Electrophysiological Recording of Voltage Responses of Drosophila Retinal Photoreceptors to Light Stimuli
Transcrição
When operating the microelectrode amplifier, always be grounded by touching the metal surface of the Faraday cage or anti-vibration table to prevent static charges. Secure the mounted fly to the fly preparation platform pole.
Rotate the fly holder so that the fly's left eye is directly facing the investigator. Next, use a small, coarse micromanipulator to insert the blunt reference electrode gently through the fly's ocelli into the head capsule. Make certain the fly still appears healthy and moves its antennae. The preparation must be superb to merit recording from.
Now, drive the sharp recording microelectrode into the left eye through the jelly-coated opening. As it enters the tissue, the electrode tip location should be apparent by its reflectance pattern. The fragile electrode tip must be guided perfectly or it will break, which is very challenging.
Depending on the cell type being recorded from, the fly's head should be oriented slightly differently. With the electrodes in place, turn on the microelectrode amplifier. Then turn off the cold light source, unplug it from the mains, and connect it to the central ground to minimize ground-loop-induced electrical noise.
Next, adjust the gooseneck light guides so that the cardan arm system can be freely moved around the fly. Then, switch off the room lights to put the fly preparation in relative darkness. Now, measure the resistance of the recording electrode in the eye. It must be between 100 and 250 megaohms.
Next, set the amplifier to current clamp or bridge, and zero the voltage between the electrodes. After the fly has been dark-adapted for a few minutes, drive the recording electrode tip into the eye in 0.1 to 1-micron steps using a piezo stepper or using gentle rotation of the fine-resolution knob.
At each step, stimulate the eye with a 1 to 10-millisecond light flash. Each light flash will cause a brief dip in the voltage or the ERG.
