Novel object recognition test

The NOR test was conducted for the assessment of visuospatial memory. The test was carried out as described previously.30 A standard squared four-compartment mildly illuminated OF box was employed (LE800SC, Panlab Harvard Apparatus, 90 × 90 × 40 cm). Before performing the test, animals were habituated to the box over 15 min; these data were employed to evaluate their motor spontaneous activity and stereotypes as described below. The test consists of two different stages; during the first one (habituation phase) two identical objects were placed into the box, symmetrically separated from each other, and each mouse was allowed to explore them for 5 min. After a delay of 24 h, the mouse was placed again in the cage and exposed for 5 min to one familiar object in the same position and to a novel object placed in a new location (NOR 24 h phase). All trials were video recorded, and the total time spent exploring each object was measured manually with a stopwatch. To avoid the presence of olfactory trails, the apparatus and the objects were thoroughly cleaned after each trial. The DI was calculated as a percentage according to this equation: (Exploration time of the novel object/Total exploration time) × 100. Consequently, a ratio of 50% reflects equal exploration of the familiar and the novel object, indicating no learning retention, and ratios above 50% are indicative of visuospatial learning retention.