A Behavioral Test Battery for the Repeated Assessment of Motor Skills, Mood, and Cognition in Mice

Neurodegenerative disease often manifests with devastating behavior symptoms, such as cognitive impairment, mood dysfunctions, or even motor dysfunctions. This study allows a behavioral phenotypes to be assessed accurately with a high level of repeatability. We design a comprehensive battery that can be repeatedly utilized in behavior analysis in mice.

This test asses motor functions, social interaction, emotional function, and cognitive function. By organizing the order and the interval of tests in this battery, we increased the testing efficiency and decreased the interference among tests and the stress on mice from frequent handling. To begin, arrange the behavioral test in the order described in the text protocol.

Perform the open field test in a non-transparent, white, plastic arena. Start the camera recording, and gently put the mouse next to the middle of a wall of the arena, facing the wall. Record the mouse’s behavior for 10 minutes, and return it to its home cage.

Next, divide the arena into four by four squares on the computer screen. Count the number of times the mouse crosses the gridlines in the arena to assess locomotor function. To gauge anxiety, measure the amount of time that the mouse spends in the central area of the arena.

Conduct the elevated plus maze test on the same day. Once all mice finish the open field test, place the mouse at the junction of the open and closed arms. Record the mouse’s behavior for five minutes before returning the mouse to its home cage.

After two consecutive days of open field test, set up the novel object recognition test according to the text protocol. Start the camera, and place the mouse in the arena, facing the middle of the wall, to familiarize the mouse. Record the mouse’s behavior for 10 minutes.

Then return the mouse to its home cage. 24 hours after the familiarization, repeat the test with one novel object. Measure the time that the mouse interacts with a new object and a familiar object separately.

Then calculate the mouse’s memory as the preference to the novel object. For the social interaction test, use an open field arena with two transparent chambers with holes on the surface. Place the subject mouse in the arena for a three-minute objects exploration period to habituate the mouse to the chambers.

Then return the subject mouse to its home cage and clean the arena. Put a novel mouse into one of the chambers. Then reintroduce the subject mouse to the arena and record for three minutes.

While reviewing the video, gauge social interaction by measuring the ratio of time the subject mouse spends interacting with the novel mouse chamber or the empty chamber. Next perform the accelerating rotarod test. In each trial, place the mouse on the static rod facing the wall opposite to the machine.

Habituate the mice to the rotarod by training it for three days in the naive mice before the onset of possible symptoms, so that the mouse equally learns the scale. Stop the device when the mouse drops, and record the time the mouse spent on the rod. Repeat this process two more times before returning the mouse to its home cage.

Next put the water maze in the center of a behavioral room, and mark the position of the maze to ensure the position of the water maze remains constant. Divide the maze into four imaginary quadrants. Hang visual cues in the center of each quadrant.

Then place a platform in the center of the fourth quadrant and mark its location. Fill the maze with water, colored white with milk powder, until the water level is one centimeter higher than the platform. At the beginning of each training trial, start the video recording, and gently put the mouse in the maze.

Stay out of sight of the mouse. Return the mouse to its home cage when the mouse cannot locate the platform within 60 seconds or if the mouse locates the platform within 60 seconds and stays on it for 10 seconds. After the test is over, stop the video, and put the mouse in its home cage under an infrared light.

Finally perform the probe phase of the water maze test as previously described, without the platform. Next prepare the cylindrical tank for the forced swim test. Start the video recording, and gently put the mouse in the water in the center of the tank.

Record the mouse for six minutes before putting the mouse back in its home cage. Review the video of the forced swim test. Measure the immobility time in the last five minutes of the video.

This protocol was to study behavioral changes in mice after respiratory exposure to silica nanoparticles. The results in the open field test and the accelerating rotarod test demonstrated that silica nanoparticles did not affect locomotor or motor function in mice. Social interaction activity was affected after one-month exposure to silica nanoparticles, however the open field test, elevated plus maze test, and the forced swim test did not indicated any anxiety or depression after one month of exposure.

A two-month exposure to silica nanoparticles did result in anxiety, as demonstrated by the results of the elevated plus maze test. Cognitive impairment was also detected in the water maze test after two months of exposure. This battery is designed for the comprehensive behavior analysis in neurodegeneration in the C57 mouse strain.

It is most suitable for neurodegeneration-related longitudinal studies in toxicity of neurotoxin or potential risk factors, or drug development, which often features long-term administration and repeated testing.