Two hours after withdrawal from the chronic ethanol diet for 16 days, the elevated plus maze test was carried out for 5 min immediately after acupuncture or intra-NAc infusions of β-endorphin using an automated video tracking method as described earlier (16). For validation of open arm entries as an index of anxiety in our elevated plus maze apparatus (44), ethanol-naïve rats were given an intraperitoneal injection of yohimbine (5 mg/kg), an anxiogenic compound, 30 min before the elevated plus maze test. The elevated plus maze test was carried out after 2 hours of withdrawal from the chronic ethanol diet for 16 days (Fig. 5A), according to a previous method (16) with slight modifications. The elevated plus maze was made of dark acrylic consisted of two closed arms (50 cm × 10 cm) with 50-cm-high dark walls and two open arms (50 cm × 10 cm) with 0.5 cm high ledges. The maze was positioned 50 cm above floor under a condition of low-intensity light (1.5 to 2.0 lux) and white noise (70 dB). Before maze test, rats were habituated to the dark anteroom for 2 hours. Each rat was placed into the center of the maze facing a closed arm, and then the time spent in the open arms was measured for 5 min using an automated video-tracking method (EthoVision, Noldus Information Technology BV, Wageningen, The Netherlands). The percentage of time spent in the open arms was used as an index of anxiety (45).

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