Using a Y-shaped maze, several experiments were developed to assess conditions of associative learning among fruit fly larvae. Prior to the start of the experiments, some fruit fly larvae were fed a diet of all-trans-retinal (ATR), which is a co-factor for csChrimson activation (eliciting escape behavior in flies which perceive red light). A subgroup of larvae who were fed ATR also received cycloheximide (CXM), an inhibitor of protein synthesis.

Fruit flies naturally avoid carbon dioxide (CO2)—to train larvae to prefer carbon dioxide gas, the investigators activated reward neurons in larvae brains while filling an entire Y-maze with the gas. Variations on the training protocol included spacing of training (massed or spaced), varying the concentration of CO2, activating reward neurons when only air was present, and presenting a red light stimulus along with carbon dioxide gas. After completing their training cycles, larvae were placed in a Y-maze with one corridor that contained CO2 while the other contained air. Navigating to the corridor with CO2 was scored as "Approach" and navigating to the corridor with air only was scored as "Avoid". Learning was evaluated through video recordings of multiple trials conducted over the course of an hour. A subset of larvae were also tested on the following day to evaluate long-term retention. Data is available for each subject for decisions made pre-training and post-training (including next day, if applicable).