TITLE: Behavioral analyses in mice 
AUTHORS: Nicolas Giguère, louis-eric.trudeau Trudeau

[DESCRIPTION]
This collection of protocols describes different behavioural assays performed in mice including, grip strength test, pole test, rotarod, open field and nose poking learning task.

[GUIDELINES]
This protocol needs prior approval by the users' Institutional Animal Care and Use Committee (IACUC) or equivalent ethics committee

[STEPS]
SECTION: Grip strength test
1. A grip strength apparatus (BioSeb instruments, BIO-GS3, France) was used to evaluate paw force of 10-12-week-old male or female mice.

Procedure:

SECTION: Grip strength test
1.1. Habituate the mouse in the testing room for at least 60 min. 
SECTION: Grip strength test
1.2. Hold the mouse firmly by the tail and slowly lower it on the grid of the grip strength apparatus until the forepaws grasp the middle of the grid. 
SECTION: Grip strength test
1.3. Lower the mouse to a horizontal position and slowly pull following the axis of the sensor until the mouse releases its grasp on the grid. 
SECTION: Grip strength test
1.4. Repeat the test 3 times with 30 min resting period between each trial. 

Repeat the test for 30 min (1/3). 
Repeat the test for 30 min (2/3). 
Repeat the test for 30 min (3/3).

SECTION: Grip strength test
1.5. Once testing is completed, place the mouse back into its home cage and clean the apparatus before testing the next mouse.
SECTION: Pole test
2. The test was conducted with a homemade 48 cm metal rod of 1 cm diameter covered with adhesive tape to facilitate traction. Male or female mice of 10-12-weeks of age were used.

Procedure:

SECTION: Pole test
2.1. Habituate the moude in the testing room for at least 60 min.

SECTION: Pole test
2.2. Hold the mouse on the surface of your hand and bring it up to the top of the pole.
SECTION: Pole test
2.3. Position the mouse head-up at the top of the pole.
SECTION: Pole test
2.4. Record the time required to turn (t-turn) and to climb down completely. 
SECTION: Pole test
2.5. Once testing is completed, place the mouse back into its home cage and clean the metal rod before testing the next mouse.
SECTION: Rotarod
3. Motor coordination was evaluated with a rotarod apparatus (Harvard Apparatus, LE8205, USA) on 10-12-week-old male or female mice.

Mice were pre-trained on the rotarod for two consecutive days to reach a stable performance and then tested on the third day.

Procedure:

SECTION: Rotarod
3.1. Day 1

Habituate the mouse in the testing room for at least 60 min. 
Place the mouse on the rotarod at 4 rpm for up to 1 min or a maximum or 3 trials.
Once testing is completed, place the mouse back into its home cage and clean the apparatus before testing the next mouse.

SECTION: Rotarod
3.2. Day 2

Habituate the mouse in the testing room for at least 60 min. 
Place the mouse on the rotarod with an accelerated rotation of 4 rpm-40 rpm for 10 min.
Repeat 2 more times with 30 minutes resting period between each trial.

- Repeat for 30 min (1/2).
- Repeat for 30 min (2/2).

SECTION: Rotarod
3.3. Day 3

Habituate the mouse in the testing room for at least 60 min. 
Place the mouse on the rotarod with an accelerated rotation of  4 rpm-40 rpm for 10 min.
Repeat 2 more times with 30 minutes resting period between each trial.

- Repeat for 30 min (1/2).
- Repeat for 30 min (2/2).

Record the latency to fall of all 3 trials.
SECTION: Open field
4. The locomotor behavior of 11-12-week-old male or female mice was recorded using an infrared actimeter (Superflex sensor version 4.6, Omnitech) using the Fusion software (v5.6 Superflex Edition, RRID:SCR_017972). A chamber partition was used to test two mice at the same time. Subjects were not given time to acclimate to the chamber and spent a total of 60 min in the chamber with the following protocol:

SECTION: Open field
4.1. Habituate the mouse in the testing room for at least 60 min. 
SECTION: Open field
4.2. Place the mouse in the chamber and record basal locomotion for 20 min.
SECTION: Open field
4.3. After 20 min, pause the recording and administer drugs or saline. In the present study, this was one of the following substances:

0.9% sodium chloride saline solution (Halyard, #cat 116)
Cocaine hydrochloride 20 mg/kg (Medisca, cat# 53-21-4, Canada)
D-amphetamine sulfate at 5 mg/kg (Tocris, 2813, UK)
SCH23390-HCl 50 µg/kg (Sigma, D-054, Canada)
Quinpirole-HCl 0.2 mg/kg (Sigma, Q-102, Canada)
Raclopride L-tartrate 1 mg/kg (Sigma, R-121, Canada)
SECTION: Open field
4.4. Immediately place the mouse back into the chamber and record locomotion for another 40 min.
SECTION: Open field
4.5. Once testing is completed, place the mouse back into its home cage and clean the open field boxes before testing the next mouse.
SECTION: Nose poking operant learning task
5. Appetitively motivated operant learning was assessed using the open source Feeding Experimentation Device 3 (FED3) (https://doi.org/10.7554/eLife.66173). Individually housed animals were evaluated in a 12h/12h light/dark cycle.

Procedure:

SECTION: Nose poking operant learning task
5.1. Place the feeding devices in the home cages under a free feeding paradigm (automatically dispenses a new pellet each time the mouse collects the pellet in the well) for 2 days.
SECTION: Nose poking operant learning task
5.2. After 2 days, change the device mode to a fixed ratio (FR1) paradigm (the mouse learns to nose poke on the active port to receive a single pellet).
SECTION: Nose poking operant learning task
5.3. Record nose-poking rate and poke efficiency for 3 consecutive days.
SECTION: Nose poking operant learning task
5.4. After 3 days, change the device mode to a repeating progressive ratio schedule (nose-poking requirement begins at FR1 and increases by 1 poke each time a pellet is earned)

o When mice fail to poke on either active or inactive port for 30 min, the ratio is automatically reset to FR1. 
SECTION: Nose poking operant learning task
5.6. Once testing is completed, remove the feeding device from the home cage and clean it before testing the next session.
SECTION: Nose poking operant learning task
5.5. Record nose-poking rate and poke efficiency for 3 consecutive days.