Exposure to MβCD enhances the electrical–mechanical response
Glass electrodes positioned in scala media were used to inject electrical currents (Fig. ). In response to positive current, which depolarizes outer hair cells, Hensen’s cells moved toward scala vestibuli, the +10 μA current step in Fig. evoking a 30-nm position shift. This position shift closely followed the input waveform, although the onset and offset of the mechanical responses were slower than the command voltage applied to the constant current stimulator. Having reached the plateau, the amplitude remained stable for the duration of the current step. After applying 1 mM of MβCD, which depletes membrane cholesterol, the magnitude of the position shift increased to 125 nm (Fig. ). In addition, a slow displacement increase was observed with the Hensen’s cells continuing to move toward the scala vestibuli until the end of the current step. On average, the magnitude of the position shift increased from 56
9 nm to 163
23 nm during MβCD perfusion (mean ± S.E.M., n
0.0003 by the Wilcoxon rank sum test).
Fig. 2 Electromechanical response of the apical cochlear partition. a–c Response to positive and d–f negative 10-μA current injections. Control responses (a, d) and the response from the same preparation after perfusion with 1 mM (more ...)
Figure shows that the step amplitude was stable before MβCD application. The increase of the response was evident minutes after its application, and the maximal increase generally occurred after 20–30 min. In several preparations, the electrically evoked responses started declining thereafter, a change reflected as an increased standard error for the late time points in Fig. . Control amplitudes in the absence of MβCD were stable, although a tendency to a decrease toward the end of the recording period was evident (Fig. , lower thick line). The stability of current-evoked responses in the absence of MβCD is evident from the small standard errors of the normalized control amplitudes throughout the experiment.
Responses to negative currents were also altered by MβCD perfusion. In the absence of the drug, a 10-μA current produced an ~20-nm position shift directed at scala tympani (Fig. ), which increased to 60 nm after 20 min of MβCD perfusion (Fig. ). A slow drift in position was evident also in this recording, but this effect persisted beyond the duration of the current step. Note that Hensen’s cells did not return to their baseline position at the end of the current step but rather overshot it by some 10 nm, a pattern seen in several preparations. The time course of changes in response magnitude was similar for positive and negative currents (compare Fig. ). On average, negative current responses increased from 39
9 to 89
10 nm due to removal of membrane cholesterol (p
To ascertain whether the kinetics of electrically evoked mechanical responses was affected by cholesterol depletion, the data were fit with a low-pass filtered version of the command voltage driving the constant current generator. In the case shown in Fig. , the time constant was 5.4 ms with an amplitude of 33 nm. The time constant became slightly slower (7.3 ms, Fig. ) during MβCD perfusion and as noted above, the amplitude increased by a factor close to 4. Due to the slowly changing position during the plateau, we could not fit this part of the response using this simple function alone. Overall, the response kinetics showed no significant change due to MβCD perfusion (before MβCD, 11.5
1.6 ms, mean ± S.E.M.; 9.1
1 ms during MβCD; p
0.26 by the Wilcoxon rank sum test, n
As evident from the data presented above, positive currents resulted in larger responses than negative ones. The ratio was about 1.5, increasing to ~1.8 during MβCD perfusion (Fig. ). The time course of this change was similar to that seen in Fig. . MβCD not only changed the magnitude of the electrically evoked response, but also the relation between applied current and the step size. In the absence of the drug, the current step amplitude relation was nearly linear, with a slight tendency to a shallower slope in the negative current region. This tendency to response saturation was more pronounced after MβCD, but positive currents resulted in responses that grew linearly with a larger slope than the controls (Fig. ).
Fig. 3 The magnitude of the response to positive current is greater than the response to negative current and salicylate blocks the electromechanical response. a Summary plot of the ratio of the magnitude of the response to positive current to the magnitude (more ...)
Figure shows the response to a +10-μA current injection before and after exposure to MβCD and NaSal. At 25 min after initiating perfusion with MβCD, the perfusion reservoir was rinsed three times and perfusion with a 10-mM NaSal solution was begun. The electromechanical response was rapidly reduced to less than the pre-MβCD value consistent with the effect of sodium salicylate on the cochlear amplifier [15
]. Salicylate did not completely block the step response as a small, ~10 nm, displacement remained. Rapidly rinsing and replacing the sodium salicylate solution in the perfusion reservoir with normal MEM solution when the response block was first observed resulted in the electromechanical response returning to near the peak values of the post-MβCD response (data not shown).
Mechanical and electrical response is similar before and after MβCD exposure
A possible explanation for the increased electrically evoked responses shown in Figs. and is a reduction in the stiffness of the organ of Corti. Because the force-generating outer hair cells are embedded in a matrix of supporting cells, a change in the stiffness of those surrounding structures will result in an altered electromechanical response. In the absence of an endocochlear potential, sound-evoked mechanical responses are largely determined by the stiffness, mass, and friction of the cochlear structures [32
]. An indirect but useful measure of these parameters can be obtained by measuring sound-evoked mechanical responses in the absence of current. Figure shows the mechanical response to a sound stimulus containing five frequencies centered on the best frequency of the recording location. The waveform acquired during MβCD perfusion is similar to the control, except for a minor increase in the noise level. Spectral analysis (Fig. ) using the Fourier transform corroborates this impression: Although small changes occur at some frequencies, these are within the noise floor. Aside from a slight shift towards higher frequencies, the cochlear microphonic potential also shows little change (Fig. ). Minor increases in cochlear microphonic potentials were seen in some preparations during MβCD perfusion, and small decreases were noted in others. Overall, MβCD did not affect the amplitude or tuning of these potentials. The data shown in Fig. indicate that the passive mechanics of the organ of Corti are unaffected by MβCD, and the lack of change in cochlear microphonics is evidence that forward transduction is impervious to the reduction in cholesterol in the cell bodies of outer hair cells.
Fig. 4 Cochlear partition tuning and cochlear microphonics are similar before and after exposure to MβCD. a Time domain and b spectral analysis of sound-evoked mechanical response. c Cochlear microphonics from the same preparation as a and b are similar (more ...)
Figure demonstrates how exposure to MβCD can result in an increase of the acoustically evoked response in the presence of a positive current. Positive current, which restores the endocochlear potential in the excised preparation, often results in larger acoustically evoked responses than either no current or negative current and MβCD could greatly enhance the increase. There was considerable variation in the increase observed following MβCD. In some cases, there would be no increase even with a major enhancement of the electromotile response. In other preparations, there was a modest increase when MβCD was present (Fig. ). Figure show a preparation in which MβCD resulted in a 6-dB boost at best frequency. The increase of sound-evoked motion amplitudes during MβCD may be seen in Fig. and is analyzed in more detail in Fig. . Responses near the best frequency (200 Hz) are more than doubled, but smaller changes are seen at 300 Hz, and no change at all at 600 Hz. Thus, although positive current increases sound-evoked responses and sharpness of tuning during MβCD perfusion, there is no shift in the best frequency. In summary, while electromotility is clearly an important component of the cochlear amplifier [14
], an increase in electromotility does not automatically produce increased amplification of sound-evoked motion.
Figure shows another example of increased cochlear amplification following MβCD perfusion. Time-resolved confocal imaging and optical flow computation [22
] was used to measure sound-evoked responses. Acoustically evoked motions were nearly perpendicular to the reticular lamina during negative current injections, with a peak amplitude of approximately 300 nm (black trajectory in Fig. ). Positive current increased perpendicular vibrations to ~360 nm (gray trajectory in Fig. ). The parallel motion component, directed along the horizontal axis of the image, appears more responsive to positive current and consequently shows a larger increase than the vertical component.
Fig. 5 Confocal imaging of sound-evoked vibrations confirms a larger effect of positive current injection following MβCD. Confocal image on the left of outer hair cell recorded in situ during simultaneous sound and electrical stimulation. Vibrations (more ...)
After 10 min of MβCD perfusion at 1 mM, negative current vibrations were unchanged, as seen by comparing the black trajectories in Fig. . The peak amplitude remained close to 300 nm, and the major axis of the trajectory remains in the same orientation. Thus, during negative currents, sound-evoked vibrations are quite stable and not significantly affected by MβCD. However, sound-evoked responses during positive current were increased. Note that the amplitude increases more for movements parallel to the reticular lamina giving the trajectory a more elliptic shape (gray trajectory in Fig. ). This is an important change, as it would be expected to be more effective in deflecting hair cell stereocilia.
The cellular components of the cochlear partition remain intact following MβCD exposure
Short-term perfusions with MβCD (<1 h) did not produce obvious morphological changes in the organ of Corti, as evidenced by confocal imaging of the measurement site after loading cells with the fluorescent membrane dye RH795. In Fig. , note that Reissner’s membrane retains its normal honeycomb configuration and that supporting cells near the measurement site all appear intact. The cell membranes of supporting cells were clearly labeled, and the lipid droplets inside Hensen’s cells are also visible. Fragmentation of lipid droplets, a common sign of acute cellular stress in the cochlear apex, was not observed. On focusing deeper into the organ of Corti (Fig. ), it is seen that outer and IHC appear normal with clearly delineated cell membranes showing no signs of swelling or other structural abnormalities. The section through the organ of Corti is oblique and the full length of the OHC bodies cannot be inspected in this image. However, preparations with long-term exposure (>1 h) to MβCD frequently, but not always, showed pathology, most commonly OHC shrinkage.
Fig. 6 Cochlear histology following MβCD exposure. a, b Confocal images of the cochlea after 10 min of perfusion with RH795. a Optical section through Reissner’s membrane (RM) and Hensen’s cells (HeC) near the site of vibration (more ...)
The antifungal macrolide filipin is highly fluorescent and binds specifically to membranes containing cholesterol and can therefore be used for visualizing such membranes. It was used to image the organ of Corti in control samples (Fig. ) as well as samples treated with MβCD (Fig. ). While there was a decrease in staining intensity in MβCD-treated samples, labeling was still observed in all cell types in the organ of Corti, confirming that removal of cholesterol with MβCD is incomplete. Preparations that were not exposed to filipin had autofluorescence that was at least one order of magnitude smaller than the fluorescence intensity observed in filipin treated samples (Fig. ). The MβCD-induced decrease in intensity should be interpreted with caution given the difficulty in quantifying fluorescent labels such as filipin.