The overall goal of the following experiment is to determine startle induced and long-term spontaneous locomotion in a rotenone induced Parkinson's disease model of drosophila, this is achieved by rearing drosophila on rotenone supplemented food to induce the phenotype. As a second step, flies are subjected to the startle response assay and a double vial apparatus and long-term spontaneous locomotion assay. In a drosophila activity monitor, the results show that exposure to rotenone causes locomotion defects in drosophila based on quantification of startle and long-term spontaneous locomotion.
These methods can help to answer key questions in the field of drosophila, models of neurodegenerative and locomotion disorders, such as defects in startle induced locomotion, and long-term spontaneous locomotion. To begin, add 1.5 grams of instant drosophila medium. In a standard fly vial constitute the medium with five milliliters of deionized water containing the appropriate rotenone dose.
Next sedate, eight to 12 male flies aged one to three days old using carbon dioxide and transport them to vials containing the drug. Supplemented food allow flies to recover from sedation for 20 minutes with the vial in a horizontal position. Then place the vial upright in a 12 hour light dark incubator at 25 degrees Celsius for the remainder of the experiment.
Next, invert a standard fly vial on top of another vial and seal the seam with tape to create a double vial apparatus. Then divide the double vial into three equal sections of 6.33 centimeters by marking circles around the vials with a permanent marker. After three days of drug exposure, transfer flies into the double vial apparatus.
Replacing the tape over the seam allow flies to acclimate to the new environment for 15 minutes. Then place the vials on a white background and set up the digital camera. Ensure the entire apparatus is visible in a single picture frame and that the double vial apparatus and timer are in view and all flies are in focus.
Mark the location of the camera and vial to maintain consistent frames between trials, clearly displayed the trial number and drug treatment in the camera view. Next firmly tap the double vial apparatus against the countertop three times and ensure that all flies fall to the bottom of the vial and start the timer immediately. Take a picture of the apparatus every five seconds for one minute.
Then allow flies to recover undisturbed for one minute. Repeat the assay two more times with one minute recovery times between each trial. Review the pictures and record the number of flies in each section over time to make the calculations reconstitute three grams of instant oph, a medium with 15 milliliters of deionized water and the appropriate rotenone dose.
After five minutes, carefully place the tubes vertically into transparent vials and load the firm food one centimeter high by twisting the tube. Next, gently push the plastic cap on the end of the tube, avoiding the creation of air bubbles Sedate. One day old male flies with carbon dioxide and carefully insert one fly into each tube with a paintbrush.
Then plug the end of the tube with a small cotton ball. Allow flies to recover from carbon dioxide with the tubes in a horizontal position for 15 minutes and ensure that all flies are alive and active. Insert the tubes into the drosophila activity monitor or DAM, and check that all the tubes are in the same position relative to the DAM.
Place the DAM in a 12 hour light dark incubator at 25 degrees Celsius. Next, connect the DAM to the data collection system. Then open the DAM software under preferences.
Select a 10 minute bin length start data collection, and allow the program to collect data for seven days. Next, open the DAM file scan program and access the monitor data by clicking select input data. Then select the appropriate monitor range and set the bin length to 10 minute intervals.
Next in output file type, choose channel files and leave all the other options at their defaults. Then click scan data and save the files to a designated folder. Finally, import data into circadian data analysis software to obtain counts per minute.
After three days of exposure to different dosages of roone wild type Canton s flies exposed to increased dosages showed a deficiency in startle response. A lower percent of flies were in the top section and a higher percent in the bottom section of the apparatus after 30 seconds indicative of a deficiency in saral response, a similar trend was seen for flies in the spontaneous locomotion assay. Counts per minute were measured in the drosophila activity monitor or DAM.
On the fourth day after exposure flies exposed to 250 micromolar and 500 micromolar of rotenone exhibit a significant reduction in counts per minute. These assays can be conveniently adapted to characterize locomotion defects in other esophagal models of disease and efficacy of therapeutic agents.
