T-Maze Test

The spontaneous exploratory behavior of mice was tested in a T-shaped maze (with arms 25 cm in length). The animal was placed inside the ‘vertical’ arm of the maze with its head facing the end wall. The performance was evaluated by determining with a chronometer the time elapsed until the animal crossed (four paws criteria) the intersection of the three arms. Defecation was also measured.

Cognitive function

Cognitive function was assessed in three paradigms of ‘learning and memory’ of increasing difficulty in the Morris water maze: a cue learning task of a visible platform to assess visual perceptual learning and memory, followed 24 h later by a place learning task using a hidden platform in a reversed position to assess spatial reference learning and memory, and a final trial for long-term memory where the platform had been removed. During each trial of the learning tasks, the escape latency was measured by means of a computerized tracking system (SMART, Panlab S.A., Barcelona, Spain).

On days 1-2 (Cue 1 and Cue 2), animals were tested for visual perceptual learning and memory. Mice were trained to locate a platform (7 cm diameter, NW position) elevated 1 cm above the water level with its position indicated by a visible striped flag (5×8×15 cm) in a circular pool (Intex Recreation Corp. CA, USA; 91 cm diameter, 40 cm height, 25°C opaque water). The cue learning task consisted of four trials 20 min apart per day (ITT, intertrial time), during two consecutive days. In each trial, the mouse was gently released (facing the wall) from a randomly selected starting point (N, S, E, or W), and was allowed to swim until it escaped onto the platform.

On days 3–7, five place tasks (PT1–PT5) for spatial reference learning and memory were administered, followed by one probe trial, usually called ‘removal’ (RM) for long-term memory. In the PTs, the hidden platform was always located in a reversed position (the middle of the SE quadrant) and the mouse was trained to locate the platform thanks to four distal visual cues. As before, the acquisition task consisted of four trial sessions per day, with 20 min of ITT. In both tasks, mice that failed to find the platform within 60 s were placed on it for 10 s, the same period allowed for the successful animals.

On day 8, the platform was removed from the maze and the mouse performed a probe trial of 60 s. The computerized system allowed to measure the time spent swimming in the Place Task trained platform quadrant of the pool (P), adjacent right quadrant (Ar), opposite quadrant (O), and adjacent left (Al).