TITLE: Behavior Protocol
AUTHORS: Sasha Burwell

[DESCRIPTION]
This protocol details the associative learning assay used to test extinction and conditioning behavior in mice.


[GUIDELINES]
Mice: 

Surgerized with a headbar for head-fixation
Singly or pair housed
Acclimated for 1 week to a 12-hr reverse light/dark cycle; perform all habituation/ behavior during the dark portion of the light/dark cycle

[STEPS]
SECTION: Habituation day 1
1. Begin water restriction: remove ad libitum water from mice.
SECTION: Habituation day 1
2. Record starting body weight for all mice.
SECTION: Habituation day 1
3. Calculate allotted daily water for all mice: 50 µL water per gram of weight.

SECTION: Habituation day 1
4. Calculate goal weight for all mice:
SECTION: Habituation day 1
4.1. Within 80-85% starting body weight.
SECTION: Habituation day 1
4.2. If mice drop below 77% starting body weight at any point during behavior, supplement the allotted daily water by a few hundred µL until weight is restored.
SECTION: Habituation day 1
4.3. If mice drop below 70% starting body weight at any point during behavior, end water restriction and return ad libitum water.
SECTION: Habituation day 1
5. Run the Matlab code 
 
 to get the counterbalancing order for all days of habituation and behavior.
SECTION: Habituation day 1
6. Headfix habituation outside of behavior apparatus (in today’s counterbalance order):
SECTION: Habituation day 1
6.1. Headfix for 2 min, short break, then 5 min .

SECTION: Habituation day 1
6.2. Put back in home cage for a longer break.
SECTION: Habituation day 1
6.3. Headfix for 5 min, short break, then 10 min .

SECTION: Habituation day 1
7. Give allotted daily water: give water in a petri dish to the mouse, placed by itself in a mouse cup (without bedding so water does not get absorbed). Leave for 30 min, then record quantity drunk.

SECTION: Habituation day 2
8. Weigh mice.
SECTION: Habituation day 2
9. Headfix habituation (in today’s counterbalance order):
SECTION: Habituation day 2
9.1. Outside of behavior apparatus: 10 min .

SECTION: Habituation day 2
9.2. Put back in home cage for a break

SECTION: Habituation day 2
9.3. Inside behavior apparatus, on treadmill but with treadmill prevented from moving:10 min .

SECTION: Habituation day 2
10. Give allotted daily water, and record quantity drunk
SECTION: Habituation day 3
11. Weigh mice.

SECTION: Habituation day 3
12. Headfix habituation (in today’s counterbalance order):
SECTION: Habituation day 3
12.1. Inside behavior apparatus, on moving treadmill: 15 min .

SECTION: Habituation day 3
13. Give allotted daily water, and record quantity drunk.
SECTION: Days 1 -&gt; 9: Training
14. 
 

Calibrate solenoid: fill the water reservoir with 50 µL of fresh 10% sucrose water, and use the Matlab code 
 
to run the solenoid until the sucrose is at the end of the lick port, and to then determine the millisecond solenoid opening needed to deliver 5 µLof reward per click .

 

SECTION: Days 1 -&gt; 9: Training
15. Weigh mice.
SECTION: Days 1 -&gt; 9: Training
16. Headfix first mouse on treadmill in behavior apparatus.
SECTION: Days 1 -&gt; 9: Training
16.1. Ensure treadmill can move freely.

SECTION: Days 1 -&gt; 9: Training
16.2. Ensure mouse can reach the reward delivery spout with its tongue


 

SECTION: Days 1 -&gt; 9: Training
17. Run behavior using the code 
 
, where 
 
 is the ms value calculated in step 14. 
SECTION: Days 1 -&gt; 9: Training
17.1. GUI input: day type = train, probe = no probe, rewarded tone = 2500.
SECTION: Days 1 -&gt; 9: Training
18. Afterwards:
SECTION: Days 1 -&gt; 9: Training
18.1. Clean treadmill by wiping it down with Trifectant or a similar veterinary disinfectant.

SECTION: Days 1 -&gt; 9: Training
18.2. Top off sucrose reservoir if it has dropped below 50 µL.

SECTION: Days 1 -&gt; 9: Training
18.3. Record number of rewarded trials the mouse received.
SECTION: Days 1 -&gt; 9: Training
19. Repeat steps 16-18 until all mice have undergone their daily behavior.
SECTION: Days 1 -&gt; 9: Training
20. Give allotted daily water minus the number of rewarded trials times 5 (as each mouse has received 5 µL of water per rewarded trial already that day), and record quantity drunk.

SECTION: Days 1 -&gt; 9: Training
21. Clean the solenoid, reservoir, and attached tubing by using the 
 
 code to flush MilliQ water through the system.
SECTION: Day 10: Training with Probes
22. 
 

Calibrate solenoid: fill the water reservoir with 50 µLof fresh 10% sucrose water, and ensure that the solenoid is delivering 5 µL of reward per click.

SECTION: Day 10: Training with Probes
23. Weigh mice.

SECTION: Day 10: Training with Probes
24. Headfix first mouse on treadmill in behavior apparatus.
SECTION: Day 10: Training with Probes
24.1. Ensure treadmill can move freely.

SECTION: Day 10: Training with Probes
24.2. Ensure mouse can reach the reward delivery spout with its tongue.
SECTION: Day 10: Training with Probes
25. Run behavior using the code 
 
, where 
 
 is the ms value calculated in step 22.

SECTION: Day 10: Training with Probes
25.1. GUI input: day type = train, probe = probe, rewarded tone = 2500.
SECTION: Day 10: Training with Probes
26. Afterwards:
SECTION: Day 10: Training with Probes
26.1. Clean treadmill by wiping it down with Trifectant or a similar veterinary disinfectant.

SECTION: Day 10: Training with Probes
26.2. Top off sucrose reservoir if it has dropped below 50 µL.

SECTION: Day 10: Training with Probes
26.3. Record number of rewarded trials the mouse received.
SECTION: Day 10: Training with Probes
27. Repeat steps 24-26 until all mice have undergone their daily behavior.
SECTION: Day 10: Training with Probes
28. Give allotted daily water minus the number of rewarded trials times 5 (as each mouse has received 5uL of water per rewarded trial already that day), and record quantity drunk.

SECTION: Day 10: Training with Probes
29. Clean the solenoid, reservoir, and attached tubing by using the 
 
 code to flush MilliQ water through the system.
SECTION: Day 11: Testing 1
30. 
 

Calibrate solenoid: fill the water reservoir with 50 µL of fresh 10% sucrose water, and use the Matlab code 
 
 to run the solenoid until the sucrose is at the end of the lick port, and to then determine the millisecond solenoid opening needed to deliver 5 µL of reward per click.

SECTION: Day 11: Testing 1
31. Weigh mice.

SECTION: Day 11: Testing 1
32. Headfix first mouse on treadmill in behavior apparatus.
SECTION: Day 11: Testing 1
32.1. Ensure treadmill can move freely

SECTION: Day 11: Testing 1
32.2. Ensure mouse can reach the reward delivery spout with its tongue
SECTION: Day 11: Testing 1
33. Run behavior using the code 
 
, where 
 
 is the ms value calculated in step 30.
SECTION: Day 11: Testing 1
33.1. GUI input: day type = flip, rewarded tone = 2500.
SECTION: Day 11: Testing 1
34. Afterwards:
SECTION: Day 11: Testing 1
34.1. Clean treadmill by wiping it down with Trifectant or a similar veterinary disinfectant.

SECTION: Day 11: Testing 1
34.2. Top off sucrose reservoir if it has dropped below 50 µL.

SECTION: Day 11: Testing 1
34.3. Record number of rewarded trials the mouse received.
SECTION: Day 11: Testing 1
35. Repeat steps 32-34 until all mice have undergone their daily behavior.
SECTION: Day 11: Testing 1
36. Give allotted daily water minus the number of rewarded trials times 5 (as each mouse has received 5uL of water per rewarded trial already that day), and record quantity drunk.
SECTION: Day 11: Testing 1
37. Clean the solenoid, reservoir, and attached tubing by using the 
 
code to flush MilliQ water through the system.
SECTION: Day 12: Testing 2
38. 
 

Calibrate solenoid: fill the water reservoir with 50 µL of fresh 10% sucrose water, and use the Matlab code 
 
 to run the solenoid until the sucrose is at the end of the lick port, and to then determine the millisecond solenoid opening needed to deliver 5 µL of reward per click. 

SECTION: Day 12: Testing 2
39. Weigh mice.

SECTION: Day 12: Testing 2
40. Headfix first mouse on treadmill in behavior apparatus.
SECTION: Day 12: Testing 2
40.1. Ensure treadmill can move freely.

SECTION: Day 12: Testing 2
40.2. Ensure mouse can reach the reward delivery spout with its tongue.
SECTION: Day 12: Testing 2
41. Run behavior using the code 
 
, where 
 
 is the ms value calculated in step 38.

SECTION: Day 12: Testing 2
41.1. GUI input: day type = test, rewarded tone = 11000.
SECTION: Day 12: Testing 2
42. Afterwards:
SECTION: Day 12: Testing 2
42.1. Clean treadmill by wiping it down with Trifectant or a similar veterinary disinfectant.

SECTION: Day 12: Testing 2
42.2. Top off sucrose reservoir if it has dropped below 50 µL .

SECTION: Day 12: Testing 2
43. Repeat steps 40-42 until all mice have undergone their daily behavior.
SECTION: Day 12: Testing 2
44. Clean the solenoid, reservoir, and attached tubing by using the 
 
code to flush MilliQ water through the system.

SECTION: Day 12: Testing 2
45. Perfuse all mice for histology (see histology protocol).