Optical Recording of Membrane Potential on Inner Ear Cells Using a Voltage-Sensitive Dye.

使用电压敏感染料光学记录内耳细胞膜电位。

基本信息

批准号：
07457407
负责人：
YAMASHITA Toshio
金额：
$ 3.9万
依托单位：
Kansai Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

项目摘要

1. Optical measurement of membrane potential is a method to quantify changes in the absorption of light by voltage-sensitive dyes or immunofluorescence corresponding to membrane potential changes evoked in cells. An optical measurement system (ARGUS-50/PDA ; Hamamatsu Photonics K.K., Hamamatsu, Shizuoka) was set up and used for recording a membrane potential.2. An absorptional dye for membrane potential measurement (NK3041 ; Nippon Kankoh Shikiso Kenkyusyo, Okayama, Japan) was used as the voltage-sensitive dye. To stimulate cells into depolarization, a high K^+ solution was used as the perfusion.3. We used acutely dissociated outer hair cells from guinea pigs initially, but responses to the stimuli could not detected because of its motility while depolarizing. When cultured spiral ganglion cells (SGCs) and vestibular ganglion cells (VGCs) from newborn mice were tested, responses were detectable. Cultured cells were considered to be stable in the perfusion system, allowing measurement.4. Ultimately, it was in only 22.2% of the cells tested that we were successful in detecting optical changes. The occurrence of substantial noises in the optical measurement with stimulation by replacement of the physiological standard solution and that the change in absorbance of NK3041 as the signal is as little as 0.3%, leading to an extremely small S/N ratio, are possible factors that would seem to account for this low success rate. This results indicated that it would be difficult to detect minute membrane potential changes under the conditions of measurement in isolated SGCs and VGCs with the optical measurement system using NK3041.5. Further study is necessary in order to establish sensitive dyes that show greater optical reactions to membrane potential changes. We had also fried to investigate to detect the spacial patterning of the voltage change producted by electrical stimulation of the auditory nerve in brain stem slices using optical measurement.

1。膜电位的光学测量是一种通过电压敏感染料或免疫荧光来量化光吸收变化的方法，与细胞中引起的膜电位变化相对应。建立了光学测量系统（Argus-50/PDA； Hamamatsu Photonics K.K.，Hamamatsu，Shizuoka）并用于记录膜电位。2。用于膜电位测量的吸收染料（NK3041； Nippon Kankoh ShikiSo Kenkyusyo，Japan，Japan）用作电压敏感的染料。为了刺激细胞进入去极化，将高的K^+溶液用作灌注3。我们最初使用急性分离的外毛细胞与豚鼠，但由于其去极化时的运动能力，无法检测到对刺激的反应。当测试了新生小鼠的培养的螺旋神经节细胞（SGC）和前庭神经节细胞（VGC）时，可以检测到反应。培养的细胞在灌注系统中被认为是稳定的，允许测量4。最终，只有22.2％的细胞在测试的细胞中成功地检测光学变化。通过替换生理标准溶液刺激的光学测量中发生大量噪声，并且NK3041的吸光度变化是信号的少于0.3％，导致S/N比极小，是可能考虑到这一较低成功率的可能因素。该结果表明，使用NK3041.5使用光学测量系统，在分离的SGC和VGC中测量条件下，很难在测量条件下检测微小的膜电位变化。为了建立对膜电位变化的光学反应更大的敏感染料，需要进一步研究。我们还油炸以检测通过使用光学测量的听觉神经对脑干切片中听觉神经的电刺激产生的电压变化的空间模式。