Project protocol - Contents

• Workflow and sampling
• Equipment and supplies
• Reagents and solutions
• Procedure: Open field test (for novelty-related measures)
• Procedure: Novelty-preference test
• Procedure: Hole board test
• Procedure: Surgical catheterization and intravenous cocaine self-administration (FR1 and PR schedules)
• Data

Workflow and sampling

Step	Procedure	Equipment	Data collected
1	Open field test on day 1	Open field arena	Distance traveled, time in center of arena
2	Novelty preference testing on day 2	Open field arena with partition, giving two chambers (one with a novel wall)	Time spent in chamber with novel wall
3	Hole board test on day 3	Open field arena with hole board insert	Number of hole pokes
4	Catheterization surgery, followed by 3-day recovery	Surgical equipment and indwelling catheters	-
5	Intravenous cocaine self-administration (fixed ratio 1 (FR1))	Operant conditioning chamber	Number of infusions, active lever presses, inactive lever presses
6	Intravenous cocaine self-administration (progressive ratio (PR))	Operant conditioning chamber	Number of infusions (breakpoint), active lever presses, inactive lever presses

Equipment and supplies

• VersaMax Animal Activity Monitors (AccuScan Instruments, Inc., Columbus OH)
  • Each square arena (39 x 39 x 39) is constructed of clear acrylic
  • Infrared detectors enabling automated data collection are positioned around the bottom edges of the arena walls
  • Arenas are housed in rectangular room (3 x 4.5 m)
  • Illumination is maintained at 150-200 lx
  • Open field test: empty arena
  • Novelty preference test: an insert separates each arena into two equally sized rectangular halves (one with clear walls and the other with black walls)
  • Hold board test: a separate insert consisting of 16 holes positioned in a 4 x 4 grid
• Surgical equipment and sutures
• Indwelling catheters (jugular vein)
• External vascular access harness (Instech Laboratories, Inc., Plymouth Meeting PA)
• Med Associates Operant conditioning chambers (307W)
  • Enclosed in sound-attenuating cubicles (ENV-022MD)
  • Two retractable response levers (ENV-310W) are mounted on the front wall
  • A stimulus light (ENV-321W) is mounted above each lever
  • A house light (ENV-315W) is centrally mounted on the rear wall
  • A 25-gauge single-channel stainless steel swivel is mounted to a counterbalance lever arm attached to the outside of the chamber
  • Tubing is used to connect a syringe mounted on the infusion pump to the swivel and to connect the swivel to a vascular access harness
• Med Associates control unit using MED-PC IV software

Reagents and solutions

• Cocaine hydrochloride (National Institutes of Health / NIDA Drug supply program); filtered through 22 µm syringe filters
• Heparinized saline (Fisher Scientific (Pittsburgh PA)
• Oxygen
• Isoflurane
• 70% ethanol for cleaning between testing sessions

Procedure: Open field test (for novelty-related measures)

1. Mice are habituated to the testing room for at least 48 h prior to testing.
2. Mice are placed into the center of the empty arena and allowed to explore for 20 min (investigators chose to use a relatively short duration test because the environment becomes less novel over time).
3. Total distance traveled and time in center are recorded.

Procedure: Novelty-preference test

1. A partition is placed into the empty arena to create two equally-spaced halves.
2. Mice are placed into one side of the two-sided arena (the familiar side) and allowed to explore for 10 min. The wall color of the familiar side (black or clear) was randomized for each mouse. Access to the other side of the chamber (novel side) is blocked.
3. Following the initial 10-min familiarization phase, the mice are allowed to access both the familiar and novel sides of the arena for 10 min.
4. The time spent in each side of the arena during this 10-min testing phase is recorded.

Procedure: Hole board test

1. The hole board insert is placed in the empty arena.
2. Mice are placed into the center of the arena and allowed to explore for 10 min.
3. Total number of exploratory hole pokes is recorded.

Procedure: Surgical catheterization and intravenous cocaine self-administration (FR1 and PR schedules)

1. An indwelling catheter is implanted into the right external jugular vein for each mouse under oxygen-isoflurane anesthesia; the catheter is inserted 12 mm into the jugular vein and anchored with sutures; the catheter travels subcutaneously to an incision at the mid-scapular region and attaches to an external vascular access harness.
2. Mice are allowed 3 days of post-surgical recovery.
3. To verify and maintain patency, the mice are briefly removed from the chamber once daily, at which point, blood is aspirated and catheters flushed with 25 µL of heparin lock solution (100 U/mL heparin/saline). Mice are dropped from the study if catheters become non-patent.
4. IVSA testing begins at 0900h each day; during the entire IVSA study, the mice are continuously housed in the operant conditioning chambers with free access to food and water.
5. Mice are tested for 5 consecutive days on a cocaine IVSA paradigm using a fixed ratio 1 (FR1) schedule at a dose of 1.0 mg/kg/infusion (infusion rate is 11.76 µL per second and infusion duration is 2 s).
6. Each day the mice are tested for two 2-h sessions separated by a 2-h time-out, resulting in a total of ten testing sessions.
7. Each 2-h session begins with the extension of the two response levers. A left (active) lever press results in a cocaine infusion and the illumination of both stimulus lights for 2 s with house light turned off. This is followed by a 20-s time-out, during which, the house light is turned off and lever presses are recorded but have no consequences.
8. The following are collected during each 120-min session: number of infusions, number of responses on the active lever, number of responses on the inactive lever.
9. Following the acquisition stage of cocaine IVSA testing, all mice are tested on a progressive ratio (PR) schedule for a single session to assess motivation to take cocaine; the schedule is identical to the FR1 schedule with the exception that, following an earned cocaine infusion, the number of active lever presses required to receive a subsequent cocaine infusion is increased within the session as determined by the equation [5^{(infusion number x 0.02)}] - 5.
10. The breakpoint is reached when 60 min elapses without earning a cocaine infusion and corresponds to the last completed step (i.e., the total number of infusions earned during the session). If the breakpoint is not reached within 6h, the session is terminated.

Data collected by investigator

• Open field test
  • Total distance traveled
  • Duration in center of arena
• Novelty-preference test
  • Time spent in familiar and novel sides of the arena
• Hold board test
  • Number of exploratory hole pokes
• IVSA: FR1
  • Number of infusions
  • Number of active lever presses
  • Number of inactive lever presses
• IVSA: PR
  • Number of infusions (breakpoint)
  • Number of active lever presses
  • Number of inactive lever presses

MPD computed means for number of infusions, active lever presses, and inactive lever presses for IVSA FR1 over 10 sessions.

All data for all sessions are available in the Dickson2 download file.