High performance liquid chromatography (HPLC) analysis of DA and serotonin and their metabolites

The HPLC experimental details are documented in our previous publication [44]. In brief, male mice (22 weeks old) were decapitated 10 min after receiving an injection of L-DOPA (L-DOPA 10 mg/kg and carbidopa 5 mg/kg, i.p.). The dorsal striatum was dissected according to a mouse brain atlas, rapidly frozen using dry ice, and stored at –80°C. Tissues were homogenized in 1 mM oxalic acid and then centrifuged at 15,000 g for 40 min at 4°C. Supernatants were filtered through a 0.22-μm syringe filter (Millipore, Bedford, MA, USA) and injected into the HPLC system for the measurement of DA, 5-HT, and their metabolites [DA metabolites: DOPAC and HVA; serotonin metabolite: 5-HIAA]. Neuronal activity was estimated by calculating DA and 5-HT turnover rates: DA turnover rate=(DOPAC + HVA) / DA, and 5-HT turnover rate = 5-HIAA / 5-HT. Linear regression analysis on a standard curve (10–1,000 nM) was used to determine DOPAC, HVA, DA, 5-HIAA, and 5-HT concentrations by comparing sample measurements with standard values.

The HPLC system was composed of a TSKgel ODS-80TM C18 column (Tosoh, Japan), an LC-10AD pump (Shimadzu, Japan), and an electrochemical detector (Coulochem II, ESA, Chelmsford, MA, USA) equipped with 5010 analytical cells and 5020 guard cells. Analytical cell voltages were set at 40 and 250 mV for detection potential, while the guard cell (mobile phase cell) was maintained at 350 mV. The mobile phase (MDTM mobile phase, ESA) consisted of 75 mM sodium dehydrogenate phosphate (monohydrate), 1.7 mM 1-octanesulfonic acid (sodium salt), 100μl/L triethylamine, 25μM EDTA, and 10% acetonitrile, at a pH of 3.00. It was delivered at a flow rate of 1.0 ml/min.