The overall goal of this procedure is to monitor neuronal activity by calcium imaging simultaneously with observation of feeding behavior. This is accomplished by first inserting a fly into the flies apparatus. Next, the fly head is dissected to expose the brain.
The fly's apparatus with the dissected fly is then positioned on the microscope stage. The final step of the procedure is stimulation of pro Bois with a sugar wick. Ultimately, results can be obtained that show activation of an identified neuron through calcium imaging correlated to Probus extension through videotaping of behavior.
I first had the idea for this method when I thought about eyes of beating beers which swim on the surface of the water. We beers have split eyes and can see both above and below the surface of water. A tricky point of my experiment is that the brain and the SCUs are close to each other, but if they're separated nicely like eyes of we beers, we can keep the SCUs dry while the brain is exposed in the satellite.
This I made a very simple partition just above the SCUs to separate the dry face from the width place, Shape a platform from the lid of a 35 millimeter falcon dish by melting the sidewall and carving it to form an appropriate angle and thickness. Drill a hole in the platform to accept the fly head, such that the mouth parts are freely exposed to the outside of the chamber. Cut the end of a pipette man tip just wide enough to accept the fly's body, and then glue the tip to the platform.
After starving an adult fly for 24 hours at 25 degrees Celsius. Anesthetize the fly by placing it in a 15 milliliter plastic tube, standing on ice using forceps. Insert a fly into the chamber of the flies and gently push the fly in until it is unable to move.
Seal the surrounding parts of the proximal probos or rostrum to the inner edge of the hole by applying a small amount of light curing glue to the sides and above the rostrum, using an eyelash or similar tool to carefully spread the glue. Also apply the glue from the inside of the flies to the space between the dorsal part of the head and the inner edge of the hole. Finally, cure the glue with the weakest illumination of blue light sufficient for curing to avoid damaging the fly.
To stabilize the fly's head and to expose the ventral part of the SOG, attach a thread to hold the rostrum partially lifted. With the fly in place, fill the surface of the platform with sucrose free saline. Open the head capsule to expose the SOG using a tungsten blade and forceps with sharpened tips we call the sharpened forceps.
Scis zips. This is a demonstration of scis zips, cutting a fiber from a Kim wipe for comparison. This is first performed with iris scissors in the visual field, and then in front of a one millimeter graduated ruler.
First, using the tungsten blade, make an incision through the posterior edge. Then cut through the side and the anterior edges using the sharpened forceps. Lift the cut cuticle piece with the forceps and remove it.
Cut the antennae and antenna nerves using the forceps. Then selectively remove air sacks between the SOG and cuticle in order to stabilize the brain and prevent movement artifacts. Next, cut the esophagus at the end towards the pharynx and at the end going into the SOG in order to remove the connection between the pharynx and the brain.
Then after identifying and pulling out muscle 16, detach any trachea connecting the brain and the cuticle. Place the flies on the stage of a spinning disc confocal microscope, and switch to the water immersion lens for imaging. Then take a single optical section at four hertz with an exposure time of 122 milliseconds using 491 nanometer excitation laser focusing at the region of interest.
Use a syringe needle with a small wick of Japanese. Was she paper protruding from the tip to offer sucrose to the fly? Discharge a small drop of sucrose solution onto the wick, then aspirate it with an injector connected to the needle through a flexible tubing using a joystick manipulator to hold the needle.
Apply the soaked washy paper wick to the tip of the probs, but only for an instant. To avoid satiation. Monitor the PROBUS extension response or PER behavior using a CCD camera attached to a dissection microscope simultaneously with calcium imaging, and be sure to collect the data within one hour of dissection.
Shown here is a typical prob Bois extension response to stimulation with a WIC containing 100 millimolar sucrose. Aqueous solution imaged using a 491 nanometer laser for G CAMP fluorescence. Simultaneous calcium imaging during prosa extension response behavior shown here first before and then after stimulation allows for the visualization of sucrose induced GAMP 3.0 responses in the motor neuron for the protractor of rostrum muscle.
In the star of state quantification of the sucrose induced GAMP 3.0 response shows that the motor neurons respond to a sucrose stimulus by a sharp increase in fluorescence, followed by a restoration phase of several seconds to the baseline. The platform flies allows for other methods like electrophysiology or optogenetic or psychogenetic, or chemical activation for neurons or TimeLapse lab imaging of GFP labeled structures in order to answer additional questions like correlation between synaptic plasticity and memory.
