Four-Way Olfactometer Assay: A Method to Assess Odorant-Cued Responses in Drosophila

- A four-way olfactometer is a closed chamber with one-way ports at each corner, through which it can deliver up to 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 to clean air and observe their movements to make sure that no odorants are contaminating the olfactometer and affecting fly behavior. Then, prepare the test odorants in separate vials and connect them to the main chamber through the one-way ports. Expose the flies to the odorants for a pre-defined period, removing the stimulus after the trial has ended.

Flies tend to avoid quadrants with repellent odors while moving towards and settling in 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 to analyze fly behavior in response to ethyl propionate, a repellent odorant, and apple cider vinegar, which is attractive.

- At least five minutes in advance, switch on the temperature controller and set it to 25 degrees Celsius. Then, connect the odorant chambers to the arena via a plastic tube. Check the airflow rate in 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 to 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 to three times and allow them to fully air-dry before proceeding. Next, clamp the glass plates to the arena. Then, transfer the flies into the arena without anesthesia. Using gravity, let them enter through the hole in 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 to each arena corner. Close the door of the chamber and let the flies acclimatize to the new environment for 10 to 15 minutes.

After the acclimation period, run a 5 to 10 minute control experiment in which flies are exposed to four control air streams. It is essential to now analyze the data immediately. If the flies are not evenly distributed in the arena, the arena must be reset.

- It is critical to test the behavior of control flies to 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. Continue 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 to the setup using a three-way valve, or reconnect the tubing and run a test experiment for 5 to 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 to continually flush the system. For efficiency, it is good to have two arenas so cleaning can be done during runs.

- If several experiments are run on the same day, pay extreme care to ensure that no odor is left in 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 to a 24-hour gap between experiment runs may be needed.