2.1 Animals and housing
Male Sprague-Dawley rats weighing 250-300 g were obtained from Taconic Laboratories (Germantown, NY). Animals were individually housed, with sucrose and water available ad libitum in their home cage (rats undergoing sucrose reinstatement were placed on restricted diets, as outlined below). A 12/12 hr light/dark cycle was used throughout these experiments with the lights on at 7:00 a.m. All experimental procedures were performed during the light cycle. The experimental protocols were all consistent with the guidelines issued by the U.S. National Institutes of Health and were approved by the Boston University School of Medicine and the University of Pennsylvania School of Medicine Institutional Animal Care and Use Committee Institutional Animal Care and Use Committees.
All experiments used Med-Associates (Georgia, VT) modular testing instrumentation enclosed within ventilated, sound-attenuating chambers (22”W × 22”H × 16”D). Each operant box had the following interior dimensions: 12.0”L × 9.5”W × 8.25”H. The operant boxes were equipped with response levers, sucrose pellet dispensers, and injection pumps for infusing drugs intravenously. Each operant chamber had two response levers, only one of which was active or paired with delivery of a reinforcer.
Cocaine was obtained from the National Institute on Drug Abuse (Rockville, MD) and dissolved in bacteriostatic 0.9% saline. Scopolamine hydrobromide (Sigma-Aldrich, St. Louis, MO) was dissolved in sterile 0.9% saline. Doses of scopolamine were selected based on systemic doses known to activate striatal dopamine levels (Chapman et al., 1997
; Forster and Blaha, 2000
) and intracranial doses used to assess cocaine reinforcement (Ikemoto and Goeders, 2000
) or food intake (Pratt and Kelley, 2005
Prior to surgery, the rats were anesthetized with ketamine (80 mg/kg, i.p.; Sigma-Aldrich, St. Louis, MO) and xylazine (12 mg/kg, i.p.; Sigma-Aldrich, St. Louis, MO). An indwelling silastic catheter (inner diameter 0.33 mm, outer diameter 0.64 mm) was inserted into the right jugular vein and sutured in place. The catheter was then threaded subcutaneously over the shoulder blade, and was routed to a mesh backmount platform (CamCath, Cambridge, UK) that secured the placement. Catheters were flushed daily with 0.3 ml of the antibiotic Timentin (ticarcillin disodium/potassium clavulanate, 0.93 mg/ml; Henry Schein, Melville, NY) dissolved in heparinized saline. The catheters were sealed with plastic obturators when not in use.
Following catheter insertion, animals were mounted in a stereotaxic apparatus and four
stainless steel guide cannulae (14 mm, 22 gauge) for microinjections were implanted bilaterally 2 mm dorsal to the nucleus accumbens core and shell in each animal
. Cannulae were cemented in place by affixing dental acrylic to stainless steel screws secured in the skull. The coordinates for the ventral ends of the guide cannulae, relative to bregma according to the atlas of Paxinos and Watson (1997)
were as follows: nucleus accumbens shell: +1.0 mm A/P, ±1.0 mm M/L, −5.0 mm D/V; nucleus accumbens core: +1.0 mm A/P, ±2.5 mm M/L, −5.0 mm D/V. An obturator (14 mm, 33 gauge stainless steal wire) was inserted into each guide cannula in order to prevent occlusion.
2.5 Cocaine self-administration and extinction training
After surgery, rats were allowed seven days to recover before behavioral testing commenced. Initially, rats were placed in operant chambers and allowed to lever press for intravenous infusions of 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 (i.e. little or none of this non-contingent injection reached the systemic circulation). Rats were allowed to self-administer a maximum of 30 injections per 120-minute operant session under the FR1 schedule. Once an animal achieved at least 20 infusions of cocaine in a single daily session under the FR1 schedule, the subject was 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.
Daily operant sessions (5 days/week) under the FR1 and FR5 schedules of reinforcement were continued for a total of 21 days. Following cocaine self-administration, drug-seeking behavior was extinguished by replacing the cocaine with 0.9% saline. The extinction phase 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 8 days for rats to meet this criterion.
2.6 Cocaine Priming-Induced Reinstatement
Following extinction, animals entered the reinstatement phase of the experiment. During reinstatement, satisfaction of the response requirements for each ratio resulted in saline rather than cocaine infusion. Each reinstatement session was followed by extinction sessions until responding was again less than 15% of the response rate maintained by cocaine self-administration. Generally, only a single day of extinction was necessary in order to reach extinction criterion between reinstatement test sessions. The FR5 schedule was used throughout the extinction and reinstatement phases of these experiments.
2.7 Microinjection procedures
Prior to a reinstatement test session, obturators were removed from the guide cannulae and 33 gauge stainless steel microinjectors (Small Parts Inc.) were inserted. These microinjectors were cut to a length that extended 2 mm below the ventral end of the guide cannulae and into the nucleus accumbens core and shell. Bilateral infusions were performed simultaneously over a 120 second time period in a total volume of 0.5 μl per hemisphere. Following microinfusion of scopolamine or vehicle, the microinjectors were left in place for 60 seconds in order to allow the solution to diffuse away from the tips of the microinjectors before they were removed. A systemic priming injection of cocaine (10 mg/kg, i.p.) was administered 10 min following microinjections and animals were placed in the operant chambers and the reinstatement session began immediately. The goal of the experimental design was to have each animal served as its own control and received up to three microinjections per brain region (i.e. two doses of scopolamine plus vehicle for a maximum of three microinjections per brain region). However, we were frequently forced to deviate from this experimental design when technical difficulties (i.e. blocked microinjection cannula or loss of catheter patency) made it impossible to test all doses of the muscarinic acetylcholine receptor antagonist plus vehicle in an entire cohort of subjects. Therefore, a mixed-factors design was used in these behavioral experiments. In every case, however, an animal received at a minimum, treatment of one drug dose and its vehicle. In order to control for potential order effects of drug and vehicle administrations, all drug and vehicle treatments were counterbalanced across reinstatement sessions.
Using this experimental design, subjects underwent a series of alternating extinction and reinstatement sessions that lasted approximately 16 days. During this period, extinction of the ability of priming injections of cocaine to induced reinstatement is a concern. However, we have previously shown that reinstatement of cocaine seeking persists for at least 20 days after the initial extinction of cocaine self-administration behavior (Anderson et al., 2008
; Famous et al., 2008
). Moreover, since the scopolamine treatments were counterbalanced across reinstatement days, we were able to assess the magnitude of reinstatement across sessions in the current experiments. All subjects demonstrated stable drug seeking throughout the reinstatement phase of these experiments.
2.8 Sucrose self-administration and reinstatement
Potential nonspecific rate-suppressing effects of systemic or intra-accumbens infusions of the muscarinic acetylcholine receptor antagonist scopolamine were evaluated by assessing the influence of scopolamine on the reinstatement of sucrose-reinforced responding. Rats were trained initially to press a lever under an FR1 schedule of reinforcement for sucrose pellet (Research Diets, Inc, New Brunswick, NJ) delivery in daily 1-hour sessions. The animals were restricted to 4 pieces of lab chow (Harlan Teklad, Wilmington, DE) per day for the duration of these experiments. Each piece of lab chow weighed on average 5.41±0.16 g. Once animals had acquired self-administration on the FR1 schedule (less than 15% variation in responding on 2 consecutive days), the rats were switched to an FR5 schedule of reinforcement. Sucrose reinforced subjects spent an average of 5.31±0.30 days training on the FR1 schedule before transitioning to an FR5 schedule of reinforcement. Animals were limited to 30 sucrose pellets within a 1-hour session.
After 3 weeks of sucrose-maintained responding on the FR5 schedule (21 total days of responding under FR1 and FR5 schedules of reinforcement), rats underwent an extinction phase wherein the sucrose pellets were removed from the dispenser, and responding no longer resulted in sucrose delivery. After lever pressing decreased to 10% or less of the responding maintained by contingent sucrose reinforcement, animals began reinstatement testing the following day. Each reinstatement session began with the issue of a non-contingent sucrose pellet prime. The experimenter remotely administered one sucrose pellet every 2 min thereafter for the first 10 min of the reinstatement session. Similar to the extinction phase of the experiment, lever responses were recorded but did not results in delivery of sucrose pellets. Each sucrose reinstatement session was followed by extinction sessions until responding was again less than 10% of the response rate maintained by sucrose.
2.9 Behavioral Experiment 1
The effect of systemic administration of the muscarinic acetylcholine receptor antagonist scopolamine (Sigma-Aldrich, St. Louis, MO) on cocaine priming-induced reinstatement of drug seeking was assessed in 11 subjects (4 out of an initial 15 subjects lost catheter patency during the experiment and were subsequently dropped from the study). Scopolamine (0.5 [n=10] or 5.0 [n=7] mg/kg, i.p.) or vehicle (n=11) was administered 10 min prior to a 10 mg/kg (i.p.) cocaine priming injection. In all of the following behavioral experiments, the animals were placed in the modular testing chambers immediately after the systemic priming injection of cocaine. Also, in all experiments, the administration of vehicle and all doses of scopolamine were counterbalanced across reinstatement sessions in order to avoid any potential rank-order effects of drug administration.
2.10 Behavioral Experiment 2
The effect of systemic administration of muscarinic acetylcholine receptor antagonist scopolamine on sucrose priming-induced reinstatement was assessed in 7 rats. Scopolamine (5.0 mg/kg, i.p.; n=5) or vehicle (n=7) was administered 10 min prior to a sucrose reinstatement session. Reinstatement sessions began with delivery of a non-contingent sucrose pellet prime. Subsequent priming pellets were delivered remotely every 2 min thereafter for the first 10 min of the reinstatement session. Scopolamine data points were excluded from two subjects because one did not reinstate to sucrose pellets in a subsequent test of sucrose reinstatement and another subject was lethargic on the scopolamine test day. Therefore, due to behavioral and health confounds, scopolamine-induced attenuation data were excluded from this experiment for these two animals.
2.11 Behavioral Experiment 3
A total of 26 rats were used for this experiment However, catheter patency was lost in 3 subjects who were ultimately dropped from this study. The effect of intracranial administration of scopolamine on systemic priming injections of cocaine was assessed in 22 animals (data from 2 subjects were excluded due to misplaced guide cannulae in the accumbens shell or core and 3 animals were excluded due to significant tissue damage around the injection sites). Scopolamine (3.6 [n=6] and 36.0 [n=6] μg/0.5 μl) or vehicle (n=9) was injected into either the nucleus accumbens core or shell 10 min prior to a 10 mg/kg (i.p.) priming injection of cocaine. Animals were placed immediately into the operant chambers following a priming injection of cocaine and the reinstatement session ensued.
2.12 Behavioral Experiment 4
The effect of intracranial administration of scopolamine on sucrose reinstatement was assessed in 18 subjects (data from 2 subjects were excluded due to misplaced guide cannulae in either the accumbens shell or core). Animals received 3.6 or 36.0 μg/0.5 μl scopolamine or vehicle injections into either the nucleus accumbens core or shell. Ten minutes following drug treatment, the animals were placed in the operant chambers and the session began with the issue of a non-contingent sucrose pellet prime. Over the first 10 min of the reinstatement session, the experimenter remotely administered a non-contingent sucrose pellet every two minutes. Each reinstatement session was followed by extinction sessions until responding was again less than 10% of the response rate maintained by sucrose self-administration.
2.13 Verification of cannulae placements
Following the completion of all microinjection experiments, the animals were given an overdose of pentobarbital (100 mg/kg, i.p.) and perfused intracardially with 0.9% saline followed by 10% formalin. The brain was removed and coronal sections (100 μm) were taken at the level of the nucleus accumbens with a Vibratome (Technical Products International; St. Louis, MO). The sections were mounted on gelatin-coated slides and stained with Cresyl violet. Using a light microscope, an individual blinded to the animals’ behavioral response identified microinjection sites as well as potential drug- or cannula-induced neuronal damage. Cell death and associated gliosis also were assessed in these sections. Animals with cannula placements outside of the accumbens core or shell subregions, or with excessive mechanical damage, were excluded from subsequent data analyses.
2.14 Behavioral Experiment Data Analyses
In the reinstatement experiments, a mixed-factors design was used in which we aimed to administer each dose of a drug plus the drug vehicle to all subjects in a cohort during the reinstatement phase. However, technical difficulties (loss of catheter patency and clogging of guide cannulae, etc.) regularly resulted in missing cells. We therefore used a statistical model (a mixed-model multivariate analysis of variance or MANOVA) that accommodates missing cells in the within-subjects aspect of an experimental design without assigning place-keeper values (such as the treatment mean) using the residual maximum likelihood approach (Berk, 1987
). The systemic and intra-accumbens scopolamine cocaine priming-induced reinstatement data (total lever responses per session) were analyzed with a two-way mixed-model MANOVA. The two factors were lever (active-inactive) and drug treatment. The systemic scopolamine food reinstatement control experiment was analyzed with an unpaired t-test. The intra-accumbens scopolamine food reinstatement control data were analyzed with a one-way mixed-model MANOVA. Time course data were analyzed by mixed-factor MANOVAs with repeated measures over time. Pairwise comparisons for all significant effects as revealed by MANOVAs were made using Fisher’s LSD post hoc