An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila

The overall goal of the following experiment is to assess the sensitivity to ethanol sedation in fruit flies. This is achieved by placing flies in vials and exposing them to ethanol vapor. The second step is to determine the number of flies that are sedated in each vial at recurring time points until all of the flies are sedated.

Next, the time point is interpolated. When 50%of the flies are sedated for each vial, the results show an index of the time required to sedate fruit flies with ethanol demonstrating this procedure today will be Simran Sindu, a student in my laboratory. To begin the experiment, collect flies in fresh food vials in groups of 11 of a single sex for up to five minutes of CO2, allow the flies to recover overnight in food vials in an environmentally controlled space.

Prepare ethanol solution by diluting pure 100%ethanol in purified water to a final concentration of 85%Allow the solution to return to room temperature overnight before the assay prepare a clean, empty food vial, which will serve as the testing vial. A new cellulose acetate plug, a silicone stopper and one milliliter of ethanol solution for each vial of flies to be tested, adjust the room temperature to 20 to 25 degrees Celsius and the relative humidity to 55 to 65%Designate another worker to assign a unique code to each group of vials and record the code for later place coded vials with flies in the testing room to acclimate for a few minutes. Next, label the empty testing vials to match the codes on the fly vials.

Construct a hard copy testing log by entering the codes into columns with one column per vial in a spreadsheet. Using the testing log as a guide. Arrange the coded food vials with flies and the empty testing vials into matching arrays in the testing room, transfer the flies from the food vials into the matched and labeled testing vials, one at a time.

Then immediately insert cellulose acetate plugs into the testing vials until the cellulose acetate plugs are two centimeters below the vial tops. For the time zero assessment. Grasp each vial individually by the thumb and forefinger.

Tap gently on the table three times to knock the flies to the bottom of the vial. Wait 30 seconds and count the number of flies that are immobile and or dead. Record the number of immobile dead flies for each vial at time.

Zero minutes in the hard copy testing log. Start the timer at time zero and immediately add one milliliter of ethanol to the cellulose acetate. Plugs in a circular motion at five second intervals for the first set of four vials in the order.

They will be tested when the ethanol has been added to all four testing vials in the set. Insert a silicone plug in each vial to seal it at 1, 2, 3, 4, and five minutes. Add one milliliter of ethanol to the second, third, fourth, fifth, and sixth sets of four vials respectively.

Continue inserting silicone stoppers after adding ethanol to each set of four vials LS at six minutes. Test the first set of four vials by grasping each vial with a thumb and a forefinger and tapping gently on the table three times to knock flies to the bottom of the vial. Wait 30 seconds, then count and record the total number of flies that are sedated.

Score flies as sedated if they, one, stand on the floor of the vial, but do not walk or two lie on their backs with or without flapping their wings. Handle each vial within the set at five second intervals using this schedule at time. 7, 8, 9, 10, and 11 minutes.

Test the second, third, fourth, fifth, and sixth sets of vials respectively as done for the first set at 12 minutes. Test the first set of four vials again and continue testing the second, third, fourth, fifth, and sixth sets of vials at 13, 14, 15, 16, and 17 minutes respectively. Continue testing the flies until they are all sedated.

Enter the total number of flies in each vial in the hard copy testing. Log sensor immobile or dead flies at time zero from the total number of flies. Raw data from the ethanol sedation assay were converted to the percent of active flies as a function of time.

Sensitivity to ethanol sedation from the primary data was quantitated as sedation time 50 or ST 50. The time required for 50%of the flies to become sedated or area under the curve or a UC.The assay detects the effects of RNAi against calnexin 14 D in the nervous system, which blunted ethanol sensitivity relative to controls, mutation of scab and rouser, enhanced ethanol sedation sensitivity and mutation of happy hour. Blunted ethanol sedation sensitivity compared to controls Once mastered.

This technique can be done in one and a half hours if performed properly.