Drosophila Y-maze Assay: A Method to Assess Olfactory Responses in Flies
Transcrição
– A Y-maze consists of a Y-shaped connector with three fly vials connected para 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 para the tapered tubes. The tapered tubes limit the ability of the flies para return para the center of the maze and slows the diffusion of the volatile chemicals.
Next, chill starved flies para 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 para the final side of the maze. Leave the flies para 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 de 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 para 20 experimental lines para glass vials with moisture provided from a wet towel. The flies must be tested de 16 para 18 hours, or they could die due para desiccation or starvation. The next day, assemble the Y-maze. Start by joining the Y-shaped connector para 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 para a loading tube, made from the large portion of a pipette tip, and allows passage from the loading vial para 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 para transfer the flies para 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 para explore the maze for 24 hours, so a maximum number of flies move para one or the other glass vial. The number of flies present de the different tubes serves para calculate the olfactory index. Standard statistical methods should be applied para this value, gathered from the behavior of up para 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 de tap water and a final rinse de DI water before air drying.
