that after anoxia. (A) Photoinactivation extending roughly one cycle basal to the traveling wave's peak sharply reduced sensitivity and lowered the cumulative gain by an order of magnitude. The stimulus intensity for all lines was 50 dB SPL. (B) Although focal inactivation well to the base of the peak locally interrupted the accumulation of gain, the sensitivity rose and gain accumulated to nearly the control level by the peak. The stimulus intensities for the red, blue, and black lines were respectively 40 dB, 40 dB, and 60 dB SPL. (C) Focal inactivation just basal to the peak significantly diminished sensitivity; the accumulation of gain nonetheless resumed apical to the perturbation. The stimulus intensities for the red, blue, and black lines were respectively 50 dB, 50 dB, and 60 dB SPL. See also Movie S1.

Figure 5. Reconstructions of Local Cochlear Impedance

For each of the three animals in Figure 4, the real part of the complex impedance, which corresponds to the dissipative force of viscous damping, is plotted for traveling waves under control conditions (top), after targeted photoinactivation (middle), and following anoxia (bottom). The regions that display net negative damping and hence active gain are shaded orange; green coloration indicates positive damping. (A) Although irradiation of a substantial segment of the cochlear partition largely abolished negative damping, anoxia exacerbated the effect. (B) Local inactivation of somatic motility basal to the traveling wave's peak diminished amplification. Subsequent anoxia revealed, however, that a portion of the active process persisted after photoinactivation. (C) Inactivation of somatic motility near the peak of the traveling wave had less effect, for most of the gain had already accumulated at more basal locations. See also Figure S3.