Name: an assay for measuring the effects of ethanol on the locomotion speed of caenorhabditis elegans
Uploaded: 2015-04-09T13:00:00
Description: Watch this Scientific Journal Video about An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans at JoVE.com

These studies were supported by grants from the National Institutes of Health, National Institute for Alcoholism and Alcohol Abuse: R01AA016837 (JCB) and P20AA017828 (AGD and JCB).

1. Steps to Perform on the Day before the Assay
<ol>
	<li>Pick L4 stage worms to fresh nematode growth medium (NGM) plates seeded with a lawn of OP50 E. coli, and culture them at 20 &#730;C O/N. Each assay condition requires 10 worms; pick an excess of worms to allow for O/N loss of worms.
	<ol>
		<li>Only assay animals that are first-day adults; many mutants grow at a slower speed than wild type. Adjust the timing of picking for strains that have developmental delays so that all animals tested are first-day a...

<ol>
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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=Conserved+role+of+unc-79+in+ethanol+responses+in+lightweight+mutant+mice.">Conserved role of unc-79 in ethanol responses in lightweight mutant mice.</a> PLoS Genet. 6, e1001057 (2010).</li><li>Thiele, T. E., Badia-Elder, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+neuropeptide+Y+in+alcohol+intake+control:+evidence+from+human+and+animal+research.">A role for neuropeptide Y in alcohol intake control: evidence from human and animal research.</a> Physiol. Behav. 79, 95-101 (2003).</li><li>Treistman, S. N., Martin, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BK+Channels:+mediators+and+models+for+alcohol+tolerance.">BK Channels: mediators and models for alcohol tolerance.</a> Trends Neurosci. 32, 629-637 (2009).</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> Am. J. Hum. Genet. 93, 1027-1034 (2013).</li><li>Kendler, K. 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=Genomewide+association+analysis+of+symptoms+of+alcohol+dependence+in+the+molecular+genetics+of+schizophrenia+(MGS2)+control+sample.">Genomewide association analysis of symptoms of alcohol dependence in the molecular genetics of schizophrenia (MGS2) control sample.</a> Alcohol. Clin. Exp. Res. 35, 963-975 (2011).</li><li>Schuckit, M. 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=Autosomal+linkage+analysis+for+the+level+of+response+to+alcohol.">Autosomal linkage analysis for the level of response to alcohol.</a> Alcohol. Clin. Exp. Res. 29, 1976-1982 (2005).</li><li>Alaimo, J. T., 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+metabolism+and+osmolarity+modify+behavioral+responses+to+ethanol+in+C.+elegans.">Ethanol metabolism and osmolarity modify behavioral responses to ethanol in C. elegans.</a> Alcohol. Clin. Exp. Res. 36, 1840-1850 (2012).</li><li>Morgan, P. G., Sedensky, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+affecting+sensitivity+to+ethanol+in+the+nematode,+Caenorhabditis+elegans.">Mutations affecting sensitivity to ethanol in the nematode, Caenorhabditis elegans.</a> Alcohol. Clin. Exp. Res. 19, 1423-1429 (1995).</li><li>Mitchell, P. H., 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=The+concentration-dependent+effects+of+ethanol+on+Caenorhabditis+elegans+behaviour.">The concentration-dependent effects of ethanol on Caenorhabditis elegans behaviour.</a> Pharmacogenomics J. 7, 411-417 (2007).</li><li>Vidal-Gadea, 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=Caenorhabditis+elegans+selects+distinct+crawling+and+swimming+gaits+via+dopamine+and+serotonin.">Caenorhabditis elegans selects distinct crawling and swimming gaits via dopamine and serotonin.</a> Proc. Natl. Acad. Sci. U S A. 108, 17504-17509 (2011).</li><li>Bettinger, J. C., Leung, K., Bolling, M. H., Goldsmith, A. D., Davies, A. 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The simple neurobiology and genetic tools available in C. elegans make the worm an excellent model in which to study the molecular bases of the effects of ethanol on behavior. Here, we describe an assay that has been used to identify several molecular and environmental mediators of the acute behavioral response to ethanol8,10,20,24,25. This method allows the differentiation and simultaneous examination of two different ethanol response behavior phenotypes, initial sensitivity and the development of ac...

Alcohol use disorders (AUD) are widespread and produce serious health, social, and economic problems. In humans, the susceptibility to developing an AUD is heavily influenced by both genetics and the environment1,2. A strong physiological predictor of abuse liability is the initial level of response (LR) to alcohol (ethanol) that is exhibited by na&#239;ve drinkers3-5. This LR phenotype is influenced by genetics and non-genetic components6. Determining the molecular mechanisms that influence the LR to ethanol is an important goal of the study of ethanol response behaviors.
The nematode, Caenorhabditis elegans, has been increasingly used as a model for studying the effects of ethanol on behavior7-9. There is strong molecular conservation in the machinery of nervous system function between worms and mammals, and several genes that have been shown to influence the LR to ethanol in worms have been shown to influence LR to ethanol in mammals10-16, and have been implicated in abuse liability in humans17-19. 
Ethanol intoxicates worms, which is reflected in a decrease in their locomotion speed. Several different laboratories have developed behavioral assays that differ in several ways, for example, the locomotion behavior that they study (crawling versus swimming11,12,14,20,21) or in the composition of the solutions in which the assays are performed (nematode growth medium versus Dent&#8217;s saline20,22). Interestingly, these diverse assays have yielded somewhat different dose response profiles for the effects of ethanol. These results have pointed to important differences in the underlying behaviors of crawling and swimming9,23, as well as a role for the environmental variable osmolarity in ethanol responses20, and have highlighted the importance of describing experimental detail of the various assays.
An assay to measure the acute effects of ethanol on crawling behavior is presented here. This assay has been used extensively to study the genetic and environmental influences on the LR to ethanol8,10,20,24,25. The mammalian LR phenotype is a composite of at least two components, initial sensitivity to ethanol and acute functional tolerance to ethanol26,27. In worms, the LR phenotype has been shown to be separable into these two components through the use of this behavioral assay. The influences of genetic and environmental manipulations on both phenotypes can be examined using this single assay. Importantly, these two phenotypes are genetically separable.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>C. elegans strains</td>
			<td>Caenorhabditis Genetics Center</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>60 x15 mm Petri plates, triple vented</td>
			<td>Greiner Bio-One</td>
			<td>628161</td>
			<td>Other plate brands will suffice.</td>
		</tr>
		<tr>
			<td>NGM agar</td>
			<td>Various</td>
			<td></td>
			<td>NaCl (3g/L), agar (17g/L), peptone (2.5g/L), 1 mL cholesterol (5mg/mL in ethanol), 1 mL (1M) MgSO4, 1 mL (1M) CaCl2, 25 mL (1M) KPO4, pH=6, 975 mL H2O</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Various</td>
			<td></td>
			<td>e.g. Fisher Scientific #10300</td>
		</tr>
		<tr>
			<td>37&#176;C Incubator</td>
			<td>Various</td>
			<td></td>
			<td>For drying agar</td>
		</tr>
		<tr>
			<td>Digital balance</td>
			<td>Various</td>
			<td></td>
			<td>For determining plate weights and agar volume</td>
		</tr>
		<tr>
			<td>Copper rings</td>
			<td>Plumbmaster</td>
			<td>STK#35583 (48 cap thread gasket)</td>
			<td>1.6 cm inner diameter, 1.8 cm outer diameter copper rings</td>
		</tr>
		<tr>
			<td>100% ethanol</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm M</td>
			<td>Bemis</td>
			<td>PM996</td>
			<td></td>
		</tr>
		<tr>
			<td>CCD camera</td>
			<td>QImaging</td>
			<td>RET-4000R-F-M-12</td>
			<td>This camera has a large field of view.</td>
		</tr>
		<tr>
			<td>Stereomicroscope with C-mount and 0.5X objective</td>
			<td>Leica</td>
			<td>MZ6</td>
			<td>Discontinued model, M60 is current equivalent.</td>
		</tr>
		<tr>
			<td>Light source</td>
			<td>Schott</td>
			<td>A08923</td>
			<td>3&#8221;x3&#8221;&#160; backlight for even illumination across the field of view</td>
		</tr>
		<tr>
			<td>Imaging and tracking software</td>
			<td>Media Cybernetics</td>
			<td>ImagePro-Plus v6.0-6.3</td>
			<td>Newer versions of the software have tracking functions.</td>
		</tr>
	</tbody>
</table>

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.

Representative data (Figure 1) from several different genotypes and their paired controls are presented8,24; data were specifically chosen that highlight differences in the assayed animals. The degree of effect at 10 min of exposure is considered the initial sensitivity of a strain, which is shown on the left axes in Figure 1B-G. Mutant strains with a relative speed larger than the control at 10 min&#160;are considered to be ethanol resistant (Figure 1F, G), w...

Watch this Scientific Journal Video about An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans at JoVE.com

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.

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 ...

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