*2.4. Monoamine Analysis*

To evaluate the levels of dopamine and its metabolites (DOPAC and HVA) in the brain, tissue was homogenized in 0.2 M perchloric acid (PCA, Wako, Tokyo, Japan) containing 100 μM EDTA·2Na (Sigma-Aldrich, St. Louis, MO, USA). After centrifugation, the supernatant was analyzed by high-performance liquid chromatography (HPLC) using an EICOMPAK SC-5ODS column and PREPAK column (Eicom, Kyoto, Japan) with an electrochemical detection (ECD) unit. The mobile phase consisted of 83% 0.1 M acetic acid in citric acid buffer (pH 3.5), 17% methanol (Wako), 190 mg/ml of sodium 1-octanesulfonate sodium (Wako), and 5 mg/mL EDTA·2Na. For ECD, the applied voltage was 750 mV vs. an Ag/AgCl reference electrode.
