ERK measurement in vitro
Adult male C57Bl6 mice were decapitated after anaesthesia and cervical dislocation, and brain slices were freshly prepared according to the protocol previously described (Arcuriet al. , 2018; Marti et al. , 2012). The brains were rapidly removed and put on a cool glass plate filled with ice-cold sucrose-based dissecting solution (87 mM NaCl, 2.5 mM KCl, 7 mM MgCl2, 1 mM NaH2PO4, 75 mM sucrose, 25 mM NaHCO3, 10 mM D-glucose, 0.5 mM CaCl2, 2 mM kynurenic acid), carbogenated (95% O2, 5% CO2) and subsequently mounted on the vibratome stage (Vibratome, VT1000S-Leica Microsystems); 200-μm-thick slices were cut and transferred into a brain slice chamber (Brain slice chamber-BSC1, Scientific System design Inc., Mississauga, ON, Canada) and allowed to recover for 1 h at 32°C, with a constant perfusion of carbogenated artificial CSF (ACSF: 124 mM NaCl, 5 mM KCl, 1.3 mM MgSO4, 1.2 mM NaH2PO4, 25 mM NaHCO3, 10 mM D-glucose, 2.4 mM CaCl2). The D1 receptor agonist SKF-38393 (100 μM) was applied for 10 min in the presence of AT-403 (30 nM), CCG203920 (500 nM) or vehicle. After fixation in 4% paraformaldehyde (PFA) for 15 min at room temperature, slices were rinsed three times in PBS and cryoprotected in 30% sucrose solution overnight at 4°C. On the following day, slices were further cut into 18-μm-thick slices using a cryostat (Leica CM1850) and mounted onto SuperFrost Plus slides (Thermo Scientific). Immunohistochemistry was performed as previously described (Papaleet al. , 2016): 1 h after blocking in 5% normal goat serum and 0.1% Triton X-100 solution, slices were incubated overnight at 4°C with anti-phospho-p44/42 MAP kinase (Thr202/Tyr204) (1:1000, Cell Signaling Technology cat. #4370 L). Sections were then incubated with biotinylated goat anti-rabbit IgG (1:200, Vector Laboratories, cat. #BA 1000) for 2 h at room temperature. Detection of the bound antibodies was carried out using a standard peroxidase-based method (ABC-kit, Vectastain, Vector Labs), followed by a 3,3’-diamino-benzidine (DAB) and H2O2 solution. Images were acquired from the striatum at 40× magnification using a brightfield microscope (Leica Macro/Micro Imaging System), and the number of pERK positive cells in the striatum was counted in each slice.