A total of 17 experimentally-naive adult male rhesus monkeys (Macaca mulatta) weighing between 7.7 and 13 kg (mean ± SD, 10.2 ± 1.32) at the start of the study served as subjects. All procedures were performed in accordance with established practices as described in the National Institutes of Health Guide for Care and Use of Laboratory Animals. In addition, all procedures were reviewed and approved by the Animal Care and Use Committee of Wake Forest University. Monkeys were individually housed in stainless steel cages with water ad libitum; animals had physical and visual contact with each other. Their body weights were maintained at approximately 90–95% of free-feeding weights by banana-flavored pellets earned during the experimental sessions and by supplemental feeding of Lab Diet Monkey Chow, provided no sooner than 30 min post-session. In addition, they were given fresh fruit or peanuts at least three times per week. Each monkey was weighed once a week, and if necessary, their diets were adjusted to maintain stable weights.
Experimental sessions were conducted in ventilated and sound-attenuated operant chambers (1.5 × 0.74 × 0.76 m; Med Associates Inc., East Fairfield, VT) designed to accommodate a primate chair (Model R001; Primate Products, Redwood City, CA). The chamber contained an intelligence panel (48 × 69 cm), which consisted of two retractable levers (5 cm wide) and three stimulus lights. The levers were positioned within easy reach of the monkey sitting in the primate chair. Banana-flavored food pellets (1 g; Bio-Serv, Frenchtown, NJ) were delivered from a feeder located on top of the chamber. A peristaltic infusion pump (7531-10; Cole-Parmer Co., Chicago, IL) was used to deliver drug injections at a rate of approximately 1 ml per 10 s to those animals self-administering cocaine. Operation of the chambers and data acquisition was accomplished with a Power Macintosh computer system with an interface (Med Associates Inc.).
All monkeys, including controls, were surgically prepared, under sterile conditions, with indwelling intravenous catheters and vascular access ports (Model GPV; Access Technologies, Skokie, IL). Monkeys were anesthetized with a combination of ketamine (15 mg/kg, i.m.) and butorphanol (0.03 mg/kg, i.m.) and an incision was made near the femoral vein. After blunt dissection and isolation of the vein, the proximal end of the catheter was inserted into the vein for a distance calculated to terminate in the inferior vena cava. The distal end of the catheter was threaded subcutaneously to an incision made slightly off the midline of the back. The vascular access port was placed within a pocket formed by blunt dissection near this incision. Monkeys were given 24–48 h recovery time before returning to food-reinforced responding. Approximately 5 days before the terminal procedure, each monkey was implanted with a chronic indwelling catheter into the adjacent femoral artery for collection of timed arterial blood samples. The surgical procedures were identical to those described for the venous catheters. On the day of the final session, a terminal cerebral glucose metabolism study was conducted in which monkeys were injected with 2-[14
C]deoxyglucose (2-DG) approximately 2 min after the end of the session and blood samples were obtained through the arterial catheter over a 45 min period (see Beveridge et al, 2006
for details). Metabolism data from these studies are not presented here.
Monkeys were initially trained to respond on one of two levers by reinforcing each response on the correct lever with a food pellet. Over approximately a 3-week period the interval between availability of food pellets was gradually increased until a 3-min interval was achieved (ie fixed-interval 3-min schedule of reinforcement; FI 3-min). Under the final schedule conditions, the first response on the lever after 3 min resulted in the delivery of a food pellet; sessions ended after 30 food presentations. At the end of each session, the response levers were retracted, houselights and stimulus lights were extinguished, and animals remained in the darkened chamber for approximately 30 min before they were returned to their home cages. All monkeys responded under the FI 3-min schedule of food presentation for at least 20 sessions and until stable performance was obtained (± 20% of the mean for three consecutive sessions, with no trends in response rates). When food-maintained responding was stable, the feeder was unplugged and the effects of extinction on responding were examined for five consecutive sessions, after which responding was reestablished and maintained by food presentation.
After baseline performance had been established, all monkeys were surgically prepared with venous catheters, as described above, and randomly assigned to one of three groups. One group of monkeys served as controls and continued to respond under the FI 3-min schedule of food presentation for a total of 100 sessions (N = 6). The remaining 11 monkeys were assigned to the cocaine self-administration groups (0.3 mg/kg per injection). Because 0.3 mg/kg cocaine per injection was considered a high dose for previously cocaine-naive monkeys, for most animals this dose was achieved within two sessions by first allowing the monkey to self-administer 0.1 mg/kg cocaine. Food-maintained performance was allowed to stabilize after surgery (approximately 4–6 days) before cocaine self-administration sessions were begun. Before each experimental session, the back of the animal was cleaned with 95% ethanol and betadine scrub and a 22 gauge Huber Point Needle (Model PG20-125) was inserted into the port leading to the venous catheter, connecting an infusion pump containing the cocaine solution to the catheter. Before the start of the session, the pump was operated for approximately 3 s, filling the port with the dose of cocaine that was available during the experimental session. Sessions ended after 30 injections; as under control conditions, monkeys remained in the darkened chamber for approximately 30 min. At the end of each session, the port was filled with heparinized saline (100 U/ml) to help prevent clotting.
Experimental sessions were conducted at approximately the same time each day and continued for a total of 100 sessions. Following the completion of the 100 sessions, an abstinence period of 30 or 90 days was introduced during which time catheters were flushed daily with heparinized saline, but no cocaine or food self-administration sessions were conducted. For the control group, abstinence periods of 30 days were imposed in four animals and 90 days in the two others. For the cocaine group, abstinence periods of 30 days were imposed on eight animals and 90 days on three animals. At the end of the period of abstinence one final self-administration session (food control or cocaine) was conducted and the 2-DG procedure was initiated immediately following the session. In two controls and four cocaine self-administration animals in the 30-day abstinence group, no cocaine was received at the final session. Animals were humanely killed with an overdose of pentobarbital (100 mg/kg, i.v.) at the end of the 45 min tracer uptake period.
After killing, brains were immediately removed, blocked, and frozen in isopentane at –35 to –55°C and then stored at –80°C. The tissue blocks containing the striatum were then cut in a cryostat at –20°C in the coronal plane into 20 μm sections, collected onto electrostatically charged slides, desiccated under a vacuum overnight at 4°C, then stored at –80°C until processed for autoradiography. Brain sections were collected from the portions of the caudate nucleus, putamen, and nucleus accumbens that lie rostral to the anterior commissure. This region is referred to as the precommissural striatum. Further, rostral and caudal levels of the precommissural striatum were designated with reference to the nucleus accumbens. The rostral precommissural striatum is the region where the nucleus accumbens is not differentiated into distinct shell and core subcompartments. The caudal precommissural striatum is the region congruent with the appearance of the shell and core of the nucleus accumbens, which is posterior to the emergence of the olfactory tubercle. For each of the binding studies, two adjacent sections were taken at each of five levels (two rostral and three caudal) through the precommissural striatum for a total of 10 sections per animal.
D1 Receptor Binding
DA D1 receptor binding site densities were determined with [3
H]SCH 23390 (specific activity—85 Ci/mmol; PerkinElmer, Boston, MA) by quantitative in vitro
receptor autoradiography according to procedures adapted from Lidow et al (1991)
and Nader et al (2002)
. Sections were preincubated for 20 min in buffer (50 mM Tris, 120 mM NaCl, 5 mM KCl, 2 mM CaCl2
, 1 mM MgCl2
, pH 7.4, 25°C) to remove endogenous DA, cocaine and [14
C] from the 2-DG procedure. Sections were then incubated for 30 min in the same buffer, pH 7.4, 25°C, containing 1 mM ascorbic acid, 40 nM ketanserin, and 1 nM [3
H]SCH 23390. After incubation, sections were rinsed twice for 20 s in buffer containing 1 mM ascorbic acid at pH 7.4, 4°C, then dipped in distilled water at 4°C, and dried under a stream of cool air. Nonspecific binding was defined by incubation of adjacent sections in the incubation solution in the presence of 5 mM (+)-butaclamol. Sections, along with calibrated [3
H] autoradiographic standards (Amersham, Piscataway, NJ), were apposed to Kodak Biomax MR film (Fisher Scientific, Pittsburgh, PA) for 6 weeks.
D2 Receptor Binding
The density and distribution of DA D2 receptor binding sites were determined with [3
H]raclopride (specific activity, 87 Ci/mmol; PerkinElmer) according to procedures adapted from Lidow et al (1991)
and Nader et al (2002)
. Sections were preincubated for 20 min in buffer (50 mM Tris, 120 mM NaCl, 5 mM KCl, pH 7.4, 25°C) to remove endogenous DA, cocaine, and [14
C] from the 2-DG procedure. Slides were then incubated for 30 min in the same buffer, containing 5 mM ascorbic acid and 2 nM [3
H]raclopride. Sections were rinsed 3 × 2 min in buffer at pH 7.4, 4°C, then dipped in distilled water at 4°C, and dried under a stream of cool air. Nonspecific binding was defined by incubation of adjacent sections in the incubation solution in the presence of 1 μ
M (+)–butaclamol. Sections, along with calibrated [3
H] autoradiographic standards, were apposed to Kodak Biomax MR film for 8 weeks.
Dopamine Transporter Binding
The density of DAT binding sites was determined using [3
H]WIN 35,428 (specific activity, 87 Ci/mmol; PerkinElmer) autoradiography according to procedures adapted from Canfield et al (1990)
and Letchworth et al (2001)
. Tissue sections were preincubated in buffer (50 mM Tris, 100 mM NaCl, pH 7.4, 4°C) for 20 min to remove any residual DA, cocaine, and [14
C] from the 2-DG procedure. Sections were then incubated for 2 h at 4°C in the same buffer containing 5 nM [3
H]WIN 35 428. Sections were rinsed for a total of 2 min in buffer at 4°C, then dipped in distilled water at 4°C, and dried under a stream of cold air. Nonspecific binding was defined by incubation of adjacent sections in the incubation solution in the presence of 30 μ
M cocaine. Sections, along with calibrated [3
H] autoradiographic standards, were apposed to Kodak Biomax MR film for 6 weeks.
Densitometry and Data Analysis
Films were developed with Kodak GBX developer, stopbath and Rapid Fixer (VWR, West Chester, PA), and then rinsed. Analysis of autoradiograms was conducted by quantitative densitometry with a computerized image processing system (MCID, Imaging Research; InterFocus Imaging Ltd, Cambridge, UK). Optical density values were converted to fmol/mg (of wet-weight tissue) by reference to the calibrated [3
H] standards. Specific binding was determined by digitally subtracting images of nonspecific binding from superimposed adjacent images of total binding. Structures were identified by Nissl staining of sections adjacent to those analyzed for receptor binding. Data from each assay were analyzed independently by means of a one-way analysis of variance followed by least-square differences post hoc
tests for multiple comparisons. Each region consisted a separate analysis. Because binding data obtained from the control animals abstinent for 30 and 90 days were not significantly different from one another, similar to previous studies (Nader et al, 2002
), data from the control groups were combined. In addition, there were no significant differences between the data from those animals who received cocaine and those that did not at their final session, so data from these groups were also combined.