Name: A Simple Way to Measure Ethanol Sensitivity in Flies (Video) | JoVE Journal
Uploaded: 2011-02-19T17:00:00.000+00:00
Duration: 11 min 15 s
Description: Learn a simple assay to measure ethanol sensitivity in Drosophila using the loss of righting reflex. This method quantifies median sedation time to compare alcohol responses across fly strains and mutants.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

We thank Aylin Rodan for critical reading of the manuscript, and members of the lab for helpful discussion. This research is supported by the NIH, R01AA019526.

1. Creating the exposure chamber
<ol>
<li>Cut a vial plug in half using a razor blade. Alternatively, use cotton balls.</li>
<li>Push the vial plug half, or the cotton ball, to the bottom of a food vial to make a smooth surface that flies will not get stuck in.</li>
<li>If making multiple exposure chambers, make sure that the vial plug and food take up the same volume in each chamber. A mark on the side of the vial can facilitate this.</li>
</ol>
2. Exposing the flies
<ol>
<li>Collect 8 flies of one sex, age 1-5 days, the day before exposure into regular food vials without yeast.</li>
<li>Label them in code, such that the experimenter is blinded to the actual genotype/strain used.</li>
<li>Transfer the eight flies into the exposure chambers by tapping. Do not anesthetize.</li>
<li>Add a small amount of food dye, e.g. acid blue 9, to 200 proof ethanol to make it easily visible.</li>
<li>Coat the bottom of a vial plug, or cotton ball, with 0.5 ml ethanol.</li>
<li>Insert the ethanol plug into the exposure vial. Ensure that the alcohol is facing into the vial, and not towards the wall of the vial.</li>
<li>Start a timer counting up.</li>
</ol>
3. Record ethanol-induced intoxication
<ol>
<li>Each minute after the timer is started, tap the exposure chamber on the bench to startle the flies and observe them for ten seconds.</li>
<li>Record the number of stationary flies for each minute. Flies are stationary if they cannot get off their backs, remain in one location, or rapidly vibrate their wings throughout the entire ten-second observation-period. The latter flies are considered stationary because the vast majority of these flies are stationary between periods of rapid wing movement. Flies are NOT stationary if they can get off their backs and walk away and/or up the vial wall.</li>
<li>An exposure trial is finished when 4 or more flies are recorded as stationary.</li>
<li>Discard flies and let the exposure chamber air out for later reuse.</li>
</ol>
4. Determine the time to 50% sedation

The time to 50% sedation (ST50) is the time after the start of the ethanol exposure when four of the eight flies remain stationary. This is determined as follows: 
Either: the first measurement minute when exactly four flies are stationary. 
Or: if less than four flies (Y1) are stationary at one time point (minute X), and more than four (Y2) at the next measuring time point (minute X+1), then linearly interpolate the ST50 using the formula: 
ST50 = (4 - Y1)/(Y2 - Y1) + X


5. Repeat exposure to determine tolerance

Exposing the flies
<ol>
<li>Determine the ST50(wt) for the wild-type control strain used in your experiment.</li>
<li>Expose the experimental groups of flies to ethanol as described above.</li>
<li>Measure their ST50(1), but continue to expose until you reach the time 2x ST50(wt).</li>
<li>Transfer the flies back into an empty food vial, and hold for 4 hours.</li>
<li>Re-expose the flies, and determine the ST50(2) of exposure 2.</li>
</ol>
Determine tolerance
Calculate tolerance in % as (ST50(2)/ ST50(1) - 1)x100.

6. Representative Results:
Shown here, are some representative results we obtained with 6-8 vials of flies per genotype and dose. If your standard error is substantially larger, then a systematic problem likely occurred. Make sure that the ethanol faces into the exposure chambers, and that the chambers are the same volume.
 
<img src="/files/ftp_upload/2541/2541fig1.jpg" alt="Bar graph comparing ST50 times across different ethanol concentrations by gender." data-altupdatedat="1788235205000" data-alt="Figure 1"> 
Figure 1. Dose response curve of wild type flies exposed to increasing percentages of ethanol. Eight vials of flies were exposed per dose and sex. Male and female flies show the same ST50 at each dose (p&gt;0.22, 2-way ANOVA), while the ST50 across doses decreases significantly with increasing ethanol dose (p&lt;0.001). Note that flies from over-crowded bottles can be substantially smaller than those from healthy bottles, and this size difference can affect the ST50. Seed each bottle with 15-20 females laying for 1-2 days to get optimal larval density per bottle.
<img src="/files/ftp_upload/2541/2541fig2.jpg" alt="Bar graph comparing ST50 values for wild type and white rabbit at 90% and 100% ethanol concentration." data-altupdatedat="1788235205000" data-alt="Figure 2"> 
Figure 2. Exposing flies to either 90%, or 100% ethanol reproduces the previously described ethanol-resistance caused by the white rabbit1 mutation2 (p&lt;0.001, 2-way ANOVA).
<img src="/files/ftp_upload/2541/2541fig3.jpg" alt="Bar chart comparing ST50 time after exposure 1 and 2 (t+4 hrs); data analysis result." data-altupdatedat="1788235205000" data-alt="Figure 3"> 
Figure 3. Repeat exposure to 100% ethanol, spaced by a 4 hour recovery interval, causes a significant increase in ST50 after the second exposure (p&lt;0.001, paired t-test). Note that the flies were exposed for 24 minutes during the first exposure (corresponding to twice their na ve ST50). Exposure for a shorter amount of time (12 minutes) did not cause a significant increase in ST50 upon re-exposure (data not shown).

<ol><li>Bainton, R. J., Tsai, L. T. -Y., Singh, C. S., Moore, M. S. Curr. Biol. 10, 187-194 (2000).</li>
<li>Rothenfluh, A., Threlkeld, R. J., Bainton, R. J., Tsai, L. T. -Y. Cell. 127, 199-211 (2006).</li>
<li>Devineni, A. V., Heberlein, U. Curr. Biol. 19, 2126-2132 (2009).</li>
<li>Scholz, H., Ramond, J., Singh, C. S., Heberlein, U. Neuron. 28, 261-271 (2000).</li>
<li>Allen, M. J., Godenschwege, T. A., Tanouye, M. A., Phelan, P. Semin. Cell Dev. Biol. 17, 31-41 (2006).</li>
<li>Feany, M. B., Bender, W. W. Nature. 404, 394-398 (2000).</li>
</ol>

No conflicts of interest declared.

In this presentation, we have outlined a simple way to determine ethanol-induced intoxication based on previous assays described by Bainton et al. 20001, and Rothenfluh et al. 20062. Since alcohol-induced sedation is correlated with alcohol drinking preference3, this assay is relevant for the study of alcohol use disorders. The assay described is based on startle-induced negative geotaxis, and how an alcohol exposure interferes with this innate response. Before starting the exposures, make sure all your flies show a normal response to such a startle, i.e. after tapping down the flies in the food vial, they should all move up from the bottom within a 10 second observation time. Examples of flies having reduced negative geotaxis include bang-sensitive seizure mutants5, and locomotion-deficient flies caused by degeneration of dopamine neurons6.
Even though the described assay is relatively simple, two variables merit the most attention to ensure reproducible results. First, all exposure vials should be the same volume. This includes making sure that the bottom of the vial contains the same volume of food + vial plug/cotton ball, as well as inserting the ethanol-containing plug the same distance for every vial. A completely empty vial can be used for this assay, but it leads to longer ST50 values, decreasing the throughput of the assay.
This assay is also suitable to determine the amount of tolerance flies develop upon repeat ethanol exposure. A number of variables should be considered when doing repeat exposures. First, the inter-exposure interval is here described as 4 hours. This variable can be changed, but we recommend it not be lower than 2 hours, to ensure clearance of ethanol from the first exposure. See Scholz et al. 20004 for the kinetics of ethanol tolerance development and decay. The second variable to consider is the duration of the first exposure. This obviously has to be the same for the control and the experimental flies. However, this may not result in the same amount of ethanol delivered to both these fly strains. Sedated flies absorb more ethanol than active flies2, therefore a strain that sedates earlier may receive a higher dose than a later-sedating strain, and thus develop more tolerance due to a higher initial dose. We strongly caution the reader when comparing tolerance development of strains that show differences in the initial ST50(1).
The assay we describe here is a useful tool in easily and reproducibly determining flies behavioral responses to alcohol.

<table><tbody><tr><td>Exposure vials</td><td>www.flystuff.com</td><td>32-116</td><td></td></tr><tr><td>Plugs</td><td>www.flystuff.com</td><td>49-102</td><td></td></tr><tr><td>Acid Blue 9</td><td>Sigma-Aldrich</td><td>861146</td><td></td></tr></tbody></table>

a simple way to measure ethanol sensitivity in flies

A Simple Way to Measure Ethanol Sensitivity in Flies

Low doses of ethanol cause flies to become hyperactive, while high doses are sedating. The sensitivity to ethanol-induced sedation of a given fly strain is correlated with that strain s ethanol preference, and therefore sedation is a highly relevant measure to study the genetics of alcohol responses and drinking. We demonstrate a simple way to expose flies to ethanol and measure its intoxicating effects. The assay we describe can determine acute sensitivity, as well as ethanol tolerance induced by repeat exposure. It does not require a technically involved setup, and can therefore be applied in any laboratory with basic fly culture tools.

A simple assay to measure the sedating effects of ethanol on Drosophila flies, based on the loss of righting reflex, is described.

Low doses of ethanol cause flies to become hyperactive, while high doses are sedating. The sensitivity to ethanol-induced sedation of a given fly strain ...

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.

a-simple-way-to-measure-ethanol-sensitivity-in-flies

Research

JoVE Journal

Neuroscience

Drosophila has emerged as a significant model system for dissecting the cellular and molecular underpinnings of behavioral responses to alcohol. Here we present a protocol for the collection of alcohol sensitivity data in a circadian context that can be easily applied to other experiments and is well-suited for undergraduate research.

The FlyBar: Administering Alcohol to Flies

Straightforward assays for measuring ethanol sensitivity and rapid tolerance in Drosophila facilitate the use of this model organism for investigating these important ethanol-related behaviors. Here, a relatively simple, scalable assay for measuring ethanol sensitivity and rapid tolerance in flies is described.

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

C. elegans is a useful model for studying the effects of ethanol on behavior. We present a behavioral assay that quantifies the effects of ethanol on the locomotion speed of crawling worms; both initial sensitivity and the development of acute functional tolerance to ethanol can be measured with this assay.

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

The CApillary FEeder (CAFE) assay is a simple, budget-friendly, highly reliable method for investigating mechanisms underlying food intake. Used with the highly versatile genetic model organism Drosophila melanogaster, it provides a powerful means of gaining new insights into regulatory mechanisms of food intake.

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

Current methods to measure alcohol sensitivity in Drosophila are designed to test groups of flies. We present a simple, low-cost, high-throughput assay for assessing alcohol sedation sensitivity in large numbers of single flies. The method does not require specialized tools and can be performed in any laboratory using common materials.

High-Throughput Method for Measuring Alcohol Sedation Time of Individual Drosophila melanogaster

The Flypub assay measures the behaviors that the fruit fly Drosophila melanogaster displays under the influence of ethanol. The assay can be readily mastered by experimenters at all levels and applied to various vaporized stimuli, facilitating substance abuse and addiction studies.

Flypub To Study Ethanol Induced Behavioral Disinhibition and Sensitization

The Vermicelli and Capellini Handling Tests of forepaw dexterity take advantage of the natural inclination of rodents to manipulate food items using skillful forepaw and digit movements. Animals are videotaped while handling short strands of uncooked dry pasta. Slow motion video playback allows for the quantification of forepaw adjustments.

The Vermicelli and Capellini Handling Tests: Simple quantitative measures of dexterous forepaw function in rats and mice

Mice and rats, due to their innate cautiousness, are initially slow in consuming a novel food, particularly in a novel place. This hyponeophagia can readily be measured in the laboratory, even though laboratory animals are much less anxious than their wild...

Hyponeophagia: A Measure of Anxiety in the Mouse

A method to determine the time course of ethanol concentration in the brains of rats during operant ethanol self-administration is described.  Gas chromatography with flame ionization detection is used to quantify ethanol in the dialysate samples, because it has the sensitivity required for the small volumes that are generated.

Microdialysis of Ethanol During Operant Ethanol Self-administration and Ethanol Determination by Gas Chromatography

Learn a simple assay to measure ethanol sensitivity in Drosophila using the loss of righting reflex. This method quantifies median sedation time to compare alcohol responses across fly strains and mutants.

A Simple Way to Measure Ethanol Sensitivity in Flies

Explore More Videos