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.