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.
