These studies were supported by grants from the National Institutes of Health, National Institute for Alcoholism and Alcohol Abuse to M.G. (P20AA017828, R01AA020634, P50 AA022537). The authors thank Jill Bettinger for helpful discussions and Jacqueline DeLoyht for technical assistance.

1. Day Before Assay
<ol>
	<li>Collect flies into fresh food vials in groups of 11 (single sex) under brief (1-5 min) CO2.</li>
	<li>Allow flies to recover O/N in food vials in an environmentally controlled space (typically 25 &#176;C, 60% relative humidity, 12 hr light/dark cycle).</li>
	<li>Prepare ethanol solution(s) by diluting pure (100%) ethanol in purified (&#8805;18 M&#937;) water to final concentration(s) appropriate for the planned experiment. Allow solution(s) to return to RT O/N. 
	Note: Dilution of ethanol is exothermic.</li>
</ol>
2. Day of Assay
<ol>
	<li>For each vial of flies to be tested, prepare (i) a clean, empty food vial; i.e., testing vial, (ii) a new cellulose acetate plug, (iii) a silicone stopper and (iv) 1 ml of ethanol solution (see Table 3).</li>
	<li>Prepare testing room by adjusting temperature to 20-25 &#176;C and relative humidity to 55-65%.</li>
	<li>Have another worker assign a unique code to each group of vials and record the code for later. Place coded vials with flies in testing room to acclimate for a few min.</li>
	<li>Label empty testing vials to match codes on fly vials from 2.3.</li>
	<li>Construct a hard copy testing log by entering the codes into columns (one column/vial) in a spreadsheet similar to Table 1.</li>
	<li>Using the testing log as a guide, arrange coded food vials with flies and empty testing vials into matching arrays in the testing room. 
	Note: A reasonable maximum number of vials to test is 24 (i.e.,&#160;6 sets of 4 vials each).</li>
	<li>Transfer flies from food vials into matched/labeled empty testing vials and immediately insert cellulose acetate plugs into testing vials until cellulose acetate plugs are 2 cm below the vial tops.</li>
	<li>Hereafter, handle each row of four vials as a set at staggered 1 min&#160;intervals.</li>
	<li>For the time 0 assessment, grasp each vial individually with thumb and forefinger, tap gently on the table three times to knock flies to the bottom of the vial, wait 30 sec and then count the number of flies that are immobile/dead. Record the number of immobile/dead flies for each vial at time 0 min in the hardcopy testing log.</li>
	<li>Start timer counting up continuously at time 0 and immediately begin adding 1 ml of ethanol to cellulose acetate plugs in the vials for the first row/set of 4 vials. Add 1 ml of ethanol to the cellulose acetate plugs in the vials at 5 sec intervals in the order they will be tested. Add ethanol to the cellulose acetate plugs in a circular motion so that the ethanol is absorbed evenly throughout the cellulose acetate plugs. When ethanol has been added to all 4 testing vials in the set, insert a silicone plug in each vial to seal it.</li>
	<li>At times 1, 2, 3, 4 and 5 min, add 1 ml of ethanol to the second, third, fourth, fifth and sixth sets of 4 vials, respectively. Continue inserting silicone stoppers after adding ethanol to each set of 4 vials.</li>
	<li>At time 6 min, test the first set of 4 vials by grasping each vial with thumb and forefinger, tapping gently on the table three times to knock flies to the bottom of the vial, waiting 30 sec and then counting and recording the total number of flies that are sedated. Score flies as sedated if they (i) stand on the floor of the vial but do not walk or (ii) lie on their backs with or without flapping their wings.</li>
	<li>Handle each vial within the set at 5 sec intervals using the schedule in Table 2.</li>
	<li>At times 7, 8, 9, 10 and 11 min, test the second, third, fourth, fifth and sixth sets of vials, respectively, as done for the first set.</li>
	<li>At time 12 min, test the first set of 4 vials again as described in 2.12 and continue testing the second, third, fourth, fifth and sixth sets of vials at 13, 14, 15, 16 and 17 min, respectively.</li>
	<li>Continue testing flies as described in 2.12 until all flies are sedated.</li>
	<li>Enter the total number of flies in each vial in the hard copy testing log. Censor immobile/dead flies at time 0 from the total number of flies.</li>
	<li>Calculate the percent active flies at each assessment time-point and plot data as % active flies (y-axis) vs. time (x-axis). Quantitate ethanol sedation by interpolating Sedation Time 50 values (ST50, time to 50% sedation) from third-order polynomial or sigmoidal curve fits or calculating area under the curve.</li>
	<li>Compile data from decoded vials and perform statistical analyses (e.g.,&#160;one-way ANOVA with Bonferroni multiple comparison test) as appropriate for the experimental design.</li>
</ol>

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The authors have nothing to disclose.

Straightforward assays that reproducibly quantitate meaningful phenotypes are of great value for the analysis of behavior. The work described here addresses several practical aspects of an assay for measuring ethanol sedation sensitivity and rapid tolerance in Drosophila. Although not a focus of this work, behavioral analyses are facilitated by maintaining the environment and genetic background constant for test subjects within a study. Furthermore, comparisons should typically be made between groups of flies re...

Alcohol use disorder (AUD) is an enormous health problem worldwide (reviewed in 1). Although the mechanisms driving the development of AUD are complex, these disorders have a major genetic component (e.g., 2). The large heritability of AUD&#160;and the conserved behavioral responses to ethanol across many species (reviewed in 3,4) have generated strong interest in using genetic model organisms to investigate the involvement of specific genes in ethanol-related behaviors toward better understanding the molecular basis of AUD. The fruit fly, Drosophila melanogaster, has emerged as a leading model organism for exploring molecular-genetic mechanisms of ethanol-related behaviors (reviewed in 3,4). Studies in flies have highlighted roles for several signaling pathways in behavioral responses to ethanol (reviewed in 5). Intriguingly, some of the genes and pathways that influence behavioral responses to ethanol in flies have also been implicated in rodent ethanol-related behaviors and/or human AUD&#160;(e.g.,&#160;6-14). The conservation of mechanisms driving ethanol-related behaviors across species, coupled with the suite of genetic tools available in the Drosophila model system, underscore the utility of the fruit fly model for investigating the genetics of behavioral responses to ethanol.
Sensitivity 15,16 and tolerance (reviewed in 17) to ethanol in humans is linked to the development of AUD. Both of these behavioral responses to ethanol can be modeled in flies via a variety of laboratory assays (reviewed in 3,4). All of the fly assays known to the authors are based on either time-dependent ethanol-induced sedation/incoordination or time-dependent recovery from ethanol sedation.
In a previous article from our group on the genetics of ethanol sensitivity and rapid tolerance in Drosophila, a behavioral assay based on ethanol vapor-induced sedation of flies was used 18. Testing in this assay was initiated by transferring live adult flies without anesthesia to empty food vials, trapping the flies in the vials with a cellulose acetate plug, adding ethanol to the top (i.e.,&#160;non-fly side) of the cellulose acetate plug, and sealing the vial containing flies, cellulose acetate plug and ethanol with a silicone stopper (see schematic in Figure S3, reference 18). Multiple vials representing different groups of flies were assessed in parallel, increasing throughput of this assay. Vials were given an anonymous code and experimenters were blinded to treatment group to prevent unintended bias in the assessment of sedation. In a standard experiment, flies in vials were tapped gently at 6 min intervals and, after a 30 sec recovery, the number of sedated flies in each vial was counted and converted to percent active flies. Flies absorbed ethanol vapor from the cellulose acetate plug in a time-dependent fashion, causing progressive increases in internal ethanol18 and sedation (c.f. reference 18 and Figure 1A and 1B in this report). Sedation in this assay was operationally defined as flies (i) standing in the absence of walking or (ii) lying on their backs with or without flapping their wings. Here, this ethanol sedation assay is described in detail, further operational optimization relevant to using it is provided, and the assay is used to address the contribution of food supplementation options on fly sedation sensitivity.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>food vials</td>
			<td>VWR</td>
			<td>89092-772</td>
			<td>narrow</td>
		</tr>
		<tr>
			<td>Flugs</td>
			<td>Genesee/flystuff.com</td>
			<td>49-102</td>
			<td>narrow</td>
		</tr>
		<tr>
			<td>silicone stopper</td>
			<td>Fisher Scientific</td>
			<td>09-704-1l</td>
			<td>#4</td>
		</tr>
		<tr>
			<td>ethanol</td>
			<td>Pharmaco-Aaper</td>
			<td>111000200</td>
			<td>200 proof</td>
		</tr>
	</tbody>
</table>

an inexpensive, scalable behavioral assay for measuring ethanol sedation sensitivity and rapid tolerance in drosophila

Alcohol use disorder (AUD) is a serious health challenge. Despite a large hereditary component to AUD, few genes have been unambiguously implicated in their etiology. The fruit fly, Drosophila melanogaster, is a powerful model for exploring molecular-genetic mechanisms underlying alcohol-related behaviors and therefore holds great promise for identifying and understanding the function of genes that influence AUD. The use of the Drosophila model for these types of studies depends on the availability of assays that reliably measure behavioral responses to ethanol. This report describes an assay suitable for assessing ethanol sensitivity and rapid tolerance in flies. Ethanol sensitivity measured in this assay is influenced by the volume and concentration of ethanol used, a variety of previously reported genetic manipulations, and also the length of time the flies are housed without food immediately prior to testing. In contrast, ethanol sensitivity measured in this assay is not affected by the vigor of fly handling, sex of the flies, and supplementation of growth medium with antibiotics or live yeast. Three different methods for quantitating ethanol sensitivity are described, all leading to essentially indistinguishable ethanol sensitivity results. The scalable nature of this assay, combined with its overall simplicity to set-up and relatively low expense, make it suitable for small and large scale genetic analysis of ethanol sensitivity and rapid tolerance in Drosophila.

The raw data from this ethanol sedation assay are the numbers of flies that are sedated as a function of ethanol vapor exposure time. Raw data are converted to the percent active flies as a function of time (primary data, Figures 1A, B, D-F). Sensitivity to ethanol sedation from the primary data can be quantitated as Sedation Time 50 (ST50), the time required for 50% of flies to become sedated or aea under the curve (AUC), via interpolation from curve fi...

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

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

Alcohol use disorder (AUD) is a serious health challenge. Despite a large hereditary component to AUD, few genes have been unambiguously implicated in ...

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10.2K Views.  Virginia Commonwealth University. 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.