Drosophila Y-maze Assay: A Method to Assess Olfactory Responses in Flies

- A Y-maze consists of a Y-shaped connector with three fly vials connected to it by straight or tapered tubes. Flies move through the maze toward the side with the stimulus that they prefer. To start the experiment, place a tissue paper with the solution containing the volatile chemicals into each vial. Then, connect them to the tapered tubes. The tapered tubes limit the ability of the flies to return to the center of the maze and slows the diffusion of the volatile chemicals.

Next, chill starved flies to slow them down. Do not use chemical anesthesia, as this may affect the outcome of the assay. Quickly connect the vial with the chilled flies to the final side of the maze. Leave the flies to explore the maze. To reduce any preference caused by bright light, carry out this step under far-red light, which flies do not see. Finally, count the flies on each side of the maze. Their location in the maze indicates their preference toward that volatile chemical scent.

In the example protocol, we will see a setup of the Y-maze assay with wild type Drosophila.

- Before conducting the assay the following morning, at the end of the day, transfer cohorts of 10 to 20 experimental lines to glass vials with moisture provided from a wet towel. The flies must be tested in 16 to 18 hours, or they could die due to desiccation or starvation. The next day, assemble the Y-maze. Start by joining the Y-shaped connector to two glass vials using foam connectors with trimmed pipette tips which make the conduits. Their tapering shape makes these one-way passages.

The third branch of the "Y" is attached to a loading tube, made from the large portion of a pipette tip, and allows passage from the loading vial to the choice point of the maze. Beyond each tapering tunnel is a glass vial, one of which must be loaded with an odorant. Load these as 40-microliter boluses soaked into 28-millimeter squares of tissue paper. Which of the two contains the odorant should alternate randomly.

To conduct the test, first cool the flies on ice briefly, so the flies are slowed down. This should be under far-red light from LED bulbs and at 25 degrees Celsius. Gently drop the cohort of flies into the loading vial and load the Y-maze with the test flies. Do not use gas anesthesia.

- It is really critical to transfer the flies to the loading vial both quickly and gently. It is actually the most difficult step of this experiment because flies have to wake up rapidly before the diffusion of odors in the Y-maze.

- The flies should be allowed to explore the maze for 24 hours, so a maximum number of flies move to one or the other glass vial. The number of flies present in the different tubes serves to calculate the olfactory index. Standard statistical methods should be applied to this value, gathered from the behavior of up to 10 flies.

If 10-fly cohorts are not providing consistent results, the trials can be run with 20-fly cohorts. To clean up the maze components, soak them, dismantled, in RBS 35 MD overnight. Follow this with rinses in tap water and a final rinse in DI water before air drying.