Mechanical responses of the organ of corti to acoustic and electrical stimulation in vitro

Abstract

The detection of sound by the cochlea involves a complex mechanical interplay among components of the cochlear partition. An in vitro preparation of the second turn of the jird's cochlea provides an opportunity to measure cochlear responses with subcellular resolution under controlled mechanical, ionic, and electrical conditions that simulate those encountered in vivo. Using photodiode micrometry, laser interferometry, and stroboscopic video microscopy, we have assessed the mechanical responses of the cochlear partition to acoustic and electrical stimuli near the preparation's characteristic frequency. Upon acoustic stimulation, the partition responds principally as a rigid plate pivoting around its insertion along the spiral lamina. The radial motion at the reticular lamina greatly surpasses that of the tectorial membrane, giving rise to shear that deflects the mechanosensitive hair bundles. Electrically evoked mechanical responses are qualitatively dissimilar from their acoustically evoked counterparts and suggest the recruitment of both hair-bundle- and soma-based electromechanical transduction processes. Finally, we observe significant changes in the stiffness of the cochlear partition upon tip-link destruction and tectorial-membrane removal, suggesting that these structures contribute considerably to the system's mechanical impedance and that hair-bundle-based forces can drive active motion of the cochlear partition.

FIGURE 1

Vertical response of the cochlear partition to acoustic stimulation. (A) In the in vitro preparation of the jird's cochlea, the cochlear partition is suspended between its natural insertions at the spiral lamina, which forms its inner (modiolar) attachment, and at the spiral ligament, which constitutes its outer boundary. The velocity of glass beads attached to the tectorial and basilar membranes was measured by laser Doppler interferometry. The numbers correspond to the locations indicated in B–E. (B) In response to a 58-dB SPL acoustic stimulus at 700 Hz, the resonant frequency of this preparation, tectorial-membrane displacement generally increased as a function of distance from the spiral lamina. (C) The phase of tectorial-membrane displacement relative to that measured at the innermost point varied only slightly with radial position. (D) In response to a 58-dB SPL acoustic stimulus, basilar-membrane displacement also grew with increasing distance from the spiral lamina. Because the preparation was mounted with its apical end down to permit access to the basilar membrane, the stimulus frequency of 1000 Hz significantly exceeded the second-order resonant frequency imposed by the increased volume of perilymph in the lower compartment of the experimental chamber. (E) The phase of basilar-membrane displacement relative to the displacement at the innermost point varied little with radial position, except for a phase lag near 50° at the outermost position. In general, the movements of the tectorial and basilar membranes were essentially in phase with one another. Here and in Figs. 2, 4, 5, and 8, the schematic illustration of the cochlear partition exaggerates the 100-μm region of the basilar membrane overlain by the organ of Corti. The additional 100–150 μm of basilar membrane between the Hensen's cells and the spiral ligament was not visible in the preparation and is not depicted to scale. (BM, basilar membrane; HC, Hensen's cell; IHC, inner hair cell; IPC, inner pillar cell; OHC, outer hair cell; OPC, outer pillar cell; SLa, spiral lamina; SLi, spiral ligament; and TM, tectorial membrane.) The points and vertical bars in B–E represent the means and standard deviations of the measurements.

FIGURE 2

Radial response of the reticular lamina to acoustic stimulation. (A) The displacements of the cellular structures on the reticular lamina indicated with dashed lines were measured with the photodiode projection system. Numbers correspond to the locations cited in B and C. (B) In response to a 58-dB SPL acoustic stimulus at 800 Hz, the resonant frequency of this preparation, the inward radial displacement of cellular structures at the reticular lamina decreased as a function of distance from the spiral lamina; the greatest displacement was observed for the hair bundles of inner hair cells. (C) Points increasingly distant from the spiral lamina exhibited a growing phase lead, with the inner boundary of the third row of outer hair cells leading the hair bundle of an inner hair cell by ∼90°. (BM, basilar membrane; IHC, inner hair cell; IPC, inner pillar cell; OHC, outer hair cell; OPC, outer pillar cell; SLa, spiral lamina; SLi, spiral ligament; and TM, tectorial membrane.) The points and vertical bars in B and C represent the means and standard deviations of the measurements.

FIGURE 3

Electrically evoked movement of the cochlear partition. (A) The displacement of the hair bundle on an inner hair cell (black) was recorded in response to a 30-μA transepithelial current stimulus (red) in the form of a 300–3000 Hz frequency sweep (left) or a 50-ms pulse (right). Upward deflections correspond to positive polarization of the upper compartment and inward hair-bundle displacement toward the modiolus, which was accompanied by outward, or positive, bundle deflection toward the tallest row of stereocilia. (B) An overlay plot of the low-frequency end of the frequency-sweep stimulus and mechanical response reveals that positive polarization of the upper compartment was in phase with hair-bundle displacement away from the modiolus, or negative hair-bundle deflection. (C) The phase spectrum of inward hair-bundle displacement relative to positive upper-compartment polarization demonstrates a consistent phase difference slightly in excess of ∼180° across the entire range of frequencies. (D) The frequency spectrum displays no resonant behavior. The gradual attenuation of displacement amplitude with increasing frequency likely reflects passive electrical filtering. (E) Hair-bundle displacement scaled linearly with the electrical stimulus over a range of 40 dB, with a power-law slope of 0.96 (r² = 1.00; note the logarithmic axes).

FIGURE 4

Vertical response of the cochlear partition to electrical stimuli. (A) The velocity of glass beads attached to the tectorial and basilar membranes was measured by laser Doppler interferometry. Numbers correspond to the locations indicated in B–E. (B) In response to a 1-kHz, 10-μA transepithelial electrical stimulus, tectorial-membrane displacement largely increased as a function of distance from the spiral lamina. (C) The phase of upward tectorial-membrane velocity did not vary significantly with radial position, and consistently lagged the electrical stimulus by 90°, indicating that downward displacement of the tectorial membrane was in phase with positive polarization of the upper compartment. (D) In response to a 1-kHz, 30-μA electrical stimulus, basilar-membrane displacement was limited and displayed no apparent radial trend. (E) In contrast to the tectorial-membrane results, the phase of upward basilar-membrane velocity varied substantially with radial position. The innermost position lagged the electrical stimulus by almost 90°, whereas the upward velocity of the outermost position, below the outer hair cells, was nearly in phase with the electrical stimulus. In general, however, the basilar membrane moved toward the scala tympani in response to positive polarization of the upper compartment. (BM, basilar membrane; IHC, inner hair cell; IPC, inner pillar cell; OHC, outer hair cell; OPC, outer pillar cell; SLa, spiral lamina; SLi, spiral ligament; and TM, tectorial membrane.) The points and vertical bars in B–E represent the means and standard deviations of the measurements.

FIGURE 5

Radial response of the reticular lamina to electrical stimulation. (A) The displacements of the cellular structures on the reticular lamina indicated by dashed lines were measured with the photodiode projection system. In this instance, the inner edge of the inner pillar cells was also monitored. Numbers correspond to the locations indicated in B and C. (B) Displacements were measured in response to electrical pulses to provide an unambiguous polarity for each response. For the ordinate, positive displacement corresponds to radially inward movement, which reflects outward (positive) hair-bundle deflection, in response to 30-μA positive polarization of the upper compartment. (C) Frequency-sweep stimuli were used in a different preparation to obtain a more precise phase profile. The phase of radially inward displacement is given relative to the phase of positive polarization of the upper compartment. This result represents a single representative preparation; the precise location of the phase inflections varied slightly from one cochlea to another, but occurred consistently within this intermediate region. (BM, basilar membrane; IHC, inner hair cell; IPC, inner pillar cell; OHC, outer hair cell; OPC, outer pillar cell; SLa, spiral lamina; SLi, spiral ligament; and TM, tectorial membrane.) The points and vertical bars in B represent the means and standard deviations of the measurements.

FIGURE 6

Stroboscopic video analysis of electrically and acoustically evoked motion. (A) A 50-μA, 1-kHz sinusoidal electrical stimulus elicited movement of the hair bundles atop inner hair cells against a nearly stationary background. Superimposed upon a differential-interference-contrast image frame captured from a stroboscopic video (Supplementary Material, Fig. S3) is the optical flow field determined with the parameter values σ_S = 1.5, σ_T = 1, τ = 1, σ_W = 1, and ρ = 0.02. The red highlights correspond to radially outward movement of the hair bundles (to the right) at ∼16 μm · s⁻¹. (B) This pseudocolor scale calibrates the optical flow calculated with the Lucas-Kanade algorithm. Each color in D and F corresponds to motion in the direction indicated by the hue in this scale and at a speed indicated by the corresponding saturation. (C) A differential-interference-contrast image displays the preparation in A under the same electrical-stimulation conditions. (D) Optical-flow measurements reveal opposing directions of movement across the reticular lamina, with a line of inflection occurring at the pillar cells. The image was acquired at the phase of greatest basilar-membrane velocity toward the lower compartment. At this phase of stimulation, the apical surfaces of the inner hair cells moved radially outwards (to the right) at ∼50 μm · s⁻¹, whereas the tops of the first row of outer hair cells translated toward the modiolus (to the left) at a similar speed. The image was acquired at the phase of greatest basilar-membrane velocity toward the lower compartment. (E) Differential-interference-contrast microscopy shows the same preparation during subsequent acoustic simulation. (F) Optical-flow measurements indicate that acoustic stimulation elicited reticular-lamina motion that was in the same direction, radially outwards (to the right), throughout the field of view. The optical-flow parameter values for D and F were σ_S = 1, σ_T = 1, τ = 0.2, σ_W = 25, and ρ = 0.05.

FIGURE 7

Stiffness of the cochlear partition. (A) The resonant frequency of the displacement response of the hair bundle on an inner hair cell was measured with varying total amounts of perilymph in the lower compartment in a freshly dissected preparation in the presence of K⁺ endolymph (black); after a 10-min treatment with 5 mM BAPTA (red), which destroys tip links; and again after removal of the tectorial membrane (green). Each of these two treatments was found to decrease the resonant frequency. The same color code applies to the remaining panels. (B) The slopes of the mass-frequency relations from the data in A indicate that the stiffness of the cochlear partition was decreased sequentially by BAPTA treatment and tectorial-membrane removal. In K⁺ endolymph, this stiffness was 19.5 N · m⁻¹; BAPTA treatment reduced it to 11.9 N · m⁻¹, and tectorial-membrane removal lowered it further, to 11.0 N · m⁻¹. (C) Frequency spectra of the hair bundle's displacement response with a 7-mm column of perilymph in the lower compartment demonstrate both downward shifts in the resonant frequency (f₀) and decreases in the quality factor (Q₀) after BAPTA treatment and then tectorial-membrane removal. (D) Lorentzian fits to the data of C emphasize the effects. In the presence of K⁺ endolymph, the data yielded values of f₀ = 612 Hz and Q₀ = 2.6; BAPTA treatment lowered these to f₀ = 558 Hz and Q₀ = 1.9, and tectorial-membrane removal decreased them further to f₀ = 482 Hz and Q₀ = 1.1. In C and D, the response amplitude is normalized to the peak value for each condition.

FIGURE 8

Motion of the cochlear partition in response to acoustic and electrical stimulation. With an exaggerated distance scale, this schematic illustration depicts the movements inferred from the measurements made in this study. In each panel, the cochlear partition is shown white-on-gray at one extreme of its motion; the other extreme is displayed with a red outline. Orange arrows emphasize the directions and relative magnitudes of movement of specific structures. (A) In response to an acoustic stimulus in the form of increased pressure in the lower compartment, the cochlear partition moves upwards as a unit. The vertical movement of the basilar membrane (BM) exceeds that of the tectorial membrane (TM), and displacements at the reticular lamina (RL) occur toward the spiral lamina (SLa) at all points. Both the spiral lamina and spiral ligament (SLi) remain stationary. (B) In contrast, electrical stimulation elicits complex modes of motion within the cochlear partition. In response to positive polarization of the lower compartment, the cochlear partition moves upwards. In contrast to the acoustic response, the vertical motion of the tectorial membrane exceeds that of the basilar membrane. The direction of radial motion at the reticular lamina varies with location: whereas the innermost and outermost points are displaced toward the spiral lamina, an intermediate region moves toward the spiral ligament. Positive polarization of the lower compartment depolarizes the apical membrane and hyperpolarizes the basolateral membrane of each hair cell. The electrically evoked response depicted here is therefore likely to result from the simultaneous recruitment of electromechanical transduction forces in the form of outward deflections of hair bundles and elongation of outer-hair-cell somata (yellow arrows).