mu opioid agonists potentiate amphetamine- and cocaine...

Mu Opioid Agonists Potentiate Amphetamine- and Cocaine-Induced Rotational Behavior in the Rat1

HEATHER L. KIMMEL and STEPHEN G. HOLTZMAN

Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia

Accepted for publication April 15, 1997

ABSTRACTOpioids modulate brain dopaminergic function in various ex-perimental paradigms. This study used the rotational model ofbehavior in rats with unilateral 6-hydroxydopamine-induced le-sions of the nigrostriatal pathway to investigate this interaction.Doses of two presynaptically acting dopaminergic drugs, am-phetamine and cocaine, were coadministered with severaldoses of the mu opioid agonist, morphine. Morphine, at 3.0mg/kg, potentiated rotational behavior induced by each dose ofthe stimulants. To determine the receptor specificity of theactions of morphine, the mu opioid agonists buprenorphine,fentanyl, levorphanol, meperidine, and methadone, and dex-trorphan, the non-opioid isomer of levorphanol, were adminis-tered alone and with 1.0 mg/kg amphetamine. Each of thesedrugs, as well as morphine, produced circling behavior on itsown. All of the mu opioid agonists and dextrorphan increased

amphetamine-induced turning; the coadministration of dextror-phan, levorphanol, meperidine, methadone and morphine withamphetamine produced turning greater than predicted by sim-ple additivity. To determine whether an opioid receptor wasinvolved in these interactions, the opioid antagonist, naltrex-one, was administered before the amphetamine/mu opioid re-ceptor agonist combination. Naltrexone blocked the potentiat-ing effects of morphine, but not those of the other drugs.Moreover, naltrexone alone dose-dependently increased am-phetamine-induced rotational behavior. These studies showthat some mu opioid receptor agonists can potentiate stimu-lant-induced rotational behavior and that blockade of opioidreceptors can also produce a potentiation. The role of muopioid receptors in these effects remains unclear.

Opioids can modulate brain dopamine systems (Wood,1983). Of the three types of opioid receptors, mu and deltareceptor activation increases dopamine release in the me-solimbic and nigrostriatal tracts. Conversely, the stimulationof kappa opioid receptors decreases dopamine levels in thesebrain regions (Di Chiara and Imperato, 1988a,b). All threeopioid receptors have been localized within these dopaminesystems (Goodman et al., 1988; Mansour et al., 1995; Sharifand Hughes, 1989; Wood and Iyengar, 1988).

Some effects exhibited by mu opioid agonists reflect in-creases in dopaminergic activity. In mice, morphine, heroin,levorphanol and meperidine increased locomotor activity(Longoni et al., 1987; Oliverio and Castellano, 1974; Rethy etal., 1971; Shippenberg et al., 1993), which is mediated bydopaminergic neurons in the substantia nigra (Iwatsubo andClouet, 1977) and in the ventral tegmental area (Matthewsand German, 1984). In vivo microdialysis indicated that mor-phine, methadone and fentanyl increased extracellular dopa-mine levels in the nucleus accumbens (Di Chiara and Im-

perato, 1988b; Kalivas and Stewart, 1991). Behavioralsensitization to repeated administration of opioids was at-tributed to the indirect stimulation of dopamine cell bodies inthe ventral tegmental area and the substantia nigra (Kalivasand Stewart, 1991). These results indicate that the stimula-tion of opioid receptors influences processes thought to bemediated by brain dopamine systems.

Opioids also can modulate the effects of drugs that act viabrain dopamine systems. The effects of cocaine and amphet-amine are believed to be mediated largely by dopamine (Ka-livas and Stewart, 1991). Behavioral interactions betweenmu opioid agonists and psychomotor stimulants have beenstudied with various experimental paradigms. However, theresults have not always been consistent among these differ-ent techniques. In drug discrimination studies, heroin sub-stituted for cocaine in some rhesus monkeys (Mello et al.,1995), and morphine potentiated the discriminative stimuluseffects of cocaine in squirrel monkeys (Spealman and Berg-man, 1994). Chronic treatment with the partial mu opioidreceptor agonist buprenorphine reduced cocaine self-admin-istration in rats (Carroll and Lac, 1992) and rhesus monkeys(Mello et al., 1992, 1993, 1995). Doses of buprenorphine andcocaine, which were ineffective by themselves, induced con-

Received for publication May 9, 19961 This research was supported by Grant DA00541, Research Scientist

Award K05 DA00008 and NRSA Predoctoral Fellowship F31 DA05692–01, allfrom the National Institute on Drug Abuse, National Institutes of Health.

ABBREVIATIONS: 6-OHDA, 6-hydroxydopamine; ANOVA, analysis of variance; DAMGO, [D-Ala2NMePhe4Gly-ol5]enkephalin; NMDA, N-methyl-D-aspartate; PCP, phencyclidine

0022-3565/97/2822-0734$03.00/0THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 282, No. 2Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A.JPET 282:734–746, 1997

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ditioned-place preference in rats when administered together(Brown et al., 1991). In studies of analgesia, cocaine potenti-ated the effects of morphine and the mu opioid receptor-selective ligand DAMGO in mice (Sierra et al., 1992) and rats(Kauppila et al., 1992), and the effects of morphine andnalbuphine in rhesus monkeys (Gatch et al., 1995). The co-administration of stimulants and morphine-like compounds,commonly known as “speedballing,” is frequently seen indrug abusers (Kosten et al., 1986; Kreek, 1987). Whether thecoadministration of the drugs enhances their euphoric effectsor attenuates their dysphoric effects is not known. In sum-mary, mu opioid receptor agonists influence the effects ofcocaine, but the direction of the change in the behavioralresponse to cocaine is difficult to predict.

The effects of opioid antagonists on the behavioral effectsof cocaine and amphetamine have also been studied. In manycases, the antagonists did not alter the effects of stimulants.When the antagonists had an effect, it was usually to reducethe effects of the stimulant drugs. For example, naloxone andnaltrexone, two nonspecific opioid receptor antagonists, at-tenuated amphetamine-induced locomotor activity in a vari-ety of animal species (Adams et al., 1981; Andrews and Holtz-man, 1978; Dettmar et al., 1978; Hitzemann et al., 1982;Holtzman, 1974; Schad et al., 1995; Winslow and Miczek,1988), and blocked cocaine- and amphetamine-induced con-ditioned place preference in rats (Gerrits et al., 1995; Sala etal., 1995; Trujillo et al., 1991). These two opioid receptorantagonists also attenuated cocaine self-administration (Cor-rigall and Coen, 1991; Ramsey and van Ree, 1991). Sensiti-zation to repeated cocaine injections was attenuated by nal-trexone (Sala et al., 1995). As the opioid antagonistspresumably were blocking effects of endogenous opioids, theresults of the experiments cited above suggest that endoge-nous opioids modulate the effects of stimulants, probably viathe dopaminergic system.

To study further the relationship between opioids and do-paminergic neurons in the brain, we used the rotationalmodel of behavior in the rat. This is a method of studying thenigrostriatal dopaminergic system in which rats are given aunilateral lesion of the nigrostriatal tract with 6-OHDA, thuscreating a postsynaptic dopamine receptor supersensitivity(Ungerstedt, 1971). These animals circle toward the lesion inresponse to presynaptically acting dopaminergic agonists(i.e., amphetamine) (Lynch and Carey, 1989; Ungerstedt andArbuthnott, 1970; Zetterstrom et al., 1986a). This paradigmhas been used infrequently to investigate interactions be-tween the opioid and dopaminergic systems of the rat brain,and results have not been consistent across studies. Mor-phine produced rotational behavior in one study (Cowan etal., 1975; Pert, 1978), but amphetamine was used to deter-mine the validity of the 6-OHDA-induced lesion in this study.Apomorphine-induced turning has been a more accurate in-dicator of the magnitude of the 6-OHDA-induced lesion (Hud-son et al., 1993). In other studies, injections of morphineproduced significant turning only in those rats that had beenmade tolerant to the depressant effects of the drug by re-peated exposure to morphine (Kimmel et al., 1995; Pert1978). In one study, amphetamine-induced turning was re-duced by morphine (Blundell et al., 1976), whereas in an-other, it was reduced by naloxone (Dettmar et al., 1978).Based on the effects of mu opioid agonists on brain dopamineand on the dopamine-mediated behaviors cited above, we

hypothesized that these drugs would potentiate the effects ofpsychomotor stimulants upon rotational behavior, and woulddo so by activating the mu opioid receptor.

In this study, we first examined the effects of morphine onamphetamine- and cocaine-induced rotational behavior. Todo this, we administered various doses of morphine immedi-ately before doses of amphetamine or cocaine. Because com-binations of the highest doses of morphine and cocaine werelethal in some rats, subsequent experiments were carried outusing only amphetamine. To determine the receptor specific-ity of the effects of morphine on turning induced by amphet-amine, we tested the mu opioid receptor agonists fentanyl,levorphanol, meperidine and methadone. We also examinedthe effects of dextrorphan, the optical isomer of levorphanol,to investigate the stereoselectivity of this interaction. Wemeasured rotational behavior for a 4-hr period to capture thefull time course of drug action.

Biochemical and behavioral effects of opioids that involvemesolimbic and nigrostriatal dopamine systems can beblocked by the general opioid antagonists naloxone and nal-trexone (Di Chiara and Imperato, 1988b; Spanagel et al.,1990). Thus, to determine whether the effects of these muopioid agonists were mediated by an opioid receptor, weadministered naltrexone before amphetamine alone or thecombination of amphetamine with the mu opioid agonists.

MethodsSubjects. Male Sprague-Dawley rats (Sasco, Inc., Omaha, NE)

weighing 300 to 350 g at the time of surgery were used. All rats weregroup housed in polycarbonate cages and maintained in a tempera-ture-controlled colony room with a 12 hr light:12 hr dark lightingcycle, beginning with lights on at 7:00 A.M. Food (Purina RodentChow, Purina Mills, St. Louis, MO) and water were available adlibitum.

Stereotaxic surgery. Rats were given unilateral lesions of theright nigrostriatal pathway by a single injection of 6-OHDA. Theywere first anesthetized with 3.3 mg/kg i.p. Equithesin and thenplaced into a stereotaxic frame. Stereotaxic coordinates relative tobregma were AP 5 24.8, ML 5 22.2, DV 5 28.0 (Paxinos andWatson, 1986). A 25-ml Hamilton syringe was used to inject 8 mg/4 mlof 6-OHDA in a solution of 0.02% ascorbic acid and 0.9% saline intothe right substantia nigra at a rate of 1.0 ml/min for 4 min. Uponcompletion, the injection needle was kept in place for an additionalminute to minimize backflow of the solution. No desmethylimipra-mine pretreatment was used before the surgeries, so norepinephrinenerve terminals may have been lesioned. However, norepinephrineinnervation of the striatum is relatively low, so this should not haveaffected the experimental results.

Rotational behavior. Rotational activity was measured in stain-less steel rotometer stations (MED Associates, Inc., East Fairfield,VT). Each of eight stations consisted of a round stainless steel bowl(40.6 cm diameter and 25.4 cm high) in a transparent Plexiglascover. A spring tether connected to a direction-sensitive rotationsensor mounted above the bowl was attached to the rat by means ofa Velcro belt. Rotational activity was recorded by the Roto-Rat Ver-sion 1.2 computer program (MED Associates). Measurements weretaken of full (360°) turns in both the clockwise and counterclockwisedirection. During experiments, counts were taken in 15-min inter-vals for 4 hr, resulting in 16 time points per animal for each session.All sessions were conducted during the light phase of the lightingcycle. Rats were allowed to recover from surgery for at least 14 days,then they received 0.3 mg/kg R(2)-apomorphine s.c. twice weekly for2 weeks. Animals exhibiting at least 50 full contralateral turns/10min for 1 hr were used for further behavioral testing. The amount ofturning observed in response to apomorphine is directly correlated

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with the extent of the nigral lesion (Hudson et al., 1993). The ratsused in this study exhibited a mean (6 S.E.M.) of 471 6 56 turns/hrin tests with 0.3 mg/kg apomorphine.

Drug administration and behavioral testing. On test days,animals were weighed and placed into the test chambers and allowedto habituate for approximately 5 min before drug injection. Rats (n 516) then received a 5-min pretreatment of doses of saline or mor-phine (0.03–10 mg/kg) administered in a random sequence. Fiveminutes later, half of the rats received saline or amphetamine (0.1–1.0 mg/kg) and the other half received saline or cocaine (3.0–30mg/kg), with the dose of each drug and saline administered in arandom order. Measurements of rotational behavior began 5 minafter the second injection. Each animal was tested twice a week, witha 3- to 4-day interval between sessions, so that each animal receivedevery possible combination of opioid and either amphetamine orcocaine. Morphine, amphetamine and saline were administered s.c.,and cocaine was administered i.p.

Based on results obtained in the experiments above, a 1.0 mg/kgdose of amphetamine was selected for testing in combination with

other mu opioid receptor agonists and dextrorphan. Buprenorphine(0.01–1.0 mg/kg), dextrorphan (1.0–10 mg/kg), fentanyl (0.01–0.056mg/kg), levorphanol (0.1–1.0 mg/kg), meperidine (3.0–30 mg/kg) andmethadone (0.3–3.0 mg/kg) were injected s.c., with doses of each druggiven in a random sequence. Amphetamine was injected 5 min later.Testing was conducted in the same manner as described earlier.

To determine whether the interactions observed between the muopioid agonists and amphetamine could be blocked by naltrexone,one group of animals was given 0.1 mg/kg naltrexone followed by 3.0mg/kg morphine and 1.0 mg/kg amphetamine. The dose of each of theother agonists that produced the greatest amount of turning incombination with amphetamine was then tested with 1.0 mg/kgnaltrexone and 1.0 mg/kg amphetamine, injected in the followingorder: naltrexone, mu opioid agonist and amphetamine. Because thisdose of naltrexone was ineffective in combination with nearly all ofthe agonists, 10 mg/kg naltrexone was tested in combination with 1.0mg/kg methadone and 1.0 mg/kg amphetamine.

Statistical analysis. Total rotational count data in the initialexperiments with morphine, amphetamine and cocaine were ana-

Fig. 1. Morphine (0.03–10 mg/kg) potentiated ipsilateral turning behavior induced by amphetamine (0.1–1.0 mg/kg) in rats with unilateral lesionsof the nigrostriatal tract. Points on the time-course curves (A) represent the mean number of turns per 15 min. Only the saline curves and the 3.0mg/kg morphine curves are shown for simplicity. A three-way ANOVA of these data revealed significant F values for all factors and interactions:amphetamine: F(3,21) 5 30.78, P , .0001; morphine: F(6,42) 5 8.02, P ,.0001; time: F(15,105) 5 27.24, P , .0001; amphetamine 3 morphine:F(18,126) 5 3.95, P , .0001; amphetamine 3 time: F(45,315) 5 13.98, P , .0001; morphine 3 time: F(90,630) 5 5.13, P , .0001; amphetamine 3morphine 3 time: F(270,1890) 5 1.92, P , .0001. Dose-response data (B) show total amount of turning for the entire 4-hr session (mean 1 S.E.).A two-way ANOVA of these data revealed a significant effect by drug as well as a significant interaction between the two drugs (amphetamine:F(3,28) 5 12.87, P , .0001; morphine: F(6,168) 5 16.88, P , .0001; and amphetamine 3 morphine interaction: F(18,168) 5 3.29, P , .0001).Asterisks indicate significant differences from saline control plus that same dose of amphetamine (points above sal), **P , .01, *P , .05. Daggersindicate significant differences of amphetamine or morphine alone from saline 1 saline, 11P , .01, 1P , .05.

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lyzed using a two-way ANOVA with repeated measures on bothfactors (morphine dose and stimulant dose). A Tukey’s protected ttest was performed for multiple pairwise comparisons. Data obtainedin the experiments with additional mu opioid agonists and dextror-

phan were analyzed by a two-way ANOVA with repeated measureson both the agonist dose and the amphetamine dose. Tukey’s pro-tected t tests were performed on these data following the statisticalanalysis. Two-hour rotation totals were subjected to a one-way re-peated measures ANOVA. The Friedman’s ANOVA was used tocompare the overall theoretical additive values of the mu opioid incombination with amphetamine to the observed behavior. The Wil-coxon matched-pairs test was then performed on each pair of theo-retical and observed values for each of the mu agonist doses. In theantagonism experiments, data were analyzed with a repeated mea-sures ANOVA with one factor, followed by a Tukey’s protected t test.All time-course data were analyzed by a three-way ANOVA withrepeated measures on all three factors (morphine dose, stimulantdose, time period). Results were considered to be statistically signif-icant if the P value was #.05.

Drugs. Buprenorphine hydrochloride (National Institute on DrugAbuse, Rockville, MD), dextrorphan tartrate and levorphanol tar-trate (Roche Laboratories, Nutley, NJ) were dissolved in distilledwater. d-Amphetamine sulfate, and naltrexone hydrochloride (Sig-ma Chemical Co., St. Louis, MO), cocaine hydrochloride (NationalInstitute on Drug Abuse), fentanyl citrate (McNeil Laboratories, FortWashington, PA), methadone hydrochloride (Mallinkrodt, St. Louis,MO), meperidine hydrochloride and morphine sulfate (Penick Corp.,Newark, NJ) were dissolved in 0.9% saline. R(2)-Apomorphine hy-

Fig. 2. Morphine (0.1–10 mg/kg) potentiated ipsilateral turning behavior induced by cocaine (3.0–30 mg/kg). A three-way ANOVA of thetime-course values (A) showed that all three variables as well as all two- and three-factor interactions were significant: cocaine: F(3,21) 5 19.39,P , .0001; morphine: F(5,35) 5 21.00, P , .0001; time: F(15,105) 5 79.64, P , .0001; cocaine 3 morphine: F(15,105) 5 2.75, P , .0013; cocaine 3time: F(45,315) 5 16.57, P , .0001; morphine 3 time: F(75,525) 5 5.33, P , .0001; cocaine 3 morphine 3 time: F(225,1575) 5 2.77, P , .0001.Total full ipsilateral turning during the 4-hr experimental period (B) was influenced by cocaine (F(3,28) 5 16.55, P , .0001) and morphine (F(5,191)5 34.85, P , .0001) as well as their interaction (F(15,191) 5 2.29, P 5 .0062). Other details are as in figure 1.

Fig. 3. Turning was induced by mu opioid agonists and dextrorphan,but not naltrexone. Asterisks represent drug doses that produced turn-ing significantly greater than the vehicle control, **P , .01, *P , .05.


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drochloride (Research Biochemicals, Inc., Natick, MA) and 6-OHDAhydrobromide (Sigma) were dissolved in a solution of 0.02% ascorbicacid in 0.9% saline. All drugs except for 6-OHDA and 30 mg/kg ofmeperidine were administered in a volume of 1.0 ml/kg b.wt., with alldoses expressed as the free base. The highest dose of meperidine (30mg/kg) was given in 3.0 ml/kg b.wt. to avoid skin lesions, whichoccurred with concentrations of meperidine higher than 10 mg/ml.

ResultsEffects of morphine on stimulant-induced turning

behavior. Amphetamine alone produced dose-dependent ip-silateral turning (fig. 1). The lowest doses of the drug (0.1 and0.3 mg/kg) had only a slight effect on circling behavior,whereas 1.0 mg/kg produced approximately 250 full ipsilat-eral turns in a 4-hr observation period, significantly greaterthan saline (P , .01). In these animals, morphine aloneproduced significant ipsilateral turning as an overall drugeffect; however, no single dose elicited turning greater thanturning after saline 1 saline (fig. 1B).

Morphine modified amphetamine-induced circling in adose-dependent manner that was more pronounced as thedose of amphetamine increased. When 1.0 mg/kg amphet-amine was administered with 3.0 mg/kg morphine, the

amount of full ipsilateral turning during 4 hr increased from250 to nearly 1000 turns (fig. 1B). The time course of effectsis illustrated for the 3.0 mg/kg dose of morphine in figure 1A.The combination of 3.0 mg/kg morphine and 1.0 mg/kg am-phetamine shifted the peak circling effect from 30 min afterinjection to 90 min after injection, and increased the maxi-mum turning in a 15-min interval from 45 turns to 110 turns.In this experiment and those that follow, no contralateralturning was observed.

Cocaine alone also produced ipsilateral turning in a dose-dependent manner (fig. 2). At 30 mg/kg, the highest dosestudied, 400 turns were recorded during the 4-hr observationperiod, slightly more than the effects produced by 1.0 mg/kgamphetamine. This dose of cocaine was the only one exam-ined that produced turning significantly greater than salinealone (P , .05). Morphine produced significant ipsilateralturning, and 3.0 mg/kg produced turning significantlygreater than saline did (P , .01) (fig. 2B), in contrast to theresults described in figure 1. The effect of this dose of mor-phine had a plateau of approximately 45 turns/15 min for 150min, which tapered off during the final 90 min (fig. 2A). Theeffects of cocaine were dose-dependently affected by mor-phine. The combination of 3.0 mg/kg morphine and 30 mg/kg

Fig. 4. Methadone (0.3–3.0 mg/kg) potentiated amphetamine-induced ipsilateral turning, with a significant effect of its own. Part A shows thetime-course effects of the saline control and one test dose of methadone (1.0 mg/kg). (The others were omitted for clarity.) Each data pointrepresents the mean full ipsilateral counts during that 15-min time block (n 5 8). The P value shown represents the amphetamine 3 methadone 3time interaction determined by a three-factor ANOVA, with all doses of methadone tested. Part B shows the same data as in A, but collapsed into2-hr time blocks in the first two panels, and then total ipsilateral turns during the entire 4-hr observation time in the third panel. In the first twopanels of B, asterisks denote a significant difference between that dose of methadone and the saline control (within the saline group and theamphetamine group), **P , .01, *P , .05. In the third panel, asterisks denote a significant difference between amphetamine alone and theamphetamine 1 methadone combination, **P , .01, *P , .05. The P values shown here were determined by a two-factor ANOVA (methadonedose 3 amphetamine dose).


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cocaine was the most effective of the doses examined inproducing ipsilateral circling. The 3.0 mg/kg morphine dosedid not shift the overall peak effect of 30 mg/kg cocaine fromthe first 15-min period, but it increased the maximum turn-ing observed during this 15-min period from 75 to 125 turns(fig. 2A). Turning was also prolonged, lasting the entire 240min, rather than ceasing after 150 min, as it did after 30mg/kg of cocaine alone. This drug combination produced 1400turns/4 hr, greater than a 3-fold increase in the amount ofturning seen with this dose of cocaine alone (fig. 2B). Thecombination of a higher dose of cocaine (56 mg/kg) and 3.0mg/kg morphine was lethal in some animals (data notshown), so we discontinued testing this pair of drug doses.

Effects of mu opioid receptor agonists alone and onamphetamine-induced turning. When doses of the muagonists were tested alone, each one induced significant ip-silateral turning across all doses during the 4-hr experimen-tal session (fig. 3). Buprenorphine at 0.1 and 1.0 mg/kg pro-duced turning greater than that occurring after saline (P ,.01) and the most of any of the mu opioid receptor agonistsexamined, with a peak effect of nearly 500 turns/4 hr. Levor-phanol at 0.3 and 1.0 mg/kg also produced significant turning(P , .01), as did the 30 mg/kg dose of meperidine (P , .05).These two mu opioid agonists produced a maximum effect ofapproximately 250 turns/4 hr. No single dose of fentanyl,methadone and morphine produced turning significantlygreater than saline 1 saline, although there was a significantoverall effect. Unlike the other drugs tested, fentanyl pro-

duced severe catalepsy and rigidity. Dextrorphan producedless turning than did its optical isomer, levorphanol, even ata dose that was 10 to 30 times higher than doses of levor-phanol that produced turning. The nonselective opioid recep-tor antagonist, naltrexone, did not produce rotational behav-ior at doses of 0.1 to 10 mg/kg.

Several doses of each of the mu opioid agonists were ad-ministered along with 1.0 mg/kg amphetamine. The time-course data were analyzed to determine whether there was asignificant interaction between the mu opioid agonist of in-terest and amphetamine. The interactions between time andamphetamine and methadone (fig. 4A) and morphine (fig.5A) were significant, as determined by a three-factor ANOVA(mu opioid agonist dose 3 amphetamine dose 3 time). How-ever, in the cases of buprenorphine (fig. 6A), fentanyl (fig.7A), meperidine (fig. 8A) and levorphanol (fig. 9A) and itsstereoisomer dextrorphan (fig. 10A), this interaction termwas not significant. Nevertheless, with the exception of fen-tanyl, these drugs increased the duration of action of amphet-amine from approximately 180 min to more than 240 min.The data presented in figure 5 for morphine are derived fromthose in figure 1, and they are shown here to facilitate com-parison with the other agonists studied.

We examined the effects of the mu opioid agonists aloneand combined with amphetamine on full ipsilateral turningduring 2-hr time blocks as well as during the full 4-hr obser-vation. These data are the same as those in the time-coursegraphs, but they are collapsed into 2-hr and 4-hr blocks,

Fig. 5. Morphine (1.0–10 mg/kg) enhanced amphetamine-induced turning and had a considerable effect alone. (The data in these graphs areredrawn from the data in figure 1 for comparison with the other drugs used in this study.) Graphs are as described in figure 4.


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respectively, for further analysis. When amphetamine waspreceded by methadone (fig. 4B), morphine (fig. 5B), bu-prenorphine (fig. 6B) and dextrorphan (fig. 10B), turning wasaugmented significantly during both 2-hr periods. However,the effects of fentanyl (fig. 7B) and meperidine (fig. 8B)occurred only during the final 2 hr. Levorphanol did notsignificantly increase amphetamine-induced turning in ei-ther 2-hr time block, although there was a trend in thatdirection (fig. 9B).

A two-factor ANOVA (mu opioid agonist 3 amphetamine)indicated that there was a significant main effect of amphet-amine alone, the mu opioid agonists alone and dextrorphanalone upon turning over the full 4 hr. However, the interac-tion term with amphetamine was significant only for meth-adone (fig. 4B) and morphine (fig. 5B), which indicated thatamphetamine changed the shape of the dose-response curvesof these two mu opioid agonists. To examine whether thesedrugs significantly potentiated the effects of amphetamine,other statistical methods must be used.

Synergism of mu opioid agonists with amphetamine.To determine whether the observed potentiation of amphet-amine-induced turning by the mu opioid agonists and dex-trorphan was a simple additive effect or an effect that wasgreater than additive (i.e., synergistic), we summed the totalturns produced by each mu opioid agonist and dextrorphanalone with the total turns produced by amphetamine alonefor each individual animal. These sums were averaged toproduce a mean and standard error for each drug combina-

tion (table 1). Because the variance of the means in the tablewas not homogeneous, we used nonparametric statisticalmethods to analyze these data. These values were comparedwith the total of full ipsilateral turns observed when the twodrugs were actually coadministered.

A Friedman’s ANOVA (calculated vs. observed rotations 3dose) on these data indicated there was an overall significantdifference between the predicted and observed amounts ofturning induced by the drugs tested in combination with 1.0mg/kg amphetamine, with the exception of buprenorphineand fentanyl (table 1). A follow-up analysis of individualdoses with a Wilcoxon matched-pairs test revealed that themiddle doses of dextrorphan (3.0 mg/kg), levorphanol (0.3mg/kg), meperidine (10 mg/kg), methadone (1.0 mg/kg) andmorphine (3.0 mg/kg), as well as the higher dose of dextror-phan (10 mg/kg) in combination with amphetamine, pro-duced turning greater than predicted, P # .05 (table 1).

Antagonism of mu opioid agonist effects. Naltrexone(1.0 mg/kg) blocked the effects of 3.0 mg/kg morphine uponamphetamine-induced rotational behavior (fig. 11). Post hoctests revealed that there was no statistical difference be-tween turning induced by amphetamine alone and by nal-trexone combined with morphine and amphetamine. Furthertesting revealed that the effects of 3.0 mg/kg morphine on 1.0mg/kg amphetamine were blocked by a lower dose of naltrex-one, 0.1 mg/kg. However, 1.0 mg/kg naltrexone did not sta-tistically alter the effects of the other mu opioid agonists ordextrorphan on amphetamine-induced turning. We tested a

Fig. 6. Buprenorphine (0.01–1.0 mg/kg) potentiated turning induced by amphetamine although it had a significant effect alone. Graphs are asdescribed in figure 4.


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higher dose of naltrexone, 10 mg/kg, with the combination of1.0 mg/kg methadone and 1.0 mg/kg amphetamine, but it didnot significantly alter rotational behavior induced by thisdrug combination (data not shown).

Inspection of the data in figure 11 suggested that thecombination of 1.0 mg/kg naltrexone and 1.0 mg/kg amphet-amine often resulted in greater turning than occurred afteramphetamine alone. This apparent interaction was not sta-tistically significant within each individual drug series. How-ever, when data were pooled from all 24 subjects, 1.0 mg/kgnaltrexone significantly potentiated turning induced by 1.0mg/kg amphetamine (P ,.05) (fig. 12). The highest dose ofnaltrexone (10 mg/kg) also potentiated amphetamine-in-duced circling (P ,.01), but the lowest (0.1 mg/kg) did not(fig. 12). None of these doses of naltrexone alone producedturning behavior (fig. 3).

DiscussionThe initial experiments in this study showed that mor-

phine potentiated rotational behavior induced by both am-phetamine and cocaine by 3- to 4-fold. Both of these stimu-lant drugs are indirect dopamine agonists with differentmechanisms of action. Amphetamine preferentially releasesnewly synthesized dopamine from presynaptic cells (Arbuth-nott et al., 1991; Kuczenski and Segal, 1989; Zetterstrom etal., 1986b). Cocaine acts by blocking the presynaptic dopa-mine transporter, thus blocking the reuptake of synaptic

dopamine. Some differences have been found in the modula-tion of locomotor effects of these two drugs by the nonselec-tive opioid receptor antagonist naloxone (Jones and Holtz-man, 1994), which suggests that modulation of the effects ofamphetamine by endogenous opioids also differs from that ofcocaine. However, our data indicate that the effects of bothamphetamine and cocaine are enhanced by mu opioid recep-tor agonists, although the exact mechanism(s) by which thatoccurs is not clear. The results correspond with those ofseveral other studies in which there was an apparent synergybetween cocaine and morphine-like opioids (see the introduc-tion).

To determine whether the effects of morphine on stimu-lant-induced rotational behavior were specific to this drug orgeneral to agonists at the mu opioid receptor, we examinedthe effects of several other mu opioid agonists. Each of thesedrugs produced ipsilateral turning when administered alone,as reflected in the significant main effect of dose. Mu opioidreceptors are localized on dopaminergic nerve terminals inthe striatum (Mansour et al., 1995), and increase the releaseof dopamine from these cells upon stimulation (Kalivas andStewart, 1991). Presumably, these mu opioid agonists pro-duced ipsilateral turning by increasing synaptic levels ofdopamine in an indirect fashion. The order of potency of thesedrugs in producing turning paralleled their order of potencyin other assays of drug interactions with the mu opioid re-ceptor (Holtzman and Locke, 1988). Morphine alone pro-

Fig. 7. Fentanyl (0.01–0.056 mg/kg) potentiated amphetamine-induced circling only slightly, and had a small effect of its own. Graphs are asdescribed in figure 4.


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duced relatively little turning in the series of experimentswith amphetamine, in agreement with previous results(Kimmel et al., 1995). However, morphine had a larger effectin the series of experiments with cocaine. Each animal inthat group was exposed to cocaine 24 times during the ran-domized sequence of tests with cocaine alone, morphine aloneand morphine-cocaine combinations. It is possible that thisexposure to cocaine sensitized the animals to the effects ofmorphine more than did comparable exposure to amphet-amine, thus accounting for the different outcomes.

Methadone had a significant overall effect on rotationalbehavior, although no single dose produced significant turn-ing, and increased amphetamine-induced rotational behav-ior. A dose of 1.0 mg/kg produced effects greater thanpredicted by simple additivity. In the conditioned place pref-erence paradigm, methadone enhanced the reinforcing prop-erties of cocaine in rats (Bilsky et al., 1992). Similarly, inhumans, cocaine use was greater in methadone-treated opi-oid addicts than in buprenorphine- or naltrexone-treated pa-tients (Kosten et al., 1989). The discriminative stimulus ef-fects of cocaine in squirrel monkeys were also enhanced bymethadone (Spealman and Bergman, 1992, 1994). This muopioid agonist increases synaptic dopamine levels in the nu-cleus accumbens (Di Chiara and Imperato, 1988b). The datain the present study concur with the findings that methadoneenhances dopaminergic functions.

Buprenorphine is a partial agonist at the mu opioid recep-tor and an antagonist at the kappa receptor (Cowan et al.,

1977; Leander, 1987; Negus and Dykstra, 1988; Negus et al.,1990). Evidence for interactions of buprenorphine with drugsthat act via the dopaminergic system has been conflicting.For example, buprenorphine attenuated cocaine self-admin-istration in humans (Foltin and Fischman, 1994), rhesusmonkeys (Mello et al., 1992, 1993) and rats (Brown et al.,1991; Carroll and Lac, 1992; Dykstra et al., 1992), and atten-uated cocaine conditioned place preference in rats in twostudies (Kosten et al., 1991; Suzuki et al., 1992), but aug-mented cocaine-induced conditioned place preference in an-other study (Brown et al., 1991). Buprenorphine alone in-creased locomotor activity in mice, but did not potentiate orattenuate locomotor activity induced by cocaine (Jackson etal., 1993). Both cocaine and buprenorphine increased dopa-mine release in the nucleus accumbens when administeredseparately and together (Brown et al., 1991). In the presentexperiments, buprenorphine produced rotational behavioralone and increased amphetamine-induced circling. Theseresults lend support to the studies which indicate that bu-prenorphine increases dopamine release, producing behav-iors that are associated with this neurochemical event. Thisaugmented release of dopamine is possibly a result of bu-prenorphine’s actions on both the mu and kappa opioid re-ceptors. Based on microdialysis data, stimulation of the muopioid receptor would result in increased synaptic dopaminelevels, whereas inhibiting the kappa opioid receptor wouldremove its inhibitory effects upon dopamine release (Di Chi-ara and Imperato, 1988b). Perhaps it is because of its dual

Fig. 8. Meperidine (3.0–30 mg/kg) augmented amphetamine-induced turning behavior and induced turning by itself. Graphs are as described infigure 4.


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actions at mu and kappa opioid receptors that buprenorphineproduced the most turning of any of the opioids that wetested.

Fentanyl did not increase the effects of amphetamine inthe present study, and in the rat drug discrimination para-digm, it did not substitute for cocaine or alter the effects ofcocaine when given as a pretreatment (Broadbent et al.,1995). However, in monkeys, fentanyl potentiated the dis-criminative effects of cocaine, as evidenced by a leftward shiftof the cocaine stimulus curve (Spealman and Bergman, 1992,1994). In vivo microdialysis studies showed that this opioidagonist increased dopamine release in the nucleus accum-bens (Di Chiara and Imperato, 1988b).

In addition to examining the effects of mu opioid agonistsupon rotational behavior, we tested dextrorphan, the opticalisomer of levorphanol. Dextrorphan has little affinity for themu opioid receptors and lacks significant analgesic and otheropioid effects (Jaffe and Martin, 1985), and alone producedfewer rotations than did levorphanol alone. Surprisingly, incombination with amphetamine, dextrorphan producedslightly more turning behavior than did levorphanol. Theeffect of dextrorphan on stimulant-induced turning may bebecause of its interaction at the PCP recognition site of theNMDA glutamate receptor (Murray and Leid, 1984; Sun etal., 1986).

Based on the model of simple arithmetic additivity, dex-trorphan, levorphanol, meperidine, methadone and mor-

phine potentiated the effects of amphetamine on circling.Although this “effect-addition” model is not without limita-tions (Woolverton, 1987), it is a reasonable way to approachthe question of drug synergy in the absence of more specificstatistical methods. The fact that the mu opioid agonistsoften increased the effects of amphetamine at times when theopioid itself had little or no effect (e.g., during the second halfof the 4-hr session in the case of most of the drugs, figs. 4B,5B, 6B, 7B, 8B) suggests a true synergy.

Naltrexone, a nonspecific opioid receptor antagonist,blocked the interaction between morphine and amphet-amine, but not that between the other mu opioid agonists andamphetamine. The reason for this is not clear. However, theoutcomes might have been confounded by an interaction be-tween naltrexone and amphetamine. When 0.1 mg/kg nal-trexone was administered with amphetamine, there were nosignificant effects on the stimulant-induced turning behav-ior. However, higher doses potentiated amphetamine-in-duced circling. This observation conflicts with findings thatthe opioid antagonist naloxone can decrease amphetamine-induced locomotor activity (Hooks et al., 1992; Jones andHoltzman, 1992, 1994) and dopamine release in rats (Schadet al., 1995). Kappa opioid receptor activation decreases do-pamine release (Di Chiara and Imperato, 1988b). Perhaps byblocking kappa opioid receptors, naltrexone augments therelease of dopamine. Thus, naltrexone might have blockedthe potentiating effect of the mu opioid agonist while increas-

Fig. 9. Levorphanol (0.1–1.0 mg/kg) increased ipsilateral turning observed with amphetamine, and produced a significant amount of turning onits own. Graphs are as described in figure 4.


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ing the dopamine response to amphetamine, which resultedin no net change in turning. However, even if this speculationwas to prove correct, it would leave unresolved the reasonthat naltrexone did block the amphetamine-potentiating ef-fect of morphine.

In summary, morphine increases rotational behavior in-duced by amphetamine and cocaine. However, the role ofopioid receptors in this interaction is unresolved. Resultswith other mu opioid agonists were consistent among drugsin that they all increased amphetamine-induced circling, butthis interaction was not greater than additive in all cases. Inaddition, each of the mu opioid agonists studied induced

turning by themselves, although not always at any one dose.The opioid antagonist naltrexone blocked the effects of mor-phine but not those of the other mu opioids. Although wecannot draw conclusions about the role of opioid receptors inthe effects of mu opioid agonists on turning induced by psy-chomotor stimulant drugs, it is, nevertheless, clear thatthese opioids can enhance a dopamine-mediated behavioraleffect of amphetamine and cocaine.

Acknowledgments

The authors extend their appreciation for the generous contribu-tion of dextrorphan tartrate and levorphanol tartrate by Roche Lab-

Fig. 10. Dextrorphan (1.0–10 mg/kg), a stereoisomer of levorphanol, potentiated amphetamine-induced ipsilateral turning and had an effect oncircling behavior alone. Graphs are as described in figure 4.

TABLE 1Synergism of mu opioid agonists and amphetamineTheoretical number of full ipsilateral turns expected when the mu opioid agonist or dextrorphan was administered with 1.0 mg/kg amphetamine vs. observed turningwith the combination. All values represent the mean 6 S.E. P values in the last column denote the statistical difference between the calculated and observed valuesat all doses of one mu opioid agonist plus 1.0 mg/kg amphetamine as determined by Freidman two-way ANOVA by rank analyses.

Calculated ObservedP

valueLow Medium High Low Medium High

Buprenorphine 559 6 149 804 6 107 885 6 128 800 6 180 931 6 57 706 6 138 .313Dextrorphan 522 6 115 585 6 128 625 6 117 702 6 135 791 6 180a 1302 6 371a .007Fentanyl 209 6 46 254 6 49 217 6 57 305 6 68 402 6 124 220 6 62 .837Levorphanol 369 6 84 554 6 125 549 6 91 617 6 79 705 6 140a 742 6 124 .018Meperidine 504 6 106 490 6 110 709 6 142 475 6 112 780 6 119a 849 6 228 .005Methadone 343 6 70 453 6 76 475 6 69 493 6 72 866 6 184a 785 6 167 .003Morphine 359 6 132 444 6 98 421 6 83 652 6 157 928 6 166a 388 6 98 .002a Significantly greater than calculated, P .05.


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oratories, of fentanyl citrate by McNeil Laboratories, and of meper-idine hydrochloride and morphine hydrochloride by Penick Corp. Wealso thank Dr. Ronald J. Tallarida for helpful discussions regardingstatistical analyses of the drug interaction data.

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