Assessment of Spontaneous Alternation, Novel Object Recognition and Limb Clasping in Transgenic Mouse Models of Amyloid-β and Tau Neuropathology

The overall goal of this procedure is to measure abnormal behaviors that are common in mouse models of Alzheimer's disease in a standardized way in order to screen for compounds that may have beneficial effects. This method can help answer key questions in the field of preclinical drug discovery for Alzheimer's disease such as whether a compound is efficacious or how a compound might work. The main advantage of this technique is that it monitors a number of different behaviors using a staged approach that is both sensitive and specific.

Though this method can provide insight into effective treatments for Alzheimer's disease, it can also be applied to other mouse models of dementia or cognitive dysfunction such as frontotemporal dementia, traumatic brain injury or autism. Before using the Y-maze thoroughly clean it with an unscented bleach germicidal wipe. Then wipe it down with 70%ethanol and rinse it with water.

Prepare the data acquisition system or video cameras to properly track the mice while they run the maze. During this process record an image of a measuring stick in the maze to calibrate distances they're in. Now, very gently transfer the mouse cage to a table in close proximity to the maze.

Remove the mouse from the cage and gently place it into one arm of the Y-maze facing the center. Then move far enough away from the maze that the mouse cannot see you and immediately start the video recording. Record the mouse for 10 minutes.

After 10 minutes, gently remove the mouse and return it to its home cage. Before proceeding with the next mouse, thoroughly wash the Y-maze again using the three-step washing procedure. Prior to this test, wash the open field arena just as done with the Y-maze.

One day prior to object exposure, habituate the mice to the arena. Make certain the tracking system is working. And calibrate distances using a measuring stick as before.

Also in the tagging software, mark the corners of the arena. Now, gently place the mouse's cage on a table close to the arena. Remove the mouse and gently place it at the center of the arena.

Then quickly start the recording and allow the mouse to explore the arena for half an hour. Be sure to be out of the animal's view and do not disturb it during the test. After the habituation session return the mice to its home cage.

And clean the arena thoroughly as before prior to habituating the next mouse. Two to three hours later, execute the sample phase of the memory test. Modify the arena by placing two identical objects within it both aligned evenly to one wall.

Fix the objects in place with mounting putty. In the software, notate the positions of the two objects as well as the arena's corners. Wash the objects and arena as usual before a mouse is presented.

Conduct the test like the habituation session placing the mouse in the arena facing the objects. Now, only let the mouse explore the arena for 15 minutes. Two to three hours later, conduct the final phase of the memory test.

For this session keep one familiar object in the same location and remove the other familiar object replacing it with a newly introduced novel object. Leave enough distance between the walls and objects for the mice to freely explore the objects from all angles. And as before, secure the objects with putty.

Across all the test groups in the study, be certain to have a balance of positions for novel and familiar objects. Conduct the final session like the other two sessions but only record the mouse's behavior for 10 minutes. Limb clasping is a functional motor test that quantifies deficits in corticospinal function.

For this test, use a handheld device to video record the entire session. Begin with placing the home cage on a clean table and documenting the animal's ID.Then gently remove the mouse from its cage and hold it suspended by the tail for five to 10 seconds while focusing the video on the animal's hindpaw and forepaw movements. Handling the mouse so that it doesn't attempt escape takes practice.

After capturing at least five seconds of video return the mouse to its home cage. And return the cage to the rack. Then clean the table before repeating the procedure on the next mouse.

Later, score the behavior in the videos on a scale from zero to four. Using the Y-maze test, an aged transgenic mouse line which exhibits Alzheimer's disease-like pathology revealed an expected spontaneous alternation defect. By contrast, another Alzheimer's model mouse line showed increased spontaneous alternation that was due to extreme hyperactivity and stereotyping in the line.

These behaviors significantly interfere with the measurement of spontaneous alternation. In the novel object test, during habituation hyperactivity and other stereotype behaviors can be assessed. During the sample phase, exploration of each object was measured separately to identify animals without object bias.

Open circles show animals that showed bias and were excluded from the test phase. The test phase was analyzed in three different ways. When total exploratory time was comparable across groups then the raw exploring time of each object was analyzed.

When the compared groups explored for different amounts of time then either novelty preference or a discrimination index was used to score the test. Limb clasping which is not a cognitive measure was observed in several transgenic tail mouse models. This behavior recapitulates some of the functional motor deficits observed in late stage Alzheimer's disease.

While attempting this procedure, it's important to remember to handle the mice gently and minimize any disturbances in the testing environment such as loud noises or novel smells. Following this procedure, other methods like spatial water maze or contextual fear conditioning can be performed in order to answer additional questions like whether a compound specifically affects function of the hippocampus. After watching this video you should have a good understanding of how to screen for efficacy of compounds in mouse models of Alzheimer's disease using the Y-maze test, the novel object recognition test and the limb clasping test.