Photometric recording of transmembrane potential in outer hair cells

Abstract

Cochlear outer hair cells (OHCs) are polarized epithelial cells that have mechanoelectrical transduction channels within their apical stereocilia and produce electromotile force along their lateral wall. Phase shifts, or time delays, in the transmembrane voltage occurring at different axial locations along the cell may contribute to our understanding of how these cells operate at auditory frequencies. We developed a method to optically measure the phase of the OHC transmembrane potential using the voltage-sensitive dye (VSD) di-8-ANEPPS. The exit aperture of a fibre-optic light source was driven in two dimensions so that a 24 microm spot of excitation light could be positioned along the length of the OHC. We used the whole-cell patch-clamp technique in the current-clamp mode to stimulate the OHC at the base. The photometric response and the voltage response were monitored with a photodetector and patch-clamp amplifier, respectively. The photometric response was used to measure the regional changes in the membrane potential in response to maintained (dc) and sinusoidal (ac) current stimuli applied at the base of the cell. We used a neutral density filter to lower the excitation light intensity and reduce phototoxicity. A sensitive detector and lock-in amplifier were used to measure the small ac VSD signal. This permitted measurements of the ac photometric response below the noise floor of the static fluorescence. The amplitude and phase components of the photometric response were recorded for stimuli up to 800 Hz. VSD data at 400-800 Hz show the presence of a small phase delay between the stimulus voltage at the base of the cell and the local membrane potential measured along the lateral wall. Results are consistent with the hypothesis that OHCs exhibit inhomogeneous membrane potentials that vary with position in analogy with the voltage in nerve axons.

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 plane perpendicular to the optical axis controls the location of the spot. The resistor across the operational amplifier in the I–V converter sets the feedback resistance, either 1 or 3 GΩ.

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 each location was separated by a distance of 16.5 μm. Horizontal bar indicated 10 μm.

Figure 3

The membrane currents of an OHC before and after di-8-ANEPPS staining show no effect from the VSD. This data were recorded in the voltage-clamp mode with a holding potential of −70 mV.

Figure 4

DC response to step current injection. Both the membrane potential (as seen by the patch-clamp amplifier) and the photometric response (ΔF/F) were measured from the base of the OHC. The upper trace shows the stimulation protocol. Each trace is the average of four measurements. The data from this cell are fitted by ΔF/F;= 2.37 + 0.032 × MP (membrane potential) with an r value of 0.993 (p < 0.001).

Figure 5

AC response to sinusoidal current injection. Again, both the membrane potential and the photometric responses were measured from the base of the OHC. Each trace is the average of four measurements.

Figure 6

Representative frequency responses of the membrane potential, the noise floor of the static fluorescence (F), and the photometric response at the base of the cell (ΔF). In this case, ΔF was plotted as a voltage (instead of a ratio) in order to compare it to the noise floor voltage. Note that ΔF could be recorded below the noise floor because the lock-in amplifier was used. All measurements are peak-to-peak values. Arrows show corner frequencies for the cell membrane potential (F_c cell) and the I–V converter of photodetector (f_c PD). Sine wave current injection (3 nA peak-to-peak) was used for stimulation.