Name: swimming induced paralysis to assess dopamine signaling in caenorhabditis elegans
Uploaded: 2019-04-03T13:00:00
Description: Watch this Scientific Journal Video about Swimming Induced Paralysis to Assess Dopamine Signaling in Caenorhabditis elegans at JoVE.com

The authors would like to thank Dr. Osama Refai from Dr. Randy Blakely&#39;s lab for guidance with the automated analysis of SWIP. This work was supported by funding from NIH R01 DA042156 to LC.

1. Preparation of Solutions and Media
<ol>
	<li>Prepare M9 buffer by dissolving KH2PO4 3.0 g (22.05 mM), Na2HPO4 6.0 g (42.2 mM), and NaCl 5.0 g (85.5 mM) in 1 L of autoclaved deionized water. Add 1.0 mL of 1 M MgSO4 (12 g in a final volume of 100 mL autoclaved deionized water) after autoclaving. Mix 100 mL of the resulting 10x M9 with 900 mL of autoclaved deionized water to make a 1x solution.</li>
	<li>To make egg buffer, dissolve 6.896 g of NaCl (118 mM), 3.578 ...

<ol>
	<li>de Bono, M., Villu Maricq, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+Substrates+of+Complex+Behaviors+in+C.+elegans.">Neuronal Substrates of Complex Behaviors in C. elegans.</a> Annual Review of Neuroscience. 28 (1), 451-501 (2005).</li><li>Sawin, E. R., Ranganathan, R., Horvitz, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C.+elegans+Locomotory+Rate+Is+Modulated+by+the+Environment+through+a+Dopaminergic+Pathway+and+by+Experience+through+a+Serotonergic+Pathway.">C. elegans Locomotory Rate Is Modulated by the Environment through a Dopaminergic Pathway and by Experience through a Serotonergic Pathway.</a> Neuron. 26 (3), 619-631 (2000).</li><li>McDonald, P. W., 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=Vigorous+Motor+Activity+in+Caenorhabditis+elegans+Requires+Efficient+Clearance+of+Dopamine+Mediated+by+Synaptic+Localization+of+the+Dopamine+Transporter+DAT-1.">Vigorous Motor Activity in Caenorhabditis elegans Requires Efficient Clearance of Dopamine Mediated by Synaptic Localization of the Dopamine Transporter DAT-1.</a> Journal of Neuroscience. 27 (51), 14216-14227 (2007).</li><li>Carvelli, L., Blakely, R. D., DeFelice, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine+Transporter/Syntaxin+1A+Interactions+Regulate+Transporter+Channel+Activity+and+Dopaminergic+Synaptic+Transmission.">Dopamine Transporter/Syntaxin 1A Interactions Regulate Transporter Channel Activity and Dopaminergic Synaptic Transmission.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (37), 14192 (2008).</li><li>Carvelli, L., Matthies, D. S., Galli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+amphetamine+actions+in+Caenorhabditis+elegans.">Molecular mechanisms of amphetamine actions in Caenorhabditis elegans.</a> Molecular Pharmacology. 78 (1), 151-156 (2010).</li><li>Refai, O., Blakely, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blockade+and+reversal+of+swimming-induced+paralysis+in+C.+elegans+by+the+antipsychotic+and+D2-type+dopamine+receptor+antagonist+azaperone.">Blockade and reversal of swimming-induced paralysis in C. elegans by the antipsychotic and D2-type dopamine receptor antagonist azaperone.</a> Neurochemistry International. , (2018).</li><li>Bermingham, D. 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=The+Atypical+MAP+Kinase+SWIP-13/ERK8+Regulates+Dopamine+Transporters+through+a+Rho-Dependent+Mechanism.">The Atypical MAP Kinase SWIP-13/ERK8 Regulates Dopamine Transporters through a Rho-Dependent Mechanism.</a> The Journal of Neuroscience. 37 (38), 9288-9304 (2017).</li><li>Nass, R., 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+genetic+screen+in+Caenorhabditis+elegans+for+dopamine+neuron+insensitivity+to+6-hydroxydopamine+identifies+dopamine+transporter+mutants+impacting+transporter+biosynthesis+and+trafficking.">A genetic screen in Caenorhabditis elegans for dopamine neuron insensitivity to 6-hydroxydopamine identifies dopamine transporter mutants impacting transporter biosynthesis and trafficking.</a> Journal of Neurochemistry. 94 (3), 774-785 (2005).</li><li>Hardaway, J. 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=Forward+genetic+analysis+to+identify+determinants+of+dopamine+signaling+in+Caenorhabditis+elegans+using+swimming-induced+paralysis.">Forward genetic analysis to identify determinants of dopamine signaling in Caenorhabditis elegans using swimming-induced paralysis.</a> G3. 2 (8), 961-975 (2012).</li><li>Hardaway, J. 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=Glial+Expression+of+the+Caenorhabditis+elegans+Gene+swip-10+Supports+Glutamate+Dependent+Control+of+Extrasynaptic+Dopamine+Signaling.">Glial Expression of the Caenorhabditis elegans Gene swip-10 Supports Glutamate Dependent Control of Extrasynaptic Dopamine Signaling.</a> Journal of Neuroscience. 35 (25), 9409-9423 (2015).</li><li>Felton, C. M., Johnson, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine+signaling+in+C.+elegans+is+mediated+in+part+by+HLH-17-dependent+regulation+of+extracellular+dopamine+levels.">Dopamine signaling in C. elegans is mediated in part by HLH-17-dependent regulation of extracellular dopamine levels.</a> G3. 4 (6), 1081-1089 (2014).</li><li>Lanzo, 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=Silencing+of+Syntaxin+1A+in+the+Dopaminergic+Neurons+Decreases+the+Activity+of+the+Dopamine+Transporter+and+Prevents+Amphetamine-Induced+Behaviors+in+C.+elegans.">Silencing of Syntaxin 1A in the Dopaminergic Neurons Decreases the Activity of the Dopamine Transporter and Prevents Amphetamine-Induced Behaviors in C. elegans.</a> Frontiers in Physiology. 9 (576), (2018).</li><li>Safratowich, B. D., Lor, C., Bianchi, L., Carvelli, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphetamine+activates+an+amine-gated+chloride+channel+to+generate+behavioral+effects+in+Caenorhabditis+elegans.">Amphetamine activates an amine-gated chloride channel to generate behavioral effects in Caenorhabditis elegans.</a> The Journal of Biological Chemistry. 288 (30), 21630-21637 (2013).</li><li>Safratowich, B. D., Hossain, M., Bianchi, L., Carvelli, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphetamine+Potentiates+the+Effects+of+-Phenylethylamine+through+Activation+of+an+Amine-Gated+Chloride+Channel.">Amphetamine Potentiates the Effects of -Phenylethylamine through Activation of an Amine-Gated Chloride Channel.</a> Journal of Neuroscience. 34 (13), 4686-4691 (2014).</li><li>Carvelli, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphetamine+activates+/+potentiates+a+ligand-gated+ion+channel.">Amphetamine activates / potentiates a ligand-gated ion channel.</a> Channels (Austin). 8 (4), 294-295 (2014).</li><li>Hardaway, J. 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=et+al.An+open-source+analytical+platform+for+analysis+of+C.+elegans+swimming-induced+paralysis.">et al.An open-source analytical platform for analysis of C. elegans swimming-induced paralysis.</a> Journal of Neuroscience Methods. 232, 58-62 (2014).</li><li>L&#252;ersen, K., Faust, U., Gottschling, D. -. C., D&#246;ring, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gait-specific+adaptation+of+locomotor+activity+in+response+to+dietary+restriction+in+Caenorhabditis+elegans.">Gait-specific adaptation of locomotor activity in response to dietary restriction in Caenorhabditis elegans.</a> The Journal of Experimental Biology. 217, 2480-2488 (2014).</li><li>Porta-de-la-Riva, M., Fontrodona, L., Villanueva, A., Cer&#243;n, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+Caenorhabditis+elegans+methods:+synchronization+and+observation.">Basic Caenorhabditis elegans methods: synchronization and observation.</a> Journal of Visualized Experiments. (64), e4019 (2012).</li><li>Lamitina, S. T., Morrison, R., Moeckel, G. 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Here, we describe a step-by-step protocol to perform a behavioral assay, SWIP, in C. elegans. This protocol is simple and straightforward with no major technical hurdles making this assay very user friendly. Nevertheless, there are some critical aspects that need to be considered in order to effectively perform the assay.
Care should be taken to ensure that the worms used for the assay are well fed, since dietary restriction affects SWIP17. Gentle handling of w...

Animals perform a variety of innate and complex behaviors that are mediated by different neurotransmitters coordinated by intricate signaling processes. The neurotransmitter dopamine (DA) mediates highly conserved behaviors across species, including learning, motor function and reward processing.
The soil nematode C. elegans, with a relatively simple and well mapped nervous system consisting of only 302 neurons, shows markedly complex behaviors, including many that are regulated by DA such as mating, learning, foraging, locomotion and egg laying1. Among other features, short life cycle, ease of handling and the conservation of signaling molecules, highlight the advantages of using C. elegans as a model for studying the neural basis of conserved behaviors.
The hermaphrodite C. elegans contains eight dopaminergic neurons; In addition to these, the male contains six extra pairs for mating purposes. As in mammals, these neurons synthesize DA and express the DA transporter (DAT-1), a membrane protein found exclusively in dopaminergic neurons, which transports DA released in the synaptic cleft back into the dopaminergic neurons. Moreover, most of the proteins involved in each step of synthesis, packaging and release of DA are highly conserved between worms and humans and, like in mammals, DA modulates feeding behaviors and locomotion in C. elegans2.
C. elegans crawls on solid surfaces and swims with a characteristic thrashing behavior in water. Interestingly, mutants lacking expression of DAT-1 (dat-1) crawl normally on solid surface but fail to sustain swimming when immersed in water. This behavior was termed swimming induced paralysis, or SWIP. Previous experiments demonstrated that SWIP, in part, is caused by an excess of DA in the synaptic cleft that ultimately overstimulates the D2-like postsynaptic receptors (DOP-3). Although originally identified in dat-1 knockout animals3, SWIP is also observed in wild-type animals treated with drugs that block the activity of DAT (e.g., imipramine4) and/or induce DA release (e.g., amphetamine5). On the other hand, pharmacological or genetic manipulations averting synthesis and release of DA and blocking DOP-3 receptor function prevent SWIP6. Taken together, these already published data have established SWIP as a reliable tool to study the behavioral effects caused by mutated proteins within dopaminergic synapses3,4,7 and to be employed for forward genetic screens for the identification of novel regulatory pathways involved in DA signaling7,8,9,10,11,12. Additionally, by providing an easily quantifiable readout of drug-induced behavior in living animals, SWIP enables the elucidation of mechanisms of action of drugs like amphetamine (AMPH) and azaperone at the dopaminergic synapses5,6,13,14,15.
Protocols for performing the SWIP assays have been described before16. Here, we describe in detail the methodology and setup to perform the assay with the goal of providing a visual guide for the C. elegans community to effectively perform SWIP.

<table border="0" cellpadding="0" cellspacing="0" style="width:992px;" width="991">
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Aluminum foil</td>
			<td style="width:260px;">Reynolds wrap</td>
			<td style="width:267px;">1091835</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Amphetamine</td>
			<td style="width:260px;">Sigma</td>
			<td style="width:267px;">51-63-8&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Autoclave</td>
			<td style="width:260px;"></td>
			<td style="width:267px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Bacterial Incubator</td>
			<td style="width:260px;">New Brunswick scientific</td>
			<td style="width:267px;">M1352-0000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Bacteriological grade, Agar</td>
			<td style="width:260px;">Lab Scientific, Inc</td>
			<td>&#160;A466</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Bacto (TM) Peptone</td>
			<td style="width:260px;">BD</td>
			<td>REF 211677</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Calcium Chloride (dihydrate)</td>
			<td style="width:260px;">Sigma-Aldrich</td>
			<td style="width:267px;">C3881</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Camera&#160;</td>
			<td style="width:260px;">Thorlabs</td>
			<td style="width:267px;">U-CMAD3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Centrifuge&#160;</td>
			<td style="width:260px;">Eppendorf 5810R 15amp</td>
			<td style="width:267px;">E215059</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Cholesterol</td>
			<td style="width:260px;">Sigma-Aldrich</td>
			<td style="width:267px;">57-88-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Deionised water</td>
			<td style="width:260px;">Millipore</td>
			<td style="width:267px;">Z00QSV0WW</td>
			<td>Milli-Q</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Depression glass spot plate</td>
			<td style="width:260px;">Corning</td>
			<td style="width:267px;">Corning, Inc. 722085</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Erlenmeyer flask</td>
			<td style="width:260px;">ThermoFisher</td>
			<td style="width:267px;">4103-0250PK</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Eye lash</td>
			<td style="width:260px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Glass slide</td>
			<td style="width:260px;">Fisherbrand</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Graphing and statistical software</td>
			<td style="width:260px;">Prism</td>
			<td style="width:267px;"></td>
			<td>Graphpad 5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">HEPES</td>
			<td style="width:260px;">Sigma-Aldrich</td>
			<td style="width:267px;">RB=H3375 &#38; H7006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Hypochlorite</td>
			<td style="width:260px;">Hawkins</td>
			<td style="width:267px;">Sodium Hypochlorite 4-6%, USP&#34; 1 gal</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">LB Broth, Miller</td>
			<td style="width:260px;">Fisher</td>
			<td style="width:267px;">BP1426</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Magnesium Chloride (Hexahydrate)</td>
			<td style="width:260px;">Sigma-Aldrich</td>
			<td style="width:267px;">RB=M0250</td>
			<td>500g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Magnesium sulfate (heptahydrate)</td>
			<td style="width:260px;">Sigma-Aldrich</td>
			<td style="width:267px;">M1880</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Magnetic stir bar</td>
			<td style="width:260px;">Fisherbrand</td>
			<td style="width:267px;">16-800-510&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Microcentrifuge tubes</td>
			<td style="width:260px;">ThermoFisher</td>
			<td style="width:267px;">69715</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">NA 22 bacteria</td>
			<td style="width:260px;">CGC</td>
			<td style="width:267px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Nystatin</td>
			<td style="width:260px;">Sigma</td>
			<td style="width:267px;">1400-61-9</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Osmometer</td>
			<td style="width:260px;">Advanced Instruments, Inc</td>
			<td style="width:267px;">Model 3320</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Pasteur Pipettes</td>
			<td style="width:260px;">Fisherbrand</td>
			<td style="width:267px;">13-678-20A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Petriplates</td>
			<td style="width:260px;">Falcon</td>
			<td style="width:267px;">351007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">pH Meter</td>
			<td style="width:260px;">Orion VersaStar Pro</td>
			<td style="width:267px;">IS-68X591202-B 0514</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Polystrine conical tubes</td>
			<td style="width:260px;">Falcon</td>
			<td style="width:267px;">352095</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Potassium Chloride</td>
			<td style="width:260px;">Sigma-Aldrich&#160;</td>
			<td style="width:267px;">P9541</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Potassium dihydrogen phosphate</td>
			<td style="width:260px;">Sigma-Aldrich</td>
			<td style="width:267px;">7778-77-0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Potassium Phosphate - DIBASIC</td>
			<td style="width:260px;">Sigma-Aldrich</td>
			<td>P-8281</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Potassium Phosphate - MONOBASIC</td>
			<td style="width:260px;">Sigma-Aldrich</td>
			<td>P0662</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Serological pipettes</td>
			<td style="width:260px;">VWR</td>
			<td style="width:267px;">10ml=89130-898</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Shaker</td>
			<td style="width:260px;">Reliable Scientific</td>
			<td>55S 12x16</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Sodium Chloride</td>
			<td style="width:260px;">Fisher</td>
			<td>RB=BP358-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Sodium dihydrogen Phosphate</td>
			<td style="width:260px;">Fisher</td>
			<td style="width:267px;">RB=S381</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Spreadsheet</td>
			<td style="width:260px;">MS office</td>
			<td style="width:267px;"></td>
			<td>Microsoft Excel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Stereo Microscope</td>
			<td style="width:260px;">Zeiss</td>
			<td style="width:267px;">Model tlb3. 1 stemi2000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Sterile Pipette tips</td>
			<td style="width:260px;">Various</td>
			<td style="width:267px;">02-707-400</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Sucrose</td>
			<td style="width:260px;">Sigma-Aldrich</td>
			<td style="width:267px;">RB=S5016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Superglue</td>
			<td style="width:260px;">Loctite</td>
			<td style="width:267px;">1647358 .14 oz.</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">SwimR sofware</td>
			<td style="width:260px;">10.18129/B9.bioc.SwimR</td>
			<td style="width:267px;"></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:295px;">Tracker 2</td>
			<td style="width:260px;">Worm Tracker 2.0</td>
			<td style="width:267px;">www.mrc-lmb.cam.ac.uk/wormtracker/</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Video recording software</td>
			<td>Virtualdub</td>
			<td style="width:267px;">http://www.virtualdub.org/</td>
			<td></td>
		</tr>
	</tbody>
</table>

swimming induced paralysis to assess dopamine signaling in caenorhabditis elegans

The swimming assay described in this protocol is a valid tool to identify proteins regulating the dopaminergic synapses. Similar to mammals, dopamine (DA) controls several functions in C. elegans including learning and motor activity. Conditions that stimulate DA release (e.g., amphetamine (AMPH) treatments) or that prevent DA clearance (e.g., animals lacking the DA transporter (dat-1) which are incapable of reaccumulating DA into the neurons) generate an excess of extracellular DA ultimately resulting in inhibited locomotion. This behavior is particularly evident when animals swim in water. In fact, while wild-type animals continue to swim for an extended period, dat-1 null mutants and wild-type treated with AMPH or inhibitors of the DA transporter sink to the bottom of the well and do not move. This behavior is termed "Swimming Induced Paralysis" (SWIP). Although the SWIP assay is well established, a detailed description of the method is lacking. Here, we describe a step-by-step guide to perform SWIP. To perform the assay, late larval stage-4 animals are placed in a glass spot plate containing control sucrose solution with or without AMPH. Animals are scored for their swimming behavior either manually by visualization under a stereoscope or automatically by recording with a camera mounted on the stereoscope. Videos are then analyzed using a tracking software, which yields a visual representation of thrashing frequency and paralysis in the form of heat maps. Both the manual and automated systems guarantee an easily quantifiable readout of the animals' swimming ability and thus facilitate screening for animals bearing mutations within the dopaminergic system or for auxiliary genes. In addition, SWIP can be used to elucidate the mechanism of action of drugs of abuse such as AMPH.

We present an example of SWIP assay induced by AMPH treatment. Figure 1 shows a schematic representation of the assay setup as described above. For the manual assay, about 8-10 age synchronized late L4 stage worms are collected with an eyelash or platinum pick and placed into a glass spot plate filled with 40 &#181;L of 200 mOsm/L sucrose (control solution) or sucrose with 0.5 mM AMPH and tested for SWIP.
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Watch this Scientific Journal Video about Swimming Induced Paralysis to Assess Dopamine Signaling in Caenorhabditis elegans at JoVE.com

Swimming Induced Paralysis to Assess Dopamine Signaling in Caenorhabditis elegans

Swimming induced paralysis (SWIP) is a well-established behavioral assay used to study the underlying mechanisms of dopamine signaling in Caenorhabditis elegans (C. elegans). However, a detailed method to perform the assay is lacking. Here, we describe a step-by-step protocol for SWIP.

Swimming Induced Paralysis to Assess Dopamine Signaling in Caenorhabditis elegans

The swimming assay described in this protocol is a valid tool to identify proteins regulating the dopaminergic synapses. Similar to mammals, dopamine (DA) ...

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