title: 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.
authors.affiliation: Bobby R Alford Department of Otolaryngology-Head and Neck Surgery, Baylor College of Medicine, Houston, TX 77030, USA.
authors.forename: Takashi
authors.initials: T
authors.lastname: Nakagawa
authors.forename: John S
authors.initials: JS
authors.lastname: Oghalai
authors.forename: Peter
authors.initials: P
authors.lastname: Saggau
authors.forename: Richard D
authors.initials: RD
authors.lastname: Rabbitt
authors.forename: William E
authors.initials: WE
authors.lastname: Brownell
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D004867:Equipment Design; D019544:Equipment Failure Analysis; D005260:Female; D006168:Guinea Pigs; D018072:Hair Cells, Auditory, Outer / Q000502:physiology*; D008163:Luminescent Measurements / Q000295:instrumentation* / Q000379:methods; D008297:Male; D008564:Membrane Potentials / Q000502:physiology*; D008856:Microscopy, Fluorescence / Q000295:instrumentation* / Q000379:methods; D011726:Pyridinium Compounds; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000295:instrumentation* / Q000379:methods
chemical_list: C089669:1-(3-sulfonatopropyl)-4-(beta-(2-(di-n-octylamino)-6-naphthyl)vinyl)pyridinium betaine; D011726:Pyridinium Compounds