In vivo microdialysis

For analysis of the release of DA, animals were anesthetized with sodium pentobarbital (50 mg/kg, i.p.), and a guide cannula (CMA/7, Cupr CMA Microdialysis AB, Stockholm, Sweden) was implanted in the NAc (+1.5 mm anteroposterior, +0.8 mm mediolateral to the bregma, −4.0 mm dorsoventral to the surface of the skull)⁴³. The mice were used in the microdialysis perfusion experiments three days after cannula implantation in order to give them time to recover from the surgery and allow the anesthesia to clear their systems. The probe (CMA/7 7/1, 1 mm, Cupr CMA Microdialysis AB, Stockholm, Sweden) was inserted through the guide cannula under ether anesthetic and was perfused with artificial cerebrospinal fluid (aCSF; 150 mM NaCl, 3 mM KCl, 1.4 mM CaCl₂, 0.8 mM MgCl₂, pH = 7.4) at a flow rate of 0.8 μl/min. During the experiments, the perfusate samples, which were collected at 20 min intervals (16 μl) in freely moving mice, were directly administered using an injection (HPLC-ECD) system for separation and quantification of DA. The mobile phase contained 75 mM NaH₂PO4·H₂O, 1.7 mM 1-OSA, 25 μM EDTA, 0.714 mM triethylamine, and 10% acetonitrile, pH = 3.0 and was maintained at a flow rate of 0.6 ml/min. Basal DA levels were monitored over 3 h for stabilization. Twenty minutes prior to the injection of MAP, mice underwent pretreatment [CPZ (5 mg/kg, i.p.) or saline (0.1 ml/10 g, i.p.)], and the release of DA was measured for up to 180 min. After the completion of microdialysis experiments, the mice were deeply anesthetized with pentobarbital (60 mg/kg, i.p.) and killed by decapitation. The position of probe placement was histologically verified to be in the NAc region.