Glutamate Release in the Nucleus Accumbens Core Is Necessary for Heroin Seeking

Subjects.

Male Sprague Dawley rats (∼300 g at the time of surgery; n = 81; Charles River, Wilmington, MA) were used in this study. They were individually housed, maintained in a temperature-controlled environment (22°C) on a 12 h light/dark cycle (lights on at 7:00 A.M.) with food and water ad libitum, and given 6–7 d to acclimatize to the vivarium before undergoing surgery. Behavioral procedures began 5–6 d after surgery. All methods used were in compliance with National Institutes of Health guidelines for care of laboratory animals and were approved by the Medical University of South Carolina Institutional Animal Care and Use Committee.

Surgery.

The rats were anesthetized with ketamine HCl (87.5 mg/kg, i.m.) and xylazine (5 mg/kg, i.m.). Ketorolac (3 mg/kg, i.p.) was administered before surgery to provide analgesia. For catheter implantation, a guide cannula (22 ga; C313G; Plastics One, Wallingford, CT) was bent at a 90° angle and attached to SILASTIC tubing (0.025 inner diameter, 0.047 outer diameter; Bio-Sil; Bio-Rad, Hercules, CA) via superglue. It was then attached to Prolite mesh via dental cement. The catheter was implanted subcutaneously between the shoulder blades and exited the skin via a dermal biopsy hole (3 mm). The other end of the tube was threaded under the skin above the shoulders and into the jugular vein.

The rats were then placed in a small animal stereotaxic instrument (Kopf Instruments, Tujunga, CA). Three surgical screws were implanted into the skull as anchors, and depending on the experiment, guide cannulas were implanted and secured with dental cement. The nose bar was maintained at −3.5 mm relative to the interaural line. For the NAcore dialysis experiments, cannulas (20 ga; Plastics One) were implanted bilaterally aimed at the NAcore [anteroposterior (AP), +1.7 mm; mediolateral (ML), ±2.5 mm at 6° angle; dorsoventral (DV), −5.5 mm; all coordinates from Paxinos and Watson (2005)]. For the experiments with NAcore dialysis and prelimbic cortex microinjections, the AP coordinates for the cannulas aimed at the core were adjusted [AP, +1.3 mm (Paxinos and Watson, 2005)] to allow room for the prelimbic cannulas. A single double-barreled cannula (26 ga; Plastics One) was implanted, aimed at the prelimbic bilaterally [AP, +3.2 mm; ML, ±0.6 mm for each cannula; DV, −2.8 mm (Paxinos and Watson, 2005)]. For microinjection experiments, the guide cannulas were constructed of 23 ga stainless steel tubing cut to a length of 15.00 ± 0.02 mm and were implanted bilaterally aimed at the NAcore [AP, +1.7 mm; ML, ±1.6 mm; DV, −5.6 mm (Paxinos and Watson, 2005)]. After the surgery, the rats were retained in an incubation chamber until they recovered from the anesthesia. Obdurators were placed in all cannulas and maintained through the reinstatement testing. The rats were then returned to their home cages and checked on the days after surgery to ensure that their wounds healed. Rats were flushed with heparinized cefazolin via the catheter for 7 d after surgery and then heparin alone throughout the remaining self-administration.

Self-administration, extinction, and reinstatement procedures.

All self-administration experiments occurred in standard operant chambers with two retractable levers, a house light, and a cue light and tone generator (Med Associates, Fairfield, VT). Before drug self-administration, all rats were food deprived for 24 h and then underwent a single 15 h food-training session in which the rats were trained to press the active lever (the right lever) for a single food pellet (45 mg; Noyes, Lancaster, NH) on a fixed-ratio 1 (FR1) schedule. After that training, the rats were given a limited quantity of food (∼20 g) immediately after every self-administration session. One day after the food training, the rats began heroin self-administration, and each session lasted 3 h or until the rats had taken a maximum of 200 infusions. The self-administration program was an FR1 schedule with a 20 s timeout to prevent overdose, although the animal could still press the active lever during the timeout. All active lever presses, including those during the timeout, were recorded and are reported as “active lever presses.” Each active lever press, except for those during the timeout, produced a 0.05 ml infusion of either 50 μg (days 1 and 2) or 22.5 μg (days 3–12) of heroin (dissolved in 0.9% sterile saline; heroin kindly provided by National Institute on Drug Abuse). Concurrent with the drug infusion, a cue tone (2900 Hz) and cue light immediately above the active lever turned on for 5 s. Any rats that did not have 10+ infusions per day for the last 3 d of self-administration were excluded from analysis.

After 12 d of self-administration, rats began extinction training. Active lever presses produced no drug infusion or light/tone cues. For microinjection experiments, rats underwent a minimum of 12 d of extinction and were required to meet a criterion of an average of <30 lever presses for 2 consecutive days before undergoing reinstatement testing. For microdialysis experiments, rats underwent 12–13 d of extinction before beginning the dialysis reinstatement. For heroin prime-induced reinstatement, rats received a single injection of heroin (0.25 mg/kg, s.c.) before beginning the 3 h session, and the session program was identical to the extinction program. For cue-induced reinstatement, no injection was given, but the session program was identical to the self-administration session program (i.e., cue tone and light turned on with an active lever press). Rats did not receive intravenous drug infusions during either reinstatement.

During self-administration, some rats were yoked to heroin self-administering rats and received noncontingent infusions of heroin (yoked-heroin) or saline (yoked-saline) in the same temporal pattern as their heroin self-administering partners. Yoked rats received identical food training and the same number of self-administration and extinction sessions as their partners.

Microinjection procedures.

For the NAcore microinjections, injector needles were constructed of 30 ga dental needles connected to PE20 tubing, which was attached to 10 μl Hamilton syringes controlled by an infusion pump. The dental needles were bent to 17 mm so that they extended 2 mm beyond the end of the cannula and into the NAcore. All microinjections (0.3 μl) into the NAcore occurred over 60 s, and the injector needles were left in place for an additional 60 s to allow for diffusion from the site of the injection. After the microinjection, rats then underwent their appropriate reinstatement testing. For the drug prime reinstatement, the following drugs were used in the microinjections: CNQX (1 nmol per side), fluphenazine (Flu; 10 nmol per side), SCH 23390 (0.6 and 6 nmol per side), and sulpiride (0.3 and 3 nmol per side). For the cue-induced reinstatement, CNQX and Flu were used. Within each experiment, each rat received every dose of the drug or vehicle in a counterbalanced manner, with a minimum of 3 d of extinction and the same extinction criteria between each reinstatement trial. All drugs were dissolved in PBS, except SCH 23390, which was dissolved in sterile saline. Doses of all four drugs were chosen based on previous work indicating a lack of effect of the high dose of the drug on locomotor activity and/or reinstatement lever pressing (Neisewander et al., 1995; Cornish and Kalivas, 2000; McFarland and Kalivas, 2001; Anderson et al., 2003, 2006).

Microdialysis procedures.

Hollow fiber microdialysis tubing (molecular weight cutoff, 13000) was inserted and glued into a 24 ga internal cannula (Plastics One). The end of the tubing was sealed with epoxy glue and cut so that 2 mm of active membrane extended beyond the end of the cannula. The probes were then constructed with inlet and outlet tubing made of fused silica inserted and glued into the other end of the cannula. On the night before reinstatement testing, the probes were inserted into the NAcore, with the internal cannula extending 1 mm beyond the end of the guide cannula. The rats were left in the operant chambers overnight, with dialysis buffer (5 mm glucose, 2.5 mm KCl, 140 mm NaCl, 1.4 mm CaCl₂, 1.2 mm MgCl₂, and 0.15% PBS, pH 7.4) perfusing through the probe (0.1 μl/min). The levers were retracted throughout the night and were not extended until the reinstatement session began. The following morning, the flow rate was increased to 2 μl/min. After 2 h, baseline sample collection began at 20 min intervals. All samples were collected in 10 μl of 0.05N HCl. After 2 h of baseline collection, rats underwent reinstatement testing. For heroin-induced reinstatement, all rats were given a single systemic injection of heroin (0.25 mg/kg, s.c.). For the cue-primed reinstatement, no injection was made. Immediately before the heroin injection, some rats also received a microinjection (0.3 μl) of either baclofen–muscimol (B/M; 1 and 0.1 mm, respectively) or aCSF into the prelimbic cortex. This dose combination has been shown previously to be effective at inactivating the prelimbic cortex without affecting locomotor activity (McFarland and Kalivas, 2001). For the microinjections, double-barreled injectors (33 ga; Plastics One) were inserted into the guide cannula, extending 1 mm beyond the end of the guide cannula and into the prelimbic cortex. Injections were made over 60 s, and injector needles were left in place for an additional 60 s to allow for diffusion. Rats then received their heroin-prime injection and were returned to the operant chamber. The reinstatement session and sample collection lasted for 3 h. For some rats that had undergone heroin self-administration, during dialysis, tetrodotoxin (TTX; 1 μm) was continuously reverse dialyzed into the NAcore beginning 1 h before the heroin prime to determine whether the glutamate release was activity dependent. Samples were stored at −80°C. Three rats underwent dialysis during cue-primed reinstatement followed by dialysis (on the other side) during heroin-primed reinstatement, with 4 d of extinction between the two reinstatement sessions. Otherwise, no rats received both a cue-prime and heroin-prime reinstatement.

Quantification of dopamine and glutamate.

Before analysis, samples were thawed and divided so that they could be used to measure levels of glutamate and dopamine. Of the 50 μl in each sample, 24 μl were used for quantification of dopamine, and 20 μl were used for quantification of glutamate. (The n is lower for dopamine measurements because some samples had to be run twice for glutamate analysis because of problems encountered during the first run; e.g., see Fig. 1, compare A, B). Dopamine was quantified using an ESA (Chelmsford, MA) model 540 autosampler connected to an HPLC system with electrochemical detection (mobile phase: 150 NaH₂PO₄·H₂O, 4.76 mm citric acid, 50 μm EDTA, 2.5 mm SDS, 10% methanol, and 17% acetonitrile, pH 5.6). Separation was achieved by pumping the samples through HR-80 reversed-phase column (3 μm × 80 mm × 3.2 mm; ESA), and then samples were reduced-oxidized using coulometric detection. Three electrodes were used: a guard cell (+400 mV), a reduction analytical electrode (−100 mV), and an oxidation analytical electrode (+220 mV). Peaks were recorded, and the area under the curve was measured by a computer running ESA501 Chromatography Data System. These values were normalized by comparison with an external standard curve for dopamine.

Glutamate was measured using an autosampler (Gilson Medical Electric, Middleton, WI) that performed a precolumn derivatization with ο-phthalaldehyde. The concentration of glutamate in samples was determined using HPLC with fluorometric detection (mobile phase: 0.1 m Na₂HPO₄, 0.09 mm EDTA-Na₄, and 11% acetonitrile, pH 6.04). Amino acids were then separated with a Velosep RP-18 column (3 μm × 100 mm × 3.2 mm; Perkin-Elmer). Glutamate was detected using a fluorescence spectrophotometer (RF-10A Spectrofluorometric Detector; Shimadzu, Tokyo, Japan) using an excitation wavelength of 340 nm and an emission wavelength of 450 nm. A chart recorder recorded peaks, and peak heights were measured. The values were normalized by comparison with an external standard curve.

Histology and statistics.

Rats were overdosed with sodium pentobarbital (100 mg/kg, i.p.) and intracardially perfused with 0.9% saline. The brains were removed and stored in 10% formalin for at least 24 h. Coronal sections (75 μm thick) were made using a vibratome. Sections were mounted on gel-coated slides and then stained with cresyl violet. Sites of dialysis probes or injector needles were verified with a light microscope.

Dialysis data were analyzed using one-way or two-way repeated-measures ANOVA. Post hoc least significant difference tests were used to determine the source of significant differences between the yoked-saline, yoked-heroin, TTX, and heroin self-administration groups (p < 0.05). Lever presses during microinjection reinstatement tests were compared using Bonferroni's multiple-comparison post hoc tests, whereas those during dialysis-reinstatement tests were compared with extinction baseline using a nonparametric Mann–Whitney U test, because of the nonparametric distribution of the reinstatement data acquired during dialysis (p < 0.05).