Werner Hemmert

Abolition of the receptor potential response of isolated mammalian outer hair cells by hair-bundle treatment with elastase : a test of the tip-link hypothesis

To test the hypothesis that the tip-links of hair-cell stereocilia are essential for mechanoelectrical transduction, tip-links of isolated outer hair cells (OHCs) of the guinea-pig cochlea were eliminated with a proteolytic enzyme, elastase, and the influence on the receptor potential measured with the whole-cell patch-clamp technique. Within 45 s of immersion of the hair bundle in 20 IU/ml elastase, the receptor potential in response to direct deflection of the hair bundle was irreversibly abolished. The electrical input impedance of the cell remained unchanged, implying that the channels of the basolateral membrane were not affected by elastase. The effect of elastase on the receptor potential was comparable to changes seen after mechanically induced hair-bundle damage. As a further control, a putative transduction-channel blocker, dihydrostreptomycin (68 microM), which does not affect tip-links, was applied to the hair bundle. Although the receptor potential was also blocked by dihydrostreptomycin, the effect was reversible. The results suggest that tip-links are required for mechanoelectrical transduction of mammalian OHCs.

Dynamics of Middle Ear Prostheses – Simulations and Measurements

The efficient and systematic development of a middle ear prosthesis necessitates the use of computer models for the prosthesis itself and the reconstructed middle ear. The structure and parameters of the computer model have to be verified by specific measurements of the implant and the reconstructed ear. To obtain a realistic model of a reconstructed ear, three steps of modeling and measurements have been carried out. To get a first approach of the coupling elements a mechanical test rig representing a simplified reconstructed middle ear was built. The velocity of the stapedial footplate was measured with a laser Doppler vibrometer. The corresponding computer model was formulated, and the respective parameters were determined using the measured dynamical transfer functions. In the second step, a prosthesis was implanted into a human temporal bone without inner ear. Exciting this system with noise, the velocity of the stapes footplate was measured with the laser Doppler vibrometer. Based on the multibody system approach, a mechanical computer model was generated to describe the spatial motions of the reconstructed ossicular chain. Varying some significant parameters, simulations have been carried out. To describe the dynamical behavior of the system consisting of middle and inner ear, the computer model used in the second step has been enlarged by adding a simplified structure of the inner ear. The results were compared with in situ measurements taken from living humans.

Sound-induced displacement responses in the plane of the organ of Corti in the isolated guinea-pig cochlea

Sound-induced displacement responses in the plane of the organ of Corti were studied in the apical turn in the isolated temporal-bone preparation of the guinea-pig cochlea. Swept sinusoidal sound stimuli (100-500 Hz) were delivered closed-field to the external auditory meatus. The surface of the organ of Corti was continuously monitored using a CCD video camera. Displacement responses in the plane of the organ of Corti were determined by analyzing the change of the location of the cells (pixel-by-pixel) within the visual field of the microscope. Displacement responses followed the stimulus amplitude and were observable at Hensens cells, three rows of outer hair cells and inner hair cells. The most prominent displacement responses were over the outer hair cells; the maximum amplitude was 0.6-1.7 microns at 100 dB SPL. Tuned displacement responses were found; the Q10 dB was 1.3 +/- 0.6. The best frequency was tonotopically organized, decreasing toward the apex with a space constant of 0.4-0.9 mm/oct. The motion was directed either strial-apically or strial-basally in a frequency dependent manner. With the aid of laser interferometric measurements of the transverse displacement, it was concluded that sound stimulation does not induce slow DC motion in the organ of Corti for the isolated temporal-bone preparation.

Frequency response of mature guinea-pig outer hair cells to stereociliary displacement

Outer hair cells (OHC) were isolated from the apical two turns of the guinea-pig cochlea and their hair-bundle stimulated mechanically by a glass probe. In accordance with in vivo data (Dallos, 1985), the resting membrane potential was typically -64 mV (N = 200). The maximum amplitudes of the receptor potentials were between 0.4 and 5.2 mV peak-to-peak, with mean of 1.5 mV +/- 0.9 mV (N = 81). The sensitivity was 0.015 mV/nm or 2 mV/deg. The frequency response of the receptor potential followed a first order low-pass filter characteristic with a corner frequency of about 63 Hz. For frequencies up to at least 1.6 kHz, the frequency response of mechanoelectrical transduction was dominated by the electrical input impedance of the cell. The presence of a single time constant in the voltage response to stereociliary deflection implies that the frequency response of mechanoelectrical transduction far exceeds that of the electrical input impedance of the cell; its time constant must be faster than 100 microseconds. Under in vivo conditions, OHC should be capable of providing a sufficiently large receptor potential to supply enough energy for electromechanical feedback.

Electromotility of outer hair cells from the cochlea of the echolocating bat, Carollia perspicillata

Isolated outer hair cells (OHCs) and explants ot the organ of Corti were obtained from the cochlea of the echolocating bat, Carollia perspicillata, whose hearing range extends up to about 100 kHz. The OHCs were about 10–30 μm long and produced resting potentials between-30 to -69 mV. During stimulation with a sinusoidal extracellular voltage field (voltage gradient of 2 mV/μm) cyclic length changes were observed in isolated OHCs. The displacements were most prominent at the level of the cell nucleus and the cuticular plate. In the organ of Corti explants, the extracellular electric field induced a radial movement of the cuticular plate which was observed using video subtraction and photodiode techniques. Maximum displacements of about 0.3–0.8 μm were elicited by stimulus frequencies below 100 Hz. The displacement amplitude decreased towards the noise level of about 10–30 nm for stimulus frequencies between 100–500 Hz, both in apical and basal explants. This compares well with data from the guinea pig, where OHC motility induced by extracellular electrical stimulation exhibits a low pass characteristic with a corner frequency below 1 kHz. The data indicate that fast OHC movements presumably are quite small at ultrasonic frequencies and it remains to be solved how they participate in amplifying and sharpening cochlear responses in vivo.

Laser interferometric vibration measurements of the middle ear in healthy humans

The use of spontaneous and evoked otacoustic emissions is now a standard clinical tool for diagnosis of the function of the inner ear. However, it is not possible to extract this information over the entire, functionally relevant frequency range because of imperfect coupling of: (1) stapedial to ear-drum vibrations through the ossicular chain of the middle ear and (2) ear-drum vibrations to air in the external auditory meatus. The problem could be circumvented if it were possible to measure the vibration of the stapes and ear drum. The ear drum can be visualized non-invasively, whereas the stapes is only accessible intra-operatively. Therefore, we designed a laser-interferometric system to non-invasively measure the vibration of the human ear drum. Vibrations were measured with a laser Doppler velocimeter (Polytec OFV-302) coupled into the side arm of an operating microscope (Zeiss OPMI MDM). The wavelength was 633 nm and emitted power was less than 1 mW. Direct coupling through the optics of the operating microscope, instead of through glass fibers, enabled a larger signal-to- noise ratio (20 - 30 dB) due to collection of more reflected light. This coupling scheme avoids the problems associated with having to place a reflecting material on the ear drum. The developed vibration measurement system allows non-invasive, fast and reproducible characterization of the dynamics of the human ear drum and as such can be used for clinical diagnostics.

Laser interferometric measurement of the micromechanics of the inner ear

Recent advances in optical techniques for vibration measurement have made it feasible to purchase commercially available laser Doppler velocimeters that, in combination with a light microscope, are capable of detecting velocities of poorly reflecting biological materials down to fractions of a micrometers s-1. Thus, in our particular application, the aim is to understand the micromechanics of the inner ear, particularly in the nanometer region where the ear is first able to detect sound. With currently available devices, it is now possible to make the necessary vibration measurements without need of introducing reflecting materials onto the object of interest. This not only avoids inertial loading, but also allows focusing through cellular layers onto otherwise inaccessible structures. Here we present the results of micromechanical experiments for the in vitro inner ear of the guinea pig, the most commonly used model in auditory physiology. Our experiments concentrate on the third and fourth cochlear turns because in this region the different cellular structures of the organ of Corti are optically accessible with present technology. The mechanical responses of all examined structures, including the basilar membrane, were equally frequency selective, but linear below 90 dB SPL, suggesting that active mechanical tuning is not as pronounced in the apex as in the base of the cochlea. This has important consequences for signal processing in the inner ear.