Functional study of the olfactory sensory signal transduction mechanism using the optic fiber system for optically monitoring membrane potential.

使用光纤系统光学监测膜电位的嗅觉感觉信号转导机制的功能研究。

• 批准号: 08458269
• 负责人: HIROTA Akihiko
• 金额: $ 4.93万
• 依托单位: Shimane 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: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08458269/
• 关键词: OPTICAL METHOD; MEMBRANE POTENTIAL; OPTIC FIBER; VOLTAGE-SENSITIVE DYE; OLFACTORY BULB; RAT; RH-414; RH-704; RH795; 実体顕微鏡

We have developed optical method for monitoring the membrane potential using optic fibers and measured the electrical activity on the rat olfactory bulb following the antidromic electric stimulation of the medial olfactory tract. The olfactory bulb was stained by the voltage-sensitive dye. The Y-shaped optic fibers were made at our special request One edge designed for putting on the preparation received two kinds of optic fibers : one was connected to light housing and another to photodetector. Illumination was provided by a 100 W tungsten-halogen lamp driven by a stable DC power supply at steady current mode. Incident light was passed through a 535 * 40 nm interference filter so as to fit the peak excitation wavelength for the voltage-sensitive dye. Between the optic fiber and photodiode in the photodetector, we put a colored glass filter. This filter pass well the fluorescent light of the dye with longer wavelength but it prevent the excitation light with shorter wavelength.We have tested the four voltage-sensitive dyes, RH-414, RH-421, RH-704 and RH-795. The optical signal size of RH-414 was very large but, since this dye has somewhat phototoxic effect and bleached rapidly, it was not fit for our relatively long lasting experiment. We did not find any toxic effect from the preparation stained with other three dyes, but the signal size was small and signal averaging was needed for analyzing the wave shape. We assumed the optical signals also detect the some changes (perhaps due to the volume changes of the preparation) other than the direct membrane potential changes but synchronized to the electrical stimulation.

我们开发了一种利用光纤监测膜电位的光学方法，并测量了大鼠嗅球在对内侧嗅道进行反向电刺激后的电活动。嗅球被电压敏感染料染色。y型光纤是根据我们的特殊要求制作的，用于放置制备的一个边缘接收两种光纤：一种连接到灯罩，另一种连接到光电探测器。照明由100 W的钨卤灯提供，由稳定的直流电源在稳定电流模式下驱动。入射光通过535 * 40nm干涉滤光片，以拟合电压敏感染料的峰值激发波长。在光电探测器的光纤和光电二极管之间，我们放了一个有色玻璃滤光片。该滤光片能很好地通过波长较长的染料的荧光，但不能通过波长较短的激发光。我们测试了四种电压敏感染料，RH-414, RH-421， RH-704和RH-795。RH-414的光信号大小非常大，但由于该染料具有一定的光毒性，并且漂白速度快，因此不适合我们进行较长时间的实验。其他三种染料染色后未发现毒性作用，但信号大小较小，分析波形需要信号平均。我们假设光信号也检测到一些变化（可能是由于制备的体积变化），而不是直接的膜电位变化，但与电刺激同步。