Quantification of Dopamine and Metabolites in the Brain Tissue

The brains were removed, cooled on ice, and three brain regions were dissected, based on the mouse brain atlas (Paxinos and Franklin, 2001). Brains were placed in a mouse brain matrix (ASI-Instruments^®, Houston, TX, USA), used to provide coronal brain sections. The brains were cut and mounted on slides (SuperFrost Plus, Thermo Fisher Scientific, MA, USA). Brain punches (1.2 mm or 1.0 mm) of the PFC, NAc, and striatum were obtained with micro punches (Harris Micro-Punch, Ted Pella). Specifically, the punched area in the frontal cortex was focused in the mPFC. The brain tissues were frozen in liquid nitrogen and maintained at −80°C for later quantification of dopamine and the metabolites DOPAC (3,4-Dihydroxyphenylacetic acid) and HVA (homovanillic acid).

The tissues (PFC, NAc and striatum) were homogenized and sonicated in 0.1 M perchloric acid solution, prepared by adding 8.68 mL of concentrated perchloric acid, 200 mg of sodium metabisulphite—Na₂S₂O₅—and 200 mg of EDTA in 1.0 L of MilliQ ultrapure water, containing 28.9 ng/mL of dihydroxy-benzylamine (DHBA). The homogenates were centrifuged at 10,000 rpm for 20 min at 4°C. At the time of homogenization, the tissues were weighed (still frozen) immediately before adding the perchloric acid solution. For each mg of tissue, 15 μl of the perchloric acid solution with DHBA was added. Dopamine, DOPAC and HVA were measured by high-performance liquid chromatography with an electrochemical detector (HPLC model LC20 AD, Shimadzu, Japan and Detector Antec Decade sdc VT 03 electrochemical Flow Cell), with a C-18 column (Shimpak; ODS, 15 cm, Kyoto, Japan), and an integrator (model 20AC Chromatopac; Shimadzu). The limit of detection was 0.02 ng for DA, DOPAC and HVA.