Project protocol - Contents

• Workflow and sampling
• Equipment and supplies
• Reagents and solutions
Procedure: Pavlovian conditioned approach testing
• Data

Workflow and sampling

Step	Procedure	Equipment	Data collected
1	Mice are randomly assigned to test groups (paired and unpaired)	-	-
2	Groups are tested for sign-tracking and goal-tracking	Conditioning chambers	Sign-tracking and goal tracking (rate and duration)

Equipment and supplies

• Conditioning chambers (16 Med Associates, St. Albans VT, model ENV-307W)
  • Enclosed in sound attenuating cubicles (ENV-022V)
  • The floor of each chamber consists of bars which are completely covered by a single piece of acrylic to facilitate cleaning and mouse ambulation
  • A retractable response lever (ENV-312-2W) is mounted to the left front wall 18 mm above the chamber floor and 28 mm away from the adjacent wall; contains infrared detectors
  • A pellet dispenser (ENV-203-20) delivers 20 mg Bio-Serv (Flemington NJ) Dustless Precision Pellets (F0071) into a pellet receptacle (ENV-303W)
  • The receptacle is mounted to the right front wall 3 mm above the chamber floor, 28 mm away from the adjacent wall, and 125 mm away from the lever; contains infrared detectors
  • A house light (ENV-315W) with bulb (CM1820, Chicago Miniature, Novi MI) is mounted outside and behind the rear wall of the chamber
  • A monochrome micro video camera (Noldus Information Technology, Leesburg VA) is mounted to the ceiling of the sound attenuating cubicle directly above the chamber.
  • The camera is connected to a video capture card in a Windows PC (Noldus Information Technology) which is used to record mouse behavior during testing
• Med Associates MED-PC IV software (St. Albans VT) controls the chambers
  • The ST/GT program was written in-house in MEDState notation

Reagents and solutions

• 70% ethanol for cleaning between testing sessions

Procedure: Pavlovian conditioned approach testing

1. Mice are randomly assigned to either a paired or an unpaired condition; mice in paired condition receive PCA training in which pellet delivery is paired predictably with lever extension and retraction (pellet delivery and retraction occur simultaneously 10 s following lever extension); mice in the unpaired condition receive identical training with the exception that pellet delivery is randomized relative to lever extension and retraction.
2. Testing is conducted between 1100 and 1600h. The house light is illuminated throughout the session.  Sessions consist of 25 trials.  Each trial is followed by an inter-trial interval (ITI) of random duration ranging from 30 to 150 s.
3. The lever is extended and retracted at the beginning and end, respectively, of each 10 s trial.
4. For mice in the paired group, a pellet is delivered concurrently with lever retraction; for mice in the unpaired group, a pellet is delivered at a random time between the beginning of each ITI and the end of the subsequent trial.
5. All mice receive 25 pellets during each session.
6. The number of lever presses and pellet-receptacle head entries during the trial and ITI are recorded automatically for each session by means of infrared detectors built into the levers and pellet receptacles.
7. Because many mice show evidence of 1) lever contact that does not result in lever depression and 2) shallow pellet-receptacle head entry that does not result in an infrared beam break, the entire session is recorded for all mice of the final test day (session 15).
8. Video data are subsequently scored to quantify approach to the lever and pellet receptacle during the trial and ITI on session 15.

Data collected by investigator

• Goal-tracking rate for paired and unpaired conditions (15 sessions)
• Goal-tracking duration for paired and unpaired conditions for trial period and ITI  (25 trials of last session)
  
• Sign-tracking rate for paired and unpaired conditions (15 sessions)
• Sign-tracking duration for paired and unpaired conditions for trial period and ITI (25 trials of last session)
• Pellet head entry for paired and unpaired conditions for ITI period

All data are available in the Dickson1 supplementary data file

Summary plots are available here