The overall goal of the procedure is to study learning and memory in adult Esso Melan Gaster first to determine if the flies suffer from locomotor deficits. They are tested in the negative geo taxes. Assay positive photo tactic behavior is then verified by placing flies in a T maze and recording the latency to move toward the favored lighted chamber.
Flies with positive phototaxis are then conditioned to associate the lighted chamber with the bitter taste of quinine. Ultimately, flies are tested on how well they associate the aversive stimulus with the lighted chamber providing a measure of learning and memory. Ola, welcome to the X lab.
Today we'll be demonstrating to you two commonly used behavioral assays to study neurodegeneration and drosophila. First, we'll show you the aversive phototaxis suppression assay demonstrated to you by Wil Fredo sala, an undergraduate in our lab and myself, which will be followed by the negative geo taxes assay demonstrated to you by kaiwan, a graduate student in our lab. Maintain the flies on the standard cornmeal agar molasses yeast medium at 25 degrees Celsius on a 12 hour light dark cycle.
On the day of the occlusion, sort virgin flies into groups of 20. Virgin flies are identified by their light color and a dark greenish spot located on the underside of their abdomen. The flies should be transferred into a fresh vial every three days, and sibling flies should be used to minimize differences arising from genetic backgrounds.
10 to 20 groups per genotype or treatment should be tested in a geo taxes study. To begin sort female and male flies into groups of 10. These flies should be tested separately.
Since behavior is significantly influenced by gender. Wait at least one hour after anesthesia before proceeding to ensure that locomotor activity is unimpaired. Once the flies are prepared perfectly, align the openings of two empty polystyrene vials by joining them vertically on the lower vial.
Measure eight centimeters from the bottom and draw a circle around the circumference. Now that the apparatus has been assembled, transfer a group of 10 flies into the lower vial. Immediately rejoin the vials and acclimate the flies for one minute before testing.
After the flies have settled, gently tap them down to the bottom of the vial and measure the number of flies that climb above the eight centimeter mark by 10 seconds. The number of flies per group that passed this mark is recorded as a percentage of total flies. Repeat this procedure 10 times per group, allowing the flies to rest for one minute between trials.
Six hours before the a PS assay, sort, female flies into polystyrene vials containing water moistened filter paper. This step ensures that the flies are starved and more perceptive to the aversive taste. In this assay, a quinine solution serves as the aversive stimulus.
Prepare a working solution of one micromolar from a 0.1 molar stock. The T maze consists of a center column with a trap door and two side chambers, a dark chamber and a lighted chamber. At the two millimeter mark, use a manual saw to cut off the closed end of two 15 milliliter plastic centrifuge tubes fit connector adapters at each end and seal the connection with paraform.
Wrap one tube with aluminum foil to service the dark chamber. Assemble the TMAs by screwing the lighted and dark chambers on each side of the center column. With the trap door in the middle closed, complete the setup by connecting a light source to the adapter of the light chamber directly Before performing the assay transfer each fly from a group of 10 into an empty 15 milliliter centrifuge tube and cap lightly to prevent escape.
Next, unscrew the dark chamber from the TMAs. Transfer a single fly into the tube and immediately insert it back into the maze. Turn off the lights in the room and turn on a red lamp.
Allow the fly to acclimatize in the dark chamber for 30 seconds and then slowly turn on the light source illuminating the lighted chamber. Once lighted, open the trap door that separates the two chambers. If the fly walks to the lighted chamber within 10 seconds, it is considered positively photo tactic and is ready to be trained.
Failure to respond in this way indicates a problem in the visual system and necessitates exclusion from the assay. Once positive phototaxis has been confirmed, tap the fly back to the dark chamber, close the trap door and turn off the light during a 32nd rest period. Prepare the aversive stimulus by placing the filter paper into the lighted chamber and pipetting one milliliter of quinine solution onto the filter paper.
Ensuring all of the filter paper is completely moist with aversive stimuli. To begin conditioning slowly open the trap door and turn on the light. Allow the fly to walk into the quinine scented lighted chamber.
After one minute, tap the fly back to the dark chamber. Repeat this procedure nine times for a total of 10 conditioning trials. Typically while type flies, avoid the lighted chamber after three to five training trials immediately after training.
Conduct five test trials. If the trained fly does not walk into the light of chamber within 10 seconds, it is recorded as a pass. The pass rate over five consecutive trials is recorded and designated.
PC zero. After training and initial PC zero recordings, each fly is placed back into its original numbered food file and kept aside for six hours. Six hours after training, conduct five additional test trials as demonstrated earlier, this pass rate represents PC six and is a measure of short-term memory.
In the negative geo taxes assay, the number of flies crossing the eight centimeter mark is converted to a percentage and presented as the climbing pass rate. Over 10 trials as seen here on the right, 10 day old male or female flies over expressing. Human tau have significant locomotor deficits compared to age match GFP over expressing flies on the left.
Performance in the aversive phototaxis suppression assay is represented by the average pass rate or the percentage of successful avoidance trials. Over total trials flies over expressing tau shown here on the right pale to associate the light with a bitter taste when compared to the control group on the left, both immediately after training and when tested, six hours later Following this procedure, samples can be taken for immunohistochemical analysis to answer additional question about morphological differences. After watching this video, you should have a good understanding of how to study learning and memory and flies amigos.
And remember, it's all about the you.
