Use of an Eight-arm Radial Water Maze to Assess Working and Reference Memory Following Neonatal Brain Injury

The overall goal of this procedure is to simultaneously quantify working and reference memory without the limitations that are typical in land-based mazes. To begin, an animal is placed into the start arm of the water maze and allowed to locate a hidden platform. Entries into each arm are recorded as well as the location of the platform used for escape.

Next, the escape platform used by the animal is removed and the procedure is repeated for three more trials. In the final step. The entries are coded as working memory, correct errors, reference memory errors, or working memory incorrect errors.

Ultimately, the eight arm radial water maze can be used to evaluate working and reference memory in multiple animal models. The main advantage of this technique over existing methods like the Morriss water maze, is that it allows the experimenter to simultaneously assess both working and reference memory. The radial maze consists of eight stainless steel arms attached to a circular base.

It is placed in a tub filled with room temperature.Water. Hidden platforms are placed one centimeter below the water surface. In four of the arms, two experimenters are required.

One to record the data and one to handle the subjects. During this collaboration, communication between the experimenters is paramount. Confirmation of start times subject numbers and proper platform placement prior to each trial is essential.

A digital tablet is used for recording maize arm entries. Open a PDF writer program and import all pre-saved data recording templates for subjects in group one. The template should have a blank representation of the maze and platform locations, as well as a place to record the subject number, trial number testing day escape platform used and trial latency.

To begin, transfer the first test subject into a separate warmed cage. Next, confirm that the platforms are correctly positioned in the eight arm water maze. After checking the subject identification number, place the subject into the start arm facing the wall of the maze, and start the timer.

It is important to remain stationary during each trial in a designated position to maintain Q consistency throughout. Testing record entries by placing a mark on the corresponding arm. In the recording template, an entry is counted.

When a subject’s shoulders break the opening plane of the arm, allow a maximum of 120 seconds for the subject to locate one of the four platforms. Once the animal reaches the platform, stop the timer to complete the trial. Record the latency to reach the platform.

For each trial to maintain consistency, a subject is said to have reached the target. When its front paws are on the platform. On the first day of testing, allow the subject to sit on the platform for 10 seconds for all four trials so the subject can reference the spatial cues.

If the subject is unable to locate the platform within 120 seconds, carefully guide them to the closest platform and allow them to remain on the platform for 10 seconds. Following trial completion, remove the subject from the platform by gently lifting them by the body. Begin the timer for the 92nd inter trial interval.

To prevent hypothermia, dry the subject with a towel. Place the subject back into the individual cage over the thermo regulation pad. Record the specific platform used for escape by circling the corresponding location on the data recording template.

During the inter trial interval, remove the escape platform used by the subject in the previous trial. Open the template for the next trial, and mark all platforms that have been used for escape by the subject and thus removed during previous trials with an x. Next, confirm the placement of the platforms for the next trial.

When ready, place the subject into the start arm facing the wall of the maze and start the timer. Repeat this process for a total of four trials. Removing the escape platform reached after each trial and marking the template appropriately during and after each trial.

Once completed, place the escape platforms back in the appropriate arms of the maze and repeat the testing process for the next subject. Once testing is completed for group one, export the data recording sheets to a folder for that testing day and save it for future analysis. For each trial, count the number of first and repeat entries into any arm that previously had a platform.

These are the working memory correct errors. Reference memory errors are counted as the number of first entries into any arm that never contained a platform. The number of repeat entries into any arm that never contained a platform are working Memory incorrect errors using the eight arm radial water maze, a significant increase in the number of incorrect errors in rodents with neonatal hypoxic ischemic brain injury was revealed.

Reference memory errors were also increased in brain injured as compared to control animals. While attempting this procedure, it is important to note that animals with experimental manipulations may require more time to acquire task proficiency. This is especially true for working memory correct errors given that the rat has a natural tendency to return to a platform arm when water escape is a motivator.