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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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) ...

The overall goal of this procedure is to demonstrate Swimming-Induced Paralysis or SWIP, a behavioral assay to assess dopamine signaling in C.elegans. SWIP allows the quick identification of genes within or related to the dopaminergic synapsis. This method allows the investigation of the effects produced by an excess of extracellular dopamine that is caused for example by drugs or by mutations at the dopamine transporter.To ensure that all of the worms tested during the assay swim for the same amount of time, the animals must be transferred quickly into the solution. Demonstrating the procedure will be Sirisha Kudumala and Serene Sossi. They both work in my laboratory.To set up worm starter cultures, use a sterile spatula to cut small pieces of agar from a plate containing well fed C.elegans and transfer the agar pieces into individual NA22 E.coli plates. After incubating the plates at 20 degrees Celsius for three to four days, visually confirm the presence of gravid adults under a stereo microscope. To obtain a synchronized population of worms, squirt water onto a plate full of gravid adults.Gently swirl the plate to dislodge the worms and use a disposal pipette to transfer the worms into a 15 milliliter polystyrene conical tube. After pulling the worms from a second plate into the tube, collect the worms by centrifugation for three washes in 15 milliliters of autoclaved water per wash. The water should appear clear after the last wash.After the last centrifugation, add five milliliters of a freshly prepared sodium hypochloride sodium hydroxide solution to the worms and mix rapidly with a vortex. Incubate the tube on a rocker, place a drop of worm solution onto a glass microscope slide and check for worm lysis under a stereoscope every two minutes for four to eight minutes. When about 70%of the worms have been lysed and the eggs had been released, immediately stop the lysis with the addition of egg buffer.Then centrifuge the embryos and worm carcasses four times resuspending the pellet with fresh egg buffer between washes to remove any remaining bleach. After the last wash, the pellet should appear white. Resuspend the pellet in five milliliters of autoclaved water and five milliliters of 60%sucrose to separate the embryos from the carcasses by centrifugation.Remove the tube carefully from the centrifuge after the density gradient separation and use a glass Pasteur pipette to transfer three to four milliliters of the embryos floating at the upper meniscus into a new 15 milliliter conical tube. Wash the embryos three times with autoclaved water to remove the sucrose followed by three washes in M9 buffer. After the last wash, incubate the embryos on a shaker overnight in 10 milliliters of fresh M9 buffer to allow the eggs to hatch and remain at the L1 larval stage.After no more than 14 hours on the shaker, wash the L1 larvae three times with the autoclaved water to remove any pheromones released by the larvae and resuspend the larval pellet in one milliliter of water. Dilute an aliquot of worms to a one to 10 ratio and add 1:10 microliter drop onto a glass slide and count the worms under the stereo microscope. Repeat to obtain an average number of worms.Use a pipette to add enough droplets of the worm solution onto a room temperature NA22 plate to seed approximately one times 10 to the third worms for the culture and leave the plate half open until the drops dry out. Then incubate the plate covered and upside down at 20 degrees Celsius for about 44 to 48 hours or until the worms reach late L4 stage as confirmed visually under a stereo microscope. For manual assessment of SWIP, add 40 microliters of control solution with or without 0.5 millimolar amphetamine into a glass spot plate.Under a stereoscope, pick eight to 10 late L4 stage worms with an eyelash pick and submerge the pick in the well containing the solution until the worms move off the pick and swim into the solution. Start the timer. Note the number of worms released into the well and observe and record the number of worms exhibiting SWIP at each minute mark for a total of 10 minutes.At the end of the procedure, copy the raw data into a spreadsheet and calculate the percent of worms paralyzed by dividing the number of worms paralyzed at each minute by the total number of worms tested throughout the assay and multiplying by 100. Copy the percent values into any graphing and statistical software and plot the data with percent values of SWIP on the y-axis and time on the x-axis using the XY graph format. Statistical analysis among the control and amphetamine-treated groups and the time of treatment can be performed, for example by using two-way ANOVA and post hoc analysis.For automated analysis of swimming-induced paralysis, set up the camera, worm tracker software, and script to run the tracking software analysis and use an eyelash pick to place a single late stage L4 worm into a glass spot plate as shown earlier. Then record swimming videos of one worm at a time and use the worm tracker software to calculate the frequency of body bends, following the script provided with the tracking software to obtain the worm thrashing frequency, and to generate heat maps from the worm thrashing data. Most of the worms tested in the control solution swim continuously for at least 10 minutes.Worms exposed to either control or amphetamine do not show SWIP in the first minute of observation. However, while worms treated with the control solution continue to swim for 10 minutes, worms treated with amphetamine begin to exhibit SWIP after two minutes of treatment. And after 10 minutes, 66%of animals show SWIP.Here examples of heat maps of animals exposed to the control or to amphetamine are shown with an expected considerable increase in paralysis observed in the amphetamine-treated animals. Before attempting this assay, one should be well acquainted with the picking and developmental staging of C.elegans. The involvement of the dopaminergic system in SWIP behavior should be confirmed using animals lacking the expression of dopaminergic proteins or by the depletion of dopamine vesicles with Reserpine.SWIP can be instrumental in identifying new proteins involved in the mechanism of action of drugs acting at the dopaminergic synapsis.

swimming-induced-paralysis-to-assess-dopamine-signaling

Research

JoVE Journal

Behavior

Creating Dynamic Images of Short-lived Dopamine Fluctuations with lp-ntPET:  Dopamine Movies of Cigarette Smoking

We describe experimental and statistical steps for creating dopamine movies of the brain from dynamic PET data. The movies represent minute-to-minute fluctuations of dopamine induced by smoking a cigarette. The smoker is imaged during a natural smoking experience while other possible confounding effects (such as head motion, expectation, novelty, or aversion to smoking repeatedly) are minimized.
We present the details of our unique analysis. Conventional methods for PET analysis estimate time-invariant kinetic model parameters which cannot capture short-term fluctuations in neurotransmitter release. Our analysis - yielding a dopamine movie - is based on our work with kinetic models and other decomposition techniques that allow for time-varying parameters 1-7. This aspect of the analysis - temporal-variation - is key to our work. Because our model is also linear in parameters, it is practical, computationally, to apply at the voxel level. The analysis technique is comprised of five main steps: pre-processing, modeling, statistical comparison, masking and visualization. Preprocessing is applied to the PET data with a unique 'HYPR' spatial filter 8 that reduces spatial noise but preserves critical temporal information. Modeling identifies the time-varying function that best describes the dopamine effect on 11C-raclopride uptake. The statistical step compares the fit of our (lp-ntPET) model 7 to a conventional model 9. Masking restricts treatment to those voxels best described by the new model. Visualization maps the dopamine function at each voxel to a color scale and produces a dopamine movie. Interim results and sample dopamine movies of cigarette smoking are presented.

We present a novel PET imaging approach for capturing dopamine fluctuations induced by cigarette smoking. Subjects smoke in the PET scanner. Dynamic PET images are modeled voxel-by-voxel in time by lp-ntPET, which includes a time-varying dopamine term. The results are 'movies' of dopamine fluctuations in the striatum during smoking.

We describe experimental and statistical steps for creating dopamine movies of the brain from dynamic PET data. The movies represent minute-to-minute ...

Uncovering Beat Deafness: Detecting Rhythm Disorders with Synchronized Finger Tapping and Perceptual Timing Tasks

A set of behavioral tasks for assessing perceptual and sensorimotor timing abilities in the general population (i.e., non-musicians) is presented here with the goal of uncovering rhythm disorders, such as beat deafness. Beat deafness is characterized by poor performance in perceiving durations in auditory rhythmic patterns or poor synchronization of movement with auditory rhythms (e.g., with musical beats). These tasks include the synchronization of finger tapping to the beat of simple and complex auditory stimuli and the detection of rhythmic irregularities (anisochrony detection task) embedded in the same stimuli. These tests, which are easy to administer, include an assessment of both perceptual and sensorimotor timing abilities under different conditions (e.g., beat rates and types of auditory material) and are based on the same auditory stimuli, ranging from a simple metronome to a complex musical excerpt. The analysis of synchronized tapping data is performed with circular statistics, which provide reliable measures of synchronization accuracy (e.g., the difference between the timing of the taps and the timing of the pacing stimuli) and consistency. Circular statistics on tapping data are particularly well-suited for detecting individual differences in the general population. Synchronized tapping and anisochrony detection are sensitive measures for identifying profiles of rhythm disorders and have been used with success to uncover cases of poor synchronization with spared perceptual timing. This systematic assessment of perceptual and sensorimotor timing can be extended to populations of patients with brain damage, neurodegenerative diseases (e.g., Parkinson&#8217;s disease), and developmental disorders (e.g., Attention Deficit Hyperactivity Disorder).

Behavioral tasks that allow for the assessment of perceptual and sensorimotor timing abilities in the general population (i.e., non-musicians) are presented. Synchronization of finger tapping to the beat of an auditory stimuli and detecting rhythmic irregularities provides a means of uncovering rhythm disorders.

A set of behavioral tasks for assessing perceptual and sensorimotor timing abilities in the general population (i.e., non-musicians) is presented here ...

A Procedure to Observe Context-induced Renewal of Pavlovian-conditioned Alcohol-seeking Behavior in Rats

Environmental contexts in which drugs of abuse are consumed can trigger craving, a subjective Pavlovian-conditioned response that can facilitate drug-seeking behavior and prompt relapse in abstinent drug users. We have developed a procedure to study the behavioral and neural processes that mediate the impact of context on alcohol-seeking behavior in rats. Following acclimation to the taste and pharmacological effects of 15% ethanol in the home cage, male Long-Evans rats receive Pavlovian discrimination training (PDT) in conditioning chambers. In each daily (Mon-Fri) PDT session, 16 trials each of two different 10 sec auditory conditioned stimuli occur. During one stimulus, the CS+, 0.2 ml of 15% ethanol is delivered into a fluid port for oral consumption. The second stimulus, the CS-, is not paired with ethanol. Across sessions, entries into the fluid port during the CS+ increase, whereas entries during the CS- stabilize at a lower level, indicating that a predictive association between the CS+ and ethanol is acquired. During PDT each chamber is equipped with a specific configuration of visual, olfactory and tactile contextual stimuli. Following PDT, extinction training is conducted in the same chamber that is now equipped with a different configuration of contextual stimuli. The CS+ and CS- are presented as before, but ethanol is withheld, which causes a gradual decline in port entries during the CS+. At test, rats are placed back into the PDT context and presented with the CS+ and CS- as before, but without ethanol. This manipulation triggers a robust and selective increase in the number of port entries made during the alcohol predictive CS+, with no change in responding during the CS-. This effect, referred to as context-induced renewal, illustrates the powerful capacity of contexts associated with alcohol consumption to stimulate alcohol-seeking behavior in response to Pavlovian alcohol cues.

A procedure to study the capacity of an alcohol associated environmental context to trigger the renewal of alcohol-seeking behavior in rats is described.

Environmental contexts in which drugs of abuse are consumed can trigger craving, a subjective Pavlovian-conditioned response that can facilitate ...

Reversible Cooling-induced Deactivations to Study Cortical Contributions to Obstacle Memory in the Walking Cat

On complex, naturalistic terrain, sensory information about an environmental obstacle can be used to rapidly adjust locomotor movements for avoidance. For example, in the cat, visual information about an impending obstacle can modulate stepping for avoidance. Locomotor adaptation can also occur independent of vision, as sudden tactile inputs to the leg by an expected obstacle can modify the stepping of all four legs for avoidance. Such complex locomotor coordination involves supraspinal structures, such as the parietal cortex. This protocol describes the use of reversible, cooling-induced cortical deactivation to assess parietal cortex contributions to memory-guided obstacle locomotion in the cat. Small cooling loops, known as cryoloops, are specially shaped to deactivate discrete regions of interest to assess their contributions to an overt behavior. Such methods have been used to elucidate the role of parietal area 5 in memory-guided obstacle avoidance in the cat.

Complex locomotion in naturalistic environments requiring careful coordination of the limbs involves regions of the parietal cortex. The following protocol describes the use of reversible cooling-induced deactivation to demonstrate the role of parietal area 5 in memory-guided obstacle avoidance in the walking cat.

On complex, naturalistic terrain, sensory information about an environmental obstacle can be used to rapidly adjust locomotor movements for avoidance. For ...

Using Facial Electromyography to Assess Facial Muscle Reactions to Experienced and Observed Affective Touch in Humans

"Affective" touch is believed to be processed in a manner distinct from discriminatory touch and to involve activation of C-tactile (CT) afferent fibers. Touch that optimally activates CT fibers is consistently rated as hedonically pleasant. Patient groups with impaired social-emotional functioning also show disordered affective touch ratings. However, relying on self-reported ratings of touch has many limitations, including recall bias and communication barriers. Here, we describe a methodological approach to study affective responses to touch via facial electromyography (EMG) that circumvents the reliance on self-report ratings. Facial EMG is an objective, quantitative, and non-invasive method to measure facial muscle activity indicative of affective responses. Responses can be assessed across healthy and patient populations without the need for verbal communication. Here, we provide two separate datasets demonstrating that CT-optimal and non-optimal touch elicit distinct facial muscle reactions. Moreover, facial EMG responses are consistent across stimulus modalities, e.g. tactile (experienced touch) and visual (observed touch). Finally, the temporal resolution of facial EMG can detect responses on timescales that supersede that of verbal reporting. Together, our data suggest that facial EMG is a suitable methodology for use in affective tactile research that can be used to supplement, or in some cases, supplant, existing measures.

We describe a protocol to assess facial muscle activity in response to experienced and observed tactile stimulation using facial electromyography.

"Affective" touch is believed to be processed in a manner distinct from discriminatory touch and to involve activation of C-tactile (CT) afferent fibers. ...

Calcium Imaging in Freely Behaving Caenorhabditis elegans with Well-Controlled, Nonlocalized Vibration

Nonlocalized mechanical forces, such as vibrations and acoustic waves, influence a wide variety of biological processes from development to homeostasis. Animals cope with these stimuli by modifying their behavior. Understanding the mechanisms underlying such behavioral modification requires quantification of neural activity during the behavior of interest. Here, we report a method for calcium imaging in freely behaving Caenorhabditis elegans with nonlocalized vibration of specific frequency, displacement, and duration. This method allows the production of well-controlled, nonlocalized vibration using an acoustic transducer and quantification of evoked calcium responses at single-cell resolution. As a proof of principle, the calcium response of a single interneuron, AVA, during the escape response of C. elegans to vibration is demonstrated. This system will facilitate understanding of neural mechanisms underlying behavioral responses to mechanical stimuli.

Calcium Imaging in Freely Behaving Caenorhabditis elegans with Well-Controlled, Nonlocalized Vibration

Reported here is a system for calcium imaging in freely behaving Caenorhabditis elegans with well-controlled, nonlocalized vibration. This system allows researchers to evoke nonlocalized vibrations with well-controlled properties at nano-scale displacement and to quantify calcium currents during responses of C. elegans to the vibrations.

Nonlocalized mechanical forces, such as vibrations and acoustic waves, influence a wide variety of biological processes from development to homeostasis. ...

Using Enclosed Y-Mazes to Assess Chemosensory Behavior in Reptiles

Reptiles utilize a variety of environmental cues to inform and drive animal behavior such as chemical scent trails produced by food or conspecifics. Decrypting the scent-trailing behavior of vertebrates, particularly invasive species, enables the discovery of cues that induce exploratory behavior and can aid in the development of valuable basic and applied biological tools. However, pinpointing behaviors dominantly driven by chemical cues versus other competing environmental cues can be challenging. Y-mazes are common tools used in animal behavior research that allow quantification of vertebrate chemosensory behavior across a range of taxa. By reducing external stimuli, Y-mazes remove confounding factors and present focal animals with a binary choice. In our Y-maze studies, a scenting animal is restricted to one arm of the maze to leave a scent trail and is removed once scent-laying parameters have been met. Then, depending on the trial type, either the focal animal is allowed into the maze, or a competing scent trail is created. The result is a record of the focal animal&#39;s choice and behavior while discriminating between the chemical cues presented. Here, two Y-maze apparatuses tailored to different invasive reptile species: Argentine black and white tegu lizards (Salvator merianae) and Burmese pythons (Python bivittatus) are described, outlining the operation and cleaning of these Y-mazes. Further, the variety of data produced, experimental drawbacks and solutions, and suggested data analysis frameworks have been summarized.

Y-mazes enable researchers to determine the relevance of specific stimuli that drive animal behavior, especially isolated chemical cues from a variety of sources. Careful design and planning can yield robust data (e.g., discrimination, degree of exploration, numerous behaviors). This experimental apparatus can provide powerful insight into behavioral and ecological questions.

Reptiles utilize a variety of environmental cues to inform and drive animal behavior such as chemical scent trails produced by food or conspecifics. ...

SECONDs Administration Guidelines: A Fast Tool to Assess Consciousness in Brain-injured Patients

Establishing an accurate diagnosis is crucial for patients with disorders of consciousness (DoC) following a severe brain injury. The Coma Recovery Scale-Revised (CRS-R) is the recommended behavioral scale for assessing the level of consciousness among these patients, but its long duration&#160;of administration is a major hurdle in clinical settings. The Simplified Evaluation of CONsciousness Disorders (SECONDs) is a shorter scale that was developed to tackle this issue. It consists of six mandatory items&#160;(observation, command-following, visual pursuit, visual fixation, oriented behaviors, and arousal)&#160;and two conditional items&#160;(communication and localization to pain). The score ranges between 0 and 8 and corresponds to a specific diagnosis (i.e., coma, unresponsive wakefulness syndrome, minimally conscious state minus/plus, or emergence from the minimally conscious state). A first validation study on patients with prolonged DoC showed high concurrent validity and intra- and inter-rater reliability. The SECONDs requires less training than the CRS-R and its administration lasts about 7 minutes (interquartile range: 5-9 minutes). An additional index score allows the more precise tracking of a patient&#39;s behavioral fluctuation or evolution over time. The SECONDs is therefore a fast and valid tool for assessing the level of consciousness in patients with severe brain injury. It can easily be used by healthcare staff and implemented in time-constrained clinical settings, such as intensive care units, to help&#160;decrease misdiagnosis rates and to optimize treatment decisions. These administration guidelines provide detailed instructions for administering the SECONDs in a standardized and reproducible manner, which is an essential requirement for achieving a reliable diagnosis.

These guidelines can be used for the administration of the Simplified Evaluation of CONsciousness Disorders (SECONDs), a short behavioral tool developed to diagnose brain-injured patients in time-constrained settings. This scale examines command-following, communication, visual pursuit, fixation, pain localization, oriented movements and arousal.

Establishing an accurate diagnosis is crucial for patients with disorders of consciousness (DoC) following a severe brain injury. The Coma Recovery ...

Comparative Analysis of Experimental Methods to Quantify Animal Activity in Caenorhabditis elegans Models of Mitochondrial Disease

Caenorhabditis elegans is widely recognized for its central utility as a translational animal model to efficiently interrogate mechanisms and therapies of diverse human diseases. Worms are particularly well-suited for high-throughput genetic and drug screens to gain deeper insight into therapeutic targets and therapies by exploiting their fast development cycle, large brood size, short lifespan, microscopic transparency, low maintenance costs, robust suite of genomic tools, mutant repositories, and experimental methodologies to interrogate both in vivo and ex vivo physiology. Worm locomotor activity represents a particularly relevant phenotype that is frequently impaired in mitochondrial disease, which is highly heterogeneous in causes and manifestations but collectively shares an impaired capacity to produce cellular energy. While a suite of different methodologies may be used to interrogate worm behavior, these vary greatly in experimental costs, complexity, and utility for genomic or drug high-throughput screens. Here, the relative throughput, advantages, and limitations of 16 different activity analysis methodologies were compared that quantify nematode locomotion, thrashing, pharyngeal pumping, and/or chemotaxis in single worms or worm populations of C. elegans at different stages, ages, and experimental durations. Detailed protocols were demonstrated for two semi-automated methods to quantify nematode locomotor activity that represent novel applications of available software tools, namely, ZebraLab (a medium-throughput approach) and WormScan (a high-throughput approach). Data from applying these methods demonstrated similar degrees of reduced animal activity occurred at the L4 larval stage, and progressed in day 1 adults, in mitochondrial complex I disease (gas-1(fc21)) mutant worms relative to wild-type (N2 Bristol) C. elegans. This data validates the utility for these novel applications of using the ZebraLab or WormScan software tools to quantify worm locomotor activity efficiently and objectively, with variable capacity to support high-throughput drug screening on worm behavior in preclinical animal models of mitochondrial disease.

Comparative Analysis of Experimental Methods to Quantify Animal Activity in Caenorhabditis elegans Models of Mitochondrial Disease

This study presents protocols for two semi-automated locomotor activity analysis approaches in C. elegans complex I disease gas-1(fc21) worms, namely, ZebraLab (a medium-throughput assay) and WormScan (a high-throughput assay) and provide comparative analysis among a wide array of research methods to quantify nematode behavior and integrated neuromuscular function.

Caenorhabditis elegans is widely recognized for its central utility as a translational animal model to efficiently interrogate mechanisms and therapies of ...

A Procedure to Study Stress-Induced Relapse of Heroin Seeking after Punishment-Imposed Abstinence

The punishment-imposed abstinence procedure models the self-imposed abstinence that humans initiate due to the adverse consequences associated with drug-taking. This model has been implemented in experiments using different types of substances of abuse such as methamphetamine, cocaine, and alcohol. However, punishment-induced abstinence in heroin-trained animals has not been demonstrated. Furthermore, acute stress is a key trigger for relapse in humans and animal models. It was previously demonstrated that acute food deprivation robustly induced reinstatement of extinguished cocaine and heroin seeking. The procedure described here can be used to assess the effects of acute stress exposure on heroin seeking after punishment-imposed abstinence. A total of 8 rats were implanted with chronic intravenous (i.v.) catheters and trained to self-administer heroin (0.1 mg/kg/infusion) for 18 days under a seek-take chained schedule. Completing the seek link gave access to the take lever, which was paired with a heroin infusion. The seek lever was programmed with a variable interval 60 schedule of reinforcement (VI60), and the take lever was programmed with a fixed-ratio 1 reinforcement schedule (FR1). Following self-administration training, a mild foot shock was delivered on 30% of the completed seek links instead of the extension of the take lever. Footshock intensity was increased by 0.1 mA per daily session from 0.2 mA to 1.0 mA. Heroin-seeking tests were performed after 24 h of food deprivation (FD) or sated conditions. Rats under acute food deprivation condition robustly increased heroin seeking after punishment-imposed abstinence.

A procedure that demonstrates a robust acute food deprivation-induced relapse to heroin seeking after punishment-imposed abstinence is described. A punishment-imposed abstinence model was successfully implemented using the seek and take chain schedule for heroin self-administration. Heroin-seeking tests are then performed following 24 h of food-deprivation stress.

The punishment-imposed abstinence procedure models the self-imposed abstinence that humans initiate due to the adverse consequences associated with ...