Project protocol - Contents

• Workflow and sampling
• Equipment and supplies
• Reagents and solutions
• Procedure: Oral sucrose self administration learning sessions
• Data

Workflow and sampling

Step	Procedure	Equipment	Data collected
1	Seven days of food restriction to achieve weights that are 90-95% of free-feeding body weights in the morning, and 80-85% in the evening	-	Free-feeding body weight; daily body weight (before and after session)
2	Three days of instrumental learning (daily: FR1, VR2, VR5)	Testing chamber	Active lever presses, lever press rate, reinforcers earned

Equipment and supplies

• Extra-wide aluminum and polycarboate modular testing chambers (Med Associates, St. Albans VT)
  • Stationed inside a sound attenuating chamber equipped with a white noise generator, house light, and a tone generator
  • A horizontal array of 5 illuminable nose-poke appertures are on one side of the box
  • The opposite wall is fitted with an illuminable pellet delivery magazine with an entry-detection photocell; magazines also contain reservoirs for sucrose solution
  • A 5 mL syringe (BD and Co, Franklin Lakes NJ) containing sucrose is connected via PE-50 tubing (Stoelting Co., Wood Dale IL) to a 26-gauge metal inlet fashioned underneath the magazine
  • Sucrose is delivered to a connected reservoir by activation of a syringe pump (PHM-100, Med Associates, St. Albans VT) situated on top of the sound attenuating chamber
  • Adjacent to the magazine, on both sides, are retractable ultra-sensitive mouse levers (2 g force requirement for actuation
  • Water is made available in the chamber via a lixit-sipper which is extended to approximatedly 2" above the grid floor, angled facing away from the magazine

Reagents and solutions

• 20% sucrose solution made fresh

Procedure: Oral sucrose self administration learning sessions

1. Mice undergo a series of 3 days of instrumental learning in 8-h sessions.
2. Each session starts with a non-contingent delivery of 10 µL of sucrose and 3-s magazine illumination, followed by the extension of a single lever into the box (location relative to the magazine is counterbalanced within and across strains).
3. The first 50 rewards are delievered on a fixed ratio 1 (FR1) schedule of reinforcement; the next 50 are delivered under a variable ratio 2 (VR2; range of 1 to 3 presses required) schedule; the schedule is then switched to variable ratio 5 (VR5; range 1 to 9 presses required) for the remainder of the sesssion which is terminated after 8 h or after delivery of 500 rewards.
4. Rewards are delivered conincident with a 3-s magazine illumination; to facilitate response acquisition, the proprioceptive feedback of a successful lever actuation is supplemented by a 0.05 s tone pulse.
5. All presses (except those made during the 0.85 s pump activation during reward delivery) count towards completion of the next response schedule.
6. Mice are allowed to freely organize their instrumental response patterns: there is never a time-out period and multiple rewards can be earned prior to their retrieval.

Data collected by investigator

• Data collected on all lever press and magazine entry events (each time stamped)
• Summary measures computed for spontaneous acquisition of instrumental behavior, including total number of lever presses (across all 3 days) and presses per reinforcement schedule per day
• A binary phenotype termed 'Learned' determined (segregates subjects according to whether they made at least 50 lever presses across the 3 days of testing)
• Body weight prior to food restriction, and on a daily basis, percent body weight lost when learning sessions started and when they finished