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

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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.