TITLE: Motor behavioral assessment
AUTHORS: Beatriz E Nielsen

[DESCRIPTION]
This protocol describes motor behavioral assays used to assess motor function in a 6-OHDA mouse model of Parkinson's disease. These assays include open field, rotarod, cylinder, and balance beam tests.
Prior to each behavioral test, mice were habituated to the testing room for 30-40 min. All behavioral tests were performed 3-4 weeks following stereotaxic injections.



[STEPS]
SECTION: Cylinder test: forelimb use asymmetry
1. 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
2. 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
3. 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
4. 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
5. 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: Rotarod: balance and coordination
6. Required materials:
Rotarod (Med Associates) connected to a PC with controlling 'RotaRod' software (mouse logo).
Paper towels.
70% ethanol.
SECTION: Balance beam: balance and coordination
11. 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
12. 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
13. 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
14. 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
14.1. Place mouse in goal box for 3 min 

SECTION: Balance beam: balance and coordination
14.2. 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
14.3. Remove feces/urine on the beam, if any, while the mouse is in the goal box.
SECTION: Balance beam: balance and coordination
14.4. Repeat 14.2 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
14.5. 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
14.6. After all mice complete the training, clean the beam/goal box with 70% ethanol, and remove the bedding.
SECTION: Balance beam: balance and coordination
15. 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
15.1. Start recording the video with the camera.
SECTION: Balance beam: balance and coordination
15.2. 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
15.3. 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
15.4. Repeat the trial with same beam. 
SECTION: Balance beam: balance and coordination
15.5. Repeat steps 11.2 to 11.4 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
15.6. 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
15.7. 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
16. 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).

SECTION: Rotarod: balance and coordination
7. Set up behavior room:
Clean each lane of the RotaRod with 70% ethanol.
Cut paper towels to the size of lanes and place them there for ease of cleaning.
Turn on RotaRod and corresponding 'RotaRod' software (mouse logo). Select the appropriate protocol. Hit 'Browse' option to navigate to your folder and create a file for the first group of mice to be tested (remember to create a new file for each group of mice).

 

 
Make sure nothing is blocking lane photobeams and push “Reset” buttons on rotarod, so each lane light is off (in the software, lanes will change from red to green. All must be green to start the test). 
SECTION: Rotarod: balance and coordination
8. Habituation:
Bring mice to the behavior room in their home cages and leave undisturbed for at least 30-40 minutes.
SECTION: Rotarod: balance and coordination
9. Testing: basic procedure for 1 day, 3 trials
SECTION: Rotarod: balance and coordination
9.1. Place mouse on the immobile rod, holding its tail for a few seconds to make sure it will not jump and to let it get its balance.
SECTION: Rotarod: balance and coordination
9.2. Wait 30 s before starting rotation (first trial only).

SECTION: Rotarod: balance and coordination
9.3. Hit 'Reset' button on software. Rod starts moving at 3 rpm (initial speed).
SECTION: Rotarod: balance and coordination
9.4. Then immediately press “On/Accel” button: acceleration program begins and automatically starts the time counting for each lane.
SECTION: Rotarod: balance and coordination
9.5. Remove the mouse if it falls (the photobem detection automatically stop the timer for that lane) or if continuous passive rotations occur (entire rotation of mouse body around the rod, with mouse holding on at one spot and riding around more than one rotation, without active intention to re-orient and continue walking forward). If passive rotations occurs, trip photobeam with your hand to stop the timer for that lane and then remove the mouse.

Although latency to fall (and the speed) will be automatically recorded and saved by the software, it is recommended to annotate it in real-time. 
 

SECTION: Rotarod: balance and coordination
9.6. Place removed mice into their home cages. Trial ends when all mice are collected. If any mice complete the 5 min (300 sec) program, stop the rotarod, remove the mice and give them the maximum score of 300 sec/30 rpm.
SECTION: Rotarod: balance and coordination
9.7. Clean rotarod (feces/urine) and replace paper towels if necessary (optional between trials for the same group of mice).
SECTION: Rotarod: balance and coordination
9.8. Let mice rest in their home cages for 10 min between trials.

SECTION: Rotarod: balance and coordination
9.9. Repeat steps from 9.1 to 9.8 for trials 2 and 3, except for step 9.2 (start the accelerated program immediately after placing mice on the rotarod, without 30 s of delay).
SECTION: Rotarod: balance and coordination
9.10. Once all three trials for a group of mice are finished, clean each lane of the rotaod with 70% ethanol, replace paper towels, and create a new file for the following group of mice.
SECTION: Rotarod: balance and coordination
9.11. Run again the previously described three trials per group of mice.

SECTION: Rotarod: balance and coordination
10. Analysis: latency to fall or time to reach maximum speed is recorded for the three trials and the average of the two best trials is reported.
SECTION: Open field: locomotion
17. Required materials:
Open field chamber: 50 cm x 50 cm square box.
Overhead mounted camera.
Video analysis software.
70% ethanol.
SECTION: Open field: locomotion
18. Set up behavior room:
Set up lighting minimizing total room light, reducing shadows within the chamber, and ensuring camera is able to detect the mice.
Clean chamber with 70% ethanol.
Place chamber under camera.
Turn on camera, adjust zoom and focus.
SECTION: Open field: locomotion
19. Habituation:
Bring mice to the behavior room in their home cages and leave undisturbed for at least 30-40 minutes.
No previous habituation to the open field arena is required.
SECTION: Open field: locomotion
20. Testing:
SECTION: Open field: locomotion
20.1. Start recording video before the mouse is in the chamber to collect the background images or just save a background image before starting the video. 
SECTION: Open field: locomotion
20.2. Place mouse in chamber.
SECTION: Open field: locomotion
20.3. Record video for the desired length of time (e.g. 10 min ).

SECTION: Open field: locomotion
20.4. Remove mouse from chamber and return it to home cage.
SECTION: Open field: locomotion
20.5. Clean the chamber between mice with 70% ethanol and allow to fully dry before starting another trial. 
SECTION: Open field: locomotion
21. Post-hoc analysis:
Run tracking EthoVision software to calculate the desired measures (e.g. total distance traveled, velocity, etc.).