The Flypub assay measures disinhibited behaviors under the influence of alcohol. Thereby facilitating the mechanistic study of alcohol use disorder. Alcohol is one of the most readily available and widely abused drugs in the world.
The neurobiological mechanism underlying alcohol use disorder, however, is incompletely understood. The fruit fly, Drosophila melanogaster, has been extensively used to study ethanol's effects on euphoria, locomotion, disinhibition, and sedation. It is also used to study its long and lasting changes in behavior relevant to addiction such as tolerance, sensitization, and ethanol seeking.
Many studies in Drosophila and other animal models focus on the locomotor activating effects of ethanol. The distinct effect of ethanol is behavioral disinhibition. However, it's study is rather underrepresented.
To address this key issue, we developed the Flypub assay to measure disinhibited behavior under the influence of ethanol. To assemble the Flypub chamber, take a round bottle and cut off its bottom portion of a 25 milliliter mark with a razor blade. Create a hole at the 50 milliliter mark using a hot soldering iron.
This is the access point where flies will be introduced. Place a round mesh 54 millimeters in diameter at the 75 milliliter mark, and secure it using hot glue. Position a clear plastic sheet 70 millimeters in diameter at the open-bottom end, and secure it using hot glue.
To ensure that the plastic round is firmly attached to the bottom, pressure down using weights. Collect male flies into a group of 33. Place them on the side of a food vial.
Place them in a 25 degree Celsius incubator with at least 50%relative humidity and a 12 hour light, 12 hour dark cycle for 2 days before ethanol exposure. Prepare the Flypub assay station by turning on lights, a computer, and a camera for video recording. Prepare six pubs for a set of experiments.
Label them with a numbers one through six. Gently introduce the flies into the pub using a small funnel. Close the pub using a tape.
Acclimate the flies for 10 minutes. During acclimation, adjust camera focus and record the last five minutes of acclimation. During acclimation prepare cotton pads for ethanol delivery.
Cut a cotton pad into four equal quadrants and then trim the corners to make it fit into a Petri dish. Add a cotton pad to a Petri dish. Add one milliliter of 95%ethanol to the cotton pad.
Make sure for ethanol solution to be evenly distributed on the entire area of the pad. Quickly cover Petri dish with lab wipes. Carefully place an ethanol containing Petri dish under each pub.
Align the pubs on the stage. Begin recording and simultaneously start a timer. Record until flies stop courting or moving.
Remove the ethanol containing Petri dish when over 90%of the flies are sedated. Gently transfer the sedated flies back into food vials. Place them back into the incubator.
Repeat the previous steps every 24 hours for six consecutive days. Open the video using a media player. Then score the number of males engaged in courtship activities every 10 seconds.
Courtship activities to score include following:unilateral wing extension, courtship chain, courtship circle, abdominal bending and mounting. Shown are representative recordings of fly behavior. During acclimation where males do not show much activity.
During first exposure where males exhibit sporadic courtship. And during sixth exposure where males display activity courtship. Enter the number of males displaying courtship in a ten second time block.
Use the maximal number of courting males into three consecutive 10 second time blocks as a representative data point. The average of 10 consecutive data points having the highest value represent the percentage of intermale courtship per pub. The wild type Canton-S exhibit progressively increased activities of disinhibited courtship upon repeated ethanol exposure.
This represents behavioral sensitization to disinhibition effect of ethanol. We examined the role of octopamine in sensitization by testing the flies that are lacking tyramine beta-hydroxylase, T beta H, thus deficient in octopamine. Like Canton-S, the T beta H mutants showed escalated responses to repeated ethanol exposure, but at a reduced level.
When we compared T beta H and Canton-S, we observed that the T beta H mutant males displayed less disinhibited courtship than Canton-S males in each exposure indicating that octopamine may be important for sensitization. Now you can set up, conduct, and analyze behaviors induced by ethanol using the Flypub assay. The key points to keep in mind include, one, proper assembly of the pub.
Two, consistent ethanol delivery, and three, precise and unbiased behavioral score. The Flypub assay has many advantages. It is simple, inexpensive to set up, and easy to learn.
Also, multiple groups of flies can be assayed simultaneously, facilitating this study to identify the effects of genetic or experimental manipulations. The Flypub assay is highly attractable to experimenters at all levels including elementary through high school, undergraduate and graduate students, post docs and faculty. In summary, the Flypub assay will allow you to identify molecular players, cellular pathways, brain circuits, and risk factors that contribute to alcohol use disorder.
