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

- C. elegans moves on its side by generating sinusoidal waves along its body. The animal moves in a forward direction when the waves are propagated from the head to the tail. Reversely, when the sinusoidal waves are propagated from the tail to the head, the worm moves backward.

Backward locomotion, known as reversals, is brief and either occurs spontaneously or is triggered by a sensory stimulus, for example, a gentle touch to the anterior of the worm. To perform the anterior touch habituation assay, isolate previously synchronized worms of the desired developmental stage on unseeded NGM plates.

Using a sterilized fine hair, perform a gentle touch to the anterior part of the body. This will elicit an escape response where the worm performs a reversal. Allow a 10-second interstimulus interval for the worm to recover and repeat the touch. To assess habituation, which is a form of non-associative learning, record the number of touches until a reversal is no longer observed.

In the example protocol, we will see a demonstration of the anterior touch habituation assay.

- First, use of fire steriled 30-gauge platinum wire pick to transfer approximately 10 synchronized young adult worms to new unseeded NGM plates. Leave the worms undisturbed for five minutes to allow them time to acclimatize to the new plate. Then, sterilize an eyebrow hair attached to the end of a wooden skewer or toothpick by dipping in 70% ethanol.

Wipe with a clean, lint-free tissue and wait for one minute for the ethanol to evaporate. Gently touch the worm on the head using the eyebrow hair. Repeat the touches allowing 10 seconds in between touches to allow the worm to recover.

Continue to touch allowing 10-second interstimulus intervals until the worm no longer moves backwards. Record the number of touches required for this habituation to occur.

- Allowing a 10-second interval between touches is important, as this allows the animal recovery time and helps was obtaining consistent habituation behavior assays.