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>

<ol>
	<li>Rehm, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+burden+of+disease+and+injury+and+economic+cost+attributable+to+alcohol+use+and+alcohol-use+disorders.">Global burden of disease and injury and economic cost attributable to alcohol use and alcohol-use disorders.</a> Lancet. 373, 2223-2233 (2009).</li><li>Prescott, C. A., Kendler, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+and+environmental+contributions+to+alcohol+abuse+and+dependence+in+a+population-based+sample+of+male+twins.">Genetic and environmental contributions to alcohol abuse and dependence in a population-based sample of male twins.</a> The American journal of psychiatry. 156, 34-40 (1999).</li><li>Devineni, A. V., Heberlein, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolution+of+Drosophila+melanogaster+as+a+model+for+alcohol+research.">The evolution of Drosophila melanogaster as a model for alcohol research.</a> Annual review of neuroscience. 36, 121-138 (2013).</li><li>Scholz, H., Mustard, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invertebrate+Models+of+Alcoholism.">Invertebrate Models of Alcoholism.</a> Current topics in behavioral neurosciences. 13, 433-457 (2011).</li><li>Rodan, A. R., Rothenfluh, A., Reilly, M. T., Lovinger, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Functional Plasticity and Genetic Variation: Insights into the Neurobiology of Alcoholism. 91, (2010).</li><li>Schumann, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+association+and+genetic+functional+studies+identify+autism+susceptibility+candidate+2+gene+(AUTS2)+in+the+regulation+of+alcohol+consumption.">Genome-wide association and genetic functional studies identify autism susceptibility candidate 2 gene (AUTS2) in the regulation of alcohol consumption.</a> Proceedings of the National Academy of Sciences of the United States of America. 108, 7119-7124 (2011).</li><li>Corl, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Happyhour,+a+Ste20+family+kinase,+implicates+EGFR+signaling+in+ethanol-induced+behaviors.">Happyhour, a Ste20 family kinase, implicates EGFR signaling in ethanol-induced behaviors.</a> Cell. 137, 949-960 (2009).</li><li>Moore, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethanol+intoxication+in+Drosophila:+Genetic+and+pharmacological+evidence+for+regulation+by+the+cAMP+signaling+pathway.">Ethanol intoxication in Drosophila: Genetic and pharmacological evidence for regulation by the cAMP signaling pathway.</a> Cell. 93, 997-1007 (1998).</li><li>Scholz, H., Franz, M., Heberlein, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hangover+gene+defines+a+stress+pathway+required+for+ethanol+tolerance+development.">The hangover gene defines a stress pathway required for ethanol tolerance development.</a> Nature. 436, 845-847 (2005).</li><li>Riley, B. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alcohol+dependence+is+associated+with+the+ZNF699+gene,+a+human+locus+related+to+Drosophila+hangover,+in+the+Irish+affected+sib+pair+study+of+alcohol+dependence+(IASPSAD)+sample.">Alcohol dependence is associated with the ZNF699 gene, a human locus related to Drosophila hangover, in the Irish affected sib pair study of alcohol dependence (IASPSAD) sample.</a> Molecular psychiatry. 11, 1025-1031 (2006).</li><li>Morozova, T. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alcohol+sensitivity+in+Drosophila:+translational+potential+of+systems+genetics.">Alcohol sensitivity in Drosophila: translational potential of systems genetics.</a> Genetics. 183, 733-745 (2009).</li><li>Ogueta, M., Cibik, O., Eltrop, R., Schneider, A., Scholz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+Adh+function+on+ethanol+preference+and+tolerance+in+adult+Drosophila+melanogaster.">The influence of Adh function on ethanol preference and tolerance in adult Drosophila melanogaster.</a> Chemical senses. 35, 813-822 (2010).</li><li>Han, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrating+GWASs+and+human+protein+interaction+networks+identifies+a+gene+subnetwork+underlying+alcohol+dependence.">Integrating GWASs and human protein interaction networks identifies a gene subnetwork underlying alcohol dependence.</a> American journal of human genetics. 93, 1027-1034 (2013).</li><li>Lind, P. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genomewide+association+study+of+nicotine+and+alcohol+dependence+in+Australian+and+Dutch+populations.">A genomewide association study of nicotine and alcohol dependence in Australian and Dutch populations.</a> Twin Res Hum Genet. 13, 10-29 (2010).</li><li>Schuckit, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+level+of+response+to+alcohol+as+a+predictor+of+future+alcoholism.">Low level of response to alcohol as a predictor of future alcoholism.</a> The American journal of psychiatry. 151, 184-189 (1994).</li><li>Schuckit, M. A., Smith, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+8-year+follow-up+of+450+sons+of+alcoholic+and+control+subjects.">An 8-year follow-up of 450 sons of alcoholic and control subjects.</a> Archives of general psychiatry. 53, 202-210 (1996).</li><li>Tabakoff, B., Cornell, N., Hoffman, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alcohol+tolerance.">Alcohol tolerance.</a> Ann Emerg Med. 15, 1005-1012 (1986).</li><li>Chan, R. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contrasting+Influences+of+Drosophila+white/mini-white+on+Ethanol+Sensitivity+in+Two+Different+Behavioral+Assays.">Contrasting Influences of Drosophila white/mini-white on Ethanol Sensitivity in Two Different Behavioral Assays.</a> Alcohol Clin Exp Res. 38, 1582-1593 (2014).</li><li>Eddison, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=arouser+reveals+a+role+for+synapse+number+in+the+regulation+of+ethanol+sensitivity.">arouser reveals a role for synapse number in the regulation of ethanol sensitivity.</a> Neuron. 70, 979-990 (2011).</li><li>Wen, T., Parrish, C. A., Xu, D., Wu, Q., Shen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+neuropeptide+F+and+its+receptor,+NPFR1,+define+a+signaling+pathway+that+acutely+modulates+alcohol+sensitivity.">Drosophila neuropeptide F and its receptor, NPFR1, define a signaling pathway that acutely modulates alcohol sensitivity.</a> Proceedings of the National Academy of Sciences of the United States of America. 102, 2141-2146 (2005).</li><li>Bhandari, P., Kendler, K. S., Bettinger, J. C., Davies, A. G., Grotewiel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+assay+for+evoked+locomotor+behavior+in+Drosophila+reveals+a+role+for+integrins+in+ethanol+sensitivity+and+rapid+ethanol+tolerance.">An assay for evoked locomotor behavior in Drosophila reveals a role for integrins in ethanol sensitivity and rapid ethanol tolerance.</a> Alcohol Clin Exp Res. 33, 1794-1805 (2009).</li><li>Bhandari, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chloride+intracellular+channels+modulate+acute+ethanol+behaviors+in+Drosophila,+Caenorhabditis+elegans+and+mice.">Chloride intracellular channels modulate acute ethanol behaviors in Drosophila, Caenorhabditis elegans and mice.</a> Genes, brain, and behavior. 11, 387-397 (2012).</li><li>Gargano, J. W., Martin, I., Bhandari, P., Grotewiel, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+iterative+negative+geotaxis+(RING):+a+new+method+for+assessing+age-related+locomotor+decline+in+Drosophila.">Rapid iterative negative geotaxis (RING): a new method for assessing age-related locomotor decline in Drosophila.</a> Experimental gerontology. 40, 386-395 (2005).</li><li>Chen, J., Wang, Y., Zhang, Y., Shen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+Bacchus+reveal+a+tyramine-dependent+nuclear+regulator+for+acute+ethanol+sensitivity+in+Drosophila.">Mutations in Bacchus reveal a tyramine-dependent nuclear regulator for acute ethanol sensitivity in Drosophila.</a> Neuropharmacology. 67, 25-31 (2013).</li><li>Lasek, A. W., Giorgetti, F., Berger, K. H., Tayor, S., Heberlein, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lmo+genes+regulate+behavioral+responses+to+ethanol+in+Drosophila+melanogaster+and+the+mouse.">Lmo genes regulate behavioral responses to ethanol in Drosophila melanogaster and the mouse.</a> Alcohol Clin Exp Res. 35, 1600-1606 (2011).</li><li>Maples, T., Rothenfluh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+way+to+measure+ethanol+sensitivity+in+flies.">A simple way to measure ethanol sensitivity in flies.</a> J Vis Exp. , e2541 (2011).</li><li>Rothenfluh, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+behavioral+responses+to+ethanol+are+regulated+by+alternate+RhoGAP18B+isoforms.">Distinct behavioral responses to ethanol are regulated by alternate RhoGAP18B isoforms.</a> Cell. 127, 199-211 (2006).</li><li>Rothenfluh, A., Troutwine, B. R., Ghezzi, A., Atkinson, N. S., Nohronha, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+23.">Ch. 23.</a> Neurobiology of Alcohol Dependence. 23, 467-495 (2014).</li><li>Jones, M. A., Grotewiel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+as+a+model+for+age-related+impairment+in+locomotor+and+other+behaviors.">Drosophila as a model for age-related impairment in locomotor and other behaviors.</a> Experimental gerontology. 46, 320-325 (2010).</li><li>Martin, I., Grotewiel, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+damage+and+age-related+functional+declines.">Oxidative damage and age-related functional declines.</a> Mechanisms of ageing and development. 127, 411-413 (2006).</li><li>Devineni, A. V., Heberlein, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+ethanol+responses+in+Drosophila+are+sexually+dimorphic.">Acute ethanol responses in Drosophila are sexually dimorphic.</a> Proceedings of the National Academy of Sciences of the United States of America. 109, 21087-21092 (2012).</li></ol>

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><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...

Watch this Scientific Journal Video about An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila at JoVE.com

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.

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

Combining Behavioral Endocrinology and Experimental Economics: Testosterone and Social Decision Making

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in rodents have shown a direct link between testosterone and aggressive behavior1 and folk wisdom adapts these findings to humans, suggesting that testosterone induces antisocial, egoistic or even aggressive behavior2. However, many researchers doubt a direct testosterone-aggression link in humans, arguing instead that testosterone is primarily involved in status-related behavior3,4. As a high status can also be achieved by aggressive and antisocial means it can be difficult to distinguish between anti-social and status seeking behavior.
We therefore set up an experimental environment, in which status can only be achieved by prosocial means. In a double-blind and placebo-controlled experiment, we administered a single sublingual dose of 0.5 mg of testosterone (with a hydroxypropyl-&beta;-cyclodextrin carrier) to 121 women and investigated their social interaction behavior in an economic bargaining paradigm. Real monetary incentives are at stake in this paradigm; every player A receives a certain amount of money and has to make an offer to another player B on how to share the money. If B accepts, she gets what was offered and player A keeps the rest. If B refuses the offer, nobody gets anything. A status seeking player A is expected to avoid being rejected by behaving in a prosocial way, i.e. by making higher offers.
The results show that if expectations about the hormone are controlled for, testosterone administration leads to a significant increase in fair bargaining offers compared to placebo. The role of expectations is reflected in the fact that subjects who report that they believe to have received testosterone make lower offers than those who say they believe that they were treated with a placebo. These findings suggest that the experimental economics approach is sensitive for detecting neurobiological effects as subtle as those achieved by administration of hormones. Moreover, the findings point towards the importance of both psychosocial as well as neuroendocrine factors in determining the influence of testosterone on human social behavior.

The procedure described in this protocol shows that testosterone administration and folk beliefs about testosterone may be associated with directly opposed social behaviors.

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in ...

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 ...

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, including rats, hamsters, and mice, are active during the night and relatively inactive during the day. Many other behavioral and physiological measures also exhibit daily rhythms, but in rodents, running-wheel activity serves as a particularly reliable and convenient measure of the output of the master circadian clock, the suprachiasmatic nucleus (SCN) of the hypothalamus. In general, through a process called entrainment, the daily pattern of running-wheel activity will naturally align with the environmental light-dark cycle (LD cycle; e.g. 12 hr-light:12 hr-dark). However circadian rhythms are endogenously generated patterns in behavior that exhibit a ~24 hr period, and persist in constant darkness. Thus, in the absence of an LD cycle, the recording and analysis of running-wheel activity can be used to determine the subjective time-of-day. Because these rhythms are directed by the circadian clock the subjective time-of-day is referred to as the circadian time (CT). In contrast, when an LD cycle is present, the time-of-day that is determined by the environmental LD cycle is called the zeitgeber time (ZT).
Although circadian rhythms in running-wheel activity are typically linked to the SCN clock6-8, circadian oscillators in many other regions of the brain and body9-14 could also be involved in the regulation of daily activity rhythms. For instance, daily rhythms in food-anticipatory activity do not require the SCN15,16 and instead, are correlated with changes in the activity of extra-SCN oscillators17-20. Thus, running-wheel activity recordings can provide important behavioral information not only about the output of the master SCN clock, but also on the activity of extra-SCN oscillators. Below we describe the equipment and methods used to record, analyze and display circadian locomotor activity rhythms in laboratory rodents.

Circadian rhythms in voluntary wheel-running activity in mammals are tightly coupled to the molecular oscillations of a master clock in the brain. As such, these daily rhythms in behavior can be used to study the influence of genetic, pharmacological, and environmental factors on the functioning of this circadian clock.

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, ...

The FlyBar: Administering Alcohol to Flies

Fruit flies (Drosophila melanogaster) are an established model for both alcohol research and circadian biology. Recently, we showed that the circadian clock modulates alcohol sensitivity, but not the formation of tolerance. Here, we describe our protocol in detail. Alcohol is administered to the flies using the FlyBar. In this setup, saturated alcohol vapor is mixed with humidified air in set proportions, and administered to the flies in four tubes simultaneously. Flies are reared under standardized conditions in order to minimize variation between the replicates. Three-day old flies of different genotypes or treatments are used for the experiments, preferably by matching flies of two different time points (e.g., CT 5 and CT 17) making direct comparisons possible. During the experiment, flies are exposed for 1 hr&#160;to the pre-determined percentage of alcohol vapor and the number of flies that exhibit the Loss of Righting reflex (LoRR) or sedation are counted every 5 min. The data can be analyzed using three different statistical approaches. The first is to determine the time at which 50% of the flies have lost their righting reflex and use an Analysis of the Variance (ANOVA) to determine whether significant differences exist between time points. The second is to determine the percentage flies that show LoRR after a specified number of minutes, followed by an ANOVA analysis. The last method is to analyze the whole times series using multivariate statistics. The protocol can also be used for non-circadian experiments or comparisons between genotypes.

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.

Fruit flies (Drosophila melanogaster) are an established model for both alcohol research and circadian biology. Recently, we showed that the circadian ...

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

Drosophila melanogaster provides an excellent model to study the genetic underpinnings of alcohol sensitivity. In contrast to studies in human populations, the Drosophila model allows strict control over genetic background, and virtually unlimited numbers of individuals of the same genotype can be reared rapidly under well-controlled environmental conditions without regulatory restrictions and at relatively low cost. Flies exposed to ethanol undergo physiological and behavioral changes that resemble human alcohol intoxication, including loss of postural control, sedation, and development of tolerance. Here, we describe a simple, low-cost, high-throughput assay for assessing alcohol sedation sensitivity in large numbers of single flies. The assay is based on video recording of single flies introduced without anesthesia in 24-well cell culture plates in a set-up that enables synchronous initiation of alcohol exposure. The system enables a single person to collect individual ethanol sedation data on as many as 2,000 flies within an 8 h work period. The assay can, in principle, be extended to assess the effects of exposure to any volatile substance and applied to measure effects of acute toxicity of volatiles on other insects, including other fly species.

High-Throughput Method for Measuring Alcohol Sedation Time of Individual 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.

Drosophila melanogaster provides an excellent model to study the genetic underpinnings of alcohol sensitivity. In contrast to studies in human ...

Behavior

Flypub To Study Ethanol Induced Behavioral Disinhibition and Sensitization

Alcohol use disorder (AUD) remains a serious problem in our society. To develop effective interventions for addiction, it is important to understand the underlying neurobiological mechanisms, for which diverse experimental approaches and model systems are needed. The main ingredient of alcoholic beverages is ethanol, which causes adaptive changes in the central nervous system and behavior upon chronic intake. Behavioral sensitization (i.e., escalated responses) in particular represents a key adaptive change underlying addiction. Most ethanol-induced behavioral sensitization studies in animal models have been conducted on the locomotor activating effect of ethanol. A prominent effect of ethanol is behavioral disinhibition. Behavioral sensitization to the disinhibition effect of ethanol, however, is underrepresented. To address this issue, we developed the Flypub assay that allows measuring the escalated increase in disinhibited courtship activities upon recurring ethanol exposure in Drosophila melanogaster.&#160;Here, we report the step-by-step Flypub assay including assembly of ethanol exposure chambers, setup of the assay station, criteria for fly care and collection, ethanol delivery, quantification of disinhibited courtship activities, data processing and statistical analysis. Also provided are how to troubleshoot critical steps, overcome limitations and expand its utility to assess additional ethanol-induced behaviors. The Flypub assay in combination with powerful genetic tools in Drosophila melanogaster will facilitate the task of discovering the mechanism underlying ethanol-induced behavioral sensitization.

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.

Alcohol use disorder (AUD) remains a serious problem in our society. To develop effective interventions for addiction, it is important to understand the ...

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by stressed conspecifics due to an odorant released during stress known as the Drosophila stress odorant (dSO). Through the use of the T-maze apparatus, one can quantify the avoidance of the dSO by responder flies in a very affordable and robust assay. Conditions necessary to obtain a strong performance are presented here. A stressful experience is necessary for the flies to emit dSO, as well as enough emitter flies to cause a robust avoidance response to the presence of dSO. Genetic background, but not their group size, strongly altered the avoidance of the dSO by the responder flies. Canton-S and Elwood display a higher performance in avoiding the dSO than Oregon and Samarkand strains. This behavioral assay will allow identification of mechanisms underlying this social behavior, and the assessment of the influence of genes and environmental conditions on both emission and avoidance of the dSO. Such an assay can be included in batteries of simple diagnostic tests used to identify social deficiencies of mutants or environmental conditions of interest.

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Here, we present a protocol to quantify the avoidance of stressed individuals. This paradigm is powerful yet user-friendly and can be used to assess the influence of genes and environment on one kind of social interaction in Drosophila melanogaster.

Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by ...

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

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the development of more effective treatments is the relative lack of understanding of the molecular underpinnings of the effects of ethanol on behavior. The nematode, Caenorhabditis elegans (C.&#160;elegans), provides a useful model in which to generate and test hypotheses about the molecular effects of ethanol. Here, we describe an assay that has been developed and used to examine the roles of particular genes and environmental factors in behavioral responses to ethanol, in which locomotion is the behavioral output. Ethanol dose-dependently causes an acute depression of crawling on an agar surface. The effects are dynamic; animals exposed to a high concentration demonstrate an initial strong depression of crawling, referred to here as initial sensitivity, and then partially recover locomotion speed despite the continued presence of the drug. This ethanol-induced behavioral plasticity is referred to here as the development of acute functional tolerance. This assay has been used to demonstrate that these two phenotypes are distinct and genetically separable. The straightforward locomotion assay described here is suitable for examining the effects of both genetic and environmental manipulations on these acute behavioral responses to ethanol in C.&#160;elegans.

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

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.

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the ...

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

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

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

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.

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

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

Summary

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Chapters in this video

Combining Behavioral Endocrinology and Experimental Economics: Testosterone and Social Decision Making

A Simple Way to Measure Ethanol Sensitivity in Flies

A General Method for Evaluating Incubation of Sucrose Craving in Rats

Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents

The FlyBar: Administering Alcohol to Flies

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

Flypub To Study Ethanol Induced Behavioral Disinhibition and Sensitization

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

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

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