Examine Chemoavoidance Behavior in Transgenic Worms Using Osmotic Avoidance Assay

In nematodes, the chemosensory — ASH — neurons can sense osmolarity changes in their surroundings caused by chemical repellents. In response, these worms move away from the repellents, leading to a chemoavoidance behavior.

Abnormal protein aggregation within the ASH neurons can lead to chemoavoidance behavior alterations.

To examine the osmotic avoidance behavior in worms, begin with transgenic nematode larvae with protein aggregates in ASH neurons. Treat the nematodes with bioactive compounds that reduce protein aggregation and restore avoidance behavior in some worms.

Transfer treated worms onto a pre-prepared, food-free media plate to maintain them at the larval stage.  The plate has glycerol — a high-osmotic strength chemical — present at its center, creating an osmotic barrier to divide the plate into two zones, a normal and a trap zone.

Supplement the trap zone with anesthesia to stop the motion of worms that cross the barrier. Add a drop of butanedione into the trap zone to attract all worms, causing them to move toward it.

The worms with protein aggregation cannot sense the osmotic stress, cross the barrier line, and get anesthetized in the trap zone. The worms with restored chemoavoidance behavior move away from the glycerol and remain within the normal zone.

The number of worms in the normal zone correlates with the efficacy of bioactive compounds in restoring the chemoavoidance behavior.

For the osmotic avoidance assay, divide a food-free NGM plate into normal and trap zones, by creating an 8 molar glycerol line in the middle. Then, spread a 200-millimolar sodium azide line at around 1 centimeter away from the glycerol line to paralyze the nematodes crossing through the glycerol barrier into the trap zone.

Transfer around 200 nematodes each onto the normal zone of three replicate plates for each group. Then, add a drop of 1% butanedione onto the trap zone to attract the nematodes. Cover the lid of the Petri dish immediately, and incubate at 23 degrees Celsius for 90 minutes.

Score the number of nematodes on both the zones under a microscope, and calculate the avoidance index.