Phytocannabinoids

All phytocannabinoids and PTZ were purchased from Sigma Aldrich (Oakville, ON, Canada). AEA, 2-AG and receptor antagonists were purchased from Cayman Chemicals (Ann Arbor, MI, USA). Larvae were collected and reared according to breeding pairs. One larva (6 dpf) was added per well in a 96-well plate, and 180 µL of cannabinoid or vehicle-treated embryo media was added. The plate was incubated at 28.5 °C for 30 min and then recorded in the dark, using an infrared camera in a tracking apparatus for 30 min with a 5 min acclimation. This provided baseline movement data (vehicle treated) and the level of sedation or hyperactivity induced by phytocannabinoid treatment. In the dark, PTZ was then added to a final concentration of 2.75 mM, and the plate was recorded for 30 additional minutes after a 5 min acclimation. Activity, measured as percent pixel change, was measured for 30 min using Noldus EthoVision XT 15 software. For receptor inhibition studies, receptor antagonists were added with phytocannabinoids to a final concentration of 2.5 μM (ML-193 and PSB-CB5) and 1 μM (AM251).

Chemicals were of the purest analytical grade. Anandamide (AEA), URB597 and hFAAH were purchased from Cayman (Cayman Chemical Company, Ann Arbor, MI, USA). Phytocannabinoids and β-caryophyllene were purchased from Sigma Chemical Co. (St. Louis, MO, USA). [Ethanolamine-14-C]AEA was purchased from American Radiolabeled Chemicals (ARC, St. Louis, MO, USA).