201y gal4

Flies were kept in a standard cornmeal/agar medium with 0.4% propionic acid at 25 °C in a 12-h light/dark cycle. The following fly strains were used: wild type (Canton-S), TH-Gal4 (a gift from J. Hirsh, University of Virginia, Charlottesville, VA, USA), UAS-DD2R-RNAi (from Bloomington Drosophila Stock Center (BDSC), Bloomington, IN, USA), 201Y-Gal4 (from BDSC), and 30Y-Gal4 (from BDSC). In order to obtain third-instar larvae (92–96 h after egg-laying (AEL)) for learning assays, adult flies were placed in an egg collection bottle with an agar plate carrying yeast paste. Next, flies were allowed to lay eggs for 4 h. Each plate had roughly 100–150 eggs on the surface. The plates were incubated at 21.7 °C. In the late third stage (92–96 h AEL), the larvae were filtered from the food plates using a 500 µm sieve (Standard Test Sieve, Newark Wire Cloth Co., Clifton, NJ, USA) with 15% glucose solution. After rinsing with distilled water, larvae were used for the following tests.