Morris Water Maze

One day after treatment, learning and spatial working memory were assessed by the Morris water maze (MWM) with the same group of animals subjected to rs-fMRI scanning (n = 8–10 per group). Detailed description of the training protocols can be found in our previous study (Kan et al., 2016). Briefly, the MWM test was divided into two parts: spatial acquisition trials (day 1–5) and probe trial (day 7). On day 6, the rats were allowed for a rest. The water maze is a black painted circular tank with 150 cm in diameter and 60 cm in height, placing at the center of a quiet room. On the wall of the tank, there were four different visual clues, dividing it into four quadrants. A black curtain was held in place by a rectangular scaffold around the water maze to minimize harsh lights entering the room. A video camera mounted to the ceiling above the water tank was used to track the rat swimming speed, latency to find the platform, and target quadrant crossovers with an animal activity tracking system (Ethovision, Noldus Information Technologies, Wageningen, The Netherlands). For each training session, the tank was filled with clean water for about 35 cm in depth (23 ± 2°C) with a hidden circle platform 1.5 cm below the water surface at the third quadrant. On day 1–5, the rat was gently placed in one quadrant facing the wall of the MWM before each spatial acquisition trials. Then, the rat was allowed to swim freely for up to 60 s in the water maze to locate the platform. If the rat succeeded, it was allowed to stay for 5 s on the platform. Otherwise, the rat was physically placed on the platform for 20 s. Four spatial acquisition trials were performed on each rat per day and continued for 5 consecutive days. A 3–5 min interval was allowed between each trial. On day 7, a probe trial was conducted in which the platform was removed and the rat was placed at the quadrant opposite to the platform and allowed to swim for 30 s. The times the rat crossover the platform location and the percentage of time spent in the platform quadrant were recorded. From day 31–35, spatial acquisition trials were repeated followed by a probe trial on day 37. The time line for the MWM testing is shown in Figure 1B.