Behavior Protocol
Sasha Burwell
Abstract
This protocol details the associative learning assay used to test extinction and conditioning behavior in mice.
Steps
Habituation day 1
Begin water restriction: remove ad libitum water from mice.
Record starting body weight for all mice.
Calculate allotted daily water for all mice: 50µL water per gram of weight.
Calculate goal weight for all mice:
Within 80-85% starting body weight.
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.
If mice drop below 70% starting body weight at any point during behavior, end water restriction and return ad libitum water.
Run the Matlab code
counterbalance(cohort, numMice)
``` to get the counterbalancing order for all days of habituation and behavior.
Headfix habituation outside of behavior apparatus (in today’s counterbalance order):
Headfix for 0h 2m 0s, short break, then 0h 5m 0s .
Put back in home cage for a longer break.
Headfix for 0h 5m 0s, short break, then 0h 10m 0s .
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 0h 30m 0s, then record quantity drunk.
Habituation day 2
Weigh mice.
Headfix habituation (in today’s counterbalance order):
Outside of behavior apparatus: 0h 10m 0s .
Put back in home cage for a break
Inside behavior apparatus, on treadmill but with treadmill prevented from moving:0h 10m 0s .
Give allotted daily water, and record quantity drunk
Habituation day 3
Weigh mice.
Headfix habituation (in today’s counterbalance order):
Inside behavior apparatus, on moving treadmill: 0h 15m 0s .
Give allotted daily water, and record quantity drunk.
Days 1 -> 9: Training
Calibrate solenoid: fill the water reservoir with 50µL of fresh 10% sucrose water, and use the Matlab code
calibrateSolenoid_NI
```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 `5µL`of reward per click .
<Note title="Note" type="warning" ><span>This should be done daily, as the solenoid may get clogged or have some other issue that would change the volume being delivered per activation</span></Note>
Weigh mice.
Headfix first mouse on treadmill in behavior apparatus.
Ensure treadmill can move freely.
Ensure mouse can reach the reward delivery spout with its tongue
Run behavior using the code
lickVid_dualConditionExtinction_NI(solOpenDuration)
```, where
solOpenDuration
GUI input: day type = train, probe = no probe, rewarded tone = 2500.
Afterwards:
Clean treadmill by wiping it down with Trifectant or a similar veterinary disinfectant.
Top off sucrose reservoir if it has dropped below 50µL.
Record number of rewarded trials the mouse received.
Repeat steps 16-18 until all mice have undergone their daily behavior.
Give allotted daily water minus the number of rewarded trials times 5 (as each mouse has received 5µL of water per rewarded trial already that day), and record quantity drunk.
Clean the solenoid, reservoir, and attached tubing by using the
calibrateSolenoid_NI
``` code to flush MilliQ water through the system.
Day 10: Training with Probes
Calibrate solenoid: fill the water reservoir with 50µLof fresh 10% sucrose water, and ensure that the solenoid is delivering 5µL of reward per click.
Weigh mice.
Headfix first mouse on treadmill in behavior apparatus.
Ensure treadmill can move freely.
Ensure mouse can reach the reward delivery spout with its tongue.
Run behavior using the code
lickVid_dualConditionExtinction_NI(solOpenDuration)
```, where
solOpenDuration
GUI input: day type = train, probe = probe, rewarded tone = 2500.
Afterwards:
Clean treadmill by wiping it down with Trifectant or a similar veterinary disinfectant.
Top off sucrose reservoir if it has dropped below 50µL.
Record number of rewarded trials the mouse received.
Repeat steps 24-26 until all mice have undergone their daily behavior.
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.
Clean the solenoid, reservoir, and attached tubing by using the
calibrateSolenoid_NI
``` code to flush MilliQ water through the system.
Day 11: Testing 1
Calibrate solenoid: fill the water reservoir with 50µL of fresh 10% sucrose water, and use the Matlab code
calibrateSolenoid_NI
``` 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 `5µL` of reward per click.
Weigh mice.
Headfix first mouse on treadmill in behavior apparatus.
Ensure treadmill can move freely
Ensure mouse can reach the reward delivery spout with its tongue
Run behavior using the code
lickVid_dualConditionExtinction_NI(solOpenDuration)
```, where
solOpenDuration
GUI input: day type = flip, rewarded tone = 2500.
Afterwards:
Clean treadmill by wiping it down with Trifectant or a similar veterinary disinfectant.
Top off sucrose reservoir if it has dropped below 50µL.
Record number of rewarded trials the mouse received.
Repeat steps 32-34 until all mice have undergone their daily behavior.
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.
Clean the solenoid, reservoir, and attached tubing by using the
calibrateSolenoid_NI
```code to flush MilliQ water through the system.
Day 12: Testing 2
Calibrate solenoid: fill the water reservoir with 50µL of fresh 10% sucrose water, and use the Matlab code
calibrateSolenoid_NI
``` 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 `5µL` of reward per click.
Weigh mice.
Headfix first mouse on treadmill in behavior apparatus.
Ensure treadmill can move freely.
Ensure mouse can reach the reward delivery spout with its tongue.
Run behavior using the code
lickVid_dualConditionExtinction_NI(solOpenDuration)
```, where
solOpenDuration
GUI input: day type = test, rewarded tone = 11000.
Afterwards:
Clean treadmill by wiping it down with Trifectant or a similar veterinary disinfectant.
Top off sucrose reservoir if it has dropped below 50µL .
Repeat steps 40-42 until all mice have undergone their daily behavior.
Clean the solenoid, reservoir, and attached tubing by using the
calibrateSolenoid_NI
```code to flush MilliQ water through the system.
Perfuse all mice for histology (see histology protocol).
