Introduction

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A hallmark of physical dependence upon morphine-like opioids is an increase in sensitivity to effects of opioid antagonists. For example, the ED₅₀ of the opioid antagonist naloxone to elicit withdrawal jumping in mice that had received an s.c. morphine pellet decreased steadily from 12 mg/kg immediately after pellet implantation to 0.045 mg/kg 72 h after implantation (Way et al., 1969).

Sensitivity to opioid antagonists increases not only during prolonged exposure to an opioid agonist but also after pretreatment with only a single dose of morphine or a related drug. The potency of naloxone and other opioid antagonists (e.g., naltrexone) to decrease the rate of schedule-controlled responding maintained by food or brain stimulation increased by as much as 2 to 3 orders of magnitude in rats that had been pretreated 4 h earlier with a single dose of a morphine-like drug (Young, 1986; Adams and Holtzman, 1990; Easterling and Holtzman, 1997). The sensitizing effect of opioid agonists was reversible, stereoselective for the levorotatory isomer, and mediated centrally, primarily by µ-opioid receptors (Adams and Holtzman, 1990, 1991; Easterling and Holtzman, 1997) These and other examples of acute agonist-induced sensitization to effects of opioid antagonists have been viewed as evidence of a state of acute opioid dependence (Meyer and Sparber, 1976; Eisenberg and Sparber, 1979; White and Holtzman, 2001). The results of clinical studies support this conclusion. Naloxone induced many of the physiological manifestations and subjective symptoms of withdrawal when it was administered several hours after a single dose of a morphine-like drug to otherwise drug-free volunteers (Bickel et al., 1987; Wright et al., 1991; Greenwald et al., 1996).

Drug discrimination affords an approach for studying in animals drug effects that have relevancy to the subjective effects of the drug in humans (Holtzman, 1990). We trained rats to discriminate 4-h pretreatment with 10 mg/kg morphine and 15-min pretreatment with 0.3 mg/kg naltrexone (morphinenaltrexone) from pretreatment with saline and 0.3 mg/kg naltrexone (salinenaltrexone (Easterling and Holtzman, 1999). The discriminative effects of morphinenaltrexone were an orderly function of the dose of morphine, the dose of naltrexone, and the morphine pretreatment interval. They were maximal when morphine was administered 3 or 4 h before a session, half-maximal when morphine was administered 8 h before a session, and virtually absent when morphine was administered only 30 min before a session. When training was suspended and a continuous infusion of morphine was administered via an s.c. osmotic pump (20 mg/kg/day), naltrexone engendered dose-dependent increases in morphinenaltrexone-appropriate responding and substituted completely for morphinenaltrexone. These results suggested that stimulus control of behavior by morphinenaltrexone derived from interoceptive stimuli associated with antagonist-precipitated withdrawal from acute physical dependence upon morphine (Easterling and Holtzman, 1999).

The purpose of this study was to extend pharmacological characterization of the morphinenaltrexone discrimination by testing opioid agonists other than morphine and antagonists other than naltrexone. The morphine-like agonists examined represented a range of intrinsic efficacies, from relatively low (e.g., buprenorphine and meperidine) to relatively high (e.g., etorphine and fentanyl; Emmerson et al., 1996; Selley et al., 1998), to assess the contribution of intrinsic efficacy to stimulus control of behavior. Two µ-opioid agonists that did not substitute completely for morphine when administered acutely also were administered continuously via s.c. osmotic pump, while discrimination training was suspended; stimulus control of behavior was assessed after the administration of naltrexone. Insofar as possible, the µ-opioid agonists were tested at doses estimated to be equieffective with 10 mg/kg morphine, based upon discriminative effects in rats trained to discriminate morphine (Shannon and Holtzman, 1976; Young et al., 1991; Walker et al., 1994; Holtzman, 1997).

Acute administration of -opioid agonists induces much less sensitization to response rate-decreasing effects of naltrexone than does acute administration of µ-opioid agonists (Adams and Holtzman, 1990; Easterling and Holtzman, 1997). In this study, a relatively high dose of each of two -opioid agonists, spiradoline and U69,593, was tested in place of morphine.

Naloxone substituted fully for naltrexone in rats that were pretreated with the combination of a single dose of morphine (Easterling and Holtzman, 1999). We replicated these results and extended observations to three more opioid antagonists: diprenorphine, which has negligible intrinsic efficacy at µ-opioid receptors (Lee et al., 1999); and nalorphine and levallorphan, drugs that are weak partial µ-agonists (Emmerson et al., 1996; Selley et al., 1998).

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Subjects. Adult male rats of Sprague-Dawley descent were obtained from Charles River Laboratories, Inc. (Wilmington, MA) and were housed in pairs in a colony room that was maintained on a 12-h light/dark cycle. Food and water always were available in the home cage. Twenty-five rats met the criterion for acquisition of the discrimination (see below) and were used in the study. Three had undergone approximately 3 months of discrimination training with 5.6 mg/kg morphine and 0.3 mg/kg naltrexone versus saline and 0.3 mg/kg naltrexone; the other 22 were experimentally naive. Experiments were conducted according to a protocol approved by the Institutional Animal Care and Use Committee of Emory University and were in keeping with the 1996 Guide for the Care and Use of Laboratory Animals (National Academy of Sciences).

Training Procedure. Discrimination performance was maintained by a two-choice discrete-trial avoidance/escape procedure (Easterling and Holtzman, 1999). During the acquisition phase, daily training sessions were conducted Monday through Friday. Either saline or morphine, 10 mg/kg, was injected s.c. 4 h before a session on alternate days; naltrexone, 0.3 mg/kg, was injected s.c. 15 min before every training session. At the end of the pretreatment interval, the rats were placed in a testing chamber that was inside of a ventilated cubicle that was lightproof and sound-attenuating. A single lever was mounted in one wall of the chamber and two "choice" levers were mounted in the opposite wall. The choice levers were separated by a Plexiglas partition that extended 5.0 cm into the chamber and ran from the grid floor to the ceiling. Illumination of the house light and onset of a white noise signaled the start of a trial. Five seconds later, a constant current of 1.0 to 1.5 mA was distributed to the grid floor of the chamber in pulses of 1.0 s every 3.0 s. A rat could end the trial at any time by completing a two-response chain: pressing the single lever in one wall of the chamber and then pressing the choice lever that was correct for the substance injected before the session (i.e., morphinenaltrexone or salinenaltrexone). For half of the animals the right choice lever was correct on days that morphine was injected and the left choice lever was correct on days that saline was injected; lever assignments were reversed for the other half of the rats. A response on the first lever turned off the white noise and a response on the correct choice lever extinguished the house light and ended the trial. The next trial began 50 s later. In the absence of a correct response sequence, a trial was terminated after 30 s. Each session consisted of 21 trials; the first "warm-up" trial was excluded from data analyses. A trial was scored as correct if the rat pressed the first lever and then pressed the correct choice lever; a trial was scored as incorrect if the rat pressed the first lever and then pressed the incorrect choice lever before pressing the correct choice lever.

Stimulus-Generalization Tests. All rats were tested first with a range of naltrexone doses after pretreatment with 10 mg/kg morphine. Other drugs were then tested in an unsystematic order. In some cases the drugs were administered in place of morphine, 4 h before the session; in other cases they were administered in place of naltrexone, 15 min before the session. In most of the drug series, three doses of naltrexone (or other antagonist) and saline were tested in a random sequence and then, depending upon the results, additional drug doses were tested. To determine whether repeated exposure to morphine and other opioid agonists sensitized the rats to naltrexone, a large subgroup (16) of the rats was tested with naltrexone after saline pretreatment at various times during the study. In each animal, doses of naltrexone (0.3, 3.0, and 30 mg/kg) and saline were tested once in a random sequence over a period of 3 to 6 months

Opioid Withdrawal Syndrome. Physical signs of opioid withdrawal were assessed with the Gellert-Holtzman Global Withdrawal Rating Scale (Gellert and Holtzman, 1978). Beginning 5 min after an injection of either saline or naltrexone, individual rats were observed for 10 min while they were in a polycarbonate holding cage. Signs marked as either present or absent (i.e., "checked" signs) were diarrhea, facial fasciculation or teeth chatter, swallowing movements, salivation, chromodacryorrhea, ptosis, abnormal posture, erection or ejaculation, and irritability to handling. "Graded" signs were number of escape attempts, "wet-dog" shakes, and abdominal constrictions. Each rat was weighed just before the injection and again at the conclusion of the session, 40 to 45 min later, to determine loss of body weight.

Data Analysis. Discrimination data are presented as the average number of trials completed on the choice lever appropriate for the morphinenaltrexone condition; the remaining trials of the session were completed on the choice lever appropriate for salinenaltrexone. Means were compared with Friedman's nonparametric ANOVA for repeated measures, yielding the statistic Fr. If the data were statistically reliable, the Wilcoxin matched-pairs signed ranks test was used to compare two means. Both tests were corrected for ties. During test sessions that followed pretreatment with 10 mg/kg morphine and 0.3 mg/kg, rats completed an average of at least 18 trials on the choice lever appropriate for morphinenaltrexone. Therefore, naltrexone or the combination of an agonist other than morphine and an antagonist was considered to have substituted completely for morphinenaltrexone if the group of rats completed an average of at least 18 trials on that choice lever.

Drugs. The drugs used, their salt forms, and their sources were as follows: naltrexone hydrochloride, naloxone hydrochloride, and dextrorphan tartrate (Sigma/RBI, Natick, MA); buprenorphine, diprenorphine, etorphine, fentanyl, heroin, meperidine, all as hydrochlorides, and (5-,7-,8-)-(+)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro(4,5)dec-8-yl]-benzeneacetamide (U69,593; National Institute on Drug Abuse, Bethesda, MD); morphine sulfate (Penick Co., Nutley, NJ); levorphanol tartrate, levallorphan tartrate (Hoffmann-La Roche, Nutley, NJ); methadone hydrochloride (Mallinkrodt, St. Louis, MO); nalorphine hydrochloride (Merck Research Labs, West Point, PA); and spiradoline methane sulfonate (Pharmacia, Kalamazoo, MI). All drugs were dissolved in either physiological (0.9%) saline or distilled water, except U69,593, which was dissolved in 3 parts of 8.5% lactic acid and 2 parts of 1.0 N sodium hydroxide. Except when they were administered via SC osmotic pumps, the drugs were injected s.c. in a volume of 1.0 ml (2.0 ml for high meperidine doses) per kilogram of body weight. Doses represent the free-base form of the drug.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Baseline. The 22 rats that were experimentally naive at the beginning of the study met the criteria for acquisition of the discrimination in an average of 53 sessions (range 23-84). When 10 mg/kg morphine was administered 4 h before a test session and saline was administered 15 min before, the rats responded almost exclusively on the choice lever appropriate for salinenaltrexone. However, the combination of morphine 4 h before a session and naltrexone (0.01-1.0 mg/kg) occasioned dose-dependent responding on the morphinenaltrexone-appropriate lever that peaked at the 0.3 mg/kg training dose of naltrexone (Fig. 1). The highest dose of naltrexone, 1.0 mg/kg, resulted in slightly but significantly fewer trials being completed on the morphinenaltrexone-appropriate lever than 0.3 mg/kg did (16.8 versus 19.2 trials, p < 0.001). The ED₅₀ of naltrexone, derived from the ascending portion of the stimulus-generalization curve, was 0.051 (0.034-0.077) mg/kg. In contrast, 4-h pretreatment with saline and 15-min pretreatment with naltrexone (0.3-30 mg/kg) occasioned relatively little responding on the morphinenaltrexone-appropriate lever, with a maximum of 2.9 trials after 30 mg/kg, 100 times the training dose (Fig. 1). Nevertheless, there was a significant effect of dose when responding after salinesaline was included in the analysis (Fr = 14.61, p = 0.002).

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Comparison of stimulus generalization curves for 4-h pretreatment with 10 mg/kg morphine and 15-min pretreatment with graded doses of naltrexone and for 4-h pretreatment with saline and 15-min pretreatment with graded doses of naltrexone. The morphinenaltrexone curve (n = 25) was determined in test sessions at the beginning of the study and the salinenaltrexone curve (n = 16) was determined in test sessions scattered throughout the study. Points above Sal represent the results of test sessions in which animals received a 15-min pretreatment with saline in place of naltrexone (i.e., morphinesaline or salinesaline). The ordinate shows the number of trials completed on the choice lever appropriate for morphine (10 mg//kg)naltrexone (0.3 mg/kg; NTX) in a 20-trial session; the remaining trials were completed on the choice lever appropriate for salinenaltrexone (0.3 mg/kg). The upper and lower horizontal dashed lines indicate the performance level at which behavior was maintained during training sessions that followed pretreatment with morphinenaltrexone and salinenaltrexone, respectively.

Other Agonists Administered Acutely. Only two of seven morphine-like agonists substituted completely for morphine when administered in a single dose 4 h before a session. When combined with 0.3 mg/kg naltrexone administered 15 min before a session, 3.0 mg/kg heroin occasioned completion of an average of more than 18 trials on the choice lever appropriate for morphinenaltrexone (Fig. 2, top). Like the morphinenaltrexone curve, the curve for 3.0 mg/kg heroin and naltrexone was biphasic: doses of naltrexone higher than 0.3 mg/kg resulted in progressively fewer trials being completed on the morphinenaltrexone-appropriate lever than did 0.3 mg/kg. A lower dose of heroin (1.0 mg/kg) followed by naltrexone occasioned completion of a maximum of 12.5 trials on the morphinenaltrexone-appropriate lever, this at 3.0 mg/kg naltrexone (Fig. 2, top). The ED₅₀ of naltrexone after 3.0 and 1.0 mg/kg heroin was 0.025 (0.008-0.079) and 0.286 mg/kg, respectively.

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Stimulus-generalization curves for 4-h pretreatment with the indicated dose of heroin (top), levorphanol, or dextrorphan (bottom) and 15-min pretreatment with graded doses of naltrexone. Each point is a mean based upon one observation in each of six rats. Other details are the same as in Fig. 1.

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Stimulus-generalization curves for different pretreatment times and doses of fentanyl (top) or methadone (bottom) and 15-min pretreatment with graded doses of naltrexone. Fentanyl was administered as a single dose of 0.056 mg/kg (4-h pretreatment) or 0.1 mg/kg (2-, 3-, 4-h pretreatment). A single dose of methadone (3.0 mg/kg) was administered either 3 or 4 h before a session. Each point is a mean based upon one observation in each of six rats. Other details are the same as in Fig. 1.

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Stimulus-generalization curves for 4-h pretreatment with a µ-opioid agonist (0.01 mg/kg etorphine, 1.0 mg/kg buprenorphine, 30 mg/kg meperidine) and 15-min pretreatment with graded doses of naltrexone (top) and for 4-h pretreatment with a -opioid agonist (3.0 mg/kg spiradoline, 3.0 mg/kg U69,593) and 15-min pretreatment with graded doses of naltrexone (bottom). Each point is a mean based upon one observation in each of six rats. Other details are the same as in Fig. 1.

Fentanyl and Methadone Administered Continuously. Saline was injected 15 min before a test session into rats that had been getting a continuous s.c. infusion of either fentanyl (0.25 mg/kg/day) or methadone (10 mg/kg/day) for 5 days in the absence of training sessions. Almost all of the trials were completed on the lever appropriate for salinenaltrexone (Fig. 5, top). In contrast, naltrexone administered in incremental doses across days 7, 9, 11, and 13 of pump implantation occasioned dose-dependent increases in trials to the morphinenaltrexone-appropriate lever, substituting completely for morphinenaltrexone at either 0.1 (fentanyl) or 0.175 mg/kg (methadone). The respective ED₅₀ values for naltrexone were 0.019 (0.011-0.033) and 0.059 (0.030-0.114) mg/kg, making naltrexone approximately 3 times more potent in rats with fentanyl pumps than in those with methadone pumps (t_[7] = 3.64, p = 0.008).

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Trials completed on the morphinenaltrexone-appropriate choice lever (top) and corresponding Gellert-Holtzman global withdrawal scores (bottom) in rats tested with saline or naltrexone (15-min pretreatment) while receiving a continuous infusion of either fentanyl (0.25 mg/kg/day) or methadone (10 mg/kg/day) via s.c. osmotic pump, and 15 min after pretreatment with saline 6 or 24 h after pumps had been removed (points to right of axis break). Saline was tested on day 5 of pump implantation and naltrexone was tested on days 7, 9, 11, and 13. Pumps were removed on day 14. Each point is a mean based upon one observation in each of four (fentanyl pump) or five (methadone pump) rats. Vertical lines in the bottom panel represent ±1 S.E.M. and are absent if the S.E.M. was less than the radius of the point. *, significantly different from corresponding withdrawal score after saline administration while pumps were implanted (points above Sal), p < 0.05.

View larger version (16K): [in this window] [in a new window]   Fig. 6.   Trials completed on the morphine-naltrexone-appropriate choice lever correlate significantly with global withdrawal score in rats receiving a continuous infusion of either fentanyl (n = 4) or methadone (n = 5) by s.c. osmotic pump and tested after 15-min pretreatment with either saline or graded doses of naltrexone. Points were derived from data to the left of the axis break in Fig. 5.

Other Antagonists. The combination of 4-h pretreatment with 10 mg/kg morphine and 15-min pretreatment with either naloxone or diprenorphine occasioned dose-dependent increases in trials completed on the morphinenaltrexone-appropriate lever and substituted completely for the stimulus effects of morphinenaltrexone (Fig. 7). Naloxone was twice as potent as diprenorphine [ED₅₀ values: 0.059 (0.014-0.245) and 0.141 (0.086-0.231) mg/kg, respectively] but the difference between the drugs was not statistically reliable (t_[10] = 1.49, p = 0.168).

View larger version (20K): [in this window] [in a new window]   Fig. 7.   Stimulus-generalization curves for 4-h pretreatment with 10 mg/kg morphine and 15-min pretreatment with graded doses of one of the indicated opioid antagonists in place of naltrexone. Points above Sal represent the results of test sessions that followed 4-h pretreatment with saline in place of morphine and 15-min pretreatment with the highest dose of each antagonist that had been tested as part of the stimulus-generalization curve. Each point is a mean based upon one observation in each of six or seven (nalorphine) rats. Other details are the same as in Fig. 1.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Of the seven µ-opioid agonists tested, only two, heroin and levorphanol, substituted completely for acutely administered morphine in occasioning morphinenaltrexone-appropriate responding. The maximum number of trials completed on the morphinenaltrexone-appropriate lever and the ED₅₀ value of naltrexone were graded functions of the pretreatment dose of the agonist. The reasons that the other µ-opioid agonists did not substitute for morphine completely are probably multiple. In some cases, it was not possible to test a dose that was equieffective with 10 mg/kg morphine. Meperidine is 1/10 as potent as morphine in producing morphine-like discriminative effects (Shannon and Holtzman, 1976), implying that 100 mg/kg would be needed for complete substitution. However, a dose that high could not be tested safely. On the other hand, buprenorphine, fentanyl, and etorphine are approximately 70 to 100, 80 to 100, and 2000 to 3000 times more potent than morphine (Young et al., 1991; Walker et al., 1994; Holtzman, 1997), respectively. They were tested at doses that are at least equivalent to 10 mg/kg morphine, and well above the ED₅₀ values for suppressing food-maintained responding (Young et al., 1991; Walker et al., 1994), but did not substitute completely. Some of the agonists have durations of action shorter than that of morphine. However, shortening the pretreatment interval to 3 h for methadone and to 3 or 2 h for fentanyl did not increase the number of trials completed on the morphinenaltrexone-appropriate lever compared with 4-h pretreatment.

Naloxone precipitated signs and symptoms of opioid withdrawal in volunteers pretreated with a single dose of fentanyl or methadone (Wright et al., 1991; Greenwald et al., 1996). When those drugs did not substitute for morphine completely after acute administration, they were administered by continuous s.c. infusion at a daily dose approximately equivalent to the morphine dose infused in a previous study (Easterling and Holtzman, 1999). Under these conditions, naltrexone occasioned discriminative effects comparable with those engendered by acute pretreatment with the combination of morphine and naltrexone. Furthermore, the ED₅₀ value of naltrexone in the rats receiving infusions of fentanyl (0.02 mg/kg) or methadone (0.06 mg/kg) was similar to the one determined in rats that had pumps releasing morphine (0.04 mg/kg; Easterling and Holtzman, 1999). Thus, the discriminative effects associated with naltrexone-precipitated withdrawal from chronic fentanyl or methadone administration were equivalent to those engendered by acute pretreatment with morphinenaltrexone, whereas discriminative effects engendered by acute pretreatment with either fentanyl or methadone were not.

The specific signs of antagonist-precipitated withdrawal from morphine in rats can change qualitatively as well as quantitatively as a function of degree of physical dependence, dose of antagonist, and time after antagonist administration (Blasig et al., 1973). In this study, rats were trained with interoceptive cues that occur 4 h after 10 mg/kg morphine and 15 min after 0.3 mg/kg naltrexone. Apparently, this same cluster of interoceptive cues does not occur when naltrexone is administered after acute pretreatment with some µ-opioid agonists or at doses of naltrexone different from the training dose. The latter point would account for the biphasic nature of most of the stimulus-generalization curves.

That rats receiving a continuous infusion of either fentanyl of methadone generalized completely to naltrexone provides further evidence that stimulus control of behavior by morphinenaltrexone derives from interoceptive stimuli associated with antagonist-precipitated withdrawal from acute morphine dependence. In addition, the number of trials completed on the morphinenaltrexone-appropriate lever correlated significantly with the global withdrawal score of somatic signs. However, the concordance of the two variables was far from perfect quantitatively. "Behavioral/motivational" signs of morphine withdrawal, such as decreased food-maintained operant responding and conditioned place aversion, manifest at antagonist doses lower than those that precipitate many of the somatic signs of withdrawal, such as weight loss and diarrhea (Schulteis et al., 1994, 1999). Moreover, some somatic signs of withdrawal are mediated in the periphery (Maldonado et al., 1992) and can be blocked by drugs that do not affect behavioral/motivational signs of withdrawal (Shippenberg et al., 2000), suggesting the two classes of withdrawal signs are mediated by separate neural substrates. Therefore, although somatic withdrawal signs provide a quantitative index of severity of physical dependence, they are not necessarily predictive of nor do they account for behavioral/motivational manifestations of withdrawal, such as stimulus control of behavior by morphinenaltrexone.

The fact that rats pretreated with saline and naltrexone at doses as high as 30 mg/kg responded almost exclusively on the lever appropriate for salinenaltrexone in test sessions scattered throughout the study supports two conclusions drawn previously (Easterling and Holtzman, 1999). First, the state of acute physical dependence upon morphine is reversible; there were no detectable residual effects of the morphine training dose on days when morphine was not administered. Acute physical dependence in humans also is reversible, with a duration that reflects the half-life of the opioid agonist (Eissenberg et al., 1996; Greenwald et al., 1996). Second, morphine did not merely potentiate an existing effect of naltrexone; rather, morphinenaltrexone gives rise to a unique set of interoceptive stimuli that are absent when naltrexone is administered without morphine pretreatment.

The cellular events that underlie acute physical dependence upon opioid drugs are not known. Agonists that lack high efficacy (e.g., morphine) induce up-regulation of adenylyl cyclase and other components of the cAMP pathway while they activate µ-opioid receptors (Sharma et al., 1975; Finn and Whistler, 2001). This up-regulated second-messenger system seems to contribute to the withdrawal syndrome that emerges when morphine is displaced from the receptor by an antagonist (Nestler and Aghajanian, 1997). High-efficacy agonists (e.g., etorphine), on the other hand, cause desensitization and endocytosis of µ-opioid receptors, which prevents up-regulation of adenylyl cyclase (Sternini et al., 1996; Finn and Whistler, 2001). Therefore, lower efficacy agonists might be more likely to produce acute dependence than higher efficacy agonists, if up-regulation of the cAMP pathway is an important factor. However, intrinsic efficacy did not seem to be a determinant of whether a µ-opioid agonist substituted for morphine after acute administration. The lower efficacy agonists buprenorphine and meperidine administered before naltrexone were no more effective than etorphine was in occasioning morphinenaltrexone-appropriate responding. On the other hand, the balance between efficacy and promotion of receptor endocytosis might be the critical determinant of dependence development, with dependence induced quickest by drugs such as morphine that have reasonably high efficacy but do not cause receptor internalization (Whistler et al., 1999). Heroin is rapidly converted to monoacetylmorphine and morphine (Way, 1967). Little is known about the intrinsic efficacy of levorphanol or on the propensity of that drug to promote endocytosis of µ-opioid receptors.

Another possible mechanism for acute opioid dependence is agonist-induced constitutive activation of µ-opioid receptors, where the receptor remains coupled to G protein and intracellular signaling pathways after the agonist has dissociated from it (Chavkin et al., 2001). Prior exposure to morphine increases basal signaling activity in cell lines expressing the µ-opioid receptor; naloxone and naltrexone are inverse agonists in those expression systems, increasing intracellular levels of cAMP (Wang et al., 1994, 2001). Only those drugs that were inverse agonists in vitro precipitated withdrawal jumping in mice given a single dose of morphine 4 h earlier (Wang et al., 2001). There is little information on whether µ-opioid agonists other than morphine induce constitutive activity of µ-opioid receptors.

Given the disparate results obtained with the µ-opioid agonists, it is difficult to address the pharmacological selectivity of the morphinenaltrexone discrimination. Nevertheless, the results of this study permit some conclusions. First, stimulus control of behavior was produced stereoselectively. Rats generalized from partially to completely to levorphanol doses of 0.3 to 3.0 mg/kg followed by naltrexone. However, the combination of 3.0 mg/kg dextrorphan, the nonopioid dextrorotatory isomer of levorphanol, and a broad range of naltrexone doses occasioned responding only on the lever appropriate for salinenaltrexone. Stimulus control of behavior by morphine alone exhibits similar stereoselectivity (Shannon and Holtzman, 1976). Second, the results with U69,593 and spiradoline indicate that the combination of a -opioid agonist and naltrexone does not result in the same discriminative effects as morphinenaltrexone does. The dose tested, 3.0 mg/kg, is readily discriminated by rats and far exceeds the ED₅₀ value for suppressing food-maintained responding (Smith and Picker, 1995; Holtzman, 2000). Thus, if -opioid agonists induce acute physical dependence, the interoceptive cues associated with antagonist-precipitated withdrawal from that state are different from those arising from precipitated withdrawal from the morphine-dependent state.

The effects of the antagonists paralleled those in rats chronically dependent upon morphine and discriminating between 0.1 mg/kg naltrexone and saline (Gellert and Holtzman, 1979): naloxone and diprenorphine substituted completely for naltrexone and levallorphan and nalorphine substituted partially. Diprenorphine and naloxone, like naltrexone, are essentially devoid of intrinsic efficacy at µ-opioid receptors (Lee et al., 1999). Levallorphan and nalorphine, on the other hand, have intrinsic efficacy estimated to range from 5 to 15% that of morphine (Emmerson et al., 1996; Selley et al., 1998) and substitute partially for morphine in rats discriminating between it and saline (Shannon and Holtzman, 1977). Thus, it seems that even limited intrinsic efficacy at µ-opioid receptors is sufficient to prevent a drug from substituting completely for naltrexone in morphine-pretreated rats. The similarity of the effects of the five antagonists in rats acutely or chronically dependent upon morphine is further evidence of the commonalities shared by these two states.