Morris water maze test

In order to study spatial memory, the Morris water maze test was done as part of the behavioral battery on the same cohort (#3) of mice as previously described³⁹. Mice were acclimated to the paradigm and tested for baseline response using a visible platform test one day prior to the start of the injury paradigm. The animals were placed in a circular pool (1 m diameter) filled with opaque water containing non-toxic white paint and a clear escape platform marked by a visible rod. To assess learning, the mice were tested using a hidden platform fixed in the northeast quadrant starting one day after the last injury. Testing was conducted with four trials a day for six days in a row. On the seventh day, a probe test was completed to test memory, where the hidden platform was removed and the time spent exploring the northeast quadrant was recorded. Black and white distal extra-maze cues were positioned on the walls of the room and geometric shaped proximal extra-maze cues were positioned above the walls of the maze. The mice were placed in pseudo-randomly varied quadrants throughout testing, and the time to locate the platform was recorded. Trials were run until the mouse found the platform or was placed there after the maximum trial time of 60 s. At the conclusion of the trial, the mouse was allowed to remain on the hidden platform for 15 s to consolidate learning, followed by removal from the pool and placement onto a heating pad for 10 min. Eight to ten mice per group and condition were used. Data was analyzed blinded to condition. Data was recorded using a video-tracking system (EthoVision XT; Noldus Information Technology, Leesburg, VA).