Hi, I'm Thomas Maples from the lab of Dr.Adrian Rothen flu in the Department of Psychiatry at UT Southwestern Medical Center in Dallas. Today I will be demonstrating a simple procedure for determining the sensitivity of flies to ethanol deed sedation. This assay is based on flies, loss of riding reflex and walking ability after exposure to ethanol.
This measure can be used to determine alcohol sensitivity differences between different strains of drosophila and mutants. We can also use this assay to induce tolerance in flies, tolerance being a reduced response to alcohol after repeat exposure. Let's get started.
For This experiment, you Will need the following vials. With eight fruit flies, a piece that you have collected one or two days before. You can do the exposures with either males or females.
However, each vial should contain only one sex. An empty food vial that does not contain flies a razor, two vial plugs made of a cotton like material. If you do not have these kind of buzz plugs, you can also use cotton balls, a 1000 microliter micro pipette with tips, 200 proof ethanol that contains food dye you can get at any grocery store and A timer.
The first thing we need to do Is make an exposure chamber in which we'll expose the flies to ethanol and monitor their mobility. To create this chamber first, cut a vial plug in half using a razor blade. Then push one of the halves to the bottom of a vial covering the food.
This creates a level nonsticky surface for the flies to walk on. If you use a cotton ball, press it down smoothly and try to make it as un fuzzy as possible so that the flies don't get stuck in the cotton threads. If you are making multiple chambers, make sure that the food and vial plug take up the same amount of space in each chamber.
This ensures that the flies in each chamber are exposed to ethanol in the same volume. With practice, it is easy to expose three vials of Flies in parallel. Once the exposure Chambers are made, we can now expose the flies to ethanol.
First transfer a vial of eight flies into an exposure chamber, sealing the chamber with the plug from the vial that originally contained the flies. Next pipette 500 microliters of dyed ethanol of your chosen concentration onto one end of a cotton like vial plug. Then replace the plug of the exposure chamber with the ethanol coated plug, inserting the end coated with ethanol into the chamber.
Immediately following the addition of the ethanol coated plug to the exposure chamber, start a timer. The dye in the ethanol helps ensure that the ethanol faces into the exposure chamber and not the wall of the vial. This is especially important if you use cotton balls as opposed to buzz plugs because any side of a cotton ball can face into the vial.
In this exposure, we are using 100%alcohol, which is now diffusing into the exposure chamber where the flies are inhaling it From the air. Each minute after adding The ethanol coated plug to the exposure chamber, we will count how many flies have become stationary, meaning they're unable to turn themselves over and walk. The longer the flies remain mobile, the more resistant they are at each one minute interval.
Startle the flies by tapping the chamber on the bench to ensure that all flies that can move are moving. Observe the flies for 10 seconds after tapping them to give them time to begin moving or turn themselves over for each one minute interval. Document how many of the eight flies are stationary at the beginning of the experiment.
Most flies will quickly run up the side of the vial after being startled. After about six minutes of 100%at ethanol exposure, they start moving sluggishly and sometimes erratically by 12 minutes. Most flies will be stationary, being unable to walk or turn themselves over from lying on their backs.
Only flies that are able to write themselves And walk should be counted as mobile flies that merely move their legs or wings in place should be counted as stationary flies that remain on their backs for the entire observation period should also be counted as stationary because their inability to write themselves is a sign of intoxication. Flies that vigorously vibrate their wings throughout the entire ten second observation, time should likewise be counted as stationary, even if they change location. The vast majority of these flies are stationary between periods of rapid wing movement.
In summary, flies that are able to turn themselves over and walk are mobile flies that are unable to turn over, move their legs and wings in place, or rapidly vibrate their wings are stationary. If we apply these rules, we see that more wild type flies become stationary as the minutes of exposure to ethanol Vapors increases. The value that This assay uses to quantify ethanol sensitivity is median sedation time or MST.
We define MST as the minutes of ethanol exposure required for four of the eight flies in the exposure chamber to become stationary. This value is easily determined if exactly four flies are stationary at a specific observation period. For example, if exactly four flies are stationary after 11 minutes of exposure, then 11 is the MST.
If exactly four flies are stationary after 11 minutes of exposure and by 12 minutes the same is true, then take the first value as it is the time the flies first reach their ST.If four flies are not stationary at a specific observation period, it is necessary to linearly interpolate the time at which four flies became stationary. For example, if the number of flies stationary jumps from three to five, between nine and 10 minutes of exposure, it can be graphically determined that the MST is 9.5. The following data that was Collected using this assay shows that this experimental design is an accurate way of measuring ethanol sensitivity.
An N of six to eight vials was used in the following experiments, which is indicated within the bars. The MST of male wild type flies exposed to 100%Ethanol is roughly 11 minutes with a low standard error. Day to day variability is low and mostly not statistically significant, but we still recommend that you measure your experimentals and controls in parallel on the same day.
Also note that the flies exposed on day A and day B are not the same individuals. These are siblings representing the same genetic stock male and female flies exposed to 100%Ethanol show no difference in MST. Still, we recommend sticking to one sex.
If we expose male and female flies to increasing doses of ethanol, we get a nice dose response curve that shows a statistically significant decrease in MST as a function of increased ethanol concentration. This assay also reproduces previously published phenotypes that were measured with a more elaborate ethanol vaporization setup flies with the right rabbit one mutation showed a significant increase in MST. This increase was observed at 100%as well as 90%Ethanol.
Using this assay, We can also induce and measure ethanol tolerance and flies. Tolerance is defined as a reduction in the response to alcohol, following a pre-exposure to alcohol to induce tolerance. Expose a vial of flies to ethanol for roughly twice their MST.
For instance, we expose these male Berlin wild type stock to 100%ethanol for 24 minutes. After exposing them, gently tap the eight flies back into their food vial where you will let them recover for four hours. Record the flies original MST on their vial after four hours of recovery, re-expose the very same flies to the same dose of ethanol and record their second MST.
As you can see on this graph, the MST of the second exposure to ethanol is significantly higher than the MST of the first exposure, indicating that the flies have acquired tolerance to the sedating effects of ethanol. A measure for tolerance is calculated by dividing the second MST of a given vial of flies. By the first MST of the same flies and subtracting one, the value is expressed as percent tolerance, which in our case is about 50%Thanks for watching and good luck with your experiment.
