Tracking Freely Walking Flies: A Method to Assess Locomotion in Drosophila

- Use a covered arena-- for example, a Petri dish filled with clear silicone-- to ensure flies can walk freely, but are unable to fly inside the arena when it's covered. Place the dish on a white illuminated surface and position a camera directly above.

With the lid open, transfer anesthetized flies into the dish, cover it, and allow the subjects to recover. Flies will move freely around the arena during the trial. To assess locomotion, track the position of each fly over time and calculate parameters that characterize locomotion, such as distance traveled by each fly. In this example protocol, we will see a demonstration of the assay setup and analysis using video tracking software.

- To track fly movements in a Petri dish, first, fill a 6 centimeter dish with transparent silicone elastomer to make the tracking arena. Leave a 3 millimeter gap between the silicone and the lid, so that flies can walk freely in the gap, but not take flight. Beneath the tracking arena, have a white background.

Next, anesthetize four flies from the same treatment group and place them in the arena. Let the flies acclimate for an hour to the arena, which should be fully lit and at about 22 degrees Celsius, not hotter than room temperature. After an hour, use a charge-coupled device camera positioned above the arena to record the fly's activity for 5 minutes.

After the 5-minute recording, anesthetize the flies in the arena and return them to a new home vial. Then, dispose of the arena. Later, analyze the recordings using the freely available Ctrax software. This software generates tracking data, which can be exported in a programmable-language-compatible format, such as the MATLAB format.

From the data, calculate the distance traveled per frame, the mean walking distance for each fly, and the average distance traveled per fly. For statistical significance, expect to test about 100 flies per treatment group.