Four-Way Olfactometer Assay: A Method to Assess Odorant-Cued Responses in Drosophila
Transcrição
– A four-way olfactometer is a closed chamber with one-way ports at each corner, through which it can deliver up para four odors, creating quadrants with distinct odor profiles. Begin an experiment by transferring awake flies into the chamber. Avoid anesthesia, as this may affect their behavior during the experiment.
Expose the flies para clean air and observe their movements para make sure that no odorants are contaminating the olfactometer and affecting fly behavior. Then, prepare the test odorants de separate vials and connect them para the main chamber through the one-way ports. Expose the flies para the odorants for a pre-defined period, removing the stimulus after the trial has ended.
Flies tend para avoid quadrants with repellent odors while moving towards and settling de quadrants with attractive odors. Measure the flies' odor-cued behavioral responses by tracking their positions over time as they explore the olfactometer.
In the following protocol, we will use a four-quadrant olfactometer with an air delivery system para analyze fly behavior de response para ethyl propionate, a repellent odorant, and apple cider vinegar, which is attractive.
– At least five minutes de advance, switch on the temperature controller and set it para 25 degrees Celsius. Then, connect the odorant chambers para the arena via a plastic tube. Check the airflow rate de each quadrant of the arena using an electronic flow meter. Make sure that the control and odor and air streams are both running at 100 milliliters per minute.
– Flies are very sensitive para airflow, and it is critical that the airflow for each quadrant is verified before each experiment.
– Now, clean the arena and the glass plates using 70% ethanol. Wipe all the parts down two para three times and allow them para fully air-dry before proceeding. Next, clamp the glass plates para the arena. Then, transfer the flies into the arena without anesthesia. Using gravity, let them enter through the hole de one of the glass plates, and then cover the hole with a circular mesh.
Now, place the fly-loaded arena into the light-tight chamber. Then, connect the four control air streams para each arena corner. Close the door of the chamber and let the flies acclimatize para the new environment for 10 para 15 minutes.
After the acclimation period, run a 5 para 10 minute control experiment de which flies are exposed para four control air streams. It is essential para now analyze the data immediately. If the flies are not evenly distributed de the arena, the arena must be reset.
– It is critical para test the behavior of control flies para just clean air. This will quickly verify that all background conditions are normal. Leaking light, temperature imbalance, tilted arena, or an odor contamination can all cause problems.
– If necessary, discard the flies and clean the arena again. If an odorant contamination is suspected, replace all the tubing. Continuar repeating the control test with new cohorts until the animals show no preference for any of the four quadrants. Then, connect the test odorant chamber para the setup using a three-way valve, or reconnect the tubing and run a test experiment for 5 para 10 minutes. For longer experimental recordings, rapidly stop and restart the tracking program every 20 minutes, or the data files will be too large.
Between tests, discard the flies, clean the arena and glass plates with 70% ethanol, and replace the connector tubes. Keep the dry airflow going para continually flush the system. For efficiency, it is good para have two arenas so cleaning can be done during runs.
– If several experiments are run on the same day, pay extreme care para ensure that no odor is left de the system from a previous run. This is normally not a problem with low concentrations of odorant or with CO2. But for highly concentrated stimuli, up para a 24-hour gap between experiment runs may be needed.
