TITLE: Basic Operant Behavioral Training
AUTHORS: Alexandra Nelson, Xiaowen Zhuang

[DESCRIPTION]
This protocol includes information about the operant chamber, mouse preparation, initial shaping (training), and instrumental learning. This can be followed by other behavioral tasks, such as probabilistic reversal learning or delay discounting, which are described in other protocols. To promote consistent results, each mouse should be trained in the same operant chamber at the same time of day, five days per week, throughout the study. Male and female mice should be trained in separate operant chambers.

[STEPS]
SECTION: Operant Box Setup
1. Ensure the correct SD cards are inserted into the logging shield of each chamber.
SECTION: Operant Box Setup
2. Connect the USB cable of each Arduino to the computer.
SECTION: Operant Box Setup
3. Turn on the 12-volt power supply.
SECTION: Operant Box Setup
4. Pour 10 mL diluted milk into each falcon tube for each behavioral box.

SECTION: Operant Box Setup
5. Connect each solenoid to its behavioral chamber: one end goes into the milk solution, and the other end goes to the chamber.
SECTION: Operant Box Setup
6. Ensure that milk is pumped into the chamber. 
SECTION: Operant Box Setup
7. Upload the Arduino code to the operant chambers, depending on the training phase of each mouse.
SECTION: Operant Box Setup
8. In each Arduino window, go to “Tools”, and the select “Port” to choose the correct board number, and then click “Upload”.
SECTION: Operant Box Setup
9. In each Arduino window, go to “Tools” again, and then select “Serial monitor” to open serial monitor windows.
 

SECTION: Operant Box Setup
10. Insert the waste pan and floor into the behavioral chamber. 
SECTION: Starting an Operant Session
11. Place each mouse into their designated box and close acrylic door to the operant box.
SECTION: Starting an Operant Session
12. Allow to acclimate for 1-2 minutes.
SECTION: Starting an Operant Session
13. Turn on 5-volt breadboard power switch and press “Reset” button on the Arduino board.
SECTION: Starting an Operant Session
14. Securely close the door of the sound attenuating box.
SECTION: Cleanup
15. Replace the falcon tube with clean tap water.
SECTION: Cleanup
16. Upload the “cleaning” protocol onto each chamber.
SECTION: Cleanup
17. Allow the water to run through the entire system for at least 15 min.

SECTION: Cleanup
18. Clean the walls of operant chamber, nosepoke ports, and reward port with 75% alcohol.
SECTION: Cleanup
19. Rinse the waste pan and floor with water, and allow them to air dry 50 min. 

SECTION: Cleanup
20. Turn off the 5-volt breadboard power switch.
SECTION: Cleanup
21. Turn off the 12-volt power supply.
SECTION: Training procedure - Preparing the mice with food restriction
22. Three days before the start of Phase 1 (Magazine Training), the free-feeding (ad libitum) weight is recorded by hand in a logbook.
SECTION: Training procedure - Preparing the mice with food restriction
23. Food is removed on the same day, and the food restriction protocol is initiated. This starts with
2.5-3 g of standard food pellets per animal per day for the first few days.
SECTION: Training procedure - Preparing the mice with food restriction
24. Subsequent adjustments may be necessary based on the animals’ weights, which must be monitored daily and maintained at approximately 85%-90% of their free-feeding weights.

 

SECTION: Training procedure - Operant Training Phase 1/Magazine Training
25. Three days after starting food restriction, animals can be started on Phase 1. This phase introduces mice to the reward delivery port (1 session).
SECTION: Training procedure - Operant Training Phase 1/Magazine Training
26. A liquid reward (10 µL of milk) is delivered on a random interval schedule of 40-80 seconds for a total of 40 rewards.

SECTION: Training procedure - Operant Training Phase 1/Magazine Training
27. Each trial begins with the delivery of milk into the center port, accompanied by a 10-second illumination of the center port light.
SECTION: Training procedure - Operant Training Phase 2/Blocked Instrumental Learning
28. In this phase, the side (choice) nosepoke triggers reward delivery (6-10 sessions).
SECTION: Training procedure - Operant Training Phase 2/Blocked Instrumental Learning
29. Mice are trained to nosepoke at the side ports to obtain a reward on a fixed-ratio 1 (FR1) schedule.
SECTION: Training procedure - Operant Training Phase 2/Blocked Instrumental Learning
30. Each training session is separated into blocks, separated by a two-minute break period when the house light is turned off.
SECTION: Training procedure - Operant Training Phase 2/Blocked Instrumental Learning
31. Each block lasts 50 min. 

SECTION: Training procedure - Operant Training Phase 2/Blocked Instrumental Learning
32. Only one nosepoke (either the left or the right side) is trained in each block, and the active nosepoke switches during the break period.
SECTION: Training procedure - Operant Training Phase 2/Blocked Instrumental Learning
33. The initial nosepoke (left or right) is counterbalanced across sessions.
SECTION: Training procedure - Operant Training Phase 2/Blocked Instrumental Learning
34. Each trial begins with a stimulus cue light above the left or right nosepoke.
SECTION: Training procedure - Operant Training Phase 2/Blocked Instrumental Learning
35. If the mouse nosepokes that port within 20 s, the cue light is extinguished, and a reward    (10 µL of milk) is delivered to the center port, which is illuminated for 10 s, followed by a random 30-50 second intertrial interval. 

 

SECTION: Training procedure - Operant Training Phase 2/Blocked Instrumental Learning
36. If the mouse does not perform a nosepoke on the correct side, then the cue lights are extinguished and a timeout period (a random 30-50 second interval) begins.
SECTION: Training procedure - Operant Training Phase 2/Blocked Instrumental Learning
37. To advance to Phase 3, a mouse must perform a correct response on more than 80% of trials in the last session.
SECTION: Training procedure - Operant Training Phase 3/Self-initiated Instrumental Learning with Randomized Trials
38. This phase shares some features with Phase 2, but in this phase, the mouse must center nosepoke to trigger trial start (5-8 sessions), and choice trials are presented in a randomized (rather than blocked) fashion.
SECTION: Training procedure - Operant Training Phase 3/Self-initiated Instrumental Learning with Randomized Trials
39. Trials begin with the center port being illuminated for 10 s.   

 

SECTION: Training procedure - Operant Training Phase 3/Self-initiated Instrumental Learning with Randomized Trials
40. During this period, a trial can be initiated by a center port nosepoke.
SECTION: Training procedure - Operant Training Phase 3/Self-initiated Instrumental Learning with Randomized Trials
41. Failure to make a center nosepoke within this period is considered a center omission and is followed by a timeout period (random 30-50 second interval). 
SECTION: Training procedure - Operant Training Phase 3/Self-initiated Instrumental Learning with Randomized Trials
42. Following an initiation nosepoke, the stimulus cue lights at either the left or right port are activated for 10 s.

SECTION: Training procedure - Operant Training Phase 3/Self-initiated Instrumental Learning with Randomized Trials
43. If the mouse nosepokes that port within 10 s, the cue light is extinguished, and a reward    (10 µL of milk) is delivered to the center port, which is illuminated for 10 s, followed by a random 30-50 second intertrial interval. 

SECTION: Training procedure - Operant Training Phase 3/Self-initiated Instrumental Learning with Randomized Trials
44. Failure to poke the correct side port within 10 s is considered a side omission, and cue lights are extinguished until the next trial.

SECTION: Training procedure - Operant Training Phase 3/Self-initiated Instrumental Learning with Randomized Trials
45. Poking the opposite, uncued side is recorded as an incorrect response and does not trigger punishment. Each session lasts 90 min.