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Rats were then euthanized by decapitation. Brain tissue was removed check details and visualization of methylene blue in the lateral ventricles confirmed that the guide cannula was correctly inserted. Data for four rats, where the guide cannula had not been inserted correctly, was omitted from our study analysis. Induction of Morphine Dependency Morphine dependency was induced in 8 separate groups by subcutaneous injection of morphine in a dose-increasing manner (15-55 mg/kg) for 5 consecutive days.22 Induction and Evaluation of Morphine Withdrawal Syndrome In order to induce withdrawal symptoms, all rats in each group were injected by naloxone 3 mg/kg and behaviors of each animal were recorded by a camera for a period of 60 minutes. The recorded behavior included jumping, head shake, wet dog shake, forepaw tremor, writhing, walking sniffing, sniffing, penile liking, rearing, chewing, body grooming, face wiping, swallowing, and teeth chattering. The number of each behavior was divided by their weighing factor (table 1) and a value was obtained. The summation of these values gave the total withdrawal score (TWS). These behavioral syndromes were commutated according to the previous MK-2206 studies.22 Table 1 Weighing factors (WFs) of different withdrawal signs of morphine in the mouse Measuring the Blood Cortisol On the 10th day, after all behavioral signs had been recorded; whole blood was collected from all animals. Serum was separated from whole blood and serum cortisol levels were measured in ��g/dl by the ELISA method. Data TRIB1 Analysis All data obtained by this study were statistically analyzed using GraphPad Prism V6 software. The data was averaged in every experimental group and expressed as means��standard error of the means (SEM). In the next step, differences between controls and treatment groups were evaluated by one way ANOVA. Differences between severities of behaviors in the treatment of each group were evaluated by Dunnett post hoc test. A value with P