2.3. Morris Water Maze Test

Morris water maze (MWM) is a well-validated test for cognitive function. For the whole test period, the lab was maintained as a sound-insulated and low-light environment, while the objects in the room were kept at their original locations and other experimental conditions remained unchanged. After EA treatment, a circular water tank (80 cm in diameter and 50 cm in depth) divided into four equal quadrants (I, II, III, and IV) was used for MWM test; a circular escape platform (9 cm in diameter and 28 cm in height) was placed at the center of quadrant I. The tank was filled with warm water (23 ± 1°C) to a depth of 30 cm and edible melanin to dye the water black. Visual cues of various shapes were located on the side of each quadrant of the tank, in plain sight of the mice. A digital camera connected with an image acquisition system was used to automatically track all trials. The MWM test was performed as two stages. (1) To evaluate changes in learning ability, each animal was subjected to a trial with a hidden platform. In order to familiarize mice with the test environment, the mice used in the experiment were separately allowed to swim freely in the water tank (without platform) for 60 seconds on the day before being subjected to the hidden platform trial. Throughout the trail, mice in each group were placed in the water at a fixed position in quadrant III while the submerged platform remained fixed in location in quadrant I. If the tested animal found the hidden platform within 60 seconds and stayed on it for at least 5 seconds, we recorded the escape latency time (expressed as the time spent swimming to find the platform). If the animal failed to find the platform within 60 seconds, it was placed on the platform for 10 seconds, allowing it to observe and memorize the location, and the escape latency time was recorded as 60 seconds. The trial with a hidden platform was repeated daily for five consecutive days. (2) We conducted a probe trial on day six to measure the ability of the mice to maintain long-term memories with the hidden platform removed. The frequency with which the platform's former location was crossed was recorded together with the time spent in the target quadrant (I) during the trial.