Behavioral test (Morris Water Maze, MWM)

In order to evaluate CA extract’s impact on spatial learning (from day 15 to day 19) and reference memory (day 20), the Morris water maze test was used. The rat was positioned in the maze so that it was faced the tank wall during the trials used for spatial learning. In principal, rats can primarily escape from swimming by climbing onto the platform, and throughout the acquisition phase, they can learn the platform’s spatial location from any point around the pool’s perimeter, demonstrating that the platform provides no local cues to assist the rats’ escape behavior. The animal’s sense of orientation is facilitated by the visual cues present outside the tank. The apparatus consists of circular water pool of 150 cm in diameter and 60 cm in height. It was filled with 23 ± 1 °C water with a depth of 40 cm. Skim milk powder was added to the pool water to create an opaque effect. A white Plexiglas platform, 12 cm in diameter, with holes drilled into the top for the animals’ grasp, was submerged 1 cm below the water’s surface. The pool’s rim was sectioned up into quadrants of equivalent size. As soon as the rat was placed in the tank, a timer or computer tracking software was activated. The rat was given one minute to find the submerging platform in the tank, and its escape latency was measured by the distance it swam in the pool. Throughout every trial, the same visual images were placed in the exact same spots around the pool where the rats could see them. The timer or computer program was halted as soon as the rat touched the platform. The rat was either placed on the platform or led to it if it did not locate it within the allotted time. The rat was allowed to be on the platform for 15 sec to memorize the distal cues around the tank. Over the course of 5 days (day 15 – day 19), each rat underwent the test four times per day with 10 min interval for each session. Each day, the beginning positions were rearranged in a different order.

On day 20, the probe test was administered to evaluate the reference memory. During the test, the tank’s platform was removed. The rat was placed in a new starting position and permitted to roam the tank for one minute. The number of targeted trips, overall distance covered, and total time spent in each quadrant were all evaluated. Video cameras were used to record all trials of the spatial learning and reference memory tests, and analysis was performed with video tracking software (SMART ver3.0 Panlab Harvard Apparatus, Barcelona, Spain) that enabled both real-time and post-hoc automatic rat path monitoring. All tests were performed during light cycle with consistent timing between groups (10 min interval for each session) and performed between 0900 and 1100 a.m in a soundproof laboratory.