The following protocol is an excerpt from Kudumala&#160;et al,&#160;<a href="https://www.jove.com/video/59243/swimming-induced-paralysis-to-assess-dopamine-signaling">Swimming Induced Paralysis to Assess Dopamine Signaling in&#160;Caenorhabditis elegans</a>,&#160;J. Vis. Exp.&#160;(2019).
SWIP
NOTE:&#160;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.
<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.</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 1.1.2. 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>

<table><tbody><tr><td>Amphetamine</td><td>Sigma</td><td>51-63-8&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>Depression glass spot plate</td><td>Corning</td><td>Corning, Inc. 722085</td><td></td></tr><tr><td>Eye lash</td><td></td><td></td><td></td></tr><tr><td>Graphing and statistical software</td><td>Prism</td><td></td><td>Graphpad 5</td></tr><tr><td>Osmometer</td><td>Advanced Instruments, Inc</td><td>Model 3320</td><td></td></tr><tr><td>Spreadsheet</td><td>MS office</td><td></td><td>Microsoft Excel</td></tr><tr><td>Stereo Microscope</td><td>Zeiss</td><td>Model tlb3. 1 stemi2000</td><td></td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>RB=S5016</td><td></td></tr><tr><td>Superglue</td><td>Loctite</td><td>1647358 .14 oz.</td><td></td></tr><tr><td>SwimR sofware</td><td>10.18129/B9.bioc.SwimR</td><td></td><td></td></tr><tr><td>Tracker 2</td><td>Worm Tracker 2.0</td><td>www.mrc-lmb.cam.ac.uk/wormtracker/</td><td></td></tr><tr><td>Video recording software</td><td>Virtualdub</td><td>http://www.virtualdub.org/</td><td></td></tr></tbody></table>

swimming-induced paralysis (swip) assay: a method to quantify dopamine-mediated locomotion in c. elegans

Source: Kudumala, S., et al. <a href="https://www.jove.com/video/59243/swimming-induced-paralysis-to-assess-dopamine-signaling">Swimming Induced Paralysis to Assess Dopamine Signaling in&#160;Caenorhabditis elegans</a>.&#160;J. Vis. Exp.&#160;(2019).
Here, we introduce an assay to measure SWIP, a behavioral phenotype of&#160;C. elegans&#160;that occurs during vigorous motion like swimming.&#160; The example protocol discusses both manual and automated analysis approaches.

Swimming-Induced Paralysis (SWIP) Assay: A Method to Quantify Dopamine-Mediated Locomotion in C. elegans

Source: Kudumala, S., et al. Swimming Induced Paralysis to Assess Dopamine Signaling in&#160;Caenorhabditis elegans.&#160;J. Vis. Exp.&#160;(2019).
Here, ...

- To perform the swimming-induced paralysis, or SWIP assay, transfer L4 staged C. elegans larvae to a glass spot plate filled with liquid. Identify L4 hermaphrodites by the presence of the developing vulva. A&#160;half-moon-shaped patch at the midpoint of the body, and a whip-like tail.
To assess dopamine-mediated swimming, record the worm&#39;s movement characterized by C- shaped body bending and thrashing behavior. Usually, dopaminergic neurons release dopamine into the synaptic cleft, which in turn, binds dopamine receptors on the receiving cell.
Once the message is passed on, dopamine is released from the receptors, and unbound neurotransmitters can be taken up by the dopaminergic neuron through specialized transporter proteins and reused.
In larvae with altered dopamine signaling, caused by inactivating mutations in the dopamine transporter or drug treatments that increase dopamine release, dopamine accumulates in the synaptic cleft. Excess dopamine over stimulates the dopamine receptors interfering with the motor program and leaving the larvae paralyzed. Larvae that are unable to move will sink to the bottom of the plate.
In the example protocol, we will see detailed demonstrations of manual and automated SWIP assays in C. elegans treated with amphetamine.
- 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 8 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.

swimming-induced-paralysis-swip-assay-method-to-quantify-dopamine

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JoVE Encyclopedia of Experiments

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Caenorhabditis elegans (worm)

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2.4K Views. - To perform the swimming-induced paralysis, or SWIP assay, transfer L4 staged C. elegans larvae to 

Swimming-Induced Paralysis (SWIP) Assay: A Method to Quantify Dopamine-Mediated Locomotion in C. elegans

Abstract

Protocol

<table><tbody><tr><td>Amphetamine</td><td>Sigma</td><td>51-63-8&nbsp;&nbsp;</td><td></td></tr><tr><td>Depression glass spot plate</td><td>Corning</td><td>Corning, Inc. 722085</td><td></td></tr><tr><td>Eye lash</td><td></td><td></td><td></td></tr><tr><td>Graphing and statistical software</td><td>Prism</td><td></td><td>Graphpad 5</td></tr><tr><td>Osmometer</td><td>Advanced Instruments, Inc</td><td>Model 3320</td><td></td></tr><tr><td>Spreadsheet</td><td>MS office</td><td></td><td>Microsoft Excel</td></tr><tr><td>Stereo Microscope</td><td>Zeiss</td><td>Model tlb3. 1 stemi2000</td><td></td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>RB=S5016</td><td></td></tr><tr><td>Superglue</td><td>Loctite</td><td>1647358 .14 oz.</td><td></td></tr><tr><td>SwimR sofware</td><td>10.18129/B9.bioc.SwimR</td><td></td><td></td></tr><tr><td>Tracker 2</td><td>Worm Tracker 2.0</td><td>www.mrc-lmb.cam.ac.uk/wormtracker/</td><td></td></tr><tr><td>Video recording software</td><td>Virtualdub</td><td>http://www.virtualdub.org/</td><td></td></tr></tbody></table>

Swimming-Induced Paralysis (SWIP) Assay: A Method to Quantify Dopamine-Mediated Locomotion in C. elegans

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Name	Company	Catalog Number	Comments
Amphetamine	Sigma	51-63-8
Depression glass spot plate	Corning	Corning, Inc. 722085
Eye lash
Graphing and statistical software	Prism		Graphpad 5
Osmometer	Advanced Instruments, Inc	Model 3320
Spreadsheet	MS office		Microsoft Excel
Stereo Microscope	Zeiss	Model tlb3. 1 stemi2000
Sucrose	Sigma-Aldrich	RB=S5016
Superglue	Loctite	1647358 .14 oz.
SwimR sofware	10.18129/B9.bioc.SwimR
Tracker 2	Worm Tracker 2.0	www.mrc-lmb.cam.ac.uk/wormtracker/
Video recording software	Virtualdub	http://www.virtualdub.org/