Project protocol - Contents

• Workflow and sampling
• Equipment and supplies
• Reagents and solutions
• Procedure: Conditioned place preference (CPP)
• Data

Workflow and sampling

Step	Procedure	Equipment	Data collected
1	Habituation	-	-
2	Preconditioning (day 1)	Conditioning chamber	-
3	Phase 1 conditioning: Mice are injected two times per training day. Control mice are injected with saline only; treated mice are injected with either saline or nicotine and then after 5h are injected with the alternate treatment (days 2-4)	Conditioning chamber	-
4	Mice are tested for 15 min (day 5)	Conditioning chamber	Preference score
5	Phase 2 conditioning (same as above) (days 6-9)	Conditioning chamber
6	Mice are tested for 15 min (day 10)	Conditioning chamber	Preference score

Equipment and supplies

• Conditioning chambers (3 identical)
  • 41.4 x 21.6 x 21.6 cm, containing two distinct chambers (19.7 x 20.3 x 18.3 cm) with Plexiglas lids
  • Wall and floor of one chamber are made of gray Plexiglas, and the other chamber is made of gray Plexiglas walls with white stripes (1.3 cm wide) and a stainless steel mesh floor
  • During training, a gray Plexiglas wall separates the two chambers
  • During preconditioning and testing, the gray wall is taken out so that mice can access both sides of the chamber

Reagents and solutions

• Nicotine hydrogen tartrate salt (Sigma, St. Louis MO) dissolved in saline at 0.35 mg/g (freebase nicotine weight)

Procedure: Conditioned place preference (CPP)

1. Mice are habituated to the experimental room; mice are handled gently for ~1 min and held as if a subcutaneous injection were administered.
2. A preconditioning session is run during the first day of training where mice are randomly placed into a chamber and have free access to both chambers for 15 min. Entry into a chamber is defined as instances when the head and all four paws cross over into the chamber.
3. The amount of time spent in each chamber is measured so that nicotine can be paired with the chamber least preferred by each mouse during the preconditioning session.
4. Phase 1 training (days 2-4): Nicotine CPP groups receive a subcutaneous injection of either nicotine or saline and are immediately confined to a nicotine or saline-paired chamber, respectively, for 15 min. (To control for order effects, the schedule of injections is counterbalanced within conditioning sessions so that half of the mice receive saline and half of the mice receive nicotine during the first conditioning trial.) Mice assigned to saline CPP groups receive subcutaneous injections of saline before being placed in the chambers for 15 min.
5. Phase 1 testing (day 5): Mice are placed in the saline-paired chamber and allowed to move freely between the two chambers for 15 min. The amount of time spent in each chamber is recorded.
6. Phase 2 training (days 6-9): Same procedure as above for days 2-4.
7. Phase 2 testing (day 10): Same procedure as above for day 5.

Data collected by investigator

• Time spent in each chamber in order to compute:
  • Conditioned place preference score for test 1, saline
  • Conditioned place preference score for test 1, nicotine
  • Conditioned place preference score for test 2, saline
  • Conditioned place preference score for test 2, nicotine

Preference scores were calculated by subtracting the differnce between the amount of time spent in the nicotine-paired chamber during reconditioning from the amount of time spent in the same chamber during testing.