5.5. Novel Object Recognition

For assessing cognitive functioning, the novel object recognition test was conducted in a large open field made of gray plastic (60 × 60 × 60 cm), as previously described [103]. First, rats were habituated to the open field (no objects present) by placing them into the box for 10 min. Next, 24 h after the habituation session, the novel object recognition test was conducted, which consisted of three phases: acquisition trial, inter-trial interval, and recognition trial. In the acquisition trial, each rat was allowed to freely explore the open field containing two identical sample objects for 5 min. The objects were placed in one of the back corners of the box, with the objects situated 15 cm away from the walls. As objects, either two silver iron cylinders (5 cm in diameter, 8 cm high) or two red metal cubes (5 × 5 × 8 cm) were used in a counter-balanced manner. After the acquisition trial, the rats were returned to their home cages for 30 min, the inter-trial interval. During that time, one clean familiar object and one clean novel object were placed in the open field, where the two identical objects had been located during in the acquisition trial. After the inter-trial interval, each rat was returned to the open field for a 5 min recognition trial and allowed to freely explore the familiar and the novel object. For behavioral analyses, a digital camera (TVVR3304; ABUS, Affing, Germany) was mounted 1.5 m above the floor of the open field and connected to a personal computer for recording and data storage. Behavior was scored from video recordings by an experienced observer blind to the rat’s genotype using The Observer XT (Noldus, Wagenigen, The Netherlands). Object exploration was quantified as time spent sniffing the object and scored whenever the nose was oriented toward the object and the nose-object or front paw-object distance was 2 cm or less. Recognition memory was defined as spending more time sniffing the novel object than the familiar object. Testing was performed under white light (40 lx) conditions.