2.6. Behavioral Tests

All behavioral tests were performed in the behavior testing room. An Anymaze video-tracking system (Stoelting) equipped with a digital camera connected to a computer was used. Following behavioral tests, open field (locomotion), novel object recognition, and passive avoidance were conducted. The study was carried out in compliance with the ARRIVE guidelines. During the behavioral tests, mice were centrally administered PBS or ampelopsin A before every behavioral test, including habituation and test session (60 min before). Subsequently, mice were treated (i.p.) with saline or scopolamine before every behavioral test (30 min before).

The open-field test (locomotion) was performed as previously described with slight modifications [26]. Specifically, the mouse was located in the center of an open field chamber (40 cm length × 40 cm width × 50 cm height) and was habituated for 20 min. Each mouse was replaced in the same chamber 24 h later. The movements of the mouse were recorded for 10 min and then analyzed via a digital camera connected to the Any-Maze animal tracking system software (Stoelting, Wood Dale, IL, USA). The total distance moved (meters) and the time (seconds) spent in the center/outer of the open field were measured.

The novel object recognition test was performed as described in the previous study [27]. Specifically, the mouse was located in a square arena (40 cm length × 40 cm width × 50 cm height) equipped with a digital camera and was allowed to familiarize with the environment for 10 min before the test. During the first session (familiarization session), two identical objects were put against the center of the opposite wall and the mouse was allowed to explore the objects for 20 min. During the second session (test session), one of the identical objects was replaced by a novel object, and the mouse was allowed to explore the objects for 10 min. In the familiarization session, the mouse contacted with two yellow square-based pyramids(8 cm × 8 cm × 6.5 cm) while in the test session it was with a yellow cube (7 cm × 7 cm × 7 cm) and a yellow square-based pyramid. The amount of time that the mouse spent exploring each object was monitored and analyzed using an ANY-maze video-tracking system (Stoelting, USA). A discrimination index was calculated as (novel − familiar object exploration time)/(novel + familiar object exploration time).

The passive avoidance test was performed as previously described [28] using an Avoidance System (B.S Technolab INC., Seoul, Korea). The apparatus (48 cm length × 23 cm width × 28 cm height) consisted of light and dark chambers separated by a gate. On the first day, the mouse was allowed to explore both compartments freely for 10 min. On the following day (training), the mouse was placed in the light compartment and 60 s later the gate was opened. Once the mouse entered the dark compartment, the door was closed and an electrical foot shock (0.3 mA, 3 s) was delivered through the floor. After 24 h (probe trial), the mouse was placed again in the light compartment and then the gate was lifted 60 s later. The step-through latency, or time taken for the mouse to enter the dark compartment, was scored 300 s as the upper limit.