In vitro Dop1R2 pharmacology

Luciferase assays were performed as previously described, with minor modifications [119]. HEK293 cells in 96-well plates were grown in serum-free Dulbecco’s modified eagle medium with antibiotics. After 48 h, cells were transfected using PEI (1 μg/ml) with the following constructs: the Drosophila Dopamine 1 receptor 2 (Dop1R2) cloned into pcDNA1.1 (4 ng/well), a CRE-LUC-HCL-PEST luciferase reporter gene (5 ng/well), and β-galactosidase-encoding plasmid (5 ng/well) as a transfection control. For agonist assays, cells were treated with the indicated concentrations of dopamine hydrochloride 24 h after transfection (Product H8502, Sigma, Natick, MA). For antagonist assays, butaclamol hydrochloride (Product D033, Sigma, Natick, MA) or flupenthixol dihydrochloride (Product 4057, Tocris Bioscience, Bristol, UK) was added to cells for 15 min prior to the addition of 1 μM dopamine. For both agonist and antagonist assays, cells were treated with compound for 4 h at 37 °C. Luciferase activity was quantified as an index of Dop1R2 signaling. Activity data were normalized relative to β-galactosidase activity as a control for transfection efficiency.