Nakagawa Takashi, Oghalai John S, Saggau Peter, Rabbitt Richard D, Brownell William E
Bobby R Alford Department of Otolaryngology-Head and Neck Surgery, Baylor College of Medicine, Houston, TX 77030, USA.
J Neural Eng. 2006 Jun;3(2):79-86. doi: 10.1088/1741-2560/3/2/001. Epub 2006 Apr 11.
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.
耳蜗外毛细胞（OHCs）是极化上皮细胞，其顶端静纤毛内有机械电转导通道，并沿其侧壁产生电动力。沿着细胞不同轴向位置发生的跨膜电压的相移或时间延迟，可能有助于我们理解这些细胞在听觉频率下是如何运作的。我们开发了一种使用电压敏感染料（VSD）di-8-ANEPPS光学测量OHC跨膜电位相位的方法。光纤光源的出射孔径在二维方向上驱动，以便激发光的24微米光斑可以沿着OHC的长度定位。我们在电流钳模式下使用全细胞膜片钳技术刺激OHC的基部。分别用光电探测器和膜片钳放大器监测光度响应和电压响应。光度响应用于测量膜电位的区域变化，以响应施加在细胞基部的持续（直流）和正弦（交流）电流刺激。我们使用中性密度滤光片来降低激发光强度并减少光毒性。使用灵敏的探测器和锁相放大器来测量小的交流VSD信号。这允许在静态荧光的本底噪声以下测量交流光度响应。记录了高达800 Hz刺激下光度响应的幅度和相位分量。400 - 800 Hz的VSD数据显示，在细胞基部的刺激电压与沿侧壁测量的局部膜电位之间存在小的相位延迟。结果与以下假设一致：OHCs表现出不均匀的膜电位，其随位置变化，类似于神经轴突中的电压。
