Fly Climbing Assay: A Method to Test General Locomotion in Drosophila melanogaster

This protocol is an excerpt from Gevedon et al., In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster, J. Vis. Exp. (2019).

1. Climbing Assay (Adapted from Ali et al., J. Vis. Exp. (2011))

1. Validate the climbing assay. In this study, test the climbing assay based on previously described methodology by Ali et al., using Elav^C155>UAS-Tau^R406W (exhibiting lower climbing pass rates) and Elav^C155>UAS-GFP (control) and Elav^C155>UAS-mCherry (control) flies.
2. Flip the flies into a testing chamber, one line at a time (do not use CO₂ anesthesia) and allow them to recover for 5 min. If testing is done on different days, perform the assay at the same time of the day. In this study, all climbing assays were performed in the afternoon to control for circadian effects on locomotion.
   NOTE Avoid exposing the flies to direct sunlight when testing; this will interfere with their ability to climb.
3. Forcibly tap the chamber (marked side down) onto a mouse pad placed on a solid surface 3 times so all flies begin the assay at the bottom. Observe fly locomotion over a period of 10 s.
4. Record the number of flies that reach or pass the 5 cm line within this time, as well as the total number of flies tested. Repeat the protocol, waiting 1 min between each trial, for a minimum of 3 trial replicates per line.
   NOTE In the present study, each vial contained between 2 and 19 flies for the initial screening (male flies) and between 6 and 21 flies for the mutant deficiency analysis of crossed female lines.