DEVELOPMENT OF A METHOD FOR LONG TERM OPTICAL RECORDING OF ELECTRICAL ACTIVITY FROM INSECT NEURONS WITH VOLTAGE SENSITIVE DYES

神经活动的光学记录对于同时分析一组神经元的活动是有用的。在这个项目中，我们开发了一种方法，可以借助昆虫中枢神经系统中的电压敏感染料来长期记录神经活动。建立了一种制备该染料染色昆虫组织的有效方法。我们将RH414，一种荧光电压敏感染料直接注射到神经组织中。这种注射产生了一致的结果。荧光由高强度卤素光源诱导，由光电二极管阵列相机与奥林巴斯BX30MF荧光显微镜相结合进行检测。为了自动长期记录，我们开发了一个计算机程序，通过GPIB来调节相机-光电探测器系统的光学测量，并将图像数据存储到大容量硬盘上。执行这项工作大约需要8分钟。相机和放大器的增益、测量的时间分辨率由光电探测器系统预先设置。来自电子刺激器的重复触发脉冲重复地驱动这一系列操作。在硬盘容量范围内继续记录是可能的。我们开发了两种程序来分析海量数据。一种是计算所有记录帧中每个像素的平均亮度，另一种是统计所有记录帧中每个像素超过阈值亮度的帧数。计算的数据可由计算机以彩色图像的形式可视化。通过这些分析方法，有可能在昆虫神经系统相当广泛的区域内检测到电活动的时间变化。

Optical recordings of neural activity are useful for simultaneous analysis of activity of a population of neurons. In this project, we have developed a method which enables us a long term recording of the neural activity with the aid of voltage sensitive dye in an insect central nervous system. We established an effective method for preparation of the insect tissue stained with the dye. We injected RH414, a fluorescence voltage sensitive dye, directly into the nervous tissue. This injection yielded consistent results. The fluorescence was induced through a high intensity halogen light source and detected by a photodiode array camera combined with the Olympus BX30MF fluorescent microscope. The signals were detected and processed by the camera was fed into a photodetector (HR Deltaron, Fuji Film Co.).For automatic long term recording, we developed a computer program which regulates the optical measurements by the camera-photodetector system through GPIB and stores the image data onto a hard disk with a large capacity. Performing this job requires about 8 min. Gains of camera and amplifers, temporal resolution of measurements are preset by the photodetector system. Repetitive trigger pulses from an electronic stimulator drive the series of operation repetitively. It is possible to continue the recording within the capacity of the hard disk.We developed two kind of programs to analyze the huge body of data. One is to calculate the average brightness of each pixel through all the recorded frames, and the other is to count the number of frames which over the threshold brightness of each pixel through all the recorded frames. The calculated data can be visualized by computer as colored images. By these analying method, it is possible to detect temporal changes in electrical activity within rather a wide region of the insect nervous system.