The overall aim of these procedures is to quantitatively assess sensor motor function. In mice. We accomplish this by using a battery of sensitive behavioral tests.
These include challenging beam traversal spontaneous activity in the cylinder and the adhesive removal test, which is also known as the DOT test. Following testing, video recordings of beam traversal and spontaneous activity can be scored. Ultimately, these tests can be used to characterize sensor or motor function in novel genetic mouse models, as well as in preclinical drug studies studying potential therapeutics for disease.
So the main advantage of these tests over other tests, especially automated ones, is that we find them to be highly sensitive to motor dysfunction, more sensitive than tests like the rotor rod. In addition though, these tests are actually very cost efficient, especially compared to automated ones, which can cost up to thousands of dollars. These tests can answer key questions in the field of movement disorders in general, and they're highly useful outcome measures.
When testing potential therapeutics, The beam is set up by inverting three clean mouse cages on a tabletop or in a mouse cage. Changing fume hood, line the cages up evenly so that they can support the length of a one meter beam. Next, assemble the four sections of the beam from the widest to narrowest section and place on top of the inverted mouse cages.
Place the home cage of the mice that you plan to test on its side at the end of the beam so that the narrowest end of the beam leads right into the home cage. To begin the assisted trial, pick up the first mouse by the base of its tail and support its hind limbs with the palm of your hand. Place the mouse at the wide end of the beam and let go of its tail and remove your hand.
Let the mouse sniff and move around in order to become better oriented to the apparatus. If the mouse turns gently redirect it to the desired direction. In order to encourage the mouse down the path, lift up the home cage, keeping it in the same position on its side, and bring it close to the test mouse.
As the mouse begins to try and enter the home cage, move it back so that the mouse does not enter, but makes a step forward. Finally, allow the mouse to enter the home cage. Do the same procedure for each mouse in the cage.
Clean the beam thoroughly between cages with animal room cleaning solution within a cage. It is important to clean off any urine or fecal pellets off the beam before the next mouse is run, as this will distract the next mouse. Once all of the mice in the cage have gone through one assisted trial, pick up the first mouse again and place it at the wide end of the beam if needed.
Gently orient the mouse in the desired direction and lightly touch its backside to encourage it to move along the length of the beam towards its home cage, correct the mouse if it stops the sniff or walks off the beam. Have your hand nearby to guide the mouse to the end of the beam. Minimize stopping and exploring while on the beam.
The more this is done during training, the less the mouse tends to do it. During testing, the trial ends when the mouse places one of its four limbs into the home cage, the next mouse can then receive its second trial. Mice receive a total of five trials on the first training day, alternating between mice, so that each mouse has an inter trial interval of 30 seconds or more.
On day two, perform five more trials for each mouse using the same beam setup as in day one, the mice should not need any assistance, but may need to be corrected or touched on the backside to prevent stopping or exploring. 24 hours later, begin the test day. Set up the beam in a similar manner to the previous two training days.
However, place a mesh grid that corresponds to each beam width on top of each beam section. Use a video camera to record all beam traversal trials before each trial begins. Label a card with the essential experiment information such as date, mouse number, and trial number, and place it in front of the camera.
Record for two to three seconds prior to each trial so that it is clear which mouse is being tested and what trial it's on. For each test, place the mouse on top of the grid surface on the widest section and record as it moves along the beam record so that the entire length of the mouse's body is visible. The camera should be positioned so that the grid is in the middle of the viewer.
Run each mouse through the test five times on the grid surface beam, cleaning it between each mouse. In order to set up the spontaneous activity experiment, arrange two cages next to each other around 18 centimeters apart so that the fronts of the cages are facing the experimenter. If needed, place an additional cage behind the other two cages to serve as support for the mirror.
Next, place a piece of glass on top of the two cages and position it so the glass is supported by the outer edge of the first two cages. Then place a cylinder on top of the glass and a mirror at an angle beneath the glass. Leaning on the third cage back.
The angle can range anywhere from 30 to 45 degrees, but more importantly, the view from the camera has to include the full diameter of the cylinder. Once the experiment is set up, place the video camera in front of the mirror and adjust both the mirror and video camera until you can view the entire diameter of the bottom of the cylinder. Then set a timer for three minutes and label a card with the important experiment details as in the previous experiment video.
Record the label for two to three seconds. Then stop. To run the experiment, place a mouse in the cylinder.
Press record and start the timer. Record the mouse for three minutes. It is important that the testing area is quiet as loud noises and conversation can distract the mouse and potentially cause freezing behavior.
During the experiment, use a hand counter to count the number of rears the mouse makes while in the cylinder. A rear is defined as a vertical movement with both fall limbs off the floor so that the mouse is standing only on its hind limbs. Once the timer expires, remove the mouse and place it back into his home cage.
Clean the cylinder and glass with animal room cleaning solution between each mouse. Allow the cleaning solution to dry before moving on to the next mouse. Begin the adhesive removal test by placing the mouse in their home cage.
With the feeder bin removed, allow the mice to habituate to the testing room and the cage without the feeder. For one hour for testing, use a clean cage to place cage mates so that the test mouse is the only one in the home cage. Remove three quarters of the bedding and place it in the clean cage with the cage mates in the home cage.
Scruff the test mouse in order to restrain it using a pair of small forceps, place one adhesive label onto the sslt of the mouse. Gently press the label on the snout with the forceps and release the mouse. Place the lid on the cage and start a stopwatch when the mouse makes an attempt to remove the labels with its four pores.
Record the time if the mouse makes contact, but does not remove the label. Record the time, but keep the timer going until it does. Remove it completely.
If the mouse does not contact or remove the sticker within 60 seconds, end the trial and manually remove the sticker. After each trial, place the test mouse in the holding cage and begin testing the next mouse. All mice receive three trials alternate between mice.
Rather than doing all three trials on a mouse at one time, trials where the label falls off or is not secure on the snouts are not counted. The challenging beam spontaneous activity in the cylinder and adhesive removal tests are all highly useful assays of sensor motor function in mice on the challenging beam test, TH one alpha-synuclein mice make significantly more errors per step than wild type mice. The number of errors increased in th one alpha-synuclein mice as the beam becomes more narrow.
This was not the case in the wild type mice. This same line of mice also shows a robust decrease in hind limb steps in the cylinder test. These results show that the overexpression of human alpha in nucle in the mutated mice leads to the development of detectable sensor motor deficits.
Finally, in the adhesive removal test, PITX three akia mice with a profound decrease in nigrostriatal dopamine neurons show significantly increased time to contact compared to wild type controls. We and others use these tests routinely to assess the therapeutic potential of treatments for Parkinson's disease. These tests can also be used to characterize genetic mouse models and to determine the consequences of specific mutations on sensory motor function.
