Novel Object Recognition Test (NORT)

The NORT is used for the evaluation of recognition memory^95,96. The mice were presented with two similar objects (blue cubes) during the first (familiarization) session. Then one of the two cubes was replaced by a novel object (pink ball) during the second (test) session. It has been demonstrated that prior experience can alter the behavioral responses of mice in the NORT⁹⁷, animals were accustomed to being handled by experimenters twice a week for 1 min each session for 1 week before the beginning of the experiments. Following the protocol of Sik et al.⁹⁸, the habituation phase consisted of 5 min exposures to the testing arena per day, separated by 6 h during 3 days before the test phase. The test was conducted 10 days after surgery. Two identical cubes were placed in a cage for mice for 10 min. Then there was a second 10 min session when one cube was replaced with an unfamiliar earlier ball⁹⁵. The exploration time for both objects during the test phase was 20 s⁹⁹. For the experiments, we used a black wooden box (33 cm × 33 cm × 20 cm) and a video-tracking package. We used two asymmetric objects of the same size (cubes: 3 cm × 3 cm × 3 cm; ball: 3 cm in diameter) and odor. Since mice have difficulty in discriminating colors, we selected bright (dark blue and pink) objects. The weights of objects were heavy enough that the mice could not move them. Additionally, we used Patafix to hold the objects stuck on the floor. The mice were housed in a light-dark cycle and were tested in the dark phase (active phase between 08:00 and 20:00). The familiarization session was carried out in the morning (at 09:00). The mice were placed in the testing room 30 min before testing. The exploration was defined as follows: directing the nose toward the object at a distance of less than or equal to 2 cm. We chose to score the object exploration whenever the mouse sniffed the object or touched the object while looking at it (i.e., when the distance between the nose and the object was less than 2 cm). Climbing onto the object (unless the mouse sniffs the object it has climbed on) or chewing the object does not qualify as exploration.