Touch Habituation Assay: A Method to Study Non-Associative Learning in C. elegans

The following protocol is an excerpt from Mersha et al. Long-term Behavioral and Reproductive Consequences of Embryonic Exposure to Low-dose Toxicants, J. Vis. Exp. (2018)

1. Anterior Touch Habituation Assay
   1. Transfer approximately 10 synchronized young adult worms to new unseeded NGM plates using a fire-sterilized 30 G platinum wire pick. Leave worms undisturbed for 5 min to allow them time to acclimatize to the new plate.
   2. For the habituation response, use an eyebrow hair attached to the end of a wooden skewer or toothpick, and sterilize by dipping in 70% ethanol. Wipe with a clean lint-free tissue and wait for 1 min for ethanol to evaporate off. Gently touch the worm on the head (anterior of the pharyngeal bulb) using the eyebrow hair. Repeat the touches, allowing 10 s in between touches in order to allow the worm to recover.
   3. Continue to touch (allowing 10 s interstimulus intervals) until the worm no longer moves backwards. At this point, the worm has habituated to the stimulus. Record the num+ber of touches required for the worm to habituate.
   4. Analyze the difference between the mean habituation rates of the treated and untreated controls using one-way ANOVA followed by Tukey's post hoc test.