This article describes a method for recording electrophysiological responses from the photoreceptor cells of an immobilized butterfly. The procedure involves precise electrode placement and light stimulation to assess the cell's sensitivity to various wavelengths.
Begin with an immobilized butterfly on a platform.
Under a dissecting microscope, insert a reference electrode through the mouthparts to reach the head, and fix the electrode to the platform.
Make a small incision in the cornea and seal it to prevent drying.
Using brief lighting under a stereoscope, position a recording electrode filled with an ionic solution above the cornea.
Then, lower the electrode into the cornea using a micromanipulator to access the photoreceptor cell.
A significant voltage change across the membrane indicates the contact of the electrode with the cell.
Dark-adapt the insect by switching off the external light sources.
Initiate light flashes with a pulse generator and direct a fiber optic cable toward the eye.
Adjust the position of the electrode and fiber optic cable until a depolarizing response is obtained.
Record the cell's responses to white light and colored filters to assess its sensitivity to different light wavelengths.
To prepare the specimen, under the microscope, affix an individual butterfly inside a small plastic tube with hot wax, so the head is immobile and protruding from one end of the tube. Then, affix the proboscis, antennae, and wings with wax. After that, hold down the abdomen with a dry piece of wax. Keep the tube humidified by placing a wet tissue inside it behind the abdomen of the specimen. Make sure the specimen is completely immobile.
Next, mount the tube using a small piece of wax onto a small platform with a ball and socket joint that is attached to a magnetic base. Under a dissecting microscope, insert a silver wire into the head via the mouth part, as the reference electrode. Next, cut a small hole in the left cornea using a razor blade, and seal the hole with Vaseline to prevent desiccation. Once the cornea is cut, insert the recording electrode into the eye as quickly as possible, because hemolymph in the eye will quickly harden and make it impossible to insert an electrode. Mount the electrode on the electrode holder.
Subsequently, insert the silver wire into the potassium chloride solution in the microelectrode. Use a light source with gooseneck attachments to light the specimen under a stereoscope, while lowering the electrode into the eye. Adjust the electrode holder so the microelectrode is directly above the hole previously cut in the cornea. Then, lower the microelectrode into the eye using the micromanipulator until a circuit is completed.
Once in the eye, swing the stereoscope outside the Faraday cage, turn off the light source illuminating the specimen, and leave the insect in the dark for 10 to 20 minutes for dark adaptation. Next, activate the pulse generator to deliver a flash of light for the duration of the experiment, and direct the fiber optic cable toward the eye. Then, adjust the fiber optic cable to find the largest depolarizing signal.
Make small adjustments with a micromanipulator and use the buzz function on the amplifier as needed, to make sure the electrode is stably recording the cell. For white light stimulation, record up to 10 individual responses with the ND filter wheel set at the highest OD that still elicits a response. Then, record with other interference filters, and allow for up to 10 responses per filter.
繁體中文
