The external solution contained 150 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES and 10 mM glucose. The pH was adjusted to 7.4 with NaOH. The internal solution contained 150 mM KCl, 0.5 mM EGTA, 10 mM HEPES, 5 mM ATP-Na2, 0.1 mM cAMP and 0.1 mM GTP-Na2. The pH was adjusted to 7.3 with KOH.
2.2. Isolation of OHCs
The dissection procedure of the guinea pig OHCs was similar to that previously reported (Nakagawa et al 1990
). Albino guinea pigs (weight 150–300 g) of either sex with good Preyer’s reflexes were decapitated, and the temporal bones were removed. All procedures were performed in accordance with the Baylor College of Medicine institutional guidelines. The bulla and bony shell of the cochlea were opened, and the organ of Corti with the attached modiolus kept in the external solution saturated with 100% O2
. The preparation was treated enzymatically with 0.5 mg ml−1
trypsin (Sigma, St. Louis, MO) and 0.5 mg ml−1
collagenase (Sigma, St. Louis, MO) at 31 °C for 3–5 min. The enzyme action was halted by washing the preparation with 0.1% bovine serum albumin (Sigma, St. Louis, MO). The organ of Corti was aspirated into a Pasteur pipette of 100–150 μ
m tip diameter, and the OHCs were isolated mechanically by gentle pipetting. Isolated OHCs were dispersed onto the 35 mm glass bottom poly-D-lysine coated Microwell dishes (MatTek Co, Ashland, MA).
2.3. Electrical recordings
Recording was performed in whole-cell configuration under either voltage clamp or current clamp. Glass pipettes were fabricated from borosilicate glass (G-1.5, Narishige, Tokyo, Japan) using a two-stage vertical puller (PP-83, Narishige, Tokyo, Japan). The resistance between the recording electrode filled with internal solution and the reference electrode was 3–6 MΩ. Data were recorded with an Axopatch 200B patch-clamp amplifier and a 12 bit acquisition board (Digidata 1200) controlled by pClamp 6.1 (Axon Instruments, Foster City, CA). The pipette seal (>1 GΩ) was always made below the nucleus. A whole-cell recording was established first in the voltage-clamp mode. Pipette capacitance, series resistance and cell capacitance were measured and compensated using the internal circuit of the amplifier. The series resistance was compensated by 70–90% during the voltage-clamp condition. For current-clamp experiments, the series resistance values obtained in voltage clamp were used for bridge balance compensation at a compensation ratio of 100%. Either current or voltage data were low-pass filtered at 10 kHz and sampled at 33 kHz. For FFT analysis, a spectrum analyser (Hewlett Packard 3567A) was used for on-line analysis. In this case, data were sampled at 6.4 kHz.
2.4. Voltage-sensitive dyes
A number of fast stryl-type VSDs were used, i.e., 3-(4-(2-(6-(dibutylamino)-2-naphthyl)-trans-ethenyl)pyridinium) propanesulfonate (di-4-ANEPPS), 1-(3-sulfonatopropyl)-4-[β
[2-(di-n-octylamino)-6-naphthyl]vinyl]pyridinium betaine (di-8-ANEPPS), N-(4-sulfobutyl)-4-(4-(4-(dibutylamino) phenyl)hexatrienyl)pyridinium (RH-160), N-(4-sulfobutyl)-4-(6-(4-(dibutylamino)phenyl)butadienyl)pyridinium (RH-237), N-(3-triethylammoniumpropyl)-4-(4-(4-(diethylamino)phenyl) butadienyl)pyridinium (RH-414) and N-(4-sulfobutyl)-4-(4-(4-(dipentylamino)phenyl)butadienyl)pyridinium (RH-421), (Molecular Probes, Eugene, OR). Stock solutions (10 mM) were prepared by dissolving the dyes in DMSO, and kept in the dark at 4 °C. Fresh staining solutions were prepared immediately before the experiments by adding appropriate amounts of the gently warmed stock solution to the standard solutions (Bullen et al 1997
). The dye concentration used for staining was between 30–100 μ
M, usually 50 μ
M. The OHCs were incubated with detergent-free staining solution for 10–20 min. After staining, OHCs were rinsed well with external solution. During the experiments, the chamber was constantly perfused with fresh external solution.
2.5. Optical recordings
is a schematic illustration of the experimental setup. The apparatus was built around an inverted microscope (Axiovert 135TV, Zeiss Inc., Thornwood, NY) mounted on a vibration isolation table (Micro-g, TMC, Peabody, MA). The objective lens used was a 63× oil immersion of N.A. 1.4 (Zeiss). The light from a xenon short arc lamp (75W, Osram, Munich, Germany; operated with a Model 1600 Power Supply, Opti Quip, Highland Mills, NY) was controlled by an electromechanical shutter (Uniblitz, Vincent Assoc. Rochester, NY) and was transmitted through fibre-optic cable (FSLE 34, Technical Video). The intensity of excitation light could be changed by a filter wheel equipped with absorptive neutral density filters (77384, Oriel Instruments, Stratford, CT). The exit aperture of a fibre-optic light source was positioned at the field stop of the microscope such that the objective lens produced a 24 μm spot of excitation light on the cell (). The aperture of fibre optic was mounted on a three-dimensional hydraulic micromanipulator (MHW30, Narishige, Tokyo, Japan) so that the spot could be positioned to excite the VSD at various locations along the cell, without having to move the patch-clamped cell. The excitation filter was a 450–490 nm band-pass interference filter. The emission light was long-pass filtered above 520 nm. The fluorescence of the preparation collected by the objective lens was focused on a 1 mm × 1 mm single silicon photodiode (1336 K2G, Hamamatsu, Japan) located below the microscope. The emission light path could be manually switched between the eye pieces and the photodiode by removing 100% reflector in the microscope. During experiments, optical signals were recorded from three specific locations along the lateral wall of the OHC by moving the centre of the excitation light spot in steps of approximately 16.5 μm (see also ). The photocurrent was passed to a custom-made current-to-voltage (I–V) converter with an initial feedback resistance of 3 GΩ. It contained three 1 GΩ resistors in series to reduce the parasitic capacity. With this layout, the converter acted as a low-pass filter with a corner frequency of 120 Hz. In later experiments, the feedback resistance was reduced down to 1 GΩ and the circuit layout was improved, resulting in a corner frequency of 1.2 kHz. The voltage output was sampled either at 3 kHz using a pClamp system (Axon Instrument, Foster City, CA), at 6.4 kHz with a spectrum analyser (Hewlett Packard 3567A), or by a digital signal processing lock-in amplifier SR830DSP, Stanford Research Systems, Stanford, CT; 1 s integration time, 24 dB/octave filter. The lock-in amplifier provides the amplitude and phase voltage signal. The phase of the photometric response at the base of the cell was set to zero and the phase at the other measurement locations was referenced to the base.
Figure 1 Schematic diagram of the experimental set-up. The solid line represents the light path during VSD recordings. The position of the fibre-optic cable along the optical axis determines the spot size of the illumination spot on the OHC; its position in the (more ...)
Figure 2 OHCs stained with di-8-ANEPPS and images of an optically partitioned cell. From left to right, the excitation light was positioned in four distinct locations: base, intermediate 1, intermediate 2 and apex. Spot diameter was 24 μm. The centre of (more ...)
Care was taken to avoid phototoxicity associated with the formation of free radicals and membrane damage and cell depolarization (Grinvald et al 1988
). We therefore continuously monitored the resting potential and rejected all data where the dc zero-current potential drifted more than 10 mV from the nominal −60 mV level at the beginning of each experiment. We also discarded all cells where the lock-in phase was not stable after 20 s (10 s lock-in integration and 10 s monitoring). Phototoxicity and rapid cell depolarization was problematic when using full strength illumination, and therefore a 10% neutral density filter was used in the excitation pathway.
Paired Student t-tests were used to compare means; differences are reported as statistically significant if p < 0.05. Linear regression was performed using least-squares methods. All statistics were performed using Origin (Microcal Software, Northampton, MA).
Experimental results were compared to the passive OHC model of Halter et al (1997)
and to the piezoelectric model of Clifford et al (2006)
. Both of these models treat the OHC as a distributed voltage system, in analogy with the classical cable equation for axons. Regional differences in the membrane potential magnitude and phase arise in these models due to the presence of a relatively high axial resistance. The axial resistance is associated with restricting the axial current to flow in the model along the extracisternal space between the lateral membrane and the SSC. At low frequencies, the high axial resistance causes the magnitude of the voltage to decay with distance according to the space constant and introduces a frequency-dependent phase shift along the length. The phase
(rad) of the plasma membrane potential relative to the voltage at the base of the cell can be interpreted as a time delay δ
between the base and the measurement site (using δ
, where ω
is frequency in rad s−1
). Further, for cable equations, the time delay measured between two points separated by distance λ
can be used to estimate a phase velocity υ
of a dispersive travelling wave using υ
. It is this phase shift and associated phase velocity that is relevant to interpretation of the present data.