The overall goal of this procedure is to measure associative memory using the adult drosophila olfactory shock assay. This is accomplished by first collecting flies under carbon dioxide anesthesia. The second step is to expose the flies simultaneously to an electric shock and a first odor A in the team A.Next, the flies are exposed to the second odor B without a shock.
Finally, the flies are taken to the choice point in the team a's where they are exposed to both odor A and B simultaneously after two minutes of distribution time, the flies that chose odor A and those that chose odor B are collected separately and counted under carbon dioxide. This allows calculation of the performance index for each odor. The final PI is obtained by averaging the PI for two separate experiments, one in which odor A is shock paired, and the second in which odor B is shock paired.
This method can provide insight into the effect of the internal state of the animal and memory. For instance, by varying its age, diet and sleep, it can also be applied to other systems. For instance, it is analogous to rodent or factory mediated fear conditioning assays with the genes and mechanisms of learning being highly conserved between insects and mammals.
Visual demonstration of this method is critical as preparing the flies for the essay and then moving the flies into within and out of the teammates is difficult to learn and not easy to appreciate how to do from just a written protocol. To begin grow flies on a standard food diet under a 12 hours light dark cycle at 25 degrees Celsius, one to two days before the experiment, anesthetize the flies with carbon dioxide and separate them into four groups of 25. Store the flies overnight in food vials without yeast at 25 degrees Celsius and 70%humidity in an environmentally controlled room under a 12 hours light dark cycle.
Next, prepare a custom Perspex team MAs and regularly check the tube fitting to ensure that an airtight seal is secured during the experiment place custom made copper grids inside the training tubes. Clean the grids regularly and replace them if they become oxidized. Use crocodile clips to attach copper grids to wires running to a switch box connected to an electric stimulator.
Then apply a volt meter to ensure that the apparatus is delivering the required shock. Next, hold the maze tightly with G clamps. To avoid any air leakage, attach the T maze to tubing running to an air pump, allowing odors to be drawn across the flies and removed from the team.
A maintain mild airflow at two liters per minute, then dilute four methyl cyclo ethanol or MCH at a one to 67 ratio and three octal or OCT at a one to 100 ratio in mineral oil. Next, pipette 30 microliters of the diluted odor into the odor cup. Prepare a custom made odor cup and cover an odor block with a plastic tube with a perforated top.
Place the custom made odor cup into the odor block. This mechanism allows air to be drawn over the odor in the cup exposing flies to an odor plume. Perform all adult olfactory shock conditioning experiments under a dim red light, which allows the researcher to see, but prevents the fly from seeing and ensures that the flies concentrate on all faction as opposed to visual inputs.
Next, introduce the flies into the training tube attached to the TMAs and allow them to adapt to the tube and the airflow for 90 seconds. Present the first odor MCH with a 60 volt shock consisting of 12 1.25. Second pulses with 3.75 second interpulse intervals for a total duration of 60 seconds.
Follow the shock with a 32nd rest period. Present the second odor of OCT for 60 seconds without a shock. Follow the shock with a 32nd rest period.
Then turn the teammates on its side and gently tap the bottom on a mouse mat to move the flies from the training chamber into the central chamber. After maintaining the flies in the central chamber for 90 seconds, fit the choice tubes into the bottom of the apparatus to form the team A to measure learning. Move the flies to the choice point of the teammates and simultaneously expose them to both odors for 120 seconds.
Trap the flies in the choice tubes by sliding the central chamber up. Then collect the flies from the central compartment and each arm of the teammates into separate food vials. Clearly labeling what the flies are and their odor choice on the vial, and then counting them to measure memory.
Collect the flies after training and transfer them from the teammates to food vials without yeast. Store flies in the dark at 25 degrees Celsius and 70%humidity as described in the accompanying text protocol. Then reintroduce the flies to the team aze.
For long-term memory. Use a custom built maze that allows several batches of flies to be trained simultaneously. Administer five cycles of training.
Maintain the flies at 18 degrees Celsius and 70%humidity in the dark until testing. Prior to testing. Move the flies to 25 degrees Celsius and allow them to acclimatize for at least one hour.
Assess long-term memory. 24 hours after training. After the behavioral experiments, clean the odor cups with hot water and odorless detergent.
After drying, apply 10 microliters of sigma coat to the odor cups. Then heat the odor cups in a microwave oven to solidify the sigma coat. Perform odor acuity by introducing 100 flies into the team aze after 90 seconds, move the flies to the choice point and allow them two minutes to choose between pure odors and air.
Then collect and count the flies. For shock reactivity. Introduce the flies into the shock chamber.
After 90 seconds of rest, administer a 60 volt DC electric shock. Some of the flies will escape to a similar tube without a shock. Give the flies two minutes to choose.
Then collect and count the flies that have escaped the shock tube to elucidate learning effects. A series of experiments were performed with Canton s wild type adult flies and learning in DS mutant adult flies. Duns flies show a reduction in learning compared to wild type flies.
While attempting this procedure, it's important to remember to keep track of exactly which flies are which and what has been done to them. Beware that flies can easily be stressed while being prepared, trained and tested. Make sure you minimize stress and are consistent.
Don't forget that Joslin earning can show day-to-day variations. Therefore, take the precaution of performing your control and experimental procedures at approximately the same time of day and under the same conditions. If your controls stop learning, try to analyze what has been done different compared to the days the experiment works.
Once you isolate the variable, try to be more consistent with that variable when you perform the memory assay. In future, good luck.
