This protocol is particularly suitable for genetic screen of visually defective Drosophila mutants. This is because they allow the detection for even small abnormalities in Drosophila phototransduction, particularly in photopigment levels and tier peer-related functions. This technique is profound in vivo, it's easy to execute and highly robust, enabling the detection of small changes in the activity of phototransduction proteins.
In my opinion, fixing the flies while maintaining the viability is probably the critical part of this method. Turn on the computer and open the Clampex software. Turn on the amplifier and the Master-8 pulse generator, then turn on the xenon lamp and shutter controller.
Switch on the puller and place a glass capillary in the puller and pull it. Remove the glass capillary from the puller. Fill the glass capillary with filtered ringer solution using an elongated tip syringe.
Insert the wire electrode into the glass capillary. Ensure that the solution within the capillary is in contact with the silver wire. Insert the electrode holders into the two electrode micromanipulators.
Turn on the power supply of the soldering iron. Set the current to approximately 2.25 ampere. This current should heat the 0.25-millimeter platinum-iridium filament to approximately 55 to 56 degrees Celsius.
Place a drop of wax with a low melting temperature on the soldering iron. Anesthetize the flies within the bottle. Pour the anesthetized flies into the sleeper container, choose one fly and cover the rest of the flies with a Petri dish.
Carefully hold the fly by its wings using a sharp tweezer and place it on the fly holder. On the fly holder, place the fly lying on its side with its back toward the hand. Using tweezers, lift the fly from its wings and fix its wings to the fly holder using the soldering iron.
Connect the fly's back to the stand surface with wax using the soldering iron. Lower the soldering iron's tip to the joining point of the legs, then melt the wax to cover all legs together. Place a small drop of wax between the head and the neck in the neck area.
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 micro manipulators. 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-8 pulse generator in TRAIN mode, then give a five-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. For measuring the ERP M-potential, give a continuous blue light pulse until a steady state voltage response is reached.
Give a brief intense light flash of a wavelength between 350 to 700 nanometers using a 20-nanometer band-pass filter. Then measure the peak amplitude of the M1 phase of the M-potential response, which reflects the metarhodopsin absorption at this specific wavelength at photoequilibrium. The PDA responses were evaluated for different mutant flies.
Wild-type flies showed a saturated depolarization in the dark that appeared as prolonged corneal negative ERG, following intense blue light stimulation. The following blue lights produced small responses superimposed on the PDA, lacking the on and off transients. In mutant flies with abnormal photopigment biogenesis such as ninaE, the voltage response returned to baseline following intense blue light stimulation, and the response to an additional blue light was not suppressed and showed normal on and off transients.
The wild-type ERP of the fly is obtained by the same protocol, but an intense green flash is applied during the PDA. This green flash elicits the ERP and suppressed the PDA. The M-potential was obtained by the green flash following blue illumination, but not by a blue flash after blue illumination in the wild-type fly.
This protocol was repeated in wild type, in photopigment hypomorph mutant ninaE, and in phototransduction-deficient mutant with normal photopigment levels norpA. Relative absorption spectra of fly rhodopsin and metarhodopsin were calculated from photometric measurements of the different spectrum and the photoequilibrium spectrum. It is important for the survival of the flies to pay attention to the wax viscosity by refraining from overheating.
This procedure is suitable for genetic and via screening of defective visual mutants. Methods that randomly isolate and identify visual mutations may facilitate the discovery of novel proteins and mechanisms involved in Drosophila phototransduction. The PDA protocol enabled the isolation of important and novel visual mechanisms.
The participation would probably have not been discovered or even anticipated otherwise.
