Behavior test

A mouse was placed in a plastic cylinder (45 cm high × 20 cm in diameter) filled with water (23–24 °C; 28–30 cm deep), and allowed to swim freely. Immobility was defined as floating without movements other than those necessary for the mouse to balance its body and keep its head above the water. Immobility time was quantified during the last 4 min of the 6 min test by the SMART system (Panlab Harvard Apparatus, Barcelona, Catalonia, Spain) [20].

The maze was cleaned with 70% ethanol before each mouse was tested. Then, a mouse was placed in the center square (10 cm × 10 cm) of the maze facing one of the open arms (40 cm above the floor). The number of entries and time spent in both open arms (30 cm × 5 cm) and the enclosed arms (30 cm × 5 cm with 15 cm-high walls) were recorded by the SMART system (Panlab Harvard Apparatus) for 5 min [20]. The percentages of open arm entries and time spent in the open arms were calculated as the number of open arm entries divided by the total number of arm entries and the time spent in the open arms divided by the total time, respectively [21].

Mice were suspended individually by the tail with adhesive tape as described previously [21]. The duration of immobility over the 6 min test period was recorded. Immobility was defined as when the mouse hung passively and motionless, and immobility time was quantified during the last 4 min of the 6 min test by the SMART system (Panlab Harvard Apparatus) [20].

Mice were individually habituated to a bottle containing 1% sucrose and a second bottle containing water on either side of the cage, and the placement of the bottles was changed every day. During the first 3 days, baseline sucrose and water intake were recorded. After deprivation of both food and water for 24 h, the mice were exposed to a bottle containing sucrose and a bottle containing water, but not food, for 12 h. Sucrose preference was expressed as (sucrose/(sucrose + water)) × 100% [22].

The maze was a circular pool (120 cm in diameter) filled with water (22–23 °C; 50 cm in depth) that was made opaque with nontoxic Crayola paint. The maze was divided into four quadrants (southwest, southeast, northwest, and northeast). For hidden platform training, a 10 cm diameter platform was placed 25 cm from the wall and 2 cm below the surface of the water in the center of the southwest quadrant. The mice received four training sessions (entry locations: north, south, east, and west) daily for 5 consecutive days. The maximum time allowed per training session was 60 s. For the probe trial on day 6, the platform was removed and the mice were placed in the northeast quadrant and allowed to swim freely for 60 s. The time spent in each quadrant, the latency to reach the platform, the distance traveled to reach the platform and mean swim speed were quantified by the SMART system (Panlab Harvard Apparatus).

The OFT was a white Plexiglas box (40 cm × 40 cm × 40 cm), and the mice were allowed to move freely in the box for 6 min. The total distance traveled by the mice was recorded by the SMART system (Panlab Harvard Apparatus) [23].

The rotarod test was used to evaluate the activity of the mice and was performed according to previous studies [24].