Our lab uses the fruit fly as a model organism to study how taste information is processed through neural circuits from the detection of chemicals to the impact on behavior. We are using this imaging protocol to identify taste cells that respond to amino acids and determine if different internal states modulate how taste cells respond to these essential nutrients. An advantage of this calcium imaging approach is that it allows taste-induced neural responses to be recorded from specific cells in awake animals where internal states stay intact.
We can now identify putative taste circuits in the new Drosophila whole brain connectome and verify functional neuronal dynamics in vivo using this calcium imaging approach. To begin, place the anesthetized fly under a dissection microscope. Using dissection scissors, remove the middle and hind legs at the femoral tibial joint and the four legs at the trochanter.
Pick up the fly by the wings using blunt forceps to position it with the head above the targeted cervix slot of the imaging chamber while keeping the body below. Using the blunt side of the scissors and blunt forceps, gently push the head and thorax simultaneously into the slot. Once the fly is securely inside the slot, push it to the back and gently reposition it so that it faces the front of the chamber.
Gather a small droplet of nail polish on the end of a toothpick and apply a thin coat to secure the fly's head to the imaging chamber. Pick up the waxer with one hand and gather a small droplet of wax on the tip. Using semi-sharp forceps, in the other hand, grab one maxillary palp and gently pull out and hold the proboscis in full extension.
Touch the tip of the waxer to the chamber near the base of the proboscis until the wax starts to flow. Move to make contact with the base of the proboscis and wax halfway down the shaft, avoiding contact with the labellar sensilli. Then fully extend the proboscis as straight as possible.
Now place the mounted flies into a humidity chamber for 60 minutes to recover. Remove the flies from the humidity chamber. Using very sharp forceps, pinch off both an antennae, pinch the cuticle to create a hole, and insert one side of the sharp forceps.
Run the forceps under the cuticle to remove it from the region covering the brain area of interest. To wash the exposed brain, add approximately 100 microliters of artificial hemolymph solution, or AHL, to the head. After washing, remove the AHL, leaving a thin layer to prevent the brain from drying out.
Using sharp forceps, remove air sacks and any large debris covering the brain. To specifically image the subesophageal zone, or SEZ, cut the esophagus at the base near the proboscis and at the point where it passes through the brain, using very sharp forceps. Remove this piece to expose the SEZ.
Under the dissection microscope, position the 10-by-20 millimeter cover slip into the angled slot of the imaging chamber. Load approximately two microliters of water, or another negative control, into the capillary tube. Locate the dissected fly and focus on the labellum using a 10x air immersion brightfield objective.
Align the capillary with the labellum under the 10x view. Leave the capillary position directly in front of the labellum ensuring it is close but not touching. Move the stage to center the brain region of interest.
Switch to a higher magnification water immersion objective such as the 40x objective. Add approximately 200 microliters of AHL on top of the brain to ensure contact with the immersion objective. Switch to 488 nanometer laser power to locate GCaMP expression in the area of interest.
After collecting at least five seconds of baseline fluorescence, manually move the stimulator so that the capillary covers the labellum for five seconds. Remove the stimulus and continue capturing as long as desired. Next, remove the AHL and return to 10x brightfield to confirm that the cover slip, stimulator, and labellum remain in the correct position.
Then remove the imaging chamber and use a lint-free wipe to remove the first solution from the capillary. Flush the pipette with water, and pipette approximately two microliters of the next tastant into the capillary tube. The relative fluorescence change in Gr64f GCaMP flies was significantly higher for sucrose stimulation compared to water, demonstrating a strong and sustained response of sweet gustatory receptor neurons.
The relative peak fluorescence for sucrose stimulation was significantly greater than that for water. The fluorescence change in Gr66a GCaMP flies was significantly higher for caffeine stimulation than for water, showing response to caffeine onset and removal. The projection pattern of axon terminals in Gr66a flies was distinct from that of Gr64f, demonstrating the anatomical segregation of bitter and sugar sensing gustatory receptor neurons.
