Many cholinergic drugs have been used in the analysis of specific mutants and behaviors; however, the two that have had the most significant impact on C. elegans neurobiology are aldicarb and levamisole.
Using Wmicrotracker SMART , it is possible to easily quantify the effect and kinetic of acetylcholine channels modulators, as well as other neuronal channels involved in behavior. Below we present an example of application.
Kinetic of paralysis of C.elegans using levamisole in P35
WMicrotracker SMART is able to get and plot the path tracking of a population of worms, calculating average movement speed in real time. A kinetics plot comparing levamisole concentration treatment (0 to 200uM) is shown below. The plots show the correlation between levamisole concentration and movement speed decrease.
| Using a 5 minute acquisition lapse every hour, the WMicrotracker SMART is able to quantify the effect of levamisole paralysis on C.elegans worms. |
Protocol:
- Grow synchronized populations of adult day-1 worms in seeding NGM plates (OP50).
- Remove worms from plates using M9 buffer and transfer them in a sterile 2ml micro tube.
- Let the worms settle. Decant the supernatant taking care not to disturb the pellet.
- Perform a wash with 2 ml of M9 buffer. Briefly shake or invert the tube
- Repeat the decantation step. Throw out the supernatant.
- Add 2 ml of M9 buffer.
- Count the number of worms in 10 µl in triplicate and calculate the average.
- Prepare a suspension to get [25 worms/10 µl]
- Transfer 10 µl of worm solution to a 35mm plate. Wait until the drop is absorbed.
- 5 minutes later, register worm activity using WMicrotracker SMART during 5 minutes. Immediately before the acquisition, stimulate the worms by subjecting the plate to mechanical stimulus (tap 3x).
Microplate preparation:
- NGM was prepared following standard procedure.
- Levamisole is added to the 55°C NGM agar solution immediately after the addition of the salts and cholesterol.
- Levamisole plates were not seeded with OP50.
- The assay is performed on plates that had been allowed to template to room temperature prior to the start of the experiment.
- Before adding the worms to each plate, make a ring of 100 mM copper sulfate around the edge of each plate to prevent worms from crawling out of the agar.
- When adding the worms, be careful not to scratch the agar – worms tend to crawl into any break in the agar surface.
In this experiment we can observe the kinetic effect of Levamisole treatment in C.elegans populations cultured in NGM medium.