Neuropsychopharmacology (2003) 28, 1125–1139, advance online publication, 5 March 2003; doi:10.1038/sj.npp.1300105

Effects of Mixed-Action / Opioids on Cocaine Self-Administration and Cocaine Discrimination by Rhesus Monkeys

Carrie A Bowen1,³, S Stevens Negus1, Rushi Zong¹, John L Neumeyer¹, Jean M Bidlack² and Nancy K Mello¹

1. ¹Alcohol and Drug Abuse Research Center, McLean Hospital-Harvard Medical School, Belmont, MA, USA
2. ²Department of Pharmacology and Physiology, University of Rochester, Rochester, NY, USA
Correspondence: Dr NK Mello, Alcohol and Drug Abuse Research Center, McLean Hospital, Harvard Medical School, 115 Mill Street, Belmont, MA 02478, USA. Tel: +1 617 855 2478; Fax: +1 617 855 2519; E-mail: [email protected]
3. ³Current address: Scion Pharmaceuticals, Inc., 200 Boston Avenue, Suite 3600, Medford, MA 02155, USA

Received 17 July 2002; Revised 8 October 2002; Accepted 15 October 2002; Published online 5 March 2003.

Subjects
Three experiments were conducted in 14 adult male rhesus monkeys (Macaca mulatta). Cocaine self-administration studies were conducted in eight monkeys. Four animals were trained to self-administer cocaine using a Rapid Assessment Procedure (Caine et al, 2000) and the acute effects of agonist treatments on cocaine- and food-maintained responding were evaluated. Four additional animals were trained to self-administer cocaine under a second-order schedule using the same procedure previously used to evaluate chronic agonist effects on cocaine self-administration (Mello and Negus, 1998; Negus et al, 1997). Drug discrimination studies were conducted in a separate group of six monkeys. Two of the monkeys trained to self-administer cocaine using the Rapid Assessment Procedure were experimentally naive at the beginning of these experiments. All of the other monkeys had an experimental history involving the evaluation of dopaminergic and/or opioid compounds in assays of cocaine self-administration or cocaine discrimination. Monkeys weighed 7.0–12.0 kg and were maintained on a diet of multiple vitamins, fresh fruit, and Lab Diet Jumbo Monkey biscuits (PMI Feeds, Inc., St Louis, MO). In addition, monkeys could receive 1-g banana-flavored pellets (Precision Primate Pellets Formula L/I Banana Flavor, PJ Noyes Co., Lancaster, NH) during daily operant sessions (see below). Water was available continuously. A 12-h light–dark cycle was in effect (lights on from 7 am to 7 pm).

Animal maintenance and research were conducted according to the guidelines provided by the National Institutes of Health Committee on Laboratory Animal Resources. The research facility was licensed by the United States Department of Agriculture. Research protocols were approved by the McLean Hospital Institutional Animal Care and Use Committe. A consulting veterinarian monitored the health of the monkeys on a regular basis. Monkeys had visual, auditory, and olfactory contact with other monkeys throughout the study. To provide environmental enrichment, each animal was supplied with toys, and music or a nature videotape was played daily. In addition, the operant procedures provided opportunities for environmental manipulation (Line et al, 1989).

Cocaine Self-Administration

Surgery and catheter maintenance.

For intravenous drug administration, a chronic double-lumen catheter was implanted into a jugular or femoral vein under aseptic conditions. The double-lumen catheter permitted simultaneous i.v. cocaine self-administration and i.v. test drug administration. Monkeys were sedated with ketamine (5–10 mg/kg, i.m.) and anesthesia was induced with a ketamine (50 mg/ml)/diazepam (2.5 mg/ml) mixture (0.1–0.15 ml/kg, i.v.) to allow for tracheal intubation. Atropine (0.05 mg/kg, i.m.) was administered to reduce salivation and buprenorphine (0.05 mg/kg, i.m.) was given for intraoperative analgesia. General anesthesia was maintained with isoflurane (2% mixed with oxygen). A 20 gauge i.v. catheter was inserted into a saphenous vein for intraoperative fluid administration (0.9% NaCl, 10 ml/kg/h). Following a skin incision, blunt dissection was performed to expose the vessel. A small incision was made in the vein using Vannas scissors. A silicone rubber double-lumen catheter (inside diameter 0.71 mm, outside diameter 2.2 mm) was inserted into the vein using a catheter introducer and secured in place with sutures. The catheter was passed subcutaneously to an exit site at the mid-scapular region. The muscle overlaying the vein was closed with sutures, and the skin incision was closed with Nexaband® adhesive.

Buprenorphine (0.05 mg/kg, i.m.) and Penicillin G procaine (20 000 IU/kg, i.m.) were administered at the end of the surgical procedure. Following surgery, buprenorphine (0.1 mg/kg, i.m.) and cefazolin (30 mg/kg, i.m.) were administered twice daily for 5 days.

Each monkey was fitted with a nylon vest (Lomir Biomedical, Montreal, Canada) attached to a flexible stainless-steel cable. The other end of the cable was attached to a fluid swivel. This tether system protected the i.v. catheter and allowed freedom of movement for the animal. Catheter patency was monitored periodically by i.v. administration of ketamine (5 mg/kg) or a short-acting barbiturate, methohexital (4 mg/kg). The catheter was considered patent if i.v. ketamine or methohexital administration resulted in loss of muscle tone within 10 s.

Behavioral Apparatus

Drug self-administration.

Drug self-administration sessions occurred in each monkey's home cage (65.7 99.9 5.9 cm3). The front wall of each cage was adapted for the attachment of an operant panel (28 28 cm2). The operant panel included three square translucent response keys (6.4 6.4 cm2) arranged 2.5 cm apart horizontally and 3.2 cm from the top of the panel. Each response key could be transilluminated by red, green, or yellow stimulus lights (Superbright LEDs). A food-pellet dispenser (Gerbrands, model G5210) was mounted above each cage to deliver 1 g banana-flavored food pellets to a receptacle (6.0 5.0 3.0 cm3) located below the operant panel. Up to three infusion pumps (Model B5P-1E, Braintree Scientific, Braintree, MA; or Model 980210, Harvard Apparatus, South Natick, MA) were mounted above each cage to deliver solutions from 60-ml plastic syringes via the i.v. catheter. One infusion pump delivered response-contingent cocaine injections through one lumen of the double-lumen catheter. The other infusion pumps delivered noncontingent saline or test drug solution through the other lumen of the catheter. The schedules of reinforcement were controlled and data were collected with an IBM-compatible computer and Med Associates, Inc. (Georgia, VT) interface and power supply located in a separate room. Room lights were extinguished during all food and drug self-administration sessions.

Study 1: Acute Effects of Agonists on Responding Maintained by Cocaine and Food

Rapid assessment procedure.

The acute effects of agonists on responding maintained by cocaine and food under a fixed-ratio (FR) 30 schedule of reinforcement were examined. A Rapid Assessment Procedure was used to study self-administration of multiple unit doses of cocaine within a single experimental session (Caine et al, 2000). Each 2-h session consisted of six components separated by 10-min intercomponent time-out (TO) periods. During the first and sixth components, food pellets were available for 5 min, and the center response key was transilluminated with a red stimulus light. During the second, third, fourth, and fifth components, i.v. injections of saline or cocaine were available for 15 min. Each drug component was initiated by the 10-s transillumination of the center response key with a yellow stimulus light and the noncontingent delivery of a single 'priming' injection of saline or the unit dose of cocaine that was available for self-administration. When cocaine or saline was available, the center response key was transilluminated with a green stimulus light. A 10-s TO followed the completion of the ratio requirement for each food pellet or injection and was signaled by the transillumination of the center response key with a yellow stimulus light. During the 10-min intercomponent TO periods, all stimulus lights were turned off, and responses had no scheduled consequences. Experimental sessions began at 3 pm and were conducted 7 days/week.
Varying the unit dose of cocaine within operant sessions.

The unit dose of cocaine available during each component of the self-administration session was determined by (1) the duration of each infusion, which determined the volume and (2) the concentration of drug in the syringe. During most sessions, the syringe pumps were programmed to deliver the following volumes during the four consecutive drug components: 32 l in 0.32 s, 100 l in 1 s, 320 l in 3.2 s, and 1000 l in 10 s. A different cocaine dose range could be studied by changing the cocaine concentration in the syringe. Complete cocaine dose–effect functions were determined across sessions by studying overlapping cocaine dose ranges. An ascending dose series was used to reduce the possibility that the dose of cocaine available in one component would influence cocaine-maintained behavior in subsequent drug components.

Rapid assessment procedure training.

Initially, food was available for all six components of the session, and monkeys were trained to respond for food pellets under the FR 30, TO 10-s schedule. After food reliably maintained responding, monkeys were trained to self-administer cocaine (0.032 mg/kg/inj). Then availability of the cocaine training dose and saline was alternated until reliable extinction was observed. Training was considered complete when the following criteria were met: (1) the peaks of the overlapping cocaine dose–effect curves were within one half-log unit of each other, (2) the overall mean cocaine dose–effect function was characterized by an inverted 'U' shape, (3) saline-maintained responding was reliably lower than peak cocaine-maintained responding and (4) food-maintained responding was stable (criterion of 20% deviation from the mean of five consecutive sessions).

Rapid assessment procedure testing.

To ensure that the effects of the -opioid agonists were sustained during the entire 2-h test session, all test compounds were delivered by continuous intravenous infusion through the second lumen of the double-lumen catheter. Each opioid was delivered at a rate of 3.3 ml/h by infusing a volume of 100 l approximately every 2 min for a total of 3 h. Test drug administration began 1 h before the test session to fill the 'dead space' in the catheter (approximately 1 ml).

Four -opioid agonists were evaluated.

Enadoline (0.00032–0.01 mg/kg/h), (-)cyclorphan (0.001–0.032 mg/kg/h), Mr2034 (0.001–0.01 mg/kg/h), and MCL-101 (0.001–0.032 mg/kg/h). Usually, test sessions were interspersed between training sessions that evaluated responding maintained by (1) the cocaine training dose only, (2) saline only, and (3) a range of cocaine doses. A test was conducted only if the peak of the most recently determined cocaine self-administration dose–effect function was within one half-log unit of that previously observed. Each dose of a test compound was investigated at least once in each animal. When possible, doses were tested two to three times with overlapping dose ranges of cocaine. Tests were conducted no more than twice/week with a minimum of 48 h between test sessions.

Data analysis.

The primary dependent variables were the number of food pellets and drug injections delivered during each component of a 2-h test session. Mean food and cocaine data are presented for three doses of each agonist. Data from individual monkeys are presented to illustrate the effects of the highest dose of each agonist on food- and cocaine-maintained responding. Data were analyzed using repeated measures analysis of variance (ANOVA; SuperAnova, Abacus Concepts, Inc., Berkeley, CA) with agonist dose and cocaine dose or food component as the two within-subjects factors. Owing to the fact that multiple sessions of overlapping cocaine dose ranges were required to characterize a complete cocaine dose–effect function, a mean cocaine dose–effect function was determined for each animal under each condition and subjected to statistical analysis. The criterion for significance was set a priori at p<0.05. If the effect of agonist dose was significant, linear contrasts comparing individual means were carried out to determine which treatment dose was significantly different from saline.

Study 2: Chronic Effects of Agonists on Responding Maintained by Cocaine or Food

Cocaine self-administration training procedures.

Monkeys were trained to respond for food and cocaine (0.01 mg/kg/inj) under a second-order schedule (FR 2 [VR16:S]) during multiple daily sessions of food and cocaine availability. Procedures for the evaluation of chronic drug treatment effects on cocaine- and food-maintained responding were identical to those used in our previous studies of the effects of -opioid drugs on cocaine self-administration (Mello and Negus, 1998; Negus et al, 1997). Once monkeys received at least 50 food pellets/day for at least 3 consecutive days under the terminal second-order schedule, the intravenous double-lumen catheter was implanted as described above. After recovery from surgery for at least 1 week, key-pressing for 0.032 mg/kg/inj, i.v., cocaine was shaped under a series of increasing variable ratios identical to those used during training of food-maintained responding. The final second-order schedule response requirement was identical for food- and drug-maintained responding (FR 2 [VR 16:S]). There were four food and cocaine sessions in each experimental day. Food sessions began at 11 am, 3 pm, 7 pm, and 6 am. Cocaine sessions began at 12 noon, 4 pm, 8 pm, and 7 am. Each food or drug session lasted 1 h or until a maximum of 25 food pellets or 20 injections had been delivered, whichever occurred first. Thus, monkeys could earn a maximum of 80 cocaine injections/day and 100 food pellets/day.

As in the Rapid Assessment Procedure, the conditions of food and cocaine availability were associated with different colored stimulus lights. The center key was transilluminated with a red stimulus light during food sessions and a green stimulus light during cocaine sessions. At the onset of reinforcer delivery, and after completion of each VR component of the second-order schedule, the appropriate colored stimulus light (red for food, green for injections) was illuminated for 1 s below the center response key. Each reinforcer delivery was followed by a 10 s TO.

Monkeys were trained until they met the following criteria for stable food and cocaine self-administration under the terminal schedule: (1) 3 consecutive days during which the number of drug injections/day differed by no more than 20% from the mean number of drug injections/day during those 3 days, and there was no upward or downward trend, and (2) during the same 3 consecutive days, the mean number of both drug injections/day and food pellets/day was greater than 50.
Drug self-administration testing.

During testing, a unit dose of 0.01 mg/kg/inj cocaine was used because it was the lowest dose to maintain reliably high rates of cocaine self-administration in all monkeys and because previous studies have shown that behavior maintained by this unit dose of cocaine is especially sensitive to the effects of opioids (Mello and Negus, 1998; Negus et al, 1997). The effects of repeated treatments with saline and each dose of (-)cyclorphan (0.0032–0.032 mg/kg/h) and MCL-101 (0.0032–0.032 mg/kg/h) were examined for 7 consecutive days. agonists were administered continuously by i.v. injections through the second lumen of the double-lumen catheter every 20 min for a total of three injections/h and 66 injections/day. No injections were delivered between 9 and 11 am. During this period, -opioid agonist syringe volumes were recorded and replenished and the health status of the animals was evaluated. At the conclusion of each 7-day test period, the maintenance dose of cocaine (0.032 mg/kg/inj) and saline control treatments were reinstated for a period of at least 4 days and until the number of reinforcers/day maintained by cocaine and food returned to baseline levels. During saline control treatment, 0.1 ml of saline was delivered every 20 min for a total of 6.6 ml/22 h. agonist doses were tested in an irregular order across monkeys.

Data analysis.

The total number of cocaine injections or food pellets delivered/day was determined. Data for the effects of (-)cyclorphan and MCL-101 on self-administration of 0.01 mg/kg/inj cocaine were compared with the effects of saline treatment using a two-factor repeated measures ANOVA with agonist dose and treatment day as the two within-subjects factors. The criterion for significance was set a priori at p<0.05. If the effect of agonist dose was significant, linear contrasts comparing individual means were carried out to determine which treatment dose was significantly different from saline.

Study 3: Cocaine Discrimination Studies

Discrimination training.

The drug discrimination procedures employed in the present study were similar to those used in previous studies of -opioid effects on cocaine discrimination (Negus and Mello, 1999). In the present experiment, the monkeys had been trained previously to discriminate 0.4 mg/kg cocaine from saline (i.m.). Training sessions consisted of one to five cycles and each cycle consisted of a 15-min TO period followed by a 5-min response period. Monkeys were given an i.m. injection of either vehicle (saline) or the cocaine training dose at the beginning of the 15-min TO period. All stimulus lights were turned off and responding had no scheduled consequences during the TO period. During the response period, the right and left response keys were transilluminated red or green, and key position was counterbalanced across monkeys. Depending upon the training condition, monkeys could respond on the stimulus-appropriate key under an FR 30 schedule to obtain up to 10 food pellets/cycle. After vehicle administration, responding on only the green key resulted in the delivery of a food pellet. After cocaine administration, responding on only the red key resulted in the delivery of food. Inappropriate responses reset the FR requirement on the stimulus-appropriate key. The center key was not illuminated during operant sessions and responding had no scheduled consequences. If all available food pellets were delivered in less than 5 min, then the stimulus lights were extinguished and responding had no scheduled consequences for the remainder of the 5-min response period.

Training sessions consisted of 0–5 saline cycles followed by 0–1 drug cycles. If the training drug was administered, it was given only during the last cycle. This design assured a constant interval between drug administration and the onset of response periods during which responding on the drug-appropriate key produced food. Monkeys were considered to have acquired the discrimination when the following criteria were met for seven out of eight consecutive training sessions: (1) the percent injection-appropriate responding before the delivery of the first reinforcer was greater than or equal to 80% for all cycles, (2) the percent injection-appropriate responding over the entire response period was greater than or equal to 90% for all cycles, and (3) response rates during vehicle training cycles were greater than 0.5 responses/s. Experimental sessions were conducted 5 days/week.

Discrimination testing.

Test sessions were conducted only if the three criteria listed above were met during the training day immediately preceding the test day. If responding did not meet criterion levels of discrimination performance, then training was continued until criterion levels of performance were obtained for at least 2 consecutive days. In general, testing was conducted on Tuesdays and Fridays and training sessions were conducted on Mondays, Wednesdays, and Thursdays. Test sessions were identical to training sessions except that responding on either key produced food, and test drugs were adminstered using either a substitution or a pretreatment protocol.

In the substitution protocol, cocaine or a -opioid agonist was administered alone using a cumulative-dosing procedure. The drugs (and doses) tested included cocaine (0.013–1.3 mg/kg), enadoline (0.0001–0.0032 mg/kg), (-)cyclorphan (0.001–0.1 mg/kg), Mr2034 (0.0001–0.01 mg/kg), and MCL-101 (0.001–0.1 mg/kg). Monkeys received an injection of the test compound 5 min after the onset of the 15-min TO at the beginning of each cycle of a multiple-cycle session. In the cumulative-dosing procedure, each dose increased the total dose by 1/4 or 1/2 log units. Dose–effect curves for each compound were determined twice in each monkey using overlapping dose ranges. Each agonist was tested up to doses that eliminated responding in most of the monkeys.

In the pretreatment protocol, a -opioid agonist dose was administered 15 min before the determination of a cumulative cocaine dose–effect curve. Since preliminary studies indicated that enadoline had a relatively short duration of action, pretreatment studies with each enadoline dose were determined twice in each monkey with overlapping cocaine dose–effect curves. For one determination, a dose range of 0.013–1.3 mg/kg cocaine was evaluated in a series of five consecutive cycles, and the total test session lasted 1 h 40 min. For the second determination, a high dose range of 0.13–1.3 mg/kg cocaine was tested in a series of three consecutive cycles and the total test session lasted 1 h. This procedure assured that active cocaine doses were evaluated while enadoline was producing its behavioral effects. Pretreatment studies with Mr2034 and MCL-101 also were determined twice in each monkey with overlapping cocaine dose ranges identical to those described above for studies with enadoline. In contrast, preliminary studies indicated that (-)cyclorphan had a relatively long duration of action. Thus, the effects of each dose of (-)cyclorphan were evaluated only once in each monkey before a full dose range of 0.013–1.3 mg/kg cocaine. The drugs (and doses) tested in pretreatment experiments were enadoline (0.00032–0.0032 mg/kg), (-)cyclorphan (0.0032–0.032 mg/kg), Mr2034 (0.0032–0.032 mg/kg), and MCL-101 (0.0032–0.032 mg/kg). Each agonist was tested up to pretreatment doses that eliminated responding in most of the monkeys.