Courtship conditioning is a classic drosophila learning and memory assay based on a reduction of male courtship behavior after sexual rejection by a non receptive female. This assay can be used to test learning, short term memory, and long term memory. This method can be used to uncover fundamental mechanisms underlying learning and memory, and to investigate the effect of different genetic or environmental conditions on learning and memory.
The main advantage of this technique is that it robustly assesses learning and memory. It takes advantage of a naturally occurring form of behavioral plasticity, and it can be conducted with minimal experimental setup. Eleven days in advance of collecting the experimental male flies, start five to 20 wild type cultures, each containing a mixed sex population of 60 to 100 flies.
Prepare each culturing vial using power food with a piece of filter paper, to expand the surface area for pupation. Using power food is an essential step to producing large numbers of pre-mated females. 10 days in advance of collecting the male subjects, add filter paper to a fly food bottle, and establish at least six normal food 175 milliliter vial cultures.
Then add around 20 males and 30 to 70 virgin females to each bottle. This experiment introduces novel experimental housing blocks. Not more than two days before the experiment, prepare these blocks.
Melt power food and add half a milliliter of it to each well of a 96 well flat bottom block using a repeater pipette. After the food solidifies, cover the block with PCR adhesive film. Then poke at least four breathing holes into each well, using a 0.8 millimeter gauge needle.
Next, cut the film between the wells lengthwise, but not quite all the way. Leave an intact strip of film at one end of the plate. The blocks can be stored at four degrees Celsius, for up to two days.
One day in advance of collecting experimental male flies, two to five hours before the flies subjective dawn, remove and discard all of the adult flies from the wild type power food cultures. Then, every two to three hours, collect the freshly eclosed adults from these cultures using carbon dioxide. Pool up to 200 flies into a new power food vial supplemented with some filter paper.
Incubate these mating vials for four days to ensure that all the females have mated. Collect enough females for all the upcoming training and testing steps. The quality of this culture, is important for the success of this experiment.
Only transfer freshly eclosed flies, and do not transfer any old flies, larvae, or pupae. It is also important that there are enough males to copulate with all the females. The next day at two to three hours before the subjective dawn, clear the other set of normal food culture vials to collect male test subjects.
Anesthetize all the adults using carbon dioxide and discard them from the vial. Then over the next five to six hours, collect freshly eclosed male flies every 20 to 30 minutes. The males must be freshly eclosed showing pale pigmentation, and the presence of the meconium on their translucent abdomen.
Next, move individual males into the wells of the housing block using an aspirator. Collect up to 48 males per experimental condition being tested. Be sure to securely reseal the housing block with the PCR film.
Incubate the males in isolation until they are four days old. Then start the training. To setup the training stage of the experiment, begin by anesthetizing all of the adults from the four day old mating vials.
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 a 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, setup 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.
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 vial. 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 towards 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. The described procedure was used to test the short term and long term memory of flies with a neuron specific RNAi knockdown of dihydroxyacetonephosphate acyltransferase or Dhap-at.
Controls expressed a non specific RNAi targeted to a nemotode zinc finger gene. In both tests, trained controls had a lower median courtship index than their naive counterparts, which is the expected result for normal flies. By comparison, trained Dhap-at knockdown males, were no better than naive flies in the long term memory test.
A quantitative analysis of memory is the learning index score. This index reflects the deficit in long term memory seen in the Dhap-at knockdown males. To demonstrate the variation typically seen between days both tests were run on two or three consecutive days.
Despite day to day variation, it was still possible to reproduce the deficit on each trial day. After watching this video, you should have a good understanding of how to analyze learning, short term memory, and long term memory, using the courtship conditioning paradigm. Once mastered, courtship conditioning can be tested for six to eight genotypes in parallel when either assessing learning, short term, or long term memory.
Once the initial cultures are established it takes about 20 days for the experiments to be completed. Animal behavior can be quite variable. Therefore it is important to eliminate any potential environmental factors including differences in light, temperature, and humidity.
Likewise, it is important to handle the flies in a consistently gentle manner. Once a learning and memory deficit is identified in a specific mutant, you can use additional methods to understand the underlying mechanism. For example, through gene expression analysis, or genetic interaction experiments.
