*2.5. Behavioral Assays*

For the assessment of the behavioral aspects, open field (OF) and novel object recognition (NOR) tests were performed at days 66 and 68–69, respectively (Figure 1).

#### 2.5.1. Open Field Test

The OF test was performed in a 60 × 60 cm black-based box with transparent walls, with a height of 50 cm, to observe the animals' locomotor and exploratory behavior. The illumination in the testing room was controlled and maintained between 50 and 100 lux. After the animals were placed in the middle of the testing area, their behavior was recorded for five minutes from above. After each test, 70% ethanol was used to disinfect the apparatus. All trials were analyzed offline with EthoVision XT (version 11.5, Noldus IT, Wageningen, The Netherlands), monitoring the distance moved, the number of entries, and the time spent in the center zone (central 30 × 30 cm area), vertical activity (wall climbing, rearing), and grooming activity.

#### 2.5.2. Novel Object Recognition Test

In an empty testing chamber (60 × 60 cm), animals were habituated for 10 min, then returned to their home cage, and the chamber was cleaned as described above. In the familiarization phase, two identical novel objects constructed of wood (10 × 4 × 4 cm) were placed in the chamber (30 cm apart from each other) (Supplementary Figure S3). Object placement was chosen so that animals could walk freely around the arena's edge, as they would have done in an open field. Thus, the animals were required to move into the center of the chamber to interact with the objects rather than unintentionally encountering them as they explored the arena perimeter.

Observations of animal activity were conducted for 5 min. A nose touch was classified as an interaction with an object. For the subsequent memory test, the animals had to interact with each object for at least 2.5 s during the familiarization phase. The animals were returned to their home cages after their five-minute time frame had ended. Before each test, the chamber and objects were cleaned with a 70% ethanol solution.

The inter-trial interval between familiarization and testing was 24 h. One of the familiar objects encountered during the familiarization phase was then replaced with a novel object, which was different in color and configuration but about the same size. Across tests, the position of the novel object (i.e., left or right) was counterbalanced. Following the same procedure as the one during familiarization, animals were reintroduced to the chamber and allowed to explore it for five minutes. Time spent by the animal in exploring individual objects during familiarization phase, total time spent by the animal in exploring both objects during the test and training phase, the total distance moved, the time spent moving, and the number of rears were quantified by a computerized analysis system (EthoVision XT, version 11.5, Noldus IT, Wageningen, The Netherlands).
