2.9. Dopamine receptor autoradiography

In Expt 6 (Fig. S1), mice were decapitated and the brains dissected out, frozen on powdered dry ice, stored at −80 °C pre- and post-transport and transported on dry ice. Frozen brains were sectioned coronally at 20 μm and serial sections containing the VTA (bregma level −3.0 mm) or NAcc (bregma 1.8 mm) were collected and mounted on polysine coated slides, and stored at −80 °C prior to autoradiography; for each region, two sections were used per mouse and receptor. Autoradiography for D1 receptor binding was conducted according to (Dalton and Zavitsanou, 2011). Briefly, sections were preincubated in 0.05 M Tris-HCl buffer (pH 7.4) for 10 min, and then incubated for 1 h with 1 nM [³H]SCH-23390 (spec. act. 60 Ci/mmol, 1 mCi/ml, PerkinElmer, U.S.A.) in Tris-buffer at room temperature. Non-specific binding was assessed in the presence of 1 μM unlabelled R(+)-SCH-23390 (Sigma Aldrich, U.K.). Slides were rinsed 2x in ice-cold Tris buffer. The method used for D2 receptor autoradiography is described in (Leventopoulos et al., 2009). Briefly, sections were pre-incubated in 0.05 M Tris-HCl buffer (pH 7.6) for 20 min, and then incubated for 1.5 h with 2 nM [methoxy-³H]raclopride (spec. act. 85 Ci/mmol, 1 mCi/ml, American Radiolabeled Chemicals, Inc., U.S.A.) in Tris-buffer at room temperature. Non-specific binding was assessed using 1 μM unlabelled raclopride (Sigma Aldrich, U.K.). For D1 and D2 receptors, air-dried brain sections and ³H-microscale standards (Amersham Biosciences) were apposed to tritium-sensitive BioMax MR hyperfilm (Kodak, U.K.) for 6 weeks to generate autoradiograms, and tissue radioactivity was then quantified by densitometry using an image analysis system (MCID™, Version 7.0, Imaging Research Inc., Interfocus Ltd, Linton, U.K.). Flat-field correction and calibration of tritium standards were applied to quantify tissue radioactivity in nCi/mg. VTA and NAcc regions of interest were sampled in both the right and left hemispheres in each section and the mean value per mouse was used for statistical analysis; imperfect images with signs of tissue damage were excluded from the analysis.