Introduction

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Converging evidence suggests that the abuse-related effects of cocaine and amphetamine are mediated by enhanced mesolimbic dopamine (DA) activity as a result of inhibited DA uptake and/or stimulated DA release (see Wise and Bozarth, 1987; Koob and Bloom, 1988; and Woolverton and Johnson, 1992, for review). Morphine and other µ-opioid agonists also have been shown to stimulate release of DA in mesolimbic regions (Di Chiara and Imperato, 1988; Spanagel et al., 1990) and to enhance cocaine-induced increases in extracellular DA (Brown et al., 1991; Zernig et al., 1997; Hemby et al., 1998). Given the likely importance of DA systems in mediating the behavioral effects of stimulants and µ opioids (Wise and Bozarth, 1987; Koob and Bloom, 1988; Di Chiara and North, 1992), the possibility of reciprocal enhancement of the behavioral effects of these two classes of drugs has been explored using procedures that model different aspects of the addiction process (e.g., drug discrimination and drug self-administration: Spealman and Bergman, 1992; Mello et al., 1995). Using drug discrimination procedures, morphine and related drugs have been shown to enhance, in a largely additive manner, the discriminative stimulus (DS) effects of cocaine in squirrel monkeys (Spealman and Bergman, 1992, 1994; Rowlett and Spealman, 1998). These results support the idea that µ opioids and cocaine given in combination can result in enhanced behavioral effects, perhaps mediated by their mutual ability to augment DA neurotransmission. Other studies, however, have reported less consistent effects of stimulant-opioid combinations. In rats, for example, clear-cut enhancement of the DS effects of cocaine by morphine has been observed by Suzuki et al. (1997) and Kantak et al. (1994) but not by others (Dykstra et al., 1992; Broadbent et al., 1995). Similarly, in rhesus monkeys enhancement of the DS effects of cocaine by µ opioids has been reported in approximately half the subjects tested (Mello et al., 1995; Negus et al., 1998). In addition, several µ opioids engendered appreciable cocaine-like responding when tested alone in these latter studies. Together, these findings suggest that individual differences may play a role in the capacity of µ opioids to either mimic or modulate the DS effects of cocaine in some instances.

Although opioid modulation of the DS effects of cocaine has received considerable attention, only a few studies have specifically investigated the ability of stimulants to modulate the DS effects of opioids. In two such experiments, Suzuki et al. (1995) and Lamas et al. (1998) observed no consistent modulation by cocaine of the DS effects of either morphine or heroin in rats. On the other hand, an earlier study by Gauvin and Young (1989a) demonstrated that amphetamine typically enhanced the DS effects of morphine at a relatively low training dose in pigeons; whereas both enhancement and blockade of the DS effects of morphine were evident in individual subjects trained to discriminate higher doses of morphine. In human drug abusers, cocaine also has been found to enhance morphine- or hydromorphone-induced subjective ratings of "high" and "drug effect", whereas it reduced opioid-induced sedation and "nodding" (Foltin and Fischman, 1992; Walsh et al., 1996).

The purpose of the present study was to investigate the ability of cocaine and related drugs to mimic and/or modulate the DS effects of the prototypic µ agonist morphine in squirrel monkeys. Monkeys were trained to discriminate morphine from vehicle injections, using a procedure similar to one used previously to investigate stimulant-opioid interactions in monkeys trained to discriminate cocaine (Spealman and Bergman, 1992, 1994; Rowlett and Spealman, 1998). Cocaine, amphetamine, and several reference drugs were studied for their ability to engender morphine-appropriate responding when administered alone as well as for their capacity to alter the morphine dose-response function when administered as pretreatments. Reference drugs included selective µ-, -, and -opioid agonists; selective DA, norepinephrine (NE), and 5-hydroxytryptamine (5-HT) uptake inhibitors; and sedative/anxiolytics. Finally, antagonism studies with the opioid receptor antagonist naltrexone and the DA receptor antagonist flupenthixol were conducted to evaluate the role of opioid and DA receptor mechanisms in the DS effects of morphine and the morphine-like stimulus effects of other drugs.

	Materials and Methods

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Subjects. Five adult male squirrel monkeys (Saimiri sciureus) were studied in daily experimental sessions. Between sessions, monkeys lived in individual home cages and had unlimited access to water. Each monkey was maintained at 85 to 90% of its free-feeding body weight (750-900 g) by adjusting its access to food (Purina Monkey Chow; Ralston Purina, St. Louis, MO; Teklad Monkey Diet; Teklad Premier Laboratory Diets, Madison, WI; and fruit). With the exception of one monkey (S-288), all were experimentally naïve at the beginning of the study. Monkey S-288 had been trained previously under a punishment procedure and tested with -aminobutyric acid_A modulators. All animals were maintained in accordance with the guidelines of the Committee on Animals of the Harvard Medical School and the Guide for Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council, Department of Health, Education and Welfare Publication No. (National Institutes of Health) 85-23, as revised in 1985. Research protocols were approved by the Harvard Medical School Institutional Animal Care and Use Committee.

Apparatus. During experimental sessions, monkeys were seated in Plexiglas chairs similar to the one described by Spealman and Bergman (1992). Two response levers (model 121-05; BRS/LVE, Beltsville, MD) were mounted 15 cm apart on the wall of the chair in front of the monkey. Each press of a lever produced an audible feedback click and was recorded as a response. Red lights mounted at eye level behind the front panel of the chair could be illuminated to serve as a visual stimulus associated with consecutive components of the session (see below). Sucrose pellets (190 mg; P.J. Noyes Co., Inc., Lancaster, NH) could be delivered to a tray that was accessible through an opening in the front panel of the chair. Each chair was enclosed in a ventilated, sound-attenuating chamber, which was equipped with white noise to mask external sounds.

Drug-Discrimination Procedure. Monkeys were trained to discriminate morphine from saline, using procedures similar to those used in previous studies with cocaine (Spealman and Bergman, 1992). After i.m. injection of morphine, 10 consecutive responses [fixed ratio (FR) 10] on one lever produced food, whereas after injection with saline, 10 consecutive responses on the other lever produced food. Responses on the incorrect lever reset the FR requirement. Daily training sessions consisted of a variable number of components (n = 1-3) of the FR schedule. Each component ended after the completion of the tenth FR 10 or after 5 min had elapsed, whichever occurred first. A 20-min timeout period, during which the lights were off and responding had no programmed consequences, preceded each component. During most training sessions, saline was injected during timeout periods preceding the first n  1 components, and morphine was injected before the final component of the session. Periodically, saline was injected before each of the three components of a training session to prevent an invariant association between drug and the third component. Injections of morphine or saline were made in a thigh or calf muscle of either leg during min 5 of the 20-min timeout periods.

Drug-Testing Procedure. Drug test sessions were conducted once or twice per week with training sessions scheduled on intervening days. Test sessions consisted of three FR components, each preceded by a 20-min timeout period. In each component, completion of 10 consecutive responses on either lever produced food. Dose-response functions were determined for test drugs, using a cumulative dosing procedure similar to the one described by Spealman and Bergman (1992). Incremental doses were injected i.m. during min 5 of the 20-min timeout periods that preceded each FR component, permitting a three-point cumulative dose-response function to be determined in a single session. In most cases, four or more different doses of a drug were studied by administering overlapping ranges of cumulative doses during test sessions on different days, and the effects of each active dose typically were determined twice in each subject.

Analysis of Drug Effects. The percentage of responses on the morphine-associated lever was calculated for individual subjects in each component of a test session by dividing the number of responses on that lever by the total number of responses on both levers and multiplying by 100. The overall rate of responding in each component was computed by dividing the total number of responses in a component (regardless of lever) by the total component duration. The doses of a drug estimated to engender 50% morphine-appropriate responding (ED₅₀) were determined for individual subjects by linear regression analysis in cases where the linear portion of the log dose-response function was defined by three or more data points and by linear interpolation in cases in which the linear portion of the log dose-response function was defined best by two points.

Drugs. Morphine sulfate (Merck, Sharpe and Dohme, West Point, PA); (+)-amphetamine sulfate (Sigma, St. Louis, MO); cocaine HCl (National Institute on Drug Abuse, Rockville, MD); midazolam base (Hoffman-LaRoche, Basel, Switzerland); cis-(Z)-flupenthixol · 2 HCl, fentanyl citrate, and naltrexone HCl (Research Biochemicals, Inc., Natick, MA); SNC 80 base (Tocris Cookson, Inc., Ballwin, MO); sodium pentobarbital (Abbott Laboratories, Chicago, IL); and U50,488H methanesulfonate (Research Biochemicals, Inc.) were dissolved in sterile water or 0.9% saline solution. GBR 12909 · 2 HCl (Research Biochemicals), fluoxetine HCl (Eli Lilly and Co., Indianapolis, IN), and talsupram HCl (Lundbeck A/S, Copenhagen, Denmark) were dissolved in small amounts of warm 0.1 N HCl or acetic acid and diluted with 0.9% saline solution. BW 373U86 HCl (Research Biochemicals) was dissolved in a small amount of dimethyl sulfoxide and 0.1 M NaHCO₃ and diluted with 0.9% saline solution.

	Results

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Effects of Morphine. Morphine at the final training doses of 0.56 or 1.0 mg/kg maintained consistent control of behavior over the course of the study. Averaged across all training sessions that preceded drug test sessions (n = 44-53), individual monkeys made 94 to 99% responses on the morphine-associated lever after injection of morphine and 1 to 4% responses on this lever after injection of saline (Table 1). The average rate of responding after injection of morphine (0.8-2.9 responses/s for individual monkeys) was comparable to the average response rate after injection of saline (0.9-2.5 responses/s), and subjects typically completed all 10 FRs in each component of a training session.

View larger version (26K): [in this window] [in a new window]   Fig. 1.   Percentage of morphine-lever responding (top) and response rate (bottom) engendered by fentanyl and morphine in squirrel monkeys trained to discriminate morphine from saline. Each column of panels represents data from an individual monkey, identified at the top of each column. Morphine dose-response functions were determined at the beginning of the study () and at the end of the study (). Horizontal bars (bottom) represent mean response rates after the morphine training dose; horizontal dashed lines represent mean response rates after saline.

Effects of Other Opioids and Reference Drugs. The selective µ-opioid agonist fentanyl had DS effects that were qualitatively similar to those of morphine (Fig. 1, solid triangles). Increasing cumulative doses of fentanyl (0.003-0.01 mg/kg) engendered dose-related increases in the percentage of responses on the morphine-associated lever, with one or more doses occasioning full substitution for morphine (i.e., >80% morphine-lever responses) in each monkey. As in the case of morphine, these DS effects were observed after administration of doses of fentanyl that did not systematically alter the response rate for the group of five monkeys, although some doses of fentanyl either increased or decreased response rate in individual subjects (Fig. 1, bottom).

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Percentage of morphine-lever responding (top) and response rate (bottom) engendered by morphine in squirrel monkeys in the presence and absence of 0.3 mg/kg naltrexone. See legend of Fig. 1 for other details.

Effects of Cocaine, Amphetamine, and GBR 12909. Unlike the other drugs studied, cocaine, amphetamine, and the selective DA-uptake inhibitor GBR 12909 had qualitatively different DS effects in different subjects (Fig. 3). In three of the five monkeys (S-204, S-288, and S-221), all three drugs engendered dose-related increases in the percentage of responses on the morphine-associated lever, reaching maximums of 77 to 99% morphine-lever responses for the individual subjects. In the remaining two monkeys (S-95 and S-89), however, none of the drugs engendered substantial morphine-lever responding regardless of dose. In general, doses of cocaine, amphetamine, and GBR 12909 that engendered maximum percentages of morphine-lever responses also decreased the overall response rate (Fig. 3, bottom). GBR 12909, however, either had little effect on or increased the rate of responding in monkeys S-221 and S-95, as did cocaine and amphetamine in monkey S-95.

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Percentage of morphine-lever responding (top) and response rate (bottom) engendered by amphetamine, cocaine, and GBR 12909 in squirrel monkeys trained to discriminate morphine from saline. See legend of Fig. 1 for other details.

Effects of Morphine Combined with Cocaine and Amphetamine. In the monkeys (S-204, S-228, and S-221) for which both stimulants exhibited full or partial substitution for morphine, combined administration of either cocaine or amphetamine with morphine had qualitatively different effects compared with monkeys S-95 and S-89 (Figs. 4 and 5). In the former three monkeys, pretreatment with 0.1 to 0.3 mg/kg cocaine or 0.03 to 0.3 mg/kg amphetamine produced an overall enhancement of the DS effects of morphine such that doses of morphine <1.0 mg/kg engendered a larger percentage of morphine-lever responses in the presence of either cocaine or amphetamine than in their absence. The degree to which cocaine or amphetamine enhanced the DS effects of morphine in these monkeys depended on the particular subject and pretreatment dose, but in no case was there evidence that either cocaine or amphetamine attenuated the DS effects of morphine. In the remaining two monkeys (S-95 and S-89), however, neither cocaine nor amphetamine enhanced the DS effects of morphine regardless of dose. Instead, pretreatment with at least one dose of either stimulant attenuated the DS effects of morphine in these subjects such that doses of morphine 0.3 mg/kg engendered a reduced percentage of morphine-lever responses in the presence compared with the absence of cocaine or amphetamine. Pretreatment with cocaine or amphetamine did not result in response rates that were invariably greater than or less than those observed with morphine alone, although lower response rates were observed in most subjects after pretreatment with one or more doses of amphetamine (Fig. 5, bottom) and both increases and decreases in response rate were observed in some instances after pretreatment with cocaine (Fig. 4, bottom).

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Percentage of morphine-lever responding (top) and response rate (bottom) engendered by morphine alone and combined with cocaine in squirrel monkeys trained to discriminate morphine from saline. See legend of Fig. 1 for other details.

View larger version (27K): [in this window] [in a new window]   Fig. 5.   Percentage of morphine-lever responding (top) and response rate (bottom) engendered by morphine alone and combined with amphetamine in squirrel monkeys trained to discriminate morphine from saline. See legend of Fig. 1 for other details.

	Discussion

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Opioid Mechanisms in the DS Effects of Morphine. Morphine is thought to exert its behavioral effects largely via stimulation of µ-opioid receptors (Martin et al., 1976; Woods et al., 1992). Consistent with this view, the DS effects of morphine in the present study were mimicked fully by the selective µ agonist fentanyl and antagonized by naltrexone. In contrast, the DS effects of morphine were not mimicked by the  agonist U50,488H. These findings corroborate existing data indicating that -opioid receptors do not play a critical role in the DS effects of morphine (Teal and Holtzman, 1980a,b). The  agonists BW 373U86 and SNC 80 also did not mimic the DS effects of morphine in the present study, suggesting that -opioid receptor mechanisms do not play a critical role in transduction of the interoceptive effects of morphine. Consistent with this view, Negus et al. (1994) found that BW 373U86 similarly did not share DS effects with the µ agonist alfentanil in rhesus monkeys, and Jewett et al. (1996) demonstrated that the µ-selective peptide [D-Ala²,N-Me-Phe⁴-Met(O)⁵-(ol)]-enkephalin did not substitute for the -selective peptide [D-Pen²-D-Pen⁵-enkephalin] in pigeons. The present results, however, differ in some respects from previous findings with -opioid agonists in other experiments. For example, morphine and BW 373U86 show considerable cross-substitution in pigeons trained to discriminate either morphine or BW 373U86 from vehicle (Comer et al., 1993). Similarly, the -selective peptide [D-Ala²,D-Leu⁵]-enkephalin substituted fully for morphine in morphine-trained rats (Locke and Holtzman, 1986). Although our results provide no support for a primary involvement of  receptors in the DS effects of morphine in monkeys, the results with pigeons and rats raise the possibility that -receptor mechanisms contribute in some way to the DS effects of morphine in other species.

Individual Differences in Shared DS Effects of Stimulants and Opioids. Clear differences in the substitution profiles for cocaine and amphetamine were observed among individual subjects in our study. In three of five monkeys, both stimulants fully or partially mimicked the DS effects of morphine, but neither drug engendered appreciable morphine-appropriate responding in the other monkeys. Individual differences in the degree to which cocaine and amphetamine share DS effects with µ opioids also have been noted previously. For example, subgroups of pigeons trained to discriminate either morphine or butorphanol from vehicle showed full substitution for the training drug when tested with amphetamine or cocaine, whereas other subjects showed little or no substitution (Gauvin and Young, 1989a; Cook and Picker, 1998). Similarly, in heroin-trained rats, cocaine partially substituted for the training drug in approximately one-third of the subjects tested (Lamas et al., 1998). Although the DS effects of morphine itself appear to be mediated primarily by µ-opioid mechanisms (see above), the morphine-like DS effects of cocaine and amphetamine do not. In this regard, the DS effects of neither cocaine nor amphetamine were altered appreciably by naltrexone in the present study at doses that clearly antagonized the DS effects of morphine. This observation is concordant with previous findings that opioid-receptor antagonists do not alter the DS effects of cocaine or amphetamine in rats or monkeys trained to discriminate either of these drugs from vehicle (Mello et al., 1995; Woolfolk and Holtzman, 1996; Rowlett and Spealman, 1998).

Individual Differences in Interactions between Stimulants and Opioids. In the subgroup of monkeys for which cocaine and amphetamine substituted for morphine, the DS effects of morphine were enhanced after pretreatment with either stimulant. Gauvin and Young (1989a) similarly have shown that amphetamine enhances the DS effects of morphine in pigeons that also showed substitution of amphetamine for morphine. In the two monkeys for which the stimulants did not substitute for morphine, however, pretreatment with either cocaine or amphetamine resulted in rightward and downward shifts in the morphine dose-response functions. Although the possibility of pharmacological antagonism of the DS effects of morphine by cocaine and amphetamine cannot be ruled out, it seems more likely that the attenuated effects of morphine in these subjects was due to perceptual masking of the DS effects of morphine by the two stimulants (cf. Gauvin and Young, 1989a,b). Perceptual masking has been inferred from demonstrations of attenuation of the DS effects of a drug without concomitant attenuation of its rate-altering effects. Although morphine itself did not alter response rates systematically, rate-decreasing effects were observed in both monkeys when morphine was combined with 0.1 or 0.3 mg/kg amphetamine and in monkey S-95 when morphine was combined with 0.1 mg/kg cocaine. Enhanced rate-decreasing effects as a result of combining the drugs would not be expected if attenuation of the effects of morphine was due principally to pharmacological antagonism (e.g., as in the case of morphine combined with naltrexone).