Synaptosomal Uptake Assay.

Dopamine uptake was measured by using synaptosomes prepared from rat striata. Adult male Sprague–Dawley rats were anesthetized for 3 min with 2.5% isoflurane before decapitation. Following decapitation, striata was immediately dissected, and homogenized in ice-cold Krebs–Ringer’s buffer containing 0.32 M sucrose and protease inhibitors. The sample was centrifuged for 10 min at 1000g, the pellet was discarded, and the remaining supernatant was centrifuged for an additional 15 min at 16 000g. The resulting pellet (P2) was dissolved in uptake buffer (25 mM HEPES, 120 mM NaCl, 5 mM KCl, 1.2 mM MgSO₄, 1 μM pargyline, 2 mg/mL glucose, 0.2 mg/mL ascorbic acid, pH 7.5). The uptake of radiolabeled dopamine (50 nM) was performed for 3 min at 25 °C in uptake buffer and terminated by filtering and extensive washes with uptake buffer using a PerkinElmer Filter Mate Universal Harvester. Background was estimated in the presence of cocaine (100 μM) or GBR12909 (1 μM). Radioactivity was measured with Wallac/PerkinElmer 1450–021 Microbeta Trilux Liquid Scintillation and Luminescence.