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 g of KCl (48 mM), 0.294 g of CaCl2-2H2O (2 mM), 0.406 g of MgCl2-6H2O (2 mM) and 5.958 g (25 mM) of HEPES in 1 L of autoclaved deionized water. Adjust pH to 7.3 using NaOH.</li>
	<li>Prepare fresh sodium hypochlorite/NaOH solution by adding 1 mL of 5-6% sodium hypochlorite (bleach) and 180 &#181;L of 10 N NaOH to 3.8 mL of deionized water.</li>
	<li>Weigh 60 g sucrose and dissolve in autoclaved deionized water to a final volume of 100 mL to make 60% sucrose solution.</li>
	<li>Dissolve 0.684 g of sucrose in 10 mL of autoclaved deionized water to make 200 mM sucrose. Check and adjust to the same an osmolarity using osmometer. Make 1 mL aliquots in 1.5 mL microcentrifuge tubes and freeze at -20 &#176;C.</li>
	<li>Weigh 0.184 g of AMPH (molecular weight 184.75 g/mol) and dissolve in 10 mL of deionized water to make a 100 mM stock solution. Mix 2 &#181;L of the stock solution in 400 &#181;L of water to make 0.5 mM working solution.</li>
	<li>Prepare Nutrient growth media (NGM) plates
	<ol>
		<li>Mix 3 g of NaCl (52.65 mM), 20 g of peptone, 25 g of bacto-agar and 975 mL of deionized water in a 2 L Erlenmeyer flask. Include a magnetic stir bar and autoclave (121 &#176;C, 15 PSI) for 1 hour using liquid cycle.</li>
		<li>Cool to and maintain the temperature at about 50 &#176;C by placing the flask on a heater while stirring. Add 0.5 mL of cholesterol (5 mg/mL in ethanol), 1 mL of 1 M MgSO4, 1 mL of 1 M CaCl2 and 25 mL of 1 M potassium phosphate buffer, pH 7.4 (108.3 g of KH2PO4, 35.6 g of K2HPO4, deionized water to 1 L).</li>
		<li>Pipette 25 mL each into 100 mm x 15 mm Petri plates and allow the media to solidify. Store the plates upside down at 4 &#176;C in a box for up to 4 weeks.</li>
	</ol>
	</li>
	<li>Preparation of Lysogeny broth (LB) broth
	<ol>
		<li>Dissolve 5 g of LB powder mix in 200 mL of deionized water in an Erlenmeyer flask. Autoclave for 30 minutes utilizing the liquid sterilization cycle. Allow the broth to cool down. Store at room temperature for 1-2 weeks.</li>
	</ol>
	</li>
	<li>Preparation of NA22 bacterial plates
	<ol>
		<li>Use a sterile pipette tip or a sterile bacterial loop to streak an LB plate with a small volume of NA22 E. coli bacteria from glycerol stock and incubate the plate upside down in a 37 &#176;C incubator overnight to grow isolated colonies. Pick and introduce a single colony into 200 mL of LB broth prepared in step 1.8 and let grow overnight at 37 &#176;C on a shaking platform.</li>
		<li>To seed the plates, dispense 200 &#181;L of bacterial culture onto the NGM plates prepared earlier in step 1.7 and spread with a sterile glass hockey stick. Let the plates dry overnight or longer under a hood and store upside down in an airtight box at 4 &#176;C.</li>
	</ol>
	</li>
	<li>To make the eyelash/platinum tool to pick worms, glue a thick eyelash or a platinum filament into a glass Pasteur pipette using super glue. Cut the tip of the eyelash at an angle using a razor blade. Alternatively, a Bunsen burner can be used to melt the tip of the glass pipette around the platinum filament.</li>
</ol>
2. C. elegans Husbandry
NOTE: Culture wild-type N2C. elegans strain on Escherichia coli NA22 plates. The detailed culture methods are described below.
<ol>
	<li>Preparation of worm culture
	<ol>
		<li>To make a starter culture of worms, cut a small piece of agar from a plate containing well-fed animals and transfer it onto a NA22 E. coli bacteria plate prepared in step 1.9 using a sterile spatula. Incubate plates at 20 &#176;C for 3-4 days. Under a stereo microscope, visually confirm the presence of gravid adults.</li>
	</ol>
	</li>
	<li>Preparation of synchronized population of worms
	<ol>
		<li>Collect gravid adults from at least 2 plates by dispensing autoclaved deionized water all around the plate using a squirt bottle. Gently swirl the plate to dislodge the worms and collect the worms into a 15 mL polystyrene conical tube using a disposable plastic pipette.</li>
		<li>Spin down the tube in a centrifuge at 140 x g for 2 min to pellet the worms, then aspirate off supernatant using a vacuum pump or with built in laboratory vacuum.</li>
		<li>Resuspend and wash the worms by filling the tube with autoclaved deionized water and mix and centrifuge at 140 x g for 2 min. Aspirate the supernatant and repeat this last step two more times or until worms are clear from bacteria (water appears clear when mixed with the worms).</li>
		<li>Add 5 mL of freshly made sodium hypochlorite/NaOH solution (step 1.3) to the worm pellet and rapidly mix using a vortex. Incubate the tube on a rocker for about 4-8 min. The time of incubation with sodium hypochlorite/NaOH solution fluctuates between 4-8 minutes based on the quality of the stock sodium hypochlorite solution (bleach).</li>
		<li>Put a drop (2-50 &#181;L) of solution containing worms on a glass microscope slide and check every 2 min under the microscope for worm lysis. When about 70% of the worms are lysed and eggs are released, fill the tube with egg buffer prepared in step 1.2 and immediately centrifuge for 1 min at 140 x g to pellet the embryos and worm carcasses.</li>
		<li>Aspirate the supernatant and wash the pellet 3 more times by filling the tube each time with egg buffer. Spin down at 140 x g for 1 min and remove the supernatant each time. The pellet turns white at the end of washes.</li>
		<li>After the final wash, separate the embryos from the dead carcasses in 30% sucrose solution. Add 5 mL of autoclaved deionized water to the pellet, resuspend and add 5 mL of 60% sucrose prepared in step 1.4. Mix thoroughly and centrifuge at 160 x g for 6 min.</li>
		<li>Use a glass Pasteur pipette to transfer the embryos floating at the upper meniscus into a fresh 15 mL conical tube. Do not take more than 3-4 mL. To remove any remaining sucrose, wash the embryos 3 times with autoclaved water by centrifuging at 140 x g for 3 min, removing the supernatant and resuspending the pellet (and filling the tube) each time.</li>
		<li>Repeat the washes with 1x M9 buffer. After the final wash, resuspend the pellet in 10 mL of M9. Leave the tubes on a shaker overnight (no more than 14 h) for the eggs to hatch into L1 larvae. Worms will remain in L1 larval stage due to lack of food.</li>
		<li>Wash the L1 larvae 3 times with autoclaved water to remove any pheromones released by the larvae by centrifuging at 140 x g for 2 min. Resuspend the larvae in 1 mL of water. Make a 1:10 dilution of the worms in water, pipette a 10 &#181;L drop on a glass slide, put a coverslip on and count the number of worms under a stereoscope. Repeat this twice and average the results.</li>
		<li>Pipette the volume of worms that corresponds to about 1,000 worms onto an NA22 plate (that was previously brought to room temperature) by placing small drops on the plate. Leave the plate half-open until the drop dries out. Then cover the plate and incubate upside down in 20 &#176;C incubator for about 44-48 h or until the worms reach late L4 stage, as confirmed visually under a stereomicroscope. Now the worms are ready to be tested for SWIP.</li>
	</ol>
	</li>
</ol>
3. SWIP
NOTE: We describe the manual method of assessing SWIP in wild-type worms treated with AMPH. We also briefly discuss the tracking of worms and further analysis of worm kinetics using an automated worm tracker and a tracking software which were previously described by Hardaway et al.10.
<ol>
	<li>Manual method to test for SWIP
	<ol>
		<li>Aliquot 40 &#181;L of 200 mOsm/L sucrose solution either with or without 0.5 mM AMPH into a glass spot plate. Under the stereoscope, pick 8-10 late-L4 stage worms with an eyelash or platinum pick and submerge the pick in the plate containing the solution until worms move off the pick and swim into the solution. Note the number of worms picked into the well, start the timer, observe and record the number of worms exhibiting SWIP at each minute mark.</li>
		<li>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 total number of worms tested throughout the assay and multiply by 100. Copy the percent values into any graphing and statistical software and plot the data with percent values on the Y axis and time on X axis using the XY graph format.</li>
		<li>Perform two-way ANOVA followed by post-hoc analysis (e.g., Bonferroni post-test) to test for statistical significance among control, AMPH groups and time of treatment.</li>
	</ol>
	</li>
	<li>Automated analysis of SWIP
	<ol>
		<li>Perform automated analysis on a single worm at a time. The protocol to set up camera, the worm tracker software and script to run the tracking software analysis are described in detail in Hardaway et al.16.</li>
		<li>Briefly, place a single late L4 stage hermaphrodite into a glass spot plate utilizing an eyelash pick, as described in the manual method in section 3.1.2. Record swimming videos of one worm at the time and use the worm tracker software to calculate the frequency of body bends.</li>
		<li>Follow the script provided with the tracking software to obtain worm thrashing frequency and to generate heat maps from the worm thrashing data.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

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">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<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>
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		<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>
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		<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) ...

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

Research

JoVE Journal

Behavior

7.6K Views.  Florida Atlantic University, John D MacArthur Campus. 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.

Video: Swimming Induced Paralysis to Assess Dopamine Signaling in Caenorhabditis elegans

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 ...

Eye-Tracking Control to Assess Cognitive Functions in Patients with Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder with pathological involvement of upper and lower motoneurons, subsequently leading to progressive loss of motor and speech abilities. In addition, cognitive functions are impaired in a subset of patients. To evaluate these potential deficits in severely physically impaired ALS patients, eye-tracking is a promising means to conduct cognitive tests. The present article focuses on how eye movements, an indirect means of communication for physically disabled patients, can be utilized to allow for detailed neuropsychological assessment. The requirements, in terms of oculomotor parameters that have to be met for sufficient eye-tracking in ALS patients are presented. The properties of stimuli, including type of neuropsychological tests and style of presentation, best suited to successfully assess cognitive functioning, are also described. Furthermore, recommendations regarding procedural requirements are provided. Overall, this methodology provides a reliable, easy to administer and fast approach for assessing cognitive deficits in patients who are unable to speak or write such as patients with severe ALS. The only confounding factor might be deficits in voluntary eye movement control in a subset of ALS patients.

Cognitive deficits are common in about one third of patients with amyotrophic lateral sclerosis, a neurological condition leading to progressive impairments in speech and movement abilities. To conduct cognitive tests in patients unable to speak or write a reliable and easy to administer eye-tracking paradigm was developed.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder with pathological involvement of upper and lower motoneurons, subsequently leading to ...

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 ...

Biomechanical Analysis Methods to Assess Professional Badminton Players' Lunge Performance

Under the condition of simulating a badminton court in the laboratory, this study used the injury mechanism model to analyze the maximal right lunge movements of eight professional badminton players and eight amateur players. The purpose of this protocol is to study the differences in kinematics and joint moment of the right knee and ankle. A motion capture system and force plate were used to capture data of the joint movements of the lower extremity and the vertical ground reaction force (vGRF). Sixteen young men who did not have any sports injuries in the past 6 months took part in the study. The subjects performed a maximal right lunge from the start position with their right foot, stepping on and fully contacting with the force plate, hit the shuttlecock with an underhand stroke to the designated position in the backcourt, and then returned to the start/end position. All subjects wore the same badminton shoes to avoid a difference in impact from different badminton shoes. The amateur players showed a greater range of ankle movement and reverse joint moment on the frontal plane, and a larger internal joint rotation moment on the horizontal plane. The professional badminton players exhibited greater knee moment on the sagittal and frontal planes. Therefore, these factors should be considered in the development of the training program to reduce the risk of sports injuries in knee and ankle joints. This study simulates the real badminton court and calibrates the range of activities of each movement of the subjects so that the subjects complete the experimental action in a natural state with high quality. A limitation of this study is that it does not combine joint load and muscle activity. Another limitation is that the sample size is small and should be expanded in future studies. This research method can be applied to the lower limb biomechanical research of other footwork in the badminton project.

Here, we present a protocol to evaluate the differences in injury mechanisms between professional and amateur players when performing a badminton maximal right lunge movement by analyzing lower limb kinematics.

Under the condition of simulating a badminton court in the laboratory, this study used the injury mechanism model to analyze the maximal right lunge ...

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. ...

Assessment of Memory Function in Pilocarpine-induced Epileptic Mice

Cognitive impairment is one of the most common comorbidities in temporal lobe epilepsy. To recapitulate epilepsy-associated cognitive decline in an animal model of epilepsy, we generated pilocarpine-treated chronic epileptic mice. We present a protocol for three different behavioral tests using these epileptic mice: novel object location (NL), novel object recognition (NO), and pattern separation (PS) tests to evaluate learning and memory for places, objects, and contexts, respectively. We explain how to set the behavioral apparatus and provide experimental procedures for the NL, NO, and PS tests following an open field test that measures the animals&#8217; basal locomotor activities. We also describe the technical advantages of the NL, NO, and PS tests with respect to other behavioral tests for assessing memory function in epileptic mice. Finally, we discuss possible causes and solutions for epileptic mice failing to make 30 s of good contact with the objects during the familiarization sessions, which is a critical step for successful memory tests. Thus, this protocol provides detailed information about how to assess epilepsy-associated memory impairments using mice. The NL, NO, and PS tests are simple, efficient assays that are appropriate for the evaluation of different kinds of memory in epileptic mice.

This article presents experimental procedures for assessing memory impairments in pilocarpine-induced epileptic mice. This protocol can be used to study the pathophysiologic mechanisms of epilepsy-associated cognitive decline, which is one of the most common comorbidities in epilepsy.

Cognitive impairment is one of the most common comorbidities in temporal lobe epilepsy. To recapitulate epilepsy-associated cognitive decline in an animal ...

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. ...