Subjects
In total, 80 male Sprague-Dawley rats (280-300 g) from the Slac Laboratory Animal Center of Shanghai (Shanghai, China) were obtained and individually housed in a temperature-controlled facility with a 12 h light/dark cycle and free food/water access. All animal protocols were approved by the Animal Care and Use Committee of Wenzhou Medical University (wydw2015–0121, Zhejiang, China). At the end of the study, animals were euthanized using sodium pentobarbital (100mg/kg, IP).
Drugs
Animals were injected intravenously with propofol (10 mg/mL; Diprivan, Astrazeneca, Italy), with drug solutions being prepared immediately prior to use. The 1.7 mg/kg per infusion dose of propofol used in this self-administration model was chosen based on a previous report[29]. MK-801 was obtained from Sigma-Aldrich (USA), and was prepared using sterile water.
Apparatus
Twelve customized plexiglass operant boxes (Ningbo Addiction Research and Treatment Center, China) were used for propofol training studies, as detailed in previous reports[30]. These boxes contained two nose-poke holes at a height of 5 cm, with each hole containing a yellow LED light. In addition, a larger light was present on the wall over these holes (28 V, 0.1 mA). Drugs were delivered to animals via Tygon tubing, with a leash assembly being used to protect the tubing and a plastic swiveling apparatus being used to guide the tubing through the ceiling. Animals wore jackets containing a customized fluid connector that attached to the leash assembly. A 5 mL syringe pump was attached to the tubing, delivering fluids at a 1.2 mL/min rate. A MED Associates interface on an IBM PC was used for experimental control, which was achieved with software written internally using Borland Delphi 6.0.
Surgery
Sodium pentobarbital (50mg/ml) was used to anesthetize animals, after which incisions were made on the chest above the right jugular vein and at the mid-scapular level on the back. Next, a chronically indwelling silastic jugular cannula was implanted such that it extended from the back of the animal[30]. A total of 0.2 mL of a saline-heparin solution (25 U/mL heparin) was flushed through these cannulas each day in order to maintain patency, while infections were prevented via the daily administration of sodium penicillin for 5 days after surgery. Prior to study initiation, animals were given a 7 day post-operative recovery period[29].
Propofol self-administration
Propofol self-administration training was performed as in previous reports [30]. Briefly, after surgical recovery animals were placed into the customized operant boxes and infusion lines were attached as appropriate. At the start of each session, the yellow LED within the active nose-poke hole was lit. Each session started with the illumination of yellow light inside the active nose-poke hole, then they were administered one 1.7 mg/kg infusion of propofol following completion of the ratio requirement(fixed ratio =1) in the active nose-poke. This administration was paired to a 5-second illumination of the house light in addition to the noise of the infusion pump apparatus. After this time, a 15 second timeout period was activated during which responses were recorded but did not alter drug administration. At the end of this period, the yellow LED within the active nose-poke hole was again illuminated. There were no consequences for responses to the inactive nose-poke hole. Sessions were allowed to continue until either 3 hours had elapsed or until 100 propofol infusions had been administered.
Specific experiments
The 32 rats in this study underwent propofol self-administration training once per day (1.7 mg/kg per infusion). Once rats exhibited a stable response (±5%) for 5 consecutive days, they were intraperitoneally administered MK-801 (0, 0.1, 0.2, or 0.4 mg/kg; n=8/group) 10 minutes before starting the next session, as determined based on previous reports[31]. Animal responses over a 1 hour self-administration period were then recorded, after which animals were euthanized.
Sucrose self-administration
For sucrose administration tests, rats were placed in 30 x 20 x 24 cm operant conditioning chambers and trained according to an FR1 schedule using food reinforcement (45 mg pellets; Noyes, NH, USA) over a period of 7 days. For these chambers, two nose-poke holes were present with a light above each and a food dispenser located between the two holes. In addition, a house light was present on the opposite wall. Food pellets were delivered only in response to the pressing of the active hole during nose-poke tests, while pressing the inactive hole elicited no response. Tests were allowed to proceed either for 3 hours or until 100 food pellets had been acquired.
After rats exhibited stable sucrose acquisition responses, they were intraperitoneally injected with MK-801 (0, 0.1, 0.2, or 0.4 mg/kg) 10 minutes prior to the next session. The numbers of active/inactive pose-poke responses and the number of sucrose pellets obtained were then recorded over the course of a 1 hour session period.
General activity test
In order to explore the ability of MK-801 to non-specifically influence general activity in rats, animals were intraperitoneally administered MK-801, after which locomotion in a novel context was analyzed. In total, 24 naive rats (n=6/group) were administered MK-801 (0, 0.1, 0.2, or 0.4 mg/kg), with their total distance traveled (cm) then being measured and analyzed with the MED Associates SOF-811 Open-field Activity Software.
Statistical analysis
The number of infusions or responses for active and inactive holes during self-administration testing was analyzed using a one-way analysis of variance (ANOVA) or a two-way (hole, treatment) repeated-measures ANOVA. Newman-Keuls multiple comparison tests with an alpha level of 0.05 or 0.01 were used for post-hoc comparisons of group means.