TITLE: Motor Behavior Assays (Mouse)
AUTHORS: Kelsey Barcomb, Beatriz E Nielsen, Christopher Ford

[DESCRIPTION]
This protocol describes motor behavioral assays used to assess motor function in different models of Parkinson's disease.
These assays include open field, pole, rotarod, cylinder, balance beam tests.
It includes an example experimental time course used to perform identical behavioral assays across two institutions in a progressive DA loss model; and the assays (and its variants) conducted to examine motor function in a partial and more complete DA depletion induced by 6-OHDA (acute model).

[STEPS]
SECTION: Open Field Test
1. Required materials
Open field chambers - either clear 25 cm diameter cylinders or gray 50 cm x 50 cm square boxes (if chambers are open ended on both sides, use gray silicone mats underneath)
Overhead mounted camera
Video analysis software
SECTION: Pole Test
4. Required materials
50-60 cm pole with attachable base
Gauze
Clear chamber (optional)
Silicone mat
Camera (the camera on a laptop is sufficient)
SECTION: Pole Test
5. Basic procedure (3 day paradigm). 
For each of the following days of training/testing, the room setup is as follows:
Turn on dim lighting (bright overhead lights should remain off)
Wrap pole with gauze; stretching taut while wrapping will reduce the tendency of the nails of the mice getting caught in the fibers of the gauze
Screw pole into base
Wipe down pole, base, clear chamber, and silicone mat with 70% ethanol
Place clear chamber on silicone mat and place pole inside the chamber
Position camera in front of apparatus 
SECTION: Rotarod Test
6. Required materials
Rotarod
Camera (laptop camera is sufficient)
 

SECTION: Example Behavior Timeline - Motor Function Battery
8. The following timeline was used for behavioral testing for a Parkinson's Disease model. In this study, 25 cm clear cylinders on top of silicone mats were used for the open field testing and the pole test.
SECTION: Example Behavior Timeline - Motor Function Battery
8.1. Day 1 - Training (OFT/Pole)
Behavior room setup
30 minute room habituation
Open Field Test - 30 minute open field habituation (not analyzed)
Pole Test - Training Day 1 (see above; not analyzed)

SECTION: Example Behavior Timeline - Motor Function Battery
8.2. Day 2 - Training (OFT/Pole)
Behavior room setup
30 minute room habituation
Open Field Test - 30 minute open field habituation (not analyzed)
Pole Test - Training Day 2 (see above; recorded but not analyzed)
SECTION: Example Behavior Timeline - Motor Function Battery
8.3. Day 3 - Testing (OFT/Pole)
Behavior room setup
30 minute room habituation
Open Field Test - 20 minute open field test (measure total distance and velocity)
Pole Test - Test Day using 3 trials, 5 minutes intertrial interval (measure Total Time, Orient Time, and Descent Time)

SECTION: Open Field Test
2. Basic procedure
SECTION: Open Field Test
2.1. Set up behavior room
Set up lighting with the goals of (a) minimizing total room light, (b) reducing shadows within the chambers, and (c) ensuring camera is able to detect the mice; bright overhead lights should remain off; adding a red filter to the light source is a good option for maintaining low light
Clean chambers and silicone mats with 70% ethanol
Place chambers under camera; a 2 x 2 configuration is recommended when multiple mice are being tested at once
Turn on camera; adjust zoom and focus 
If using simultaneously tracking with EthoVision, set up the software
SECTION: Open Field Test
2.2. Habituation
Bring mice to the behavior room in their home cages. Leave undisturbed for at least 30 minutes.
SECTION: Open Field Test
2.3. Testing
Start recording video before mice are in the chamber to collect the background images
If performing a multi-day testing paradigm, it is recommended to use a Sharpie to mark the tails with the corresponding chamber number
Place mice in chambers
Collect video for desired length of time (usually 10-30 minutes, see use cases below)
Remove mice from chambers and return them to their home cage
Save video file and (if desired) count the number of fecal deposits in the chamber
SECTION: Open Field Test
2.4. Cleanup
Between groups of mice, clean chambers and silicone mats with 70% ethanol; allow to fully dry before starting another trial
At the end of the day, wipe everything down with a sterilant such as Clidox 
 


SECTION: Open Field Test
2.5. Analysis
Run tracking software to calculate the desired measures, usually total distance traveled and velocity. We typically use EthoVision or ezTrack.
 

 

Depending on main research question, you may want to gather additional parameters such as total time in center, rearing/stereotyped behaviors, total fecal deposits, or binned distance over time.
SECTION: Open Field Test
3. Common use cases for open field test
SECTION: Open Field Test
3.1. Open field test for stress
One week before OFT, move mice to a clean cage and limit interaction with other animal staff (e.g. place on "Do Not Disturb" status)
For each of the three days leading up to OFT, the researcher who will perform OCT should handle mice for 30-60 seconds
On the day of testing, set up OFT chambers as normal, using the 50 x 50 cm square chambers; if using real-time tracking, set up to measure time in center vs. borders
Record locomotion in the chamber for 30 minutes
Compare percentage of time in center vs. border zones
SECTION: Open Field Test
3.2. Open field test for parkinsonian motor deficits
Plan for a 3-day paradigm
Set up the chambers as normal each day and label the tails of the mice 1-4 with a Sharpie; each mouse should stay in its same chamber location each day
 

Days 1 and 2, habituate animals to the chamber by running a 30 minute OFT; these trials do not need to be recorded or analyzed
Day 3, run the OFT for 20 minutes; measure total distance and velocity
SECTION: Pole Test
5.1. Training Day 1 - perform three trials of training as described below; recording video and analyzing trials are not required 
Habituate mice to the room in their home cage for at least 30 minutes
Start training on the first cage and prepare the apparatus by covering the base of the pole with home cage bedding; if desired, use a Sharpie to label the tails of the mice to keep track of their order.
Perform trial one: Grab the first mouse from the first cage and place approximately halfway up the pole with their nose facing down towards the base. Allow the mouse to travel down the pole and dismount onto the base; if they are struggling or not completing the task correctly, you may need to tap their backside to nudge them in the correct direction and/or help them maintain a downward orientation. Return mouse to its home cage and repeat for each subsequent mouse in that cage; start a timer for 5 minutes.
Perform trial two: Repeat the same procedure as for trial one, but starting the mice at 3/4 of the way up the pole rather than halfway (still facing downward). 
Perform trial three: Repeat the same procedure as for trial one, but starting the mice at the top of the pole (still facing downward). 
If there are additional cages to be tested, clean apparatus by discarding the bedding and wiping down all components thoroughly with 70% ethanol.
Repeat procedure on each subsequent cage. 
After the last cage, clean everything with Clidox (or similar disinfectant).
 

SECTION: Pole Test
5.2. Training Day 2 - perform three trials of training as described below; recording video and analyzing trials are not required, but are suggested for trials 2 and 3
Habituate mice to the room for at least 30 minutes
Start training on the first cage and prepare the apparatus by covering the base of the pole with home cage bedding; if desired, use a Sharpie to label the tails of the mice to keep track of their order.
Perform trial one: Grab the first mouse from the first cage and place at the top of the pole, facing down. Allow the mouse to travel down the pole and dismount onto the base; if they are struggling or not completing the task correctly, you may need to tap their backside to nudge them in the correct direction and/or help them maintain a downward orientation. Return mouse to its home cage and repeat for each subsequent mouse in that cage; start a timer for 3-5 minutes.
Perform trial two: Repeat the same procedure as for trial one, but starting the mice at the top of the pole and facing upwards. Recording video is recommended.
Perform trial three: Repeat the same procedure as for trial one, but starting the mice at the top of the pole and facing upwards (identical to trial two). Recording video is recommended. 
If there are additional cages to be tested, clean apparatus by discarding the bedding and wiping down all components thoroughly with 70% ethanol.
Repeat procedure on each subsequent cage. 
After the last cage, clean everything with Clidox (or similar disinfectant).
 

SECTION: Pole Test
5.3. Test Day - perform three trials of training as described below; recording video and analyzing trials is required for all 3 trials
Habituate mice to the room for at least 30 minutes
Start testing on the first cage and prepare the apparatus by covering the base of the pole with home cage bedding; if desired, use a Sharpie to label the tails of the mice to keep track of their order.
Perform test trial one: Start recording video. Grab the first mouse from the first cage and place at the top of the pole, facing UP. Allow the mouse to travel down the pole and dismount onto the base; do NOT assist during the test phase. Return mouse to its home cage and repeat for each subsequent mouse in that cage; stop video and start a timer for 5 minutes.
Perform test trial two: Repeat the same procedure as for trial one.
Perform test trial three: Repeat the same procedure as for trial one.
If there are additional cages to be tested, clean apparatus by discarding the bedding and wiping down all components thoroughly with 70% ethanol.
Repeat procedure on each subsequent cage. 
After the last cage, clean everything with Clidox (or similar disinfectant).

 

SECTION: Pole Test
5.4. Analysis - For each full trial (i.e. trial in which mouse was placed on the top of the pole facing upward and not assisted in completing the trial), determine the following measures by analyzing the video recordings:
Total time on pole: number of seconds from when the mouse was placed on the pole until the mouse touched its two front paws to the base
Time to orient: number of seconds from when the mouse was placed on the pole until the mouse oriented fully downward, as defined by having all four paws rotated down
Descent time: number of seconds from when the mouse is oriented downward until the mouse touched its two front paws to the base

Depending on the desired analysis, any of these three measures can be used as the average of all three trials, the best trial (i.e. fastest run), or the sum of the two best (i.e. drop the worst). 
 

 
SECTION: Rotarod Test
7. Basic procedure for a 1 day test
SECTION: Rotarod Test
7.1. Set up behavior room
Turn on dim lighting (bright overhead lights should remain off)
Clean each slot and tray of the RotaRod with 70% ethanol, allow to fully dry before beginning test
Turn on RotaRod, select "Ramp" setting and set to 5-80 RPM over 300 seconds
Place paper towel in each tray for the slots that will be used
Position camera in front of apparatus (a laptop camera is sufficient)
SECTION: Rotarod Test
7.2. Habituate mice to behavior room in their home cage for at least 30 minutes
SECTION: Rotarod Test
7.3. Hit "Run" on the RotaRod to begin the rotation; load mice onto apparatus and begin the video recording

 
 
SECTION: Rotarod Test
7.4. Once all mice have been loaded, wait 30 seconds to make sure all of the mice continue to walk as the rod rotates, then hit "Start" to initiate the ramp process.
SECTION: Rotarod Test
7.5. Observe the mice as they perform the RotaRod task and notate the following (either in real-time or by watching the video recording later):
End point - time in seconds at which the mice stop successfully completing the task, either due to a fall or no longer continuing to keep up with the rotation (e.g. holding on at one spot and riding around the rod for one or more rotations)
Fall point - time in seconds at which the mice fall off the rod (note, this may be the same as the end point)
Depending on the analysis, you may want to make additional notations regarding number of times that the mouse rotates with the rod before falling or changes in orientation.

 

SECTION: Rotarod Test
7.6. Use an intertrial interval of 5 minutes and perform the task 2 more times for a total of 3 trials. Mice should remain in the same slot for each trial and cleaning the apparatus between trials is not required.
SECTION: Rotarod Test
7.7. After completing the three trials for a group, if another group will be run then wipe down the apparatus with 70% ethanol and replace the paper towels in the trays. At the end of the day, clean the apparatus using Clidox (or similar disinfectant).
SECTION: Example Behavior Timeline - Motor Function Battery
8.4. Day 4 - Testing (RotaRod)
Behavior room setup
30 minute room habituation
RotaRod Test - 3 trials, 5 minutes intertrial interval (measure Latency to End)
SECTION: Example Behavior Timeline - Motor Function Battery
8.5. Day 5 (optional) - Testing (RotaRod)
Behavior room setup
30 minute room habituation
RotaRod Test - 3 trials, 5 minutes intertrial interval (measure Latency to End)
SECTION: Example Behavior Timeline - Motor Function Battery
8.6. Day 6 (optional) - Testing (RotaRod)
Behavior room setup
30 minute room habituation
RotaRod Test - 3 trials, 5 minutes intertrial interval (measure Latency to End)
SECTION: Rotarod Test
7.8. Analysis - determine Latency to End (in seconds) for each mouse in each trial. 
For each mouse, the following measures of performance may be used depending on the goals of the analysis:
average of 3 trials
sum of two best trials (drop lowest)
maximum trial time
time course across all 3 trials (recommended if assessing improvement over time)
SECTION: Cylinder test: forelimb use asymmetry
9. Required materials:
Clear plastic cylinder: 10.5 cm diameter, 14.5 cm height.
Mirror
Camera
Video player software (e.g. VLC).
70% ethanol.
SECTION: Cylinder test: forelimb use asymmetry
10. Set up behavior room:
Clean cylinder with 70% ethanol.
Place the camera in front of the cylinder with mirrors behind.
Turn on camera; adjust zoom and focus and verifying an appropriate 360º vision of cylinder walls. 

SECTION: Cylinder test: forelimb use asymmetry
11. Habituation
Bring mice to the behavior room in their home cages and leave undisturbed for at least 30-40 minutes.
There must be no prior habituation to the plastic cylinder.
SECTION: Cylinder test: forelimb use asymmetry
12. Testing:
Place the mouse inside the cylinder and start video recording for 5 min
 

After recording, return the mouse to home cage and clean the cylinder with 70% ethanol, before proceeding with another mouse.
SECTION: Cylinder test: forelimb use asymmetry
13. Post-hoc scoring or analysis:
Analyze the video frame by frame and count the number of fully extended digits wall touches executed with forelimb paws, discriminating between right and left touches.
 
Calculate the percentage of touches performed with the forelimb right vs left or contralateral vs ipsilateral to the injected brain side with respect to the total paw use.

 

SECTION: Balance beam: balance and coordination
14. Required materials:
Balance beam stands.
Mirrors.
Goal cage.
Camera and tripod.
Lamp.
Beams (square): 19 mm, 12 mm and 6 mm cross section. Each beam has two lines marked in black separated by 80 cm lenght.
Video player software (e.g. VLC).
70% ethanol.
SECTION: Balance beam: balance and coordination
15. Set up behavior room:
Wipe down beams and goal box with 70% ethanol.
Assemble balance beam stand with goal box, inside a closed space (e.g. water tank).
Place some bedding (preferentially from the home cage) in the goal box to make it more enticing for the mouse.
Attach a camera/tripod to the the tank wall at the side opposite the goal box. Connect the camera to a PC.
Turn on the lamp and shine the bright light onto the initial segment of the beam, the end opposite to the goal box (this encourages the mouse to cross the beam to reach a darker goal box). 
Place mirrors on each side of the apparatus and angle them so that the camera can
capture the middle 80cm (comprised between the two black markers) of the different sized balance beams at both sides.
SECTION: Balance beam: balance and coordination
17. Training: Day 1
Video recording of training session is not required.
Only medium size beam (12 mm) is used for training.
SECTION: Balance beam: balance and coordination
17.1. Place mouse in goal box for 3 min 

SECTION: Balance beam: balance and coordination
17.2. Pick the mouse from goal box and place it on the medium size beam (12 mm) at 6 inches from the goal box. Make sure the mouse is facing the goal box and is well-balanced before you release its tail. Let the mouse walk along the beam and enter the goal box, then leave it inside for 30 s.     

 

SECTION: Balance beam: balance and coordination
17.3. Remove feces/urine on the beam, if any, while the mouse is in the goal box.
SECTION: Balance beam: balance and coordination
17.4. Repeat 17.2 step three more times by taking the mouse out of the goal box and then placing it at:
12 inches from the goal box
In the middle of the beam
At the far end of the beam
Allow the mouse to stay inside the goal box for 30 s each time.   
SECTION: Balance beam: balance and coordination
17.5. After a mouse completes the training, if another mouse from the same home cage is going to be trained, keep that same home cage bedding in goal box and just remove feces/urine on the beam.

When all mice from a given home cage were trained, clean the beam/goal box with 70% ethanol, and remove the bedding. Replace with bedding from the home cage of the following mice to train.
SECTION: Balance beam: balance and coordination
17.6. After all mice complete the training, clean the beam/goal box with 70% ethanol, and remove the bedding.
SECTION: Balance beam: balance and coordination
18. Testing: Day 2

Setup is the same as day 1, but all different sized beams are used (19 mm, 12 mm and 6 mm). 
Wipe down beams and goal box with 70% ethanol prior to start.
SECTION: Balance beam: balance and coordination
16. Habituation
Bring mice to the behavior room in their home cages and leave undisturbed for at least 30-40 minutes.
SECTION: Balance beam: balance and coordination
18.1. Start recording the video with the camera.
SECTION: Balance beam: balance and coordination
18.2. Place a mouse on the 19 mm beam (large sized beam) at the farthest position from the goal box. Make sure the mouse is facing the goal box and is well-balanced before you release its tail. Encourage the mouse to move along the beam if it does not do so on its own after a few seconds, but just until it reaches the first marker. 
The mouse must cross the 80 cm distance between the two markers on the beam in less than 1 min . The maximal score for latency to cross the beam is 60s and it is considered an unsuccessful trial.     

SECTION: Balance beam: balance and coordination
18.5. Repeat steps 18.2 to 18.4 with the 12mm (medium) and 6mm (small) beams, going from largest to smallest, for a total of 6 trials per mouse (2 trials per beam size).
SECTION: Balance beam: balance and coordination
18.3. Once crossed the beam and reached the goal box, leave the mouse inside for 30 s 
If it the maximal score 60s was reached, gently grab the mouse from the beam and place it on the goal box also for  30 s
SECTION: Balance beam: balance and coordination
18.4. Repeat the trial with same beam. 
SECTION: Balance beam: balance and coordination
18.6. Once finished testing one mouse, clean the beam/goal box with 70% ethanol, and replace the bedding (for mice from the same home cage it is not necessary to clean and change the bedding, just clean excess of urine or feces that can be on the beam).
SECTION: Balance beam: balance and coordination
18.7. After all mice complete the test, stop video recording, clean the beam/goal box with 70% ethanol, and discard the bedding.
SECTION: Balance beam: balance and coordination
19. Post-hoc analysis:
The following data is expressed as the average of the two trials per beam size
Latency to cross the beam: corresponds to the latencty to walk the 80cm between the two markers in each beam, with a maximum score of 60 seconds (unsuccessful trial).
Number of hind foot slips made along the way (If the trial is unsuccessful because the mouse remains immobile for most of the time and not engaged in the walking task, do not include the number of slips for the average).