This article details an electrophysiological method to study the phototransduction process in Drosophila. The procedure involves securing the fly, implanting electrodes, and applying light pulses to measure the resulting voltage responses.
Secure a white-eyed Drosophila fly to a holder in a recording arena.
Position a light source near the fly.
Implant the ground electrode into the fly's back.
Insert the recording electrode into the outer periphery of the fly's eye, near the retinal photoreceptor cells containing a photopigment.
Dark-adapt the fly briefly. This converts metarhodopsin, the light-activated form of the photopigment, to rhodopsin, its inactive form.
Apply a high-intensity orange light pulse. This converts any remaining metarhodopsin to rhodopsin.
Next, apply blue light pulses. The blue light converts rhodopsin to metarhodopsin, initiating a signaling cascade and opening specific ion channels.
This induces a continuous positive ion influx and sustained depolarization, even after the blue light is removed — the prolonged depolarizing afterpotential, or PDA, recorded by the electrode.
Now, reapply the orange light pulses to convert the metarhodopsin to rhodopsin.
This stops the signaling cascade, terminates the PDA, and repolarizes the photoreceptor membrane.
Place the fly holder in a dark Faraday cage on a magnet block, and ensure that the fly is approximately five millimeters from the end of the light guide. Place the recording electrode above the fly's eye, and the ground electrode over the fly's upper back using the micromanipulators. Then, insert the ground electrode into the back of the fly using the micromanipulators.
Insert the recording electrode into the outer periphery of the fly's eye, preferably using the micromanipulators. Close the Faraday cage, and turn off the lights in the room to allow adaptation to dark for five minutes. Input the parameters, starting from a pulse duration of 500 meters per second, a pulse interval of 60 seconds, and the number of pulses set to six.
For measuring the PDA, set the master rate pulse generator in Train mode. Then, give a 5-second light pulse of maximal intensity using an orange filter. Check the voltage response. Replace the orange filter with a broadband blue filter, and give three 5-second light pulses at maximum intensity. Check the voltage response.
Wait at least 60 seconds in the dark. Replace the blue filter with the previous orange filter. Then, give two 5-second light pulses with 60-second intervals. Check the voltage response.
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