Animals and Housing
Male Sprague Dawley rats (Rattus norvegicus) weighing 250–300 g were obtained from Taconic Laboratories (Germantown, N.Y., USA). Animals were single-housed with food and water available ad libitum (rats undergoing food reinstatement experiments were placed on restricted diets, as outlined below). All animals were housed in a colony maintained on a 12-hr/12-hr light/dark cycle with the lights on at 7:00 a.m. All experimental procedures were performed during the light phase. All experimental protocols were in accordance with the guidelines set forth by the National Institutes of Health and were approved by the Boston University School of Medicine Institutional Animal Care and Use Committee.
All behavioral experiments were conducted in ventilated, sound attenuating operant chambers purchased from Med-Associates Inc. (East Fairfield, VT). Each operant chamber was equipped with both inactive and active response levers, a food pellet dispenser as well as an automated injection pump for administering drug or vehicle solutions intravenously.
Rats were allowed one week to acclimate to their home cages upon arrival. Prior to surgery, the rats were anesthetized with 80 mg/kg ketamine and 12 mg/kg xylazine (Sigma/RBI, St. Louis, MO). An indwelling catheter (CamCaths; Cambridge, UK) was inserted into the right, external jugular vein and sutured securely in place. The catheter was connected to a mesh backmount, which was implanted subcutaneously above the shoulder blades. In order to prevent infection and to maintain patency, catheters were flushed daily with 0.3 ml of a solution of the antibiotic Timentin (0.93 mg/ml) dissolved in heparinized saline. When not in use, the catheters were sealed with plastic obturators.
Immediately following implantation of the indwelling catheter, some rats were mounted in a stereotaxic apparatus (Kopf Instruments, CA) and bilateral guide cannulae (14 mm 24 gauge tubing, Small Parts Inc., Roanoke, VA) were implanted 2 mm dorsal to the mPFC, 1 mm dorsal to the PPTg/LDT or 1 mm dorsal to the VTA according to the following stereotaxic coordinates from the atlas of Paxinos and Watson (1997)
: mPFC: +2.5 mm anteroposterior (A/P, relative to bregma), ±0.5 mm mediolateral (M/L, relative to bregma) and −2.0 mm dorsoventral (D/V, relative to dura): PPTg/LDT: −7.8 mm A/P, ±2.0 mm M/L and −6.2 mm D/V: VTA: −5.8 mm A/P,±0.5 mm M/L and −7.0 mm D/V. Guide cannulae were cemented in place by affixing dental acrylic to three stainless steel screws fastened to the skull. Obturators (14 mm, 33 gauge stainless steel wire, Small Parts Inc., Roanoke, VA) were inserted into each guide cannula in order to prevent occlusion.
After surgery, rats were allowed seven days to recover before behavioral testing commenced. Initially, rats were placed in operant chambers daily and allowed to lever press for intravenous cocaine (0.25 mg cocaine/59 μl saline, infused over a 5 sec period) on a fixed-ratio 1 (FR1) schedule of reinforcement. Each session began with the i.v. administration of 59 μl cocaine (0.25 mg) to fill the catheter. Rats were allowed to self-administer a maximum of 30 injections per 120-minute operant session. Stable responding on the FR1 schedule was defined as less than 15% variation in response rates over three consecutive self-administration days. After stable responding was achieved, animals were switched to a fixed-ratio 5 (FR5) schedule of reinforcement. The maximum number of injections was again limited to 30 per daily self-administration session under the FR5 schedule. For both the FR1 and FR5 schedules, a 20 second time-out period followed each cocaine infusion, during which time active lever responses were tabulated but had no scheduled consequences. Responses made on the inactive lever, which had no scheduled consequences, were also recorded during both the FR1 and FR5 training sessions.
Extinction and reinstatement of cocaine seeking
Following approximately 21 days of daily cocaine self-administration sessions, drug-seeking behavior was extinguished by replacing the cocaine with 0.9% saline. Daily two-hour extinction sessions continued until responding on the active lever was <15% of the response rate maintained by cocaine self-administration under the FR5 schedule of reinforcement. Typically, it took approximately 7 days for rats to meet this criterion.
The FR5 schedule of reinforcement was used throughout the reinstatement phase of the experiment. When an animal met the response requirement (five presses on the active lever) an intravenous infusion of saline was administered. Using a between-session reinstatement paradigm, each daily reinstatement session was followed by extinction days until responding was less than 15% of the maximum number of responses maintained by cocaine self-administration. In general, it took 1–2 days of extinction for each animal to reach criterion between reinstatement sessions. Using this experimental design, subjects underwent a series of extinction and reinstatement sessions that lasted approximately 16 days. During this period, animals may lose the ability to reinstate active lever responding following a priming injection of cocaine. However, we have previously shown that reinstatement of cocaine seeking persists for at least 20 days after the initial extinction of cocaine self-administration (Park et al., 2002
; Anderson et al., 2003
). Moreover, we were able to assess the magnitude of reinstatement by randomly administering priming injections of cocaine throughout the reinstatement phase of the experiment. All animals displayed stable drug seeking, which was operationally defined as greater than 30 active lever responses per 2-hr operant session, during the reinstatement phase of the experiment.
Once self-administration behavior was extinguished, the ability of a priming injection of cocaine (10 mg/kg, i.p.) or its vehicle (0.9% saline) to reinstate cocaine seeking was assessed. On subsequent test days, the selective D1-like dopamine receptor agonist R(+)-SKF-81297 hydrobromide (1.0 μg/0.5 μl, Sigma/RBI, St. Louis, MO) or its vehicle (0.9% saline) was microinjected into the mPFC in order to test its ability to reinstate cocaine-seeking behavior. Animals were placed into the operant chambers immediately following the intra-mPFC microinfusion of SKF-81297 and the 2-hr reinstatement session began. Additional experiments assessed the ability of an intra-PPTg/LDT microinjection of an ionotropic glutamate receptor antagonist or an intra-VTA microinjection of a nicotinic acetylcholine receptor antagonist, muscarinic acetylcholine receptor antagonist or ionotropic glutamate receptor antagonist to attenuate reinstatement of drug seeking elicited by a priming injection of cocaine (10 mg/kg, i.p.). The ionotropic glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione disodium salt (CNQX; 0.03 or 3.0 μg/0.5 μl, Tocris, Ellisville, MS) was administered directly into the PPTg/LDT ten minutes prior to a priming injection of cocaine (10 mg/kg, i.p.). Furthermore, CNQX (0.03 or 3.0 μg/0.5 μl), (−)-scopolamine hydrochloride (2.4 or 24.0 μg/0.5 μl, Sigma/RBI, St. Louis, MO) or mecamylamine hydrochloride (1.0 or 10.0 μg/0.5 μl, Sigma/RBI, St. Louis, MO) was microinjected into the VTA ten minutes prior to a systemic priming injection of cocaine (10 mg/kg, i.p.) in order to assess their ability to attenuate cocaine priming-induced reinstatement.
The dose ranges for each of the aforementioned pharmacological compounds were based on the following rat microinjection experiments: SKF-81297 (Beyer & Steketee, 2002
; Cools et al., 2002
; Schmidt et al., 2006
), CNQX (Cornish et al., 1999
; See et al., 2001
; Park et al., 2002
), (−)scopolamine hydrochloride (Ikemoto & Goeders, 2000
; Corrigall et al., 2002
; See et al., 2003
; Pratt & Kelley, 2005
) and mecamylamine hydrochloride (Sziraki et al., 2002
; Forster & Blaha, 2003
; Sharf & Ranaldi, 2006
Obturators were removed from the guide cannulae and 33 gauge stainless steel microinjectors (Small Parts Inc.) were inserted. Microinjectors were cut to a length that extended 2 mm (for microinjections targeting the mPFC) or 1 mm (for microinjections targeting the PPTg/LDT or VTA) below the ventral end of the guide. Bilateral infusions were performed simultaneously over a 120 second time period in a total volume of 0.5 μl per side. Microinjectors were left in place for 60 seconds following the microinfusions, in order to allow the drug solution or vehicle to diffuse away from the tips of the cannulae, before they were removed. Animals were placed immediately into the operant chambers following microinjection of SKF-81297 or its vehicle (0.9 % saline) and the reinstatement session began without delay. Animals pretreated intra-cranially with CNQX, scopolamine, mecamylamine or their vehicle (0.9% saline) 10 minutes prior to a priming injection of cocaine (10 mg/kg, i.p.) were placed in the operant chambers immediately following the cocaine priming injection.
The goal of the experimental design was to have each animal serve as its own control and receive up to six microinjections per brain region (i.e. two doses plus vehicle for two drugs for a maximum of 6 microinjections per brain region). However, we were frequently forced to deviate from this experimental design when technical difficulties (i.e. blocked microinjection cannulae or loss of catheter patency) made it impossible to test all doses of a compound plus vehicle in an entire cohort of subjects. In every case, however, an animal received a minimum treatment of one drug dose and its vehicle. In order to control for potential rank order effects of drug or vehicle treatments, the agonist, antagonists and vehicle were counterbalanced across reinstatement sessions and within cohorts of animals. Using this experimental design, no order effects of drug treatments were observed.
Potential nonspecific rate-suppressing effects of the pharmacological compounds tested were evaluated by assessing the influence of intra-PPTg/LDT CNQX or intra-VTA CNQX, scopolamine or mecamylamine on reinstatement of food-reinforced responding. Rats were trained initially to lever press for sucrose pellets (Research Diets, Inc., New Brunswick, NJ) on a FR1 schedule of food reinforcement during 1-hour operant sessions. Once animals achieved stable responding for food (defined as <15% variation in responding over two consecutive days) on the FR1 schedule of reinforcement, the response requirement was increased to an FR5 schedule of reinforcement. Animals were limited to 30 sucrose pellets within a 1-hour operant session and were food restricted to four pellets of lab chow (Harlan Teklad, Wilmington, DE) in their home cages for the duration of the experiment.
After two weeks of food-maintained responding on the FR5 schedule of reinforcement, responding was extinguished by inactivating the food dispenser so that every 5 lever presses had no scheduled consequences. Once lever responding decreased to <15% of the maximum number of responses completed during food self-administration, animals proceeded to reinstatement testing. Bilateral microinjections of CNQX (0.3 μg/0.5 μl) or vehicle into the PPTg/LDT or scopolamine (24.0 μg/0.5 μl), mecamylamine (10.0 μg/0.5 μl), CNQX (0.3 μg/0.5 μl) or vehicle into the VTA were administered ten minutes prior to the beginning of the reinstatement session. The experimenter remotely administered one sucrose pellet every 2 minutes for the first 10 minutes of the reinstatement session. A between-session paradigm was used so that each daily 1-hour reinstatement session was followed by an extinction session the following day until responding was again <15% of the response rate maintained by food.
Histology and Verification of Cannulae Placements
After the microinjection experiments were concluded, animals were overdosed with a systemic injection of pentobarbital (100 mg/kg) and perfused intracardially with 60 ml of 0.9% saline followed by 60 ml of 10% formalin. The brains were removed and stored in 10% formalin. Subsequently, 100–150 μm thick coronal sections were taken at the level of the mPFC, PPTg/LDT or VTA with a Vibratome (Technical Products Int., St. Louis, MO.). These sections were mounted on gel-coated slides and stained with Cresyl violet. An individual blind to the animals’ behavioral responses determined cannulae placements as well as potential drug- or cannula-induced neuronal damage. Light microscopy was used to determine cannulae placements as well as the presence and extent of cell death and associated gliosis.
Behavioral Data Analyses
Total active and inactive lever responses during the reinstatement phase of experiments with SKF-81297 in the mPFC were analyzed with unpaired t-tests. For the experiments utilizing CNQX in the PPTg/LDT, and CNQX, scopolamine and mecamylamine in the VTA, the total mean active and inactive lever responses during the reinstatement phase were analyzed with one-way analyses of variance (ANOVAs) or unpaired t-tests. Pairwise comparisons were made with Tukey’s HSD (P<0.05).
Cocaine was obtained from the National Institutes of Drug Abuse (NIDA) and dissolved in sterile 0.9% saline. R(+)-SKF-81297 hydrobromide (1.0 μg/0.5 μl), (−)-scopolamine hydrochloride (2.4 or 24.0 μg/0.5 μl), and mecamylamine hydrochloride (1.0 or 10.0 μg/0.5 μl) were purchased from Sigma/RBI (St. Louis, MO) and dissolved in sterile 0.9% saline. CNQX disodium salt (0.03 or 3.0 μg/0.5 μl) was purchased from Tocris (Ellisville, MS) and dissolved in sterile 0.9% saline.