Introduction

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Heroin is the most widely abused opioid and is associated with the highest mortality of all illicit drugs (United Nations International Drug Control Programme, 1997; Bammer et al., 1999). Although heroin can be smoked or snorted, i.v. injection continues to be the predominant method of use. After i.v. administration, addicts typically describe the subjective experience of heroin as a "rush", followed by a sense of tranquility, reduced apprehension, and euphoria (Jasinski and Preston, 1986; Comer et al., 1999). Drug discrimination procedures in laboratory animals provide a useful experimental counterpart to measures of subjective effects in humans. Despite the prevalence of heroin abuse, surprisingly few studies have assessed the discriminative stimulus (DS) effects of heroin, and to date, there are no reported investigations of the DS effects of heroin when administered by the i.v. route.

Heroin can be considered a prodrug that is transformed to several metabolites that likely contribute to its behavioral effects (Umans and Inturrisi, 1981; Corrigall and Coen, 1990). After i.v. or other peripheral routes of administration, heroin is rapidly metabolized by sequential deacetylation to 6-monoacetylmorphine (6 MAM) and morphine (Kamendulis et al., 1996). Morphine, in turn, is metabolized via glucuronidation to morphine-6--D-glucuronide (M6G) and morphine-3--D-glucuronide (M3G) (Glare and Walsh, 1991; Milne et al., 1996). Previous studies have shown that some of these metabolites may contribute to the subjective effects of heroin. For example, both 6 MAM and morphine have been shown to engender heroin-like DS effects in rats trained to discriminate heroin from vehicle by the s.c. route (Corrigall and Coen, 1990). Similarly, M6G has been found to engender drug-appropriate responding in rats trained to discriminate s.c. morphine from vehicle (Easterling and Holtzman, 1998).

Opioid receptors traditionally have been classified into µ-, -, and -subtypes (Martin et al., 1976). Although heroin displays little selectivity at these receptor subtypes, the in vivo effects of heroin have been attributed predominantly to µ-opioid receptor activation (Bertalmio et al., 1992). In some situations, however, -opioid receptors also may play a role in the effects of heroin (Rady et al., 1994; Uchihashi et al., 1996). Rady et al. (1994), for example, demonstrated that the -opioid receptor antagonist naltrindole blocked the antinociceptive and locomotor stimulant effects of heroin in rodents. Thus, the first purpose of the present study was to evaluate the role of opioid receptor mechanisms in the DS effects of i.v. heroin. This was accomplished by conducting substitution tests with selective µ-, -, and -opioid agonists. Additional antagonism studies, in conjunction with in vivo apparent pA₂ analysis, were conducted with the opioid antagonist naltrexone.

Compared with other heroin metabolites, M3G binds weakly to opioid receptors (Mignat et al., 1995; Löser et al., 1996). Moreover, M3G exhibits little analgesic activity (Gong et al., 1991; Easterling and Holtzman, 1998) and may have effects on respiration opposite to those of conventional µ-opioid agonists (Gong et al., 1991). For example, Gong et al. (1991) found that administration of morphine or M6G reduced respiratory frequency in anesthetized rats, whereas M3G increased respiratory frequency. Our second purpose, then, was to characterize the role of heroin's metabolites in its subjective effects by assessing the capacity of the heroin metabolites 6 MAM, morphine, M6G, and M3G to engender heroin-like responding.

Although opioid receptor stimulation undoubtedly plays a key role in the behavioral effects of heroin, several µ-opioid agonists have been shown to stimulate release of dopamine (DA) in mesolimbic brain regions, a mechanism that may also contribute importantly to the behavioral effects of these drugs (Di Chiara and North, 1992; Wise, 1998). Moreover, other compounds that stimulate DA release or block its uptake such as cocaine and methamphetamine have been shown to share DS effects with µ-opioid agonists under certain conditions (Lamas et al., 1998; Platt et al., 1999; Rowlett et al., 2000), raising the possibility that stimulation of DA activity contributes at least indirectly to the DS effects of heroin. Along these lines, Platt et al. (1999) previously found the morphine DS to generalize to cocaine, amphetamine, and the selective DA uptake blocker GBR 12909 in the majority of squirrel monkeys tested. The final purpose of our study, then, was to determine the contribution of DA receptor stimulation to the DS effects of heroin by investigating the ability of prototypical DA releasers and uptake blockers to mimic the DS effects of heroin.

	Materials and Methods

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Subjects and Surgical Procedure. Four male rhesus monkeys (Macaca mulatta), weighing 5.7 to 6.8 kg, were studied in daily experimental sessions (Monday to Friday). All were experimentally naïve at the beginning of the study. Between sessions, monkeys lived in individual home cages where they had unlimited access to water. Monkeys were maintained at 85 to 90% of their free-feeding body weight by adjusting their access to food in the home cage (Lab Diet 5038; PMI Nutrition International, Inc., Brentwood, MO, supplemented with fresh fruit). 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, revised 1996. Research protocols were approved by the Harvard Medical School Institutional Animal Care and Use Committee.

Apparatus. Experimental sessions were conducted in ventilated and sound-attenuating chambers. Monkeys were seated in custom-made Plexiglas primate chairs (Crist Instrument Co., Hagerstown, MD). Two response levers (model ENV-610 M, MED Associates, Georgia, VT) were mounted 16 cm apart on the wall of the chamber in front of the monkey. Each press of a lever with a minimum downward force of approximately 0.25 N produced an audible click and was recorded as a response. Food pellets (Formula 0094, 1 g; Bioserve, Frenchtown, NJ) could be delivered to a tray located between the levers. Colored lights mounted above the levers could be illuminated to serve as visual stimuli.

Heroin Discrimination Procedure. Monkeys initially were trained to respond on each of two levers under a 10-response fixed ratio (FR 10) schedule of food reinforcement. Once consistent lever pressing was established, the monkeys were implanted with intravenous catheters, and drug discrimination training was started 2 to 4 days later. The training dose of heroin initially was 0.03 mg/kg for all subjects, but subsequently was increased to 0.1 mg/kg for monkeys M-163 and M-426 to achieve consistent stimulus control of behavior. After an i.v. injection of heroin, 10 consecutive responses on one lever produced a food pellet, whereas after an i.v. injection of saline, 10 consecutive responses on the other lever produced a pellet. For half of the monkeys, responding on the right lever after an injection of heroin resulted in pellet delivery. For the other monkeys, responding on the left lever after injection of heroin was reinforced. Delivery of each pellet was followed by a 10-s timeout period. Responses on the incorrect lever (e.g., the saline-appropriate lever after heroin injection) reset the FR requirement.

Drug Testing Procedure. Once consistent stimulus control was achieved, drug test sessions were conducted once or twice per week with training sessions scheduled on intervening days. Test sessions were conducted only if 80% of responses were made on the injection-appropriate lever during at least four of the preceding five training sessions. Test sessions consisted of four FR components, each preceded by a 10-min timeout period. During 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. The drugs studied were heroin (0.001-0.1 mg/kg), 6 MAM (0.003-0.3 mg/kg), morphine (0.01-0.56 mg/kg), M3G (0.1-10.0 mg/kg), M6G (0.1-10.0 mg/kg), fentanyl (0.0003-0.018 mg/kg), methadone (0.03-1.0 mg/kg), SNC 80 (0.003-0.3 mg/kg), spiradoline (0.0003-0.03 mg/kg), cocaine (0.01-1.0 mg/kg), GBR 12909 (0.1-1.0 mg/kg), and methamphetamine (0.01-0.3 mg/kg). Under the cumulative dosing procedure, incremental doses of each drug (1/4-1/2 log increments) were injected i.v. during timeout periods that preceded sequential FR components, permitting a four-point cumulative dose-response function to be determined in a single session. When warranted, five or more different doses of a drug were studied by administering overlapping ranges of cumulative doses during test sessions on different days. The effects of most doses were determined twice, although low, inactive doses and high doses that produced adverse effects were usually studied only once in each subject. Antagonism studies were conducted by administering naltrexone (0.003-0.1 mg/kg i.m.) 5 min before the session, followed by cumulative doses of heroin, 6 MAM, morphine, M6G, and M3G as described above. In addition to establishing a cumulative dose-response function for heroin, a conventional single-dose testing procedure with varying pretreatment times (5, 20, 80, 160, and 320 min) was used to determine the time course of the DS effects of heroin (M-164 and M-216, 0.03 mg/kg heroin; M-163 and M-426, 0.1 mg/kg heroin).

Analysis of Drug Effects. Percentage of heroin-lever responding was computed for individual subjects in each component of a test session by dividing the number of responses on the heroin lever by the total number of responses on both levers and multiplying by 100. Percentage of heroin-lever responding was calculated for an individual monkey only if the response rate was >0.1 responses/s during the component. Mean percentage of heroin-lever responding and S.E.M. were then calculated for the group of monkeys at each dose. A drug was considered to substitute fully for heroin if the maximum percentage of drug-lever responding was 80%.

Drugs. Heroin hydrochloride, 6-monoacetylmorphine base, morphine-6--D-glucuronide base, and morphine-3--D-glucuronide base were provided by the National Institute of Drug Abuse (Rockville, MD). Other drugs were purchased from commercial sources: morphine sulfate, fentanyl citrate, spiradoline mesylate, (+)-methamphetamine hydrochloride, naltrexone hydrochloride, GBR 12909 dihydrochloride, and cocaine hydrochloride (Sigma Chemical, St. Louis, MO); (±)-methadone hydrochloride (Sandoz, Basel, Switzerland); and SNC 80 base (Tocris Cookson, Ballwin, MO). All drugs were dissolved in small amounts of 0.1 N HCl as required and diluted to the desired concentrations with sterile water or 0.9% saline solution.

	Results

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Intravenous Heroin Discrimination. Two monkeys (M-164 and M-216) acquired the i.v. heroin (0.03 mg/kg) discrimination after 50 and 98 sessions, respectively. The remaining two subjects (M-163 and M-426) initially acquired the i.v. heroin discrimination after 54 and 97 sessions, respectively, but then required additional training, for an average of at least 15 sessions, with 0.1 mg/kg heroin to maintain consistent stimulus control of behavior. Despite the different training doses (0.03 versus 0.1 mg/kg), no systematic differences in drug effects were noted during the study, and consequently, all data are presented as means for the group of four subjects.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Percentage of heroin-lever responding (mean ± S.E.M.) engendered by heroin in the presence and absence of naltrexone in rhesus monkeys (n = 4) trained to discriminate heroin from saline (top). Naltrexone was administered i.m. 5 min before sessions in which cumulative doses of heroin were tested. Points above Sal show the effects of saline alone or combined with different doses of naltrexone. The bottom panel shows a Schild plot for naltrexone antagonism of the DS effects of heroin. DR is the ED50 for heroin plus naltrexone divided by the ED50 for heroin alone. , heroin alone; , +0.003 naltrexone; , +0.01 naltrexone; , +0.03 naltrexone; , +0.1 naltrexone.

Effects of Metabolites of Heroin. The deacetylated metabolites of heroin, 6 MAM and morphine, had DS effects that were qualitatively similar to those of heroin (Fig. 2, top, closed symbols). Increasing cumulative doses of both 6 MAM and morphine engendered dose-related increases in the percentage of responses on the heroin-associated lever, with one or more doses substituting fully for heroin in 3 of 4 monkeys (6 MAM) and 4 of 4 monkeys (morphine). As in the case of heroin, these effects were observed after administration of doses of 6 MAM and morphine that did not markedly alter rate of responding (Table 1).

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Percentage of heroin-lever responding (mean ± S.E.M.) for 6 MAM, morphine, M3G, and M6G in the presence and absence of 0.01 mg/kg naltrexone in rhesus monkeys (n = 4) trained to discriminate heroin from saline. Naltrexone was administered i.m. 5 min before sessions in which cumulative doses of 6 MAM, morphine, M3G, and M6G were tested. , alone; , +0.01 naltrexone.

Effects of Other Opioids, DA Releasers, and DA Uptake Blockers. Fentanyl, an agonist with high selectivity for µ- over - and -opioid receptors, as well as methadone, a moderately selective µ-opioid receptor agonist, had DS effects that were qualitatively similar to those of heroin (Fig. 3). Increasing cumulative doses of fentanyl and methadone engendered dose-related increases in the percentage of responses on the heroin-associated lever, with full substitution for heroin observed in each subject at doses that did not markedly alter response rate (Table 1). In contrast, the selective -agonist spiradoline and the selective -agonist SNC 80 did not engender consistent responding on the heroin-associated lever in any subject, regardless of dose (Fig. 3). Spiradoline produced an average maximum of 33% heroin-lever responding, and SNC 80 engendered an average maximum of 19% heroin-appropriate responding at doses that severely reduced responding (Table 1).

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Percentage of heroin-lever responding (mean ± S.E.M.) for fentanyl, spiradoline, SNC 80, and methadone in rhesus monkeys (n = 4) trained to discriminate heroin from saline. Points above Sal show the effects of saline. , methadone; , fentanyl; , spiradoline; , SNC 80.

	Discussion

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Despite the well characterized subjective effects of heroin in humans and the recognized relevance of these effects for heroin addiction, comparatively little is known about the mechanism of action and time course of the DS effects of heroin in animals. In the present study, i.v. heroin was successfully established as a DS in nonhuman primates. Consistent with clinical reports of peak plasma levels of heroin occurring 5 min postinjection (Jenkins et al., 1994), the onset of the DS effects of heroin in monkeys was rapid and endured for 1 to 2 h.

Role of Metabolites in DS Effects of Heroin. The deacetylated metabolites of heroin, 6 MAM and morphine, as well as the glucuronated metabolites, M6G and M3G, substituted fully for the DS effects of heroin. The rank order of potency for heroin and its metabolites to engender heroin-lever responding was heroin > 6 MAM > morphine > M3G  M6G. This order is consistent with the rank order of potency observed for these opioids in other in vivo procedures (e.g., antinociception, respiratory depression; Umans and Inturrisi, 1981; Corrigall and Coen, 1990; Easterling and Holtzman, 1998). The observed potency differences, however, do not correspond to differences in binding affinity at the µ-opioid receptor because 6 MAM, morphine, and M6G have higher affinities at this receptor than does heroin (Bertalmio et al., 1992; Mignat et al., 1995). More likely, the potency differences reflect the ability of these compounds to penetrate the central nervous system. For example, in contrast to heroin and 6 MAM, morphine, M6G, and M3G penetrate the central nervous system more slowly after peripheral administration (Umans and Inturrisi, 1981; Bickel et al., 1996; Wu et al., 1997). The observation that several of the metabolites of heroin exhibit relatively slow brain penetration, yet high affinity at µ-opioid receptors, may explain why the DS effects of heroin persist for 1 to 2 h, even though it is metabolized rapidly itself (Jenkins et al., 1994).

Heroin-Like Effects of Morphine-3--D-Glucuronide. M3G engendered full heroin-like DS effects in all animals. Moreover, the heroin-like DS effects of M3G could be blocked by the opioid antagonist naltrexone, indicating that these effects were mediated via stimulation of opioid receptors. These findings were unexpected in light of previous reports that M3G binds only weakly at µ-opioid receptors (Löser et al., 1996) and exhibits limited µ-agonist activity. For example, M3G has been shown to engender only low levels of drug-lever responding in rats discriminating i.m. morphine from saline (Easterling and Holtzman, 1998). However, recent studies have provided evidence for µ-agonist effects of M3G in vitro. For example, in human neuroblastoma cells, M3G has been shown to inhibit cAMP formation to the same extent as morphine, and this inhibitory effect of M3G was antagonized by the opioid antagonist naloxone (Baker et al., 2000a). In a related study, Baker et al. (2000b) also demonstrated µ-agonist effects of M3G using voltage-clamp techniques to measure opioid receptor-activated channel responses. Collectively, these results suggest that M3G possesses significant µ-agonist activity in functional assays.

Opioid Mechanisms in DS Effects of Heroin. Although some of the effects of heroin have been attributed to -opioid receptor stimulation (Rady et al., 1994; Uchihashi et al., 1996), heroin is thought to exert its behavioral effects primarily via stimulation of µ-opioid receptors. In the present study, several lines of evidence support a primary role for µ-opioid receptors in the transduction of the DS effects of heroin. First, the DS effects of heroin were antagonized in a dose-dependent manner by relatively low doses of naltrexone. Increasing the dose of heroin surmounted this antagonism, resulting in rightward shifts in the heroin dose-response function. Schild analysis of these studies was consistent with competitive antagonism at a single receptor population (Tallarida et al., 1979). Moreover, the in vivo apparent pA₂ value of 8.3 obtained in the present study is similar to apparent pA₂ values obtained previously in rhesus monkeys treated with naltrexone combined with either morphine or heroin (8.3, France et al., 1990; 8.0, Rowlett et al., 1998). Finally, in substitution studies, the DS effects of heroin were mimicked fully by the µ-agonists fentanyl and methadone at doses that did not alter rates of responding. Another selective µ-agonist, dihydroetorphine, also has been found to substitute fully for the DS effects of heroin in rats (Beardsley and Harris, 1997). In contrast to these consistent findings, neither the -selective agonist SNC 80 nor the -selective agonist spiradoline engendered heroin-like DS effects at any dose. These findings suggest that µ- but not - or -opioid receptor mechanisms play a principal role in transduction of the interoceptive effects of heroin.

DA Mechanisms in DS Effects of Heroin. Although µ-opioid agonists have been found to augment release of DA in the nucleus accumbens and other brain regions implicated in the effects of abused drugs (Di Chiara and North, 1992; Wise, 1998), we found no evidence that either blockade of DA reuptake or enhancement of DA release by cocaine, GBR 12909, or methamphetamine played a major role in the DS effects of i.v. heroin. In this regard, cocaine, methamphetamine, and GBR 12909 consistently failed to engender heroin-lever responding regardless of dose in any subject. This finding differs from other studies (Lamas et al., 1998; Platt et al., 1999) that have found cocaine to substitute for the DS effects of heroin or morphine in some individual rats or squirrel monkeys. Collectively, these findings imply a possible role for dopaminergic mechanisms underlying the DS effects of i.m. morphine and i.p. heroin in at least some subjects, but not the DS effects of i.v. heroin. Although the factors underlying these different findings are not clear, one cannot rule out species or procedural differences (route of administration). Interestingly, Negus et al. (1998) recently reported that several µ-opioids including heroin substituted for the DS effects of cocaine in about one-half of the rhesus monkeys trained to discriminate cocaine from vehicle. Taken together, our findings and those of Negus et al. (1998) raise the possibility that there exists an asymmetrical generalization profile for stimulants and opioids in rhesus monkeys. It is not unprecedented that we should observe asymmetrical generalization. For example in other species, the DS effects of barbiturates generalize to benzodiazepines, but the DS effects of benzodiazepines do not generalize to barbiturates (Ator and Griffiths, 1989).

Summary. To our knowledge, the present study provides the first demonstration that intravenously administered heroin can be established as a DS in nonhuman primates. Because this route of administration is the predominant method of abuse by heroin addicts, the i.v. heroin discrimination may be a useful procedure for establishing mechanisms of action underlying the subjective effects of heroin in people. In the present study, the DS effects of i.v. heroin were largely attributable to stimulation of µ-opioid receptors, rather than stimulation of - or -opioid receptors, or DA activity. Unexpectedly, M3G, a metabolite of heroin previously thought to have little opioid activity, occasioned full heroin-like DS effects. Similarly, 6 MAM, morphine, and M6G also shared DS effects with heroin, suggesting that the acetylated and glucuronide metabolites of heroin contribute significantly to the transduction of the DS effects of heroin.