From Bozarth, M.A., & Wise, R.A. (1982). Localization of the reward-relevant opiate receptors. In L.S. Harris (Ed.), Problems of drug dependence, 1981 (National Institute on Drug Abuse Research Monograph 41, pp. 158-164). Washington, DC: U.S. Government Printing Office.
Localization of the Reward-Relevant Opiate Receptors
Michael A. Bozarth & Roy A. Wise
Center for Research on Drug Dependence
Department of Psychology
Montreal, Quebec H3G 1M8 CANADA
Recognition of the importance of the rewarding properties of opiates in establishing compulsive drug use (Eddy, 1973; Jaffe, 1975) has prompted a search for the identification of brain mechanisms subserving opiate reward. One of the first steps is the neuroanatomical localization of the reward-relevant opiate receptors, but attempts to identify the brain site of rewarding drug action have been subject to serious limitations of current paradigms (Bozarth, 1982).
The most powerful method to demonstrate that a drug is acting at a particular brain site is to show that the drug is self-administered directly into that site. This can identify the initial target of rewarding drug action and thus neuroanatomically define the population of receptors mediating this effect. Another method for studying drug reward is the conditioned place preference paradigm (Rossi & Reid, 1976). In this paradigm, animals are confined to a normally nonpreferred portion of a test chamber following drug injections. They are subsequently tested in the drug-free condition to determine if a learned preference or aversion has developed to the place where the drug was experienced. This technique can potentially reveal the affective consequences of the drug experience: increases in the amount of time spent in the portion of the test chamber associated with the drug experience suggest that the drug has positive affective consequences. This paradigm has the advantage of not making any response demands on the animal in the drugged condition, so it avoids the problem of sedative side-effects of some drug treatments.
The present paper reports the results of experiments using the intracranial self-administration and conditioned place preference paradigms. Rats quickly learned a lever-pressing response to inject morphine directly into the ventral tegmental area but did not self-administer morphine into other brain regions. This rewarding effect of morphine was confirmed using the conditioned place preference paradigm. The reward produced by systemically injected heroin, as assessed by the conditioned place preference paradigm, was blocked by dopamine receptor blockade. These studies suggest that the rewarding properties of opiates are dependent on a dopaminergic mechanism probably activated through opiate receptors in the ventral tegmental area.
The usefulness of the intracranial self-administration paradigm rests on the demonstration of behavioral, pharmacological, and anatomical specificity of the effect. First, it must be established that the animals are working for the rewarding properties of the drug injections, and that responding is not merely the consequence of nonspecific behavioral arousal. Second, it is necessary to demonstrate that the rewarding effects of central drug injections are dependent on the same receptor mechanisms that mediate systemic drug reward. Third, it must be determined if the intracranial self-administration of a drug is localized to restricted brain regions or is an effect which is demonstrable throughout the brain. It is in the localization of the reward-relevant opiate receptors that intracranial self-administration can make its most important contribution to the understanding of the mechanisms of opiate reward and abuse.
Experimentally naive, male Long-Evans rats were unilaterally implanted with 22-gauge guide cannulae stereotaxically aimed at one of several brain regions. Obturators were fitted to a depth of 0.5 mm beyond the guide cannula. The rats were tested in a 27 x 38 x 39 cm box house in a dimly illuminated, sound-attenuating chamber. An operant lever was mounted on one wall of the test box. Drug injections were delivered using an electrolytic microinfusion transducer (EMIT) system. In these experiments, a 200-mA current applied for 5 seconds was used to deliver 100 nl of drug solution. For methodological details of the EMIT system, see Bozarth and Wise (1980).
Experiment I: Reward From Ventral Tegmental Morphine Injections
The first experiment was designed to determine if experimentally naive rats would learn to press a lever to inject morphine directly into the ventral tegmental area. To control for accidental lever contacts, a yoked control procedure was used. Each morphine-reward rat was paired with a yoked-control animal such that lever-presses by the experimental rat produced concurrent infusions in both animals. Lever presses by the yoked-control rat were recorded but did not produce infusions. Using this procedure, the influence of increased locomotor activity on response measures could be assessed.
Rats implanted with guide cannulae in the ventral tegmental area were randomly assigned to either a morphine reward, yoked control, or Ringerís control group. For the morphine reward group, each lever press produced a 100-nl infusion of morphine sulfate dissolved in 100 nl of Ringerís solution. Yoked control animals received the same injections as their experimental partners. The Ringerís control group received response contingent vehicle only. The rats were tested for lever pressing in three four-hour sessions on alternate days. Rats in the morphine reward group were tested in a fourth session in which naloxone hydrochloride (10 mg/kg, i.p.) was injected one hour into the session.
The morphine reward group rapidly learned the lever-pressing response making significantly more responses than the yoked or Ringerís control groups (p < .01). Naloxone, injected one hour into the last test session, effectively blocked morphine self-administration.
Figure 1: Mean (± standard error) number of responses/hour across all three sessions. Exp. I: all rats were implanted with cannulae in the ventral tegmental area (VTA); (a) morphine reward, (b) yoked control, (c) Ringerís control, n=5/group (adapted from Bozarth & Wise, 1981). Exp. II: all rats received response contingent morphine; cannulae were implanted in the VTA, periventricular gray substance (PVG), lateral hypothalamic area (LHA), nucleus accumbens septi (NAS), or caudate nucleus (CPU), n=6 to 8/group.
These data suggest that morphine injected into the ventral tegmental area can serve as a reward for a lever-pressing response. Since the response rates of the morphine reward group were reliably greater than those of the control groups, the lever pressing was not due to nonspecific behavioral arousal. The fact that intracranial self-administration was blocked by naloxone (data not illustrated) suggests that the rewarding action of these microinjections was dependent on an opiate-receptor mechanism and not the consequence of some nonspecific changes in local osmolarity, pH, or calcium flux.
Several observations suggest that the dopaminergic cells in the ventral tegmentum mediated the rewarding effects of these microinjections. First, opiate receptors appear to be located on these dopaminergic cells or their afferents (Schwartz, 1979) and microinjections of morphine into this region cause an increase in the single unit activity of these cells (Finnerty & Chan, 1980). Second, morphine microinjected into the ventral tegmental area has been reported to increase locomotor activity (Joyce & Iversen, 1979; Pert et al., 1979), and this effect was observed in both the morphine reward and yoked control animals of the present study. Stimulation of locomotor activity seems to be dependent on a dopaminergic substrate with cell bodies located in the ventral tegmental area (Joyce & Iversen, 1979). Furthermore, since the rewarding injections were unilateral, the increased locomotor activity was asymmetrical and resulted in circling: the direction of circling was contralateral to the side of injection indicating that dopamine release was enhanced at the terminal fields of these cells (Ungerstedt, 1971a).
Experiment II: Lack of Morphine Reward From Other Injection Sites
To determine if other brain sites would support intracranial self-administration, rats were unilaterally implanted with guide cannulae in the following brain regions: ventral tegmental area, periventricular gray substance, lateral hypothalamic area, nucleus accumbens, or caudate nucleus. Experimentally naive animals were tested every other day for three four-hour sessions. Each lever press resulted in a 100-ng infusion of morphine as in the first experiment.
Acquisition of the lever-pressing response was found only in rats with cannulae in the ventral tegmental area. The lack of self-administration into the periventricular gray substance is of particular significance since it eliminates the possibility that morphine injected into the ventral tegmentum is rewarding because of diffusion up the guide cannula to the cerebral ventricles (the anterior-posterior and medial-lateral stereotaxic coordinates for these two sites were the same).
The failure to obtain morphine self-administration into the lateral hypothalamic area is in conflict with previous reports (E. Stein & J. Olds, 1977; M. Olds, 1979). There are two methodological differences that deserve particular attention. First, intraventricular self-administration of opioids has been demonstrated (Amit et al., 1976; Belluzzi & L. Stein, 1977). The studies reporting morphine self-administration into the lateral hypothalamic area used guide cannulae that were much larger than those used in the present study. This can facilitate drug spread up the guide shaft and into the cerebral ventricles (Routtenberg, 1972) resulting in diffusion to a distal site of action. Second, the reports of lateral hypothalamic self-administration have involved rats previously trained to lever press for brain stimulation reward. Lateral hypothalamic morphine injections might be capable of maintaining an already learned response but not capable of establishing a new habit; this might be expected if lateral hypothalamic injections were slowly diffusing to a distal site of action and serving as a weak reward. Regardless of the explanation for the lateral hypothalamic self-administration reported by others, it is apparent that ventral tegmental morphine injections are a much more potent reward than are microinjections into the other brain regions tested in this study.
The conditioned place preference paradigm can make several important contributions to the study of drug reward. It offers an independent method of assessing a drugís rewarding properties which is not susceptible to behaviorally debilitating effects of various drugs or lesions. Also, conditioning variables have been implicated in the maintenance of drug-seeking behavior (Schuster & Woods, 1968), and this paradigm allows a direct comparison of these conditioning variables across different drugs and parameters of conditioning. Finally, this paradigm is extremely quick and easy to use.
Place preference was measured in a shuttle box (25 x 36 x 35cm) with a wood floor on one side and a wood floor covered with wire mesh on the other. The amount of time rats spent on each side of the box was automatically recorded in 15-minute sessions for five consecutive preconditioning days. Next, the animals received daily drug injections for four days in which they were restricted to the nonpreferred side of the box for 30 minutes. Finally, the animals were retested for place preference during access to the entire shuttle box.
Experiment III: Place Preference From Central Morphine Injections
In intracranial self-administration studies, the animal controls the number of infusions and thus the total dose and volume of drug injected. Since the field of effective drug spread varies as a function of response rate, it is difficult to estimate the distance of the cannula placements to the reward-relevant receptors (Bozarth, 1982). This problem can be overcome by injecting each animal with the same volume of drug and assessing reward using the conditioned place preference paradigm. Cannula placements can then neuroanatomically map the region of the reward-relevant receptor population.
Rats were injected with 250 ng of morphine sulfate into the ventral tegmental area immediately before each of the four conditioning trials. The infusions were delivered over 28 seconds in 500 nl of Ringerís solution using the EMIT method (Bozarth & Wise, 1980).
Figure 2: Mean (± standard error) number of seconds on the side of putative conditioning. Exp. III: (a) cannulae 2.8 to 3.8 mm posterior to bregma, n=11, (b) cannulae 4.2 to 4.8 mm posterior to bregma, n=8; histological verification was based on Pellegrino et al. (1979); between group difference, p < 0.01. Exp. IV: (c) heroin plus saline, (d) heroin plus naloxone, (e) heroin plus pimozide, (f) saline plus naloxone, (g) saline plus pimozide, n=11/group.
Rats with cannulae in the same region as those supporting self-administration in the earlier experiments developed a conditioned place preference for the side of the box associated with morphine injections. Rats with cannulae placed caudal to this region failed to acquire such a preference. Preliminary estimates suggest that the caudal limit of the reward-relevant receptor population corresponds to the boundary of the ventral tegmental dopamine-cell group.
Experiment IV: Neuroleptic Challenge of Heroin Reward
The conditioned place preference paradigm avoids some of the problems of lever-pressing experiments by assessing drug reward in the drug-free state. Thus, this paradigm is not sensitive to the response depressant or excitatory effects of drugs or brain lesions. In the next experiment, the conditioned place preference produced by systemic heroin injections was challenged with the dopamine-receptor blocker pimozide and the opiate antagonist naloxone.
Five groups of rats were tested. Three groups received heroin (0.5 mg/kg, s.c.) preceded by either saline (1.0 mg/kg, i.p.), naloxone (3.0 mg/kg, i.p.), or pimozide (0.5 mg/kg, i.p.). One group was injected with naloxone alone and another with pimozide only. All drugs were injected immediately before the conditioning trials, except pimozide which was injected four hours prior to conditioning.
Animals injected with heroin plus saline showed a shift in place preference similar to that seen after intracranial morphine injections (p < .01). Pretreatment with either naloxone or pimozide blocked the development of a heroin-induced place preference, while treatment with either naloxone or pimozide alone had no effect.
These results are concordant with self-administration studies suggesting an attenuation of opiate reward following neuroleptic treatment (Hanson & Cimini-Venema, 1972; Pozuelo & Kerr, 1972). Since place preference is tested in drug-free animals, pimozideís blockade of heroin reward cannot be attributed to a general sedative effect that could confound lever-press measures.
The use of the intracranial self-administration paradigm in Experiments I and II has provided a direct demonstration of the rewarding action of morphine delivered into the ventral tegmental area. The fact that the rats learn to lever press rapidly suggests that the rewarding effects of these injections occur soon after the drug infusions. Rats would not be expected to learn this response as rapidly if the rewarding impact of the infusions was delayed, as would be the case if the rewarding effects were dependent on the diffusion of drug to a distal site of action.
The rewarding action of morphine injected into the ventral tegmental area has been confirmed using the conditioned place preference paradigm. In a similar study, place preference was established with bilateral morphine injections into the ventral tegmentum but not with injections dorsal to it (Phillips & LePiane, 1980). Experiment III extended these results showing that unilateral injections are also effective and that injections caudal to the ventral tegmental area are not effective.
The failure to find intracranial self-administration into the brain sites tested in Experiment II suggests a neuroanatomical separation of the rewarding, analgesic, and sedative properties of opiates. The periventricular gray substance has been implicated in opiate-induced analgesia (Jacquet & Lajtha, 1976; Pert & Yaksh, 1974; Sharpe et al., 1974; Yaksh et al., 1976) and sedation (Pert et al., 1978), while the rewarding properties of morphine appear to be dependent on an action in the ventral tegmental area. The site of action for opiate-induced physical dependence is less clearly defined, but it seems to involve receptors in the thalamus and periventricular gray region (Wei et al., 1973; Wei & Loh, 1976; Wei, 1981). These studies suggest that the ability of an opiate to produce analgesia, sedation, and physical dependence may be neuroanatomically separable from its rewarding properties.
The importance of dopamine-containing cells in the regulation of food, water, and brain stimulation reward is well established (Ungerstedt, 1971b, Wise, 1978, 1980). An important role for dopaminergic cells projecting to the nucleus accumbens has also been demonstrated in psychomotor stimulant reward (Roberts et al., 1980). The fact that reward from systemic heroin appears to be dependent on a dopaminergic mechanism (Experiment IV) suggests that it may share a common neural substrate with these other sources of reward. The localization of the reward-relevant opiate receptors in the ventral tegmental area makes this a likely place for opiate reward to interface with a dopaminergic substrate of reward.
This research was supported by grant DA 02285 from the National Institute on Drug Abuse. The surgical and histological assistance of Lydia Alessi and Martha Asselin is gratefully acknowledged.
Amit, Z., Brown, Z., & Sklar, L. (1976). Intraventricular self-administration of morphine in naive laboratory rats. Psychopharmacology 48: 291-294.
Belluzzi, J.D., & Stein, L. (1977). Enkephalin may mediate euphoria and drive-reduction reward. Nature 266: 556-558.
Bozarth, M.A. (1982/1983). Opiate reward mechanisms mapped by intracranial self-administration. In J.E. Smith and J.D. Lane (Eds.), Neurobiology of opiate reward processes (pp. 331-359). Amsterdam: Elsevier/North Holland Biomedial Press. [Corrected bibliographic data; publisher and publication year changed.]
Bozarth, M.A., & Wise, R.A. (1980). Electrolytic microinfusion transducer system: An alternative method of intracranial drug application. Journal of Neuroscience Methods 2: 273-275.
Bozarth, M.A., & Wise, R.A. (1981). Intracranial self-administration of morphine into the ventral tegmental area in rats. Life Sciences 28: 551-555.
Eddy, N.B. (1973). Prediction of drug dependence and abuse liability. In L. Goldberg and F. Hoffmeister (Eds.), Psychic dependence: Definition, assessment in animals and man, theoretical and clinical implications. New York: Springer-Verlag.
Finnerty, E.P., & Chan, S.H.H. (1980). Further studies of the nigro-striatonigral mechanisms and their roles in morphine suppression of caudate neuronal activities. Society for Neuroscience Abstracts 6: 809.
Jacquet, Y.F., & Lajtha, A. (1976). The periaqueductal gray: Site of morphine analgesia and tolerance as shown by 2-way cross tolerance between systemic and intracerebral injections. Brain Research 103: 501-513.
Jaffe, J.H. (1975). Drug addiction and drug abuse. In L.S. Goodman and A. Gilmans (Eds.), The pharmacological basis of therapeutics (pp. 284-324). New York: MacMillan.
Joyce, E.M., & Iversen, S.D. (1979). The effect of morphine applied locally to mesencephalic dopamine cell bodies on spontaneous motor activity in the rat. Neuroscience Letters 14: 207-212.
Olds, M.E. (1979). Hypothalamic substrate for the positive reinforcing properties of morphine in the rat. Brain Research 168: 351-360.
Pellegrino, L.J., Pellegrino, A.S., & Cushman, A.J. (1979). A stereotaxic atlas of the rat brain. New York: Plenum Press.
Pert, A., DeWald, L.A., Liao, H., & Sivit, C. (1979). Effects of opiates and opioid peptides on motor behaviors: Sites and mechanisms of action. In E. Usdin, W.E. Bunny Jr. and N.S. Kline (Eds.), Endorphins in mental health research (pp. 45-61). New York: Oxford University Press.
Pert, A., & Yaksh, T. (1975). Localization of the antinociceptive action of morphine in primitive brain. Pharmacology Biochemistry & Behavior 3: 133-138.
Phillips, A.G., & LePaine, F.G. (1980). Reinforcing effects of morphine microinjection into the ventral tegmental area. Pharmacology Biochemistry & Behavior 12: 965-968.
Pozuelo, J., & Kerr, F.W.L. (1972). Suppression of craving and other signs of dependence in morphine-addicted monkeys by administration of alpha-methyl-para-tyrosine. Mayo Clinic Proceedings 47: 621-628.
Roberts, D.C.S., Koob, G.F., Klonoff, P., & Fibiger, H.C. (1980). Extinction and recovery of cocaine self-administration following 6-hydroxydopamine lesions of the nucleus accumbens. Pharmacology Biochemistry & Behavior 12: 781-787.
Rossi, N.A., & Reid, L.D. (1976). Affective states associated with morphine injections. Physiological Psychology 4: 269-274.
Routtenberg, A. (1972). Intracranial chemical injection and behavior: A critical review. Behavioral Biology 7: 601-641.
Schuster, C.R., & Woods, J.H. (1968). The conditioned reinforcing effects of stimuli associated with morphine reinforcement. International Journal of Addiction 3: 223-230.
Schwartz, J.-C. (1979). Opiate receptors on catecholaminergic neurones in brain. Trends in Neurosciences 2: 137-139.
Sharpe, L.G., Garnett, J.E., & Cicero, T.J. (1974). Analgesia and hyperactivity produced by intracranial microinjections of morphine into the periaqueductal gray matter of the rat. Behavioral Biology 11: 303-313.
Stein, E.A., & Olds, J. (1977). Direct, intracerebral self-administration of opiates in the rat. Society for Neuroscience Abstracts 3: 971.
Ungerstedt, U. (1971a). Striatal dopamine release after amphetamine or nerve degeneration revealed by rotational behavior. Acta Physiologica Scandinavica Suppl. 367: 49-68.
Ungerstedt, U. (1971b). Adipsia and aphagia after 6-hydroxydopamine induced degeneration of the nigro-striatal dopamine system. Acta Physiologica Scandinavica Suppl. 367: 95-122.
Wei, E.T. (1981). Enkephalin analogs and physical dependence. Journal of Pharmacology and Experimental Therapeutics 216: 12-18.
Wei, E., & Loh, H. (1976). Physical dependence on opiate-like peptides. Science 24: 1262-1263.
Wei, E., Loh, H.H., & Way, E.L. (1973). Brain sites of precipitated abstinence in morphine-dependent rats. Journal of Pharmacology and Experimental Therapeutics 185: 108-115.
Wise, R.A. (1978). Catecholamine theories of reward: A critical review. Brain Research 152: 215-247.
Wise, R.A. (1980). Action of drugs of abuse on brain reward systems. Pharmacology Biochemistry & Behavior 13: 213-223.
Yaksh, T.L., Yeung, J.C., & Rudy, T.A. (1976). Systematic examination in the rat of brain sites sensitive to the direct application of morphine: Observation of differential effects within the periaqueductal gray. Brain Research 114: 83-103.