Ai 4 1

Microdialysis was performed as described previously22 (link)46 (link). Briefly, the animals were anesthetized with sodium pentobarbital, and surgery was performed to implant a guide cannula (AG-4; EICOM, Kyoto, Japan) into the nucleus accumbens. On the following day, a dialysis probe (AI-4-1, 1-mm membrane length; EICOM) was inserted through the guide cannula and was perfused with artificial cerebrospinal fluid (148 mM NaCl, 2.7 mM KCl, 1.2 mM CaCl2, and 0.85 mM MgCl2) at a flow rate of 1 μl min–1. After an equilibration period of one hour, samples were collected every 20 minutes. After collection of at least five baseline fractions, mice were administered MAP (1 mg kg–1, i.p.), and 6 more fractions of samples were collected. The concentration of dopamine in the samples was measured by HPLC on a 4.6 by 30 mm PP-ODS column (EICOM) that was maintained at 25 °C and equipped with an electrochemical detection system (HTEC-500, EICOM) and PowerChrom (EICOM).

The in vivo microdialysis experiment was performed in an electromagnetic- and sound-shielded room (length 1.2 m, width 2.2 m, height 2.3 m)18 (link). The day before the experiment, the stylet was replaced with a dialysis probe (AI-4-1 or 8-1, Eicom Co.) under light ether anesthesia. During the experiment, an artificial cerebrospinal fluid solution (147 mM NaCl, 4 mM KCl, 1.2 mM CaCl₂, and 0.9 mM MgCl₂) was infused through the dialysis probe with a 1.0-mm-long semi-permeable membrane at a rate of 1.2 μl/min using a microdialysis pump (CMA/102, Carnegie Medicin, Stockholm, Sweden) under unanesthetized, unrestrained conditions. Dialysates (24 μl) were automatically collected in an autoinjector (EAS-20, Eicom Co.) every 20 min for 24 h, and the same volume of ethylhomocholine solution (100 nM) was mixed in as the internal standard solution. This mixture was injected directly into a high performance liquid chromatography column every 20 min for the ACh assay.
After sampling, the animals were deeply anesthetized with sodium pentobarbital and perfused with 10% formalin solution. Frozen coronal sections (50 μm thick) were sequentially cut from the forebrain using a microtome (MA-101, Yamato Koki Co., Miyagi, Japan). The location of the dialysis probe was microscopically verified in the frozen sections using a brain atlas56 (Fig. 5).