Vibration of the organ of Corti within the cochlear apex in mice

Abstract

The tonotopic map of the mammalian cochlea is commonly thought to be determined by the passive mechanical properties of the basilar membrane. The other tissues and cells that make up the organ of Corti also have passive mechanical properties; however, their roles are less well understood. In addition, active forces produced by outer hair cells (OHCs) enhance the vibration of the basilar membrane, termed cochlear amplification. Here, we studied how these biomechanical components interact using optical coherence tomography, which permits vibratory measurements within tissue. We measured not only classical basilar membrane tuning curves, but also vibratory responses from the rest of the organ of Corti within the mouse cochlear apex in vivo. As expected, basilar membrane tuning was sharp in live mice and broad in dead mice. Interestingly, the vibratory response of the region lateral to the OHCs, the "lateral compartment," demonstrated frequency-dependent phase differences relative to the basilar membrane. This was sharply tuned in both live and dead mice. We then measured basilar membrane and lateral compartment vibration in transgenic mice with targeted alterations in cochlear mechanics. Prestin(499/499), Prestin(-/-), and Tecta(C1509G/C1509G) mice demonstrated no cochlear amplification but maintained the lateral compartment phase difference. In contrast, Sfswap(Tg/Tg) mice maintained cochlear amplification but did not demonstrate the lateral compartment phase difference. These data indicate that the organ of Corti has complex micromechanical vibratory characteristics, with passive, yet sharply tuned, vibratory characteristics associated with the supporting cells. These characteristics may tune OHC force generation to produce the sharp frequency selectivity of mammalian hearing.

Keywords: biomechanics; cochlea; cochlear amplifier; electromotility; hair cell; hearing; hearing loss.

Fig. 1.

Schematic depiction of the vibrometry methodology. A: with traditional vibrometry methodology, a point measurement of basilar membrane vibration in response to sound stimulation is performed. This is most commonly performed by opening the cochlea, placing a reflective glass bead on the underside of the basilar membrane, and then measuring vibrations of the bead using a laser Doppler vibrometer. B: with the optical coherence tomography (OCT) technique we used, the cochlea was left unopened. The light was scanned over the tissue in the radial direction while collecting depth profiles. Thus vibrometry data were collected from every point in the grid over the organ of Corti. We analyzed vibration of the basilar membrane in the traditional manner by referencing a point measurement (dark rectangle on basilar membrane) to the sound input measured at the ossicular chain. We analyzed the vibration of the lateral compartment of the organ of Corti (large oval) by referencing a point measurement at the apical portion (dark rectangle at top of the lateral compartment) to that of the basilar membrane (dark rectangle on basilar membrane).

Fig. 2.

Basilar membrane tuning curves in a representative CBA mouse. A: plastic-embedded section of the mouse cochlea at the location we studied. B: in vivo OCT cross-sectional image from a representative mouse. The rough locations of the key structures within the organ of Corti are outlined (yellow). TM, tectorial membrane; BM, basilar membrane; OHC, outer hair cell region; IHC, inner hair cell region; Lat, lateral compartment of the organ of Corti (which contains Hensen’s, Boettcher's, and Claudius' cells). The white dotted line denotes the beam position for BM vibration measurements. The BM response was the average vibration along a 20-μm region (pink segment). C: raw BM vibration magnitudes from the live mouse. The numbers denote the stimulus intensities in dB sound pressure level (SPL). D: raw BM vibration magnitudes from the same mouse postmortem. E: BM sensitivity ratios in the same mouse, where BM vibration magnitude was normalized to that of the ossicular chain in the living (red) and dead (black) conditions. F: BM phase responses from the same mouse in the living (red) and dead (black) conditions. The phase was referenced to the ossicular chain.

Fig. 3.

Comparison of BM sensitivity and phase measurements from five different mice. All data were normalized to the middle ear response. A: the data from the mouse presented in Fig. 2. B–E: data from four additional mice.

Fig. 4.

Representative displacement measurements across the organ of Corti in the CBA mouse. Left: anatomic image. Center: magnitude response. Right: phase response. A and B: magnitude and phase responses to 5- and 8-kHz stimuli superimposed on the anatomic OCT image using pseudo-color in a live mouse. The beam path for simultaneous phase measurements (dotted line) and the specific positions analyzed for the data in Fig. 5 (Lat and BM) are shown. Scale bar, 50 μm. C and D: magnitude and phase responses to 5- and 8-kHz stimuli superimposed on the anatomic OCT image using pseudo-color in a postmortem mouse. The estimated locations of various organ of Corti substructures, including the BM region, the IHC region, OHC region, and the Lat region, were drawn for the anatomic image in A and for all magnitude and phase images to aid data interpretation.

Fig. 5.

Comparison of BM and Lat vibratory responses. A: the magnitude of BM displacement in one representative mouse (the same mouse from Fig. 3B) after normalization to the middle ear response. B: the normalized magnitude of the Lat displacement measured simultaneously. C: the phase of the Lat and the BM measured simultaneously from the living mouse, using a stimulus intensity of 80 dB. The phase values were referenced to the phase of the ossicular chain. Around 7–8 kHz, a difference in the phase of the two locations was noted (blue arrows). D: the phase of the Lat and the BM in the same mouse postmortem. The same phase difference noted in the live mouse exists in the dead mouse (blue arrows). E and F: the difference between the phase of the Lat and the phase of the BM for both the live (E) and dead (F) conditions (red and black circles, respectively). These data were fit with a second-order band-pass filter model (lines).

Fig. 6.

Comparison of averaged BM sensitivity and Lat phase differences in CBA mice. A: BM tuning curve sharpness [best frequency divided by the bandwidth 10 dB from the peak response (Q_10dB)] was lowest in dead mice. In living mice, it increased as the stimulus intensity decreased, from 80- to 60- to 40-dB SPL. Published auditory nerve (AN) data (Taberner and Liberman 2005) collected at threshold had higher Q_10dB values and fit the exponential trend line. The Q_10dB values calculated from fits of the phase difference between the Lat and the BM in both living and dead mice were similar to the AN data. B: the center frequencies (F_c) of the Lat phase fits were between the best frequencies of dead mice and living mice measured by BM vibratory magnitudes. Values are means ± SE. Numbers of mice (or nerve fibers) are labeled in the bar graph. *P < 0.05. NS, not significant. Student's t-tests were performed after a one-way ANOVA.

Fig. 7.

BM tuning curves from representative transgenic mice. Illustrations are of organ of Corti behavior (left), BM sensitivity curves from representative mice (middle), and BM phase curves after referencing to the ossicular chain (right). These studies were performed in wild-type CBA (same mouse presented in Fig. 5; A), Prestin^499/499 (B), Prestin^−/− (C), Tecta^{C1509G/C1509G} (D), and Sfswap^Tg/Tg mice (E). For the illustrations, BM motion in the vertical direction (black arrows) leads to horizontal deflection of stereocilia (red arrows), resulting in OHC force production back in the vertical direction (cyan arrows). No OHC force production occurs in Prestin^499/499, Prestin^−/−, or Tecta^{C1509G/C1509G} mice (cyan lines without arrows). Reduced OHC length and stiffness are found in Prestin^−/− mice (dashed cyan lines).

Fig. 8.

Average BM vibratory gain, tuning curve sharpness (Q_10dB), and best frequency from transgenic mice. These studies were performed in wild-type CBA (A), Prestin^499/499 (B), Prestin^−/− (C), Tecta^{C1509G/C1509G} (D), and Sfswap^Tg/Tg mice (E). Values are means ± SE. Number of mice is indicated in the bar graphs. *P < 0.05; **P < 0.01; ***P < 0.001; Student's t-test after one-way ANOVA. ¥P < 0.05; nonpaired Student's t-test between CBA and Sfswap^Tg/Tg cohorts.

Fig. 9.

Organ of Corti measurements from representative transgenic mice. These studies were performed in wild-type CBA (A), Prestin^499/499 (B), Prestin^−/− (C), Tecta^{C1509G/C1509G} (D), and Sfswap^Tg/Tg mice (E). Scale bar, 50 μm. Pseudo-colored organ of Corti vibratory phases (first column) were collected near the best frequency of that cochlear location using an 80-dB stimulus. The displacement magnitudes of the Lat region to different stimulus intensities are provided (second column). The raw phase data from the Lat and the BM are also shown (third column). The phase differences between the Lat and the BM (circles, fourth column) and phase fits to the data (lines, fourth column) are given. All phase data are from live mice with 80-dB SPL stimuli.

Fig. 10.

Comparison of fitting parameters of the frequency-dependent phase difference between the Lat and the BM in transgenic mice. The average sharpness (Q_10dB; A) and F_c (B) from the fits are shown. Values are means ± SE. Numbers of mice are labeled in the F_c graph. *P < 0.05; **P < 0.01; Student's t-tests were performed after a one-way ANOVA.

Fig. 11.

The organ of Corti in the apical turn of CBA and Sfswap^Tg/Tg mice. A and B: top-down three-dimensional reconstruction of immunofluorescence images of the cochlear epithelium labeled with actin (green) and prestin (blue). C and D: cross-sectional images at the level of OHCs. Prestin is only in the cell membrane of OHCs. The IHCs and OHCs are labeled. The rows of OHCs are indicated by numbers. The arrows point to actin within the phalangeal process of Deiters' cells. Scale bars, 10 μm.