2.3. Morris Water Maze

Morris water maze (MWM) was performed to assess cognitive performance. The apparatus consisted of a blue plastic circular pool (180 cm in diameter) with a circular platform (10 cm in diameter, transparent plastic, submerged 1 cm below the water surface). The position of the platform was constant (in the center of SW quadrant). The pool was filled with water (21–22 °C, 28 cm deep) colored by a small amount of non-toxic grey dye. The position of the rat was recorded every 40 ms by an overhead camera connected to a digital tracking system (Tracker, Biosignal Group, New York, NY, USA). The data was later analyzed using software Carousel Maze Manager 0.4.0 [51].

The MWM testing was initiated on Day 12 of the experiment and it was conducted during four consecutive days (MWM Day 1–4) in the light phase of the day. The rats were trained to find the hidden platform. Each day the rats underwent 8 swims from different starting points located on the periphery of the pool (in pseudorandom order). Animals were released into the water facing the inner wall of the pool. If the rat did not find the platform in 60 s, it was guided to the platform by the experimenter. On the MWM Day 4, after finishing the MWM sessions, the platform was removed, and the rats underwent a 30-s probe trial. Subsequently, the platform was returned to the pool, raised 1 cm above the water surface, and provided with a contrast rim for better visibility. A 60-s visible platform trial was conducted to assess sensorimotor functions and motivation [52].

The dependent variables for training trials were total distance (m) moved by the rat to reach the hidden platform (or total distance moved in the case of unsuccessful trials) and mean distance (cm) from platform (mean of the distances of the animal from the platform, sampled in 40 ms intervals), which represents a sensitive parameter, reflecting not only the ability to locate the platform, but also the search strategy [53]. Mean daily values were calculated for each animal and used for analysis. Latency was also analyzed, but as latency and distance are generally correlated, we report only the distance, which is less sensitive to possible differences in swimming speed. Cumulative latency (sum of all latencies during MWM Day 3 and 4) was calculated to assess the best achieved performance [54]. Days 3–4 were chosen because all groups reached asymptotic performance by MWM Day 3 (within each group, there was no significant difference between MWM Day 3 and Day 4 with respect to the distance moved as well as escape latency [latency data not shown]; two-way repeated measures ANOVA with Bonferroni post hoc test).

The dependent variables for probe trial and visible platform trial were dwell time in target quadrant (=the quadrant where the platform was originally located) and latency to find the platform, respectively.

“The periphery of the pool” refers to an 18-cm wide annulus; the wall of the pool represented the outer border of the annulus.