The overall goal of this method of intracellular recording is to determine the spectral sensitivity of individual photoreceptor cells in an insect compound eye in vivo. This method can tell us whether the wavelengths of light that a cell is sensitive to are affected by filtering pigments. The main advantage of this technique is that by knowing the spectral sensitivities of individual photoreceptors, we can predict what range of colors an animal can see.
To prepare for the recording, turn on the xenon lamp at least 45 minutes before the experiment, and turn on the microelectrode puller at least 30 minutes before pulling the glass electrodes. Next, turn on all the recording equipment. Make sure the shutter is closed by default, so no light passes through the fiber optic cable.
Then, pull fine borosilicate glass microelectrodes using the microelectrode puller. Backfill the electrodes with three molar potassium chloride. 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.
Before the experiment, permanently fix the wire to the platform. 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.
If possible, perform the dissection in the rig where the recording will take place. Then, connect the head stage ground wire to the reference electrode on the specimen platform, using the alligator clips. 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 goose neck 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.
Check the resistance of the electrode by applying one nanoamp current and noting the change in voltage. Resistance should typically be in the range between 100 and 250 megaohms. 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.
Check the oscilloscope for voltage change with each light flash. A negative change in voltage signifies that the electrode has not yet entered the cell. Move the fiber optic cable around the specimen until a maximum voltage response is observed.
To generate a depolarizing light response, rotate the micromanipulator back and forth, that result in small vertical movements of the electrode, while lightly tapping the base of the electrode holder or using the buzz function on the preamplifier. Continue to make small adjustments until a depolarizing light response appears on the oscilloscope. 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 need, to make sure the electrode is stably recording the cell. A stable recording should have little to no change in resting potential, low background noise, and a consistently large depolarizing response. It is essential to have a stable recording where resting membrane potential is not noisy and does not change over time.
Responses should be large and positive with no hyperpolarization. Once the setup is stable, open the software and close out the dialogue box called Experiments Gallery"to open a popup window with four channels. Adjust the voltage scale at the top-right corner of the software window to 500 millivolts.
Then, click Start at the bottom right-hand corner to begin recording. Minimize channels three and four, and adjust the X and Y-scale. Then allow the software to run for the duration of the experiment.
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. Here, the change in response is shown at 3.0 OD and 2.0 OD.Next, record the same number of responses from 3.5 to 0.0 OD, in every combination, in order to provide a response log intensity curve. Before recording the response of the cell to all wavelengths using the interference filters, first quickly find the peak wavelength.
Without any ND filter in the light path, place a UV-transmitting filter there, and briefly observe the response amplitude. Repeat the procedure with a blue transmitting filter, a green transmitting filter, and a red transmitting filter, which should give some idea of where the peak response will be. Record under the filter wavelength that gives the max response before changing to another interference filter.
Between recording with subsequent filters, allow the cell to respond to one to two flashes of white light without any filter in the light path, in order to help monitor whether the peak response is degrading over time. Then, record with other interference filters, and allow for up to 10 responses per filter. This is an example of a large negative voltage change that is seen just before entering a cell.
Shown here is a clean recording, which has little background noise, and a large depolarizing response, typically of at least 40 millivolts. And here is an example of a poor recording, due to the negative potential change after the main peak. This is another example of a bad recording.
The resting potential is undergoing large fluctuations, and the large amount of background noise can obscure the amplitude of the response. This is a representative example of spectral sensitivity derived from a single recording. The cell types are classified by peak sensitivity at a similar wavelength, and overall shape of the sensitivity spectrum.
The same cell types are then averaged, and the mean sensitivity is plotted with standard error bars at each wavelength. This graph shows the spectral sensitivities of three typical cell types found in an insect. Once mastered, this technique can be done in about two hours.
Following this procedure, behavioral experiments can be performed in order to determine whether or not all photoreceptor cell types contribute to color discrimination.
