Elevated plus-maze (EPM) test and open field test (OFT)

The device and procedures used for EPM and OFT tests were those reported previously⁷. The EPM device consisted of two open arms (30 × 6 cm), alternating at right angles with two closed arms (30 × 6 × 15 cm). The central platform delimited by the four arms was 36 cm². The whole maze was elevated 50 cm above the floor. The OFT device consisted of a black square floor of 50 cm × 50 cm and white walls 45 cm tall. The floor was divided into 25 squares of 10 cm × 10 cm, of which those 16 squares touching a wall were designated as peripheral area and the 9 remaining squares as the central area.

The test room was maintained at 22–25 °C with continuously provided 50–55 dB white noises. Illumination levels were provided by four 36-W lamp tubes that give a light intensity of 357 lx. The tests were always performed between 8:00 and 11:00 a.m. Immediately before the test, mice were individually put in the test room for 30 min for them to habituate to the test environment. To begin EPM test, a mouse was placed on the central platform, facing an open arm, and was allowed to explore the maze for 5 min. Following a four-paw criterion, numbers of entries and time spent in each arm over the total exploration in both open and closed arms were calculated using an Any-maze software (Stoelting Co, Illinois, USA). The OFT was performed one week after the EPM test. To start the OFT test, a mouse was placed in the central area and was allowed to explore the open field for 5 min. Then the numbers of central entries, time spent in central area and distance traveled in the open field were calculated using the Any-maze software. The % of central distance (cD)/total distance (tD) of OFT was calculated by dividing the total distance a mouse traveled during the 5 min test by the distance it traveled within the central area. After each trial, the device was cleaned with 10% ethanol to effectively remove the scent of the previously tested animal.