Behavioral Tests

Behavioral tests were conducted between 9 am and 2 pm in constant white light. Before each test, all elements were cleaned with alcohol to remove olfactory clues. Tests were video recorded for subsequent analysis.

The open field test (OFT), adapted from Schiavone et al,34 was carried out in a circular arena (60 cm diameter) divided into 6 equal fields. The test was conducted 1 day after habituation (6 minutes in the arena). To perform the test, animals were placed in the arena for 6 minutes, and a trained and blinded observer counted the number of fields crossed, which led to the test result.

The novel object recognition (NOR) test was adapted from Giovannini et al.35 Animals were tested in a plastic box (28×40×22 cm). On the first day (adaptation), animals were allowed to explore the open field for 10 minutes. The next day, animals were placed in the box (training) to familiarize them with 2 identical objects for 10 minutes. After 60 minutes, animals were placed in the box again (choice trial). In the choice trial, 1 of the familiar objects was replaced with a novel object and animals were left to explore the box for 10 minutes. A trained, blinded observer measured the time spent on exploration of each object (ie, touching, sniffing, sitting in close proximity with nose directed to the object). The NOR index was calculated using the following formula: (N−F)/(N+F) (where N=time spent on exploration of the novel object during the choice trial; F=time spent on exploration of the familiar object in the choice trial).35