- 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 half-moon-shaped patch at the midpoint of the body, and a whip-like tail.
To assess dopamine-mediated swimming, record the worm'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.