HPLC was performed as published previously44 on a Jasco model PU-2080 isocratic pump (Jasco Inc., Easton, MD) with changes as outlined below. Four replicates of 10 brains/sample were run for each condition. Ten 3-day-old adult Drosophila brains were hand dissected on dry ice between 1 and 3 pm (during the middle of the light phase) with as much precision as possible to remove all trace of eye pigmentation. Brains were placed directly into 50 μL of ice-cold 50 mM citrate acetate, pH 4.5, and quickly homogenized with a Teflon pestle. Homogenates were run through a 0.22 μm spin filter (Millipore Corporation, Bedford, MA) and frozen at -80°C. The typical injection volume was 10 μL, or one brain equivalent. Standard mixes of dopamine, and serotonin (Sigma–Aldrich, St. Louis, MO) were injected at a concentration of 10 ng/mL and a calibration curve was generated based on injections containing 1, 5, 10, 50 and 100 pg.
