-First, isolate single naive, or unmated, Drosophila melanogastor males. Then, to condition the males, add a female that has already mated, and is therefore non-receptive to courtship. The male displays typical courtship behaviors such as orienting towards her, following her around, tapping her with a foreleg, and extending and vibrating his wing to produce the courtship song. He may also attempt licking and mating. No matter the efforts, the female remains non-receptive, and the male experiences a rejection. Thus, he learns not to court a previously mated female.
To test the male's memory, allow him to rest, and then expose him to another mated female. The trained male will not attempt to court this fly because he retains the memory of past rejection. In the example protocol, we will see how flies are handled during the training and testing phases of a courtship conditioning assay for short- and long-term memory assessment.
- Begin by anesthetizing all of the adults from the four-day-old mating files. Separate the sexes, and transfer single premated females into each of the 12 wells of one row of a housing block. Then, using an aspirator and no anesthesia, remove the naive males from their home wells and transfer them into the wells containing the premated females. Repeat this process in the second row to set up 12 more pairs.
To test learning or short-term memory, perform this setup at dawn, and let the training period go for one hour. To test long-term memory, set up the pairs four hours before subjective dawn, and let the training go on for eight hours.
- In order for learning and memory to be established, it is important that the males fail to copulate, so periodically check the pairs during the training period, to make sure that the males are being rejected.
When the training period is over, gently remove the males from the wells using an aspirator, not carbon dioxide anesthesia. Load the males into a new housing block. For the learning experiment, immediately test the males following the training, without a rest period. For the short-term memory experiment, allow the males an hour of rest before starting the test stage. For the long-term memory experiment, rest the males until the following subjective dawn.
To prepare for the testing stage of the experiment, anesthetize premated females and transfer them to a new normal food vile. Allow at least an hour for the flies to recover from the anesthesia. Next, prepare the testing setup. Be sure to have the video recorders ready to record activity in the courtship arenas.
To start the test, use gentle aspiration to transfer a test male from the housing block to the courtship arena with the dividers closed. Move the entry holes between the arenas until all 18 arenas are loaded with single males. Then, using the same technique, transfer individual premated females to each of the 18 arenas, but to the opposite side of the dividers. Now, carefully flip the courtship arena, and place it under the video camera with the well openings down toward the white bench surface. Then, remove the dividers and start the video recording. Record at least 10 minutes of activity. When the experiment is over, remove the flies from the arenas using a vacuum and ventilate the courtship chambers for at least 10 minutes before they are reused.