Microdialysis for dopamine detection in the mPFC

A microdialysis probe was inserted into the mPFC via the implanted guide cannula connected and perfused with Ringer’s solution (147 mM NaCl, 4 mM KCl, and 2.3 mM CaCl₂) at 2.0 μl/min so as to allow freely moving condition. A stable dialysate dopamine and serotonin concentration was usually obtained after a minimum of 2 h post-implantation of the probe. The 20 μl of microdialysate was collected in every 10 minutes using a fraction collector (EFC-82; Eicom, Japan), and then automatically injected into an HPLC-ECD system (HTEC-500; Eicom, Japan) by an autosampler (M-510; Eicom, Japan). Samples were analyzed for dopamine and serotonin concentration by the HPLC system with an EICOMPAK CAX column (2.0 mm,i.d. × 200 mm; Eicom, Japan) and a graft electrode (WE-3G; Eicom, Japan) set at 450 mV (vs Ag/AgCl reference electrode)⁶¹. The mobile phase contained 0.1 M ammonium acetate buffer, 0.05 mg/l sodium sulfate, 50 mg/l EDTA, and methanol (7:3, v/v), with a pH of 6.0. For data analysis, basal dopamine and serotonin concentration was estimated from an average of two HPLC time points before i.p. administration. At the end of each experiment, rats euthanized with pentobarbital, and the brain was removed. The position of the microdialysis probe was verified in coronal sections with Nissl staining.