The goal of this procedure is to identify cognitive deficits in a mouse model of Alzheimer's disease through the use of a Morris Water maze Protocol, tailored for use with the 1 2 9 6 FBB background strain. This is accomplished by first acclimating the mice to the task to alleviate stress-induced performance deficits. The second step is to use the visual platform task in which a platform is raised one inch above the waterline to assess visual and motoric functioning.
Next, the hidden platform trials are conducted where the platform is submerged five millimeters below the surface of the water. The final step is to use probe trials where the platform is removed to assess spatial reference memory and determine the type of search strategy used. Ultimately, this tailored water MAs procedure allows for the assessment of spatial learning and memory in a commonly used mouse model of Alzheimer's disease.
Generally, individuals new to this method will struggle because of the necessity to optimize the testing procedures, such as the number of training trials and probes used based on the background strain of the mouse line and the environment in which the mice are tested. This part of the procedure acclimate the mice to pull entry and removal. First, place the box so it is raised to a comfortable height and fill it with two liters of water at a temperature of 21 degrees Celsius and to a level of approximately one centimeter.
After bringing the mice to the testing room, remove a subject from the cage, verify the subject identification, and then mark the tail with a non-toxic permanent marker to distinguish it from mice that have not been through the procedure. Place a mouse in the transfer beaker for approximately five seconds. Then gently pour the mouse out of the beaker into the pre handling pool time the mouse in the pool for 20 seconds.
After the time has elapsed, gently place a scoop in front of the mouse without angling it. Allow the mouse to enter and explore the scoop If needed, encourage the mouse to get on the scoop by gently sliding the scoop under the mouse while slowly lifting. Take care not to frighten the mice with the scoop.
Repeat these procedures for each mouse in the cage once per day for 10 days. After each animal, pick up fecal boli and bedding from the pool, using a net being the transfer beaker between cages. Perform visible training on testing days one, two, and three with six trials each day and approximately 10 minutes between each trial.
First, set up the mouse performance tracking software and create the run sheets according to the instructions in the text portion of the protocol. Once the software is configured, set up the room by first hanging curtains so that any spacial cues are obscured. Next, set up the tub by filling with tap water at 21 degrees Celsius.
Use a thermometer to ensure that the water temperature remains within one degree of this temperature. Throughout testing. Place the platform which has a mounted flag that reaches the height of 13 centimeters and is 4.5 by 4.5 centimeters with a bold S-shaped character embossed on it in the first predetermined location of the tub so that it rises approximately one inch above the surface of the water.
Now take the first mouse from the cage. Verify the subject identification, mark the tail using a permanent marker of the correct color, and place it in a transfer beaker. Gently place the mouse into the tub.
Start the timer and ensure that the tracking software is tracking the animal. Allow the mouse up to two minutes to locate the platform. Once the mouse locates the platform, allow the mouse 20 seconds on the platform.
If the animal fails to find the platform in the two minute period, scoop the mouse and place it on the platform for 20 seconds. Following 20 seconds on the platform, remove the mouse from the platform as done in acclimation training, and place it in a heated cage lined with paper towels and warm to approximately 31 degrees Celsius by a heating pad and heat lamp for 30 seconds. Then transfer the mouse to the holding cage with only a heating pad, but no heat lamp to recover between trials.
After cleaning the tub of debris with a net, repeat these steps for each mouse in the group. When each mouse is completed, the first trial, move the platform to the second predetermined location. Perform hidden platform training from days four to 10 after hanging curtains.
As before, place cues strategically on the curtains. Ensure that the cues are large and contain contrasting colors for better visibility, and are set to distance and height where they are visible to mice from inside the tub. Configure the software according to the instructions in the written portion of the protocol.
Then set up the tub by first filling it with water so that the platform is approximately five millimeters below the surface of the water. Place the platform in the predetermined location. The location of the platform will remain the same for almost being tested across all days and trials.
It.It is essential to vary the start location so that the animals do not develop a nons spatial, non hippocampal motoric strategy. To solve the maze, use non-toxic white temperate paint to make the water opaque. Do this to ensure that the top of the platform is invisible from the animal's eye level while swimming.
After identifying and marking the mouses before, gently pour the mouse into the maze, so the ENT facing the wall. Each mouse will be placed in the same starting location for a unique trial. At the initial release of the mouse, begin the timer and stand in an area where the tester is not easily visible to the mice.
Once the animal reaches the platform, allow it to remain on the platform for 15 seconds to orient its spatial location within the room. If the mouse does not locate the platform within 60 seconds, gently scoop the mouse and place it on the platform. After 15 seconds on the platform, remove the mouse from the maze and return it to the heated cage.
After cleaning the maze of debris before, repeat these procedures for the remaining mice in the group. Thus, for testing days four through nine, conduct four trials of hidden platform training per day with approximately 20 minutes between each trial. Perform probe trials prior to platform training on day six, seven, and eight, and the final probe trial.
On day 10, conduct the probe trials in a similar manner to hidden platform training with the exception that no platform is in the maze. Release a mouse from a predetermined release point and allow the mouse 60 seconds to swim in the maze Following the probe trial on day 10, there are no additional trials conducted during the probe trials. Use the animal tracking software to track the path length of the animal, the percentage of time the animal spends in each quadrant of the maze, and the number of times an animal swims over the previously platformed area.
Open the experimental list tab and obtain the measurements from the data analysis tab and the water maze tab. Export directly to an Excel spreadsheet. Then analyze the data according to the instructions in the written portion of the protocol.
41 TP 3 0 1 L mice containing the human P 3 0 1 LTL gene and 46 controls were tested using this protocol at approximately 6.5 months of age. This represents three months of Tau P 3 0 1 L expression in the transgenics. Each group contained approximately equal numbers of males and females path length.
Invisible platform training did not differ between controls and TP 3 0 1 L mice. This lack of difference indicates that control and tau P 3 0 1 LM do not differ in visual or locomotor capability, and that performance deficits on hidden trial testing can therefore be attributable to memory impairments. However, during hidden platform training, TP 3 0 1 LM demonstrated significantly longer path lengths across all training blocks.
Finally, controls improved across the four probe trials. Whereas Tau P 3 0 1 L mice did not control mice were better able to use a spatial strategy to solve the task and had better learned to localize the platform to the target zone. When adapting this procedure for use with other mouse lines, it's important to assess the learning rate of controls, to identify the number of hitting training trials needed, and to determine when the first probe trial should be inserted.
