2.4. Morris Water Maze (MWM) Test

Spatial learning ability was tested using the Morris water maze (MWM) test after four-week intragastric administration. The water maze was an open circular black tank (120cm in diameter and 60 cm in height) and a movable circular platform (8cm in diameter). A camera two meters above the tank was connected to the computer to record the rat’s movement, and the data was analyzed by TopScanLite (CleverSys Inc). The tank was filled with water at a height of 23cm (23-25°C) and dyed with black edible pigment [37]. The tank was divided into 4 quadrants by two imaginary perpendicular axes with four cardinal points: North (N), South (S), East (E) and West (W), and the platform was placed in the middle of SW quadrant, 2cm submerged into the water surface.

MWM test was started with the place navigation trial, and each rat in all groups (n=15 in each group) underwent four trials per day with a one-minute-interval for 5 consecutive days. The rat was gently placed in the water from the starting position (location of SE, NE, SW, and NW quadrant, respectively), facing the wall of the tank. The rat was allowed to swim for 90 seconds until it found the submerged platform and stayed on the platform for a maximum of 10 seconds. If the rat failed to find the hidden platform within 90 seconds, it would be guided to the platform and remained on the platform for a maximum of 10 seconds before being removed from the pool. The escape latency was defined as the time taken to search for the platform within 90 seconds. The escape latency, swimming speed, and swimming path were recorded by the tracking system.

The spatial probe test was conducted 24 hours after the 5-day place navigation trial where the platform was removed from the pool to assess memory retention for the location of the platform. The rat was put into the pool at the NE point and allowed to swim for 90 seconds. The swimming speed, swimming path, and the frequency of rat crossing the virtual platform were recorded.