In the video tracking assay, flies must be loaded into a square observation chamber without the use of anesthesia. To do this, a small funnel must be lined up with screw holes in the chamber, so as to knock the flies into the chamber. Using this funnel, screws then seal the holes of the chamber and an air pulse is administered through the air vents to initiate locomotion.
A video camera suspended above the chamber, captures this locomotion and sends it to a computer where the paths of the flies are traced and analyzed. Hi, I'm Justin Slawson from the Griffith Lab here at the Biology Department at Brandeis University. Today we're going to show you a procedure for high resolution video tracking of locomotion.
We're gonna use this procedure in our lab to study locomotive behavior in adult flies. So let's get started. To get started with the experiment, grow flies and bottles containing yeast-free standard media.
This protocol requires a large number of flies, so crosses need to be set up accordingly. Flies should be grown in a 12 hour light dark cycle at 25 degrees Celsius. Collect the flies one to three days after Aus use a carbon dioxide diffuser to sort flies into test tubes containing yeast free media sort 10 males per test tube.
Wait at least a day or two after use of carbon dioxide before running the behavioral experiments. Once the flies have been collected and sorted, they can be run in the video tracking system. The video tracking experiments are done in an environment room, which maintains a constant temperature of 25 degrees Celsius with 70%humidity.
To videotape flies in motion, you'll need to suspend a digital video camera over the area to be recorded. With the camera facing downwards, we suspend the camera over a light box since our tracking software is contrast based. Next, using a fire wire cable, connect the camera to a computer.
We use the software program, windows movie maker to record the video directly onto the computer. Since air pulses will be administered in this protocol, an airflow source must be present in the room. Rubber tubing connects the air source to a carbon filter for air filtration.
Rubber tubing also connects the other side of the carbon filter to an erlenmeyer flask via a hollow rubber stopper. The flask should be filled with about a half inch of water, which will humidify the air. The flask should also have a small sidearm, which should be connected to a Ys shaped valve via more rubber tubing.
One branch of the Y valve connects to a flow meter while the other branch connects to the square locomotion chamber. This square locomotion chamber is based on the design from Wolf Etal 2002 with the addition of small drop trays to prevent the flies from flying within the chamber before connecting the chamber to the tracking system, remember to use 70%ethanol to sterilize all the chamber components. Allow the components to drive before use.
Now that the tracking system is ready, we can deliver the flies to the locomotion chamber to deliver the flies into the chamber. Remove the screws which normally keep the small plexiglass top of the chamber secure. Then position the plexiglass top so that the screw hole sits directly over the inner chamber.
Anesthetizing the flies immediately before conducting. A behavioral assay can potentially compromise performance. To avoid anesthesia, use a funnel with a small outlet to gently knock flies into the chamber.
To make a funnel with a small outlet, attach a P 1000 blue pipette tip to the end of the funnel. Use scissors to cut off the very end of the pipette tip to make the opening big enough for a fly to pass through. Next, take a test tube containing the flies and place it upside down over the funnel.
Then place the end of the funnel into the screw hole. Do not move the funnel as this could hurt or damage the flies. Instead, gently bang the whole chamber funnel and all until all flies are inside the chamber.
Use a mouse pad to absorb the shock of this banging. When flies are inside, remove the funnel and reposition the plexiglass top over the screw beds then scr the plexiglass top into place. Once the flies are properly loaded, place the chamber on top of a light box.
Turn off the other lights in the environment room and let the flies acclimate in the chamber for 30 minutes. After the 30 minute acclimation period, proceed with the locomotor assay to begin the assay. Switch the Y valve in the airflow system such that the air will only flow to the flow meter, turn on the air and ramp the air up to the desired speed.
Any speed within four to six liters per minute will work. We generally use five to five and a half liters per minute. Once the desired air speed is attained, switch the Y valve so that the air flows to the locomotion chamber.
Time this air pulse for 15 seconds and then abruptly turn the air off as you turn the air off. Switch the camera to record mode. This step takes practice to do all at once.
We record 32nd trials at 10 frames per second and at least eight trials per genotype. Stop the recording when the trial's complete and make sure you save the movie when the trials are complete. We can proceed with the video analysis to perform motion tracking of the locomotor assay videos.
We use the dynamic image analysis system in order to use this software. Convert the videos to an A VI file format with QuickTime 7.5 0.5. Next, use the auto trace by threshold function to track the flies based on contrast, followed by the make path from trace function to digitize.
These traces then apply the compute parameters function to output the instantaneous speeds of each fly into a database file. This function also allows for data smoothing. We use a 5 15 60 15 5 smoothing window.
Once this information is outputted as a database file, we then open and save the files in Microsoft Excel. Excel files are processed using a MATLAB script to compile the instantaneous speeds and to calculate additional parameters necessary for understanding movement dynamics. And bout structure.
Here is a representative result from this assay. The figure on the left shows traces from wild type Canton s organisms while the figure on the right shows flies null for the dask gene, which were generated by crossing two large overlapping deletions. As you can see in our paradigm, the Dask null flies show greatly decreased locomotion compared to Canton S, which has already been reported in the literature using a different type of locomotor assay.
We've just shown you how to set up and run a locomotor video tracking assay. When doing this procedure, it's important to remember to perform the assays within the same two to three hour time window each day in order to avoid circadian rhythm issues and always run wild type organisms first to make sure that behavioral responses are within the normal range on a given day. So that's it.
Thanks for watching and good luck with your experiments.
