2.3.5. T-maze Test (TM)

Two different paradigms were carried out in a T-shaped maze (woodwork; two short arms of 30 × 10 cm2 and a long arm of 50 × 10 cm2). Copying with stress strategies, risk assessment, and working memory were assessed in a spontaneous alternation task [29]. Animals were placed inside the maze's long arm with its head facing the end wall, and it was allowed to explore the maze during a maximum of 5 min. The latencies to each one of the goals in this task, namely, to move and turn (freezing behavior), then to reach the intersection, the time elapsed until the animal crossed (4 paws criteria) the intersection of the three arms, and the total time invested in exploring the three arms of the maze (test completion criteria) were recorded. The entry of an already visited arm in the trial before completing the test was considered an error. Defecation boli and urination were also noted.

The working memory paradigm was studied 24 h later and consisted of two consecutive trials: one forced choice followed, 60 s later, by one free choice (recall trial). In this case, mice were placed inside the short arm of the maze and the latencies to each one of the goals in this task, namely, to move and turn, then to reach the intersection, the time elapsed until the animal crossed (4 paws criteria) the intersection of the three arms and the time elapsed until the mice completed 20 s in the forced arm were recorded (time to reach the criteria). Sixty seconds later, the animals that completed the forced trial in less than the cut-off time (10 min) were allowed to explore the maze in a free choice trial where both arms were accessible for 5 min. The arm chosen by the mice and the time spent to reach the

correct arm during the free choice were recorded (exploration criteria). The choice of the already visited arm in the previous trial was considered as an error, and the total number was calculated. Finally, defecation boli and urination were also recorded.

#### 2.3.6. Morris Water Maze Test (MWM)

Animals were tested for spatial learning and memory in the MWM test consisting of 1 day of cue learning and 2 days of place learning for spatial reference memory. We used this short protocol, adapted from the 2 day water maze protocol [30], as more suitable in studies where the repeated swimming or the water maze situation—which is stressful for mice but not for rats—can have an impact on other variables, such as cardiovascular system or blood pressure. Mice were trained to locate a hidden platform (7 cm diameter, 1 cm below the water surface) in a circular pool for mice (120 cm in diameter and 60 cm deep, 25 ◦C opaque water). Mice that failed to find the platform within 60 s were placed on it for 10 s, the same period as was allowed for the successful animals.

Cue learning with a visible platform: On the first day, the animals were tested for the cue learning of a visual platform consisting of four trials in 1 day. In each trial, the mouse was gently released (facing the wall) from one randomly selected starting point (W-S-E-N) and allowed to swim until it escaped onto the platform, elevated 1 cm above the water level in the NE position and indicated by a visible striped flag (5.3 × 8.3 × 15 cm3). Extra maze cues were absent in the black walls of the room.

Place learning with a hidden platform: On the following day, the place learning task consisted of four trial sessions per day for 2 days with trials spaced 30 min apart. The mouse was gently released (facing the wall) from one randomly selected starting point (N-E-W-S; E-N-S-W) and allowed to swim until escaped onto the hidden platform, which was now located in the middle of the SW quadrant (reversal). Different geometric figures hung on each wall of the room were used as external visual clues.

Variables of time (escape latency), distance covered, and swimming speed were analyzed in all the tasks' trials. The escape latency was readily measured with a stopwatch by an observer unaware of the animal's genotype and confirmed during the subsequent video-tracking analysis (ANY-Maze v. 5.14, Stoelting, Dublin, Ireland).
