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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定量光吸收变化的方法，对应于细胞中诱发的膜电位变化。光学测量系统（ARGUS-50/PDA ; Hamamatsu Photonics K.K.，Hamamatsu，静冈），并用于记录膜电位.使用用于膜电位测量的吸收染料（NK 3041; Nippon Kankoh Shikiso Kenkyusyo，冈山，日本）作为电压敏感染料。为了刺激细胞进入去极化，高K^+溶液被用作灌注液。我们最初使用急性分离的豚鼠外毛细胞，但由于其在去极化时的运动性，对刺激的反应无法检测到。当测试来自新生小鼠的培养的螺旋神经节细胞（SGCs）和前庭神经节细胞（VGC）时，可检测到反应。认为培养的细胞在灌注系统中是稳定的，允许测量。最终，只有22.2%的测试细胞成功检测到光学变化。在通过替换生理标准溶液进行刺激的光学测量中出现大量噪声，以及作为信号的NK 3041的吸光度的变化小至0.3%，导致极小的S/N比，这些可能的因素似乎可以解释这种低成功率。该结果表明，在使用使用NK 3041.5的光学测量系统的测量条件下，在分离的SGCs和VGC中难以检测微小的膜电位变化。进一步的研究是必要的，以建立敏感的染料，显示更大的光学反应膜电位的变化。本实验还利用光学测量技术，对电刺激听神经引起的脑干脑片电压变化的空间模式进行了研究。