1. Drosophila Startle-induced Locomotion Assay

1. Drug Treatment
   1. Sedate to immobilize desired number (approximately 8-12) of 1-3 day-old male flies using CO₂ and transport them to vials containing the drug-supplemented food. Note: Another anesthetic e.g., ether or ice can be used to sedate flies to enable counting and handling.
   2. Allow flies to recover from sedation for 20 min (or until recovery) with the vial in a horizontal position (to prevent flies getting stuck on food) and then place the vial upright in a 12 hr dark, 12 hr light incubator at 25 °C for the remainder of the experiment.
2. Experimental Set Up
   1. Divide this double vial setup into three equal sections of 6.33 cm by marking circles around the vials with a permanent marker.
   2. After 3 days of drug exposure, transfer flies without anesthetizing into the bottom vial and quickly place the top vial over the opening. Tape the two vials together with clear tape.
   3. Allow flies to acclimate to the new environment for 15 min.
   4. Place vials on a white background and set up a digital camera at an appropriate distance from the double vial apparatus with a timer in view. Ensure the entire apparatus is visible in a single picture frame and that all flies are in focus. To maintain consistent frames between trials, mark the location of camera and vial.
3. Mobility Assay
   1. Clearly display the trial number, drug treatment, and timer in camera view.
   2. Firmly tap the double vial apparatus against the countertop 3 times and ensure that all flies fall to the bottom of the vial. Simultaneously start the timer.
   3. Every 5 sec for 1 min, take a picture of the apparatus. Note: Alternatively, a video could be captured and paused at appropriate intervals for measurements.
   4. Allow flies to recover undisturbed for 1 min.
   5. Repeat 2 more times with 1 min recovery time between each trial. Note: Each apparatus should take 5 min to complete data collection. Maintain similar force of tapping between trials. Multiple (at least 3) apparatus can be easily handled simultaneously.
4. Data Analysis
   1. Review pictures and record the number of flies in each section over time. Calculate the percentage of flies in each section over time. Notes: Repeat this entire procedure with the same flies at 2 or 3 time points of interest, for example, day 3, 5, and 7. If too many flies die throughout the experiment it is possible to scale up the original trial number to compensate for the mortality. Use appropriate statistical analysis to compare the data.

2. Drosophila Spontaneous Locomotion Assay

1. Food Preparation
   1. Reconstitute 3 g of instant Drosophila medium with 15 ml of de-ionized water and desired rotenone (or another drug of interest) dosage.
   2. Once the food mixture has become firm (about 5 min), carefully load the food to be approximately 1 cm high into manufacturer supplied transparent tubes (5 mm X 65 mm). Add the drug infused food to the tubes by carefully placing the tubes vertically in the food and twisting them until they can be removed with the food inside the tube. Note: It is helpful to place a finger on the opening of the tube to create a vacuum. Food should not contain any air bubbles or have an uneven surface as the flies can become stuck.
2. Experimental Setup
   1. Place a plastic cap on the end of tube nearest the food. Push the plastic cap on the tube as little as possible, as it can create an air bubble in the vial if pushed to forcefully.
   2. Sedate 1 day-old male flies using CO₂ and carefully insert 1 male fly into each tube with a paintbrush. Repeat depending on the number of desired trials.
   3. Plug the end of the tube farthest from the food with a small cotton ball, which can be hand rolled from larger store bought cotton balls.
   4. Allow flies to recover with the tubes in a horizontal position for 15 min and ensure that all flies are alive and active. Insert the tubes into DAM and make sure that all the tubes are in the same position relative to DAM. Note: It is possible to place them with the area of monitoring at the middle of the vial, or to push all the vials to the side, so that the end of the tube is being monitored. Note: See discussion for variations on this method.
3. Data Collection
   1. Place the DAM in a 12 hr dark, 12 hr light incubator set to 25 °C. Connect the DAM to the data collection system. Open the DAM software and under preferences select bin length to 10 min. Start data collection and allow the program to collect data for 7 days. Note: Bin length can be adjusted if required.
   2. Data Analysis
      Note: Process the data to obtain counts per min as a measure for long-term spontaneous locomotion.
      1. Open DAM file scan program and access monitor data by clicking select input data.
      2. Select appropriate monitor range and select bin length to 10 min intervals.
      3. In output file type choose channel files. Leave all other options as default.
      4. Click scan data and save to a designated folder.
      5. Import data in a circadian data analysis software to obtain counts per min. Note: For data analysis Clocklab software is commonly used. Other options are also available.