The overall aim of this procedure is to use optogenetic stimulation to activate specific neurons in freely walking flies to test their involvement in the flies escape response. This is accomplished by first starting with flies of the appropriate genotype that have been raised on food supplemented with all trans retinal. The second step is to place an individual fly in a pipette tip.
Next, the pipette tip is put inside a custom built holder surrounded by LEDs. Finally, when the fly emerges onto the platform, illuminate the blue LEDs ultimately recording. The time of takeoff shows that loom sensitive neurons in the flies optic lobe are sufficient for the fly's escape behavior.
The main advantage of this technique over existing optogenetic methods is that it allows the use of optogenetics in the visual system of the fly by eliminating the visual responses to blue light. Though this method can provide insight into escape behavior and flight initiation, it can also be applied to other behaviors in the freely walking fly. Begin by melting standard cornmeal, fly food in a microwave and let cool until warm to touch.
Once called mixed 50 microliters of previously prepared 20 millimolar all trans into each vial. Then let vials cool and keep protected from light until required. First cross UAS channel drops into flies with the appropriate GAL four.
Driver G 1 0 5. GAL four was used here for expression in fomo one neurons in the optic lobe. Both lines should be from a white plus nor pay background to eliminate the possibility of a visual response to blue light stimulation.
12 to 13 days after the cross white plus nor pay G 1 0 5, GAL four uas. CR two positive flies are obtained. Transfer adult females onto fresh food, supplemented with 10 micromolar all trans retinal for three days before performing the behavioral assay.
The base of the apparatus consists of a 17 centimeter by 25 centimeter piece of polyoxyethylene with threaded holes at each corner to fit one quarter inch NPT coolant Hose connectors attached to the base is a vertical piece of polyoxyethylene. A 10 millimeter wide groove runs the length of the holder with a thumb screw at the bottom. The platform is attached to the top of the holder with a 3.5 millimeter diameter hole aligned with the groove in the holder.
Forearms of coolant hose around 18 centimeters long are affixed to the platform base using the coolant hose connector as structural support for the LEDs. Properly spaced grooves are cut into the final piece of coolant hose hosing of each arm to affix heat sinks to the end of each arm. A blue rebel Tristar, LED is mounted to each heat sink using precut thermal adhesive tape.
This image shows the Tristar LED on the heat sink in. Close up a Carlow 18 degree tri lens is affixed to each Tristar. This image shows the lens in closeup Tristar.
LEDs are wired to the buck puck DC drivers and a power supply as specified by the manufacturer. This setup has been arranged with each buck puck powering two Tristar in series. The circuit diagram for the buck puck and LED circuit is illustrated here.
This setup with all four Tristar LEDs illuminated at 700 milliamps yielded in a radiance of 713 watts per meter square on the platform. Finally, a camera is mounted on a small tripod and focused on the top of the platform. First, briefly anesthetize the flies on ice.
Then place individual flies in P 1000 pipette tips with the tip cut to form a poor diameter of around 2.25 millimeters and seal each end with tape. Once the fly has awoken and is exploring the pipette tip, remove the tape and quickly place the tip in the groove in the vertical holder. Secure the tip in place with the thumbscrew, which closes the bottom of the tip flies.
Typically explore the tip for between 30 and 60 seconds. As the fly nears the top of the tip, start the camera recording just before the fly emerges onto the platform. After the fly has emerged onto the platform, wait one to two seconds and then turn on the blue LEDs.
Use a timer to manually measure the time until the fly initiates flight. This cumulative histogram shows the time of escape jump for experimental and control. Fly lines all flies express white plus nor pay to render them visually.
Blind fomo. One flies have the G 1 0 5 GAL four. Driver MB flies have the 2 0 1 Y gal, four driver that drives expression in the mushroom body.
When the channel adopts in two is expressed in the fomo, one neurons illumination with blue light elicits the escape response. Over 50%of the flies tested took off within one second of illumination and 75%within five seconds. In contrast, control flies, including those expressing channel redin two in the gamma lobe of the mushroom body exhibited a low rate of takeoff.
These images are high speed video frames taken of channel redsin induced escape responses. Frames are numbered sequentially with five milliseconds between frames. These images reveal that these responses were similar to the lu evoked escape behavior.
Namely, 90%of the flies filmed raised their wings prior to takeoff. Further, the ensuing flight trajectory was less stable than voluntary takeoff with the fly's body being in a more vertical orientation, but was more stable than previously reported. Giant fiber mediated responses When attempting this procedure, it's important to remember to handle the flies gently so that they're not overly excited from recent manipulation.
After watching this video, you should have a good understanding of how to use optogenetics to activate specific neurons in freely walking flies.
