Chronic stress and HPA-axis dysfunction are generally considered risk factors for the development of psychiatric disorders, including major depression. to food and water. Behavioral experiments were performed between 9:00 and 18:00. All efforts were made to minimize both the suffering of and the number of animals used. The experimental protocol was reviewed and approved Paeoniflorin by the Experimental Animal Care Committee of Kracie Pharma, Ltd. (Toyama, Japan). Plant Materials and Preparation of the Extract Ninjinyoeito is composed of twelve dried medical herbs, Paeoniflorin including rehmannia root, Japanese angelica root, Paeoniflorin atractylodes rhizome, poria sclerotium, ginseng, cinnamon bark, polygala root, peony root, citrus unshiu peel, astragalus root, glycyrrhiza, and schisandra fruit (Table ?(Table1),1), and is supplied by Kracie Pharma, Ltd. as a dried extract powder. Each plant material was identified by external morphology and authenticated by marker compounds of plant specimens according to the method of Japanese Pharmacopeia and our companys standard. The extract powder (lot no. 15112017) was suspended in distilled water immediately before use and was administered orally at a dose of 500 or 1000 mg/kg body-weight/day. Table 1 Medical herb composition of NYT. = 10), CORT-treated group (= 10), CORT + NYT (500 or 1000 mg/kg)-treated group (= 10), CORT + imipramine-treated group (= 10). Mice were administered CORT (100 Paeoniflorin g/mL; Sigma-Aldrich, St. Louis, MO, United States) in place of drinking water for 14 days. Animal were weaned with 50 g/mL CORT for 3 days and then with 25 g/mL CORT for 3 days to allow for gradual recovery of endogenous corticosterone secretion. NYT (500 or 1000 mg/kg/day) was orally administered once daily from day 21 to day 49. As a positive control, imipramine (10 mg/kg/day, intraperitoneally (i.p.); Wako Pure Chemical, Osaka, Japan) was administered once daily. Subsequent behavioral tests were performed on days 50C64 and brain samples were collected on day 65. On the days behavioral tests were performed, the drugs were administered 30 min before the tests. A 5-bromo-2-deoxyuridine (BrdU) solution (50 mg/kg/day, i.p.; Sigma-Aldrich) was administered from day 15 to day 19. Open Field Test Each mouse was placed in the periphery of the open field apparatus (width 30 cm length 30 cm height 30 cm). The total distance traveled in the market and the time spent in the center zone (width 15 cm size 15 cm) was recorded for 10 min using a video tracking system, ANY-maze (Muromachi Kikai Co., Ltd., Japan). Tail Suspension Test We performed the tail suspension test as described inside a earlier statement (Can et al., 2012). Briefly, the tails of mice were suspended with a piece of adhesive tape 50 cm above the floor with climbstoppers (obvious plastic cylinder, 3 cm size, 1 cm outside diameter, 0.5 cm MPL inside diameter), and animal behavior was recorded for 6 min. Like a test parameter, the latency to immobility and the total immobility time in the last 4 min were measured manually inside a blinded manner. Small movements that were limited to the front legs, but without the involvement of the hind legs, were counted as immobility. Additionally, oscillations and pendulum-like swings that were due to the momentum gained during the earlier mobility bouts were also counted as immobility. The latency to immobility was identified as the time required for the mouse to 1st cease all movement for 5 s. Pressured Swim Test Mice were placed in a glass cylinder (height, 30 cm; diameter, 15 cm) filled with water (23 2C) to a 15-cm depth for 6 min. Mice were judged to be immobile when they floated passively in the water, making only small movements to keep up their body balance or to keep their mind above the water. Like a test parameter, the latency to immobility and the total immobility and mobility time during the last 4 min were measured manually inside a blinded manner. The latency to immobility was identified as the time required for the mouse to 1st cease all movement for 2 s. Sucrose Preference Test Animals were habituated to drinking water from two bottles for 2 days. Mice were deprived of water for 14 h before the test, and the test was carried out on the following morning at 10:00. In the sucrose preference test, two pre-weighed bottles [one containing tap water and the additional comprising a 1% (w/v) sucrose remedy] were offered to each animal for 4 h. The position.