2.4. Novel object recognition test

Novel object recognition is a behavioral test evaluating the animal’s cognition, particularly recognition memory. The test consists of three sessions: (1) Adaptation session: the mice to be tested were placed in an empty opaque box (50 cm × 50 cm × 60 cm) for 2 days (10 min/each mouse per day) before the experiment started. (2) Familiarization session: three objects A, B, and C were selected, where A and B objects were identical and C was completely different from A and B objects. The two identical objects A and B were placed on the left and right ends of the square box and place the mouse into the box between two objects and facing the side wall, taking care to ensure that the distance from the tip of the mice’s nose to the two objects A and B was the same. The mice were put into the square box for 5 min, and the contacts between the mice and the two objects were recorded using a video camera, and the probing time within 1 cm from the tip of the mice’s nose to the objects was recorded (including the time spent licking the objects, resting the front paws on the objects and sniffing the objects with the nose, climbing onto the objects without moving and posing could not be regarded as probing the new objects). (3) Test session: replace object B in the square box with object C, which is different from B, with the same position and still place the mouse between the two objects with their backs to each other. The mice were observed in the square box for 5 min, and the images captured by the camera were analyzed by Smart 3.0 software (Panlab), and the contact between the mice and the two objects was recorded. The total exploration time and novel object preference of the familiarization and test phases were calculated separately: RI = exploration time of object C/total exploration time, which was used to judge the cognitive function of the mice.