Nicotine hydrogen tartrate salt (Sigma Aldrich, St. Louis, MO) was dissolved in 0.9 % NaCl (Hospira, Inc, Lake Forest, IL) with pH adjusted to 7 using sodium hydroxide. l-THP (base), was acquired from Wuxi Gorunje Technology Co., LTD and was dissolved in 2 % tween 80 (Sigma Aldrich, St. Louis, MO), 3 % ethanol (Sigma Aldrich, St. Louis, MO), and 95 % sterile water (Hospira, Inc, Lake Forest, IL). The reported purity of l-THP was assessed by high performance liquid chromatography where the purity was determined as 98.85 %. Varenicline tartrate was supplied by LKT Laboratories, Inc and dissolved in 0.9 % NaCl with pH adjusted to 7 using sodium hydroxide. Bupropion was supplied by Enzo Life Sciences and dissolved in 0.9 % NaCl. 3-Hydroxytyramine (3,4 Dihydroxyphenethylamine; Dopamine Hydrochloride) (Sigma Aldrich, St. Louis, MO) was diluted to 10−8 with Perchloric Acid 70 % (Sigma Aldrich, St. Louis, MO) and sterile water (Hospira, Inc, Lake Forest, IL). Ketamine HCl was supplied through NIDA pharmacy. Xylazine (Sigma Aldrich, St. Louis, MO) was dissolved in 0.9 % NaCl. Equithesin (Pentobarbital Na, Chloral hydrate and Magnesium Sulfate) was supplied by NIDA. Formalin Solution 10 % was acquired from Sigma Aldrich, St. Louis, MO.
For the behavioral experiments, male Sprague-Dawley rats (Charles River) weighing 300–325 g at the beginning of the study were individually housed and maintained in temperature- and humidity-controlled facilities fully accredited by AAALAC. Animals were housed on a 12 h/12 h dark/light cycle (lights out from 8:00 am to 8:00 pm). Behavioral experiments were conducted in the dark phase. For the microdialysis experiments, male Sprague-Dawley rats (Charles River) weighing 200–275 g at the beginning of the study were housed two to a cage and maintained in temperature- and humidity-controlled facilities fully accredited by AAALAC. Animals were housed on a 12 h/12 h light/dark cycle (lights out from 8:00 pm to 8:00 am). Microdialysis experiments were conducted in the light phase. All experimental procedures were approved and conducted in accordance with guidelines of the Institutional Animal Care and Use Committee of the Intramural Research Program National Institute on Drug Abuse, National Institutes of Health, Department of Health and Human Services and the University of Maryland, Baltimore. All treatment groups were randomly assigned for each study. Only animals meeting the specified criteria per experiment were analyzed.
Nicotine SA and reinstatement
Jugular vein catheter implantations were performed as described previously . Briefly, rats were anesthetized under ketamine/xylazine (100 mg/kg, 10 mg/kg), and implanted with a catheter in the jugular vein and a mesh-based backmount just below the shoulder blades. Rats were allowed to recuperate for at least five days.
After the recuperation period, rats began SA training (Coulbourn Instruments, Whitehall, PA). Rats were placed on a restricted diet of chow per day (~30 g) to maintain their current weight. During 2-h training sessions, nose pokes to the correct hole resulted in an infusion (Harvard Apparatus, Holliston, MA) of 0.03 mg/kg nicotine, followed by a 20-s timeout period in which house lights flashed on and off. Training began under a fixed-ratio (FR) 1 schedule of reinforcement. Once a rat received at least 10 reinforcers for three consecutive sessions, the response criteria was increased incrementally to FR 2, FR 3, and finally FR 5. Once a rat responded at or above 10 infusions for five consecutive sessions, pretreatment with saline (i.p. 30 min), l-THP (3 mg/kg, 5 mg/kg i.p. 30 min), varenicline (1 mg/kg i.p. 2-h), or bupropion (40 mg/kg i.p. 30 min) began. Repeated testing consisted of three consecutive days during which rats were pretreated with one of the aforementioned drugs and allowed to self-administer nicotine during 2-h sessions. Separate rats were used for dosing groups.
Extinction occurred in the same chambers as SA by removing nicotine-associated cues and replacing saline infusions for nicotine. Thus the infusion and 20-s timeout did not occur after the FR 5 criterion was met. Rats were trained under extinction criteria for at least five sessions or until responding was at or below 25 % of nicotine baseline responding. Once this criterion was met, reinstatement testing began.
Rats were pretreated with saline (i.p.), l-THP (3 mg/kg, 5 mg/kg i.p.), varenicline (1 mg/kg i.p.), or bupropion (40 mg/kg i.p.) 30 min before placement into chambers and received nicotine (0.3 mg/kg s.c.) 5 min before placement into chambers. Environmental cues present during nicotine SA (20 s time out, infusion of pump) were reintroduced. During reinstatement sessions nicotine was not available, infusions of 0.03 ml/kg saline were delivered after the FR 5 criterion was met.
Nicotine-induced hyperactivity testing
Nicotine-induced hyperactivity testing was conducted over the course of 14 one-hr sessions. Rats were allowed to acclimate to the chambers for two sessions. Baseline readings with injections of saline 30 min and 5 min before access to locomotor chambers were taken for four sessions. On the seventh session, pretreatment with either l-THP (3 mg/kg, 5 mg/kg i.p.) or saline (i.p.) was given 30 min before placement into the chamber followed by an injection of 1 ml/kg saline (s.c.) 5 min before the start of the session. During sessions 8–13 rats were pretreated with l-THP or saline 30 min before the start of the session, followed by and injection of 0.4 mg/kg nicotine (s.c.) 5 min before the start of the session. In session 14 (Nic Challenge) rats received only a challenge dose of 0.4 mg/kg nicotine five minutes before the start of the session.
l-THP locomotor control
Rats were allowed two sessions to acclimate to locomotor chambers (Med Associates Inc, Georgia, VT). After the acclimation period, rats were pretreated with saline or l-THP (3, 4, 5 mg/kg i.p.) 30 min before locomotor sessions began. Rats received an injection of 1 ml/kg saline (s.c.) 5 min before placement into locomotor chambers. This delivery method of treatments was done to mirror injections given during hyperactivity testing. Rats were then allowed to move freely in locomotor chambers for 1-h sessions.
l-THP food reward control
Food reward studies were conducted in behavioral chambers (Med Associates Inc, St. Albans, VT) on a separate group of rats. Rats were again food restricted to maintain their present weight. Rats were trained to nose poke under an FR 10 schedule for a delivery of sucrose pellets (Bioserv, Flemington, NJ). Sessions timed out after 1-h, 20-s time outs occurred after each pellet delivery. Once animals received the maximum number of pellets in the session (40 pellets) for three consecutive sessions, l-THP testing began. Rats were pretreated with saline, 3, 5, 7, or 9 mg/kg l-THP (i.p.) 30 min prior to food reinforcement training.
Microdialysis surgeries were performed as described previously  in a separate group of rats. Briefly, rats were anesthetized under Equithesin (20 mg/kg) and probes made of 22 g 1/2 needles with 8 mm silica, and 2 mm exposed membrane were implanted into the shell of the nucleus accumbens (A +2 mm, L +1 mm from bregma, V-8 mm to dura). Rats were placed in individual hemispheric bowls to recuperate. Experiments were performed the following day to minimize surgery-induced neurotransmitter release.
Microdialysis experiments were performed in the same hemispheric bowls in which rats stayed overnight. Rats were connected to pumps (Bioanalytical Systems Inc, W. Lafavette, IN) with ringer solution (sodium chloride: calcium chloride: potassium chloride, filtered with 25 mm 0.2um syringe filter) infused at a flow rate of 1 ul/min. DA 10−8M was used as an external standard and was tested at least twice immediately before baseline measures were taken to ensure 10 % or less variability in the system (peak of the standard concentration was 100 fmol/min). Baseline DA measures were taken every 20 min until three consecutive samples displayed variability of no more than 17 %. Once this criterion was met, rats were given 5 mg/kg l-THP (i.p.) or 0.4 mg/kg nicotine (s.c.) (control groups); or pretreated with 5 mg/kg l-THP, 40 mg/kg bupropion (i.p.) (40 min), or 1 mg/kg varenicline (i.p.) (2-h) then given 0.4 mg/kg nicotine (s.c.). Dialysis samples were taken every 20 min over 3-h post nicotine injection. Samples were injected without extraction or purification into Dionex UltiMate 3000 HPLC (Chelmsford, MA) coupled to ESA Coulochem III electrochemical detector (Chelmsford, MA), monosodiumphosphate buffer in methanol/water (10:74:16, v/v/v).
At the end of microdialysis experiments, rats were overdosed with phenobarbital. Probes were removed and brains fixed in 20 % formalin for at least two weeks. Brains were cut by vibratome 1000 plus (The vibratome company, St. Louis, MO) into serial coronal slices of 1 cm thickness to verify the placement of probes. Indentation marks left from probes were observed to ensure probe placement was within the nucleus accumbens.
Statistical analysis was carried out by GraphPad 5.00 for windows (La Jolla, CA). Student’s t-test, one-way or two-way ANOVA was used to assess significance according to experimental design. When appropriate one or two-way ANOVA was followed by Bonferroni post hoc test of significance. Significance was reported at p < 0.05 or lower. All analysis was performed on the raw data. For data presentation, a percent of control measure was used to display the observed effects of compounds on SA and reinstatement testing. The percent of control was calculated by taking the three day mean of each individual rat within the same treatment group and dividing it by the three day mean of the entire treatment group during nicotine baseline. In SA testing the test average is presented as a percentage of nicotine baseline response. In reinstatement testing extinction and reinstatement are displayed as a percentage of nicotine baseline response.