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

Protocol

The following protocol is an excerpt from Kudumala et al, Swimming Induced Paralysis to Assess Dopamine Signaling in Caenorhabditis elegans, J. Vis. Exp. (2019).

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.

1. Manual method to test for SWIP
   1. Aliquot 40 µL 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.
   2. 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.
   3. 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.
2. Automated analysis of SWIP
   1. 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.
   2. 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.
   3. Follow the script provided with the tracking software to obtain worm thrashing frequency and to generate heat maps from the worm thrashing data.

Materials

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/