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Mu opioid receptor: a gateway to drug addiction
Mu opioid receptor: a gateway to drug addiction Candice Contet, Brigitte L Kieffer and Katia Befort Mu opioid receptors mediate positive reinforcement following direct (morphine) or indirect (alcohol, cannabinoids, nicotine) activation, and our understanding of mu receptor function is central to the development of addiction therapies. Recent data obtained in native neurons confirm that mu receptor signaling and regulation are strongly agonist-dependent. Current functional mapping reveals morphine-activated neurons in the extended amygdala and early genomic approaches have identified novel mu receptor-associated proteins. A classification of about 30 genes either promoting or counteracting the addictive properties of morphine is proposed from the analysis of knockout mice data. The targeting of effectors or regulatory proteins, beyond the mu receptor itself, might provide valuable strategies to treat addictive disorders. Addresses Institut de Ge´ne´tique et de Biologie Mole´culaire et Cellulaire, CNRS/INSERM/ULP, UMR7104, Parc d’Innovation, 1 rue Laurent Fries BP 10142, 67404 Illkirch Cedex, Strasbourg, France e-mail: [email protected]
Current Opinion in Neurobiology 2004, 14:370–378 This review comes from a themed issue on Signalling mechanisms Edited by Richard L Huganir and S Lawrence Zipursky Available online 19th May 2004 0959-4388/$ – see front matter ß 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.conb.2004.05.005 Abbreviations DAMGO [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin GIRK G protein-coupled inwardly rectifying potassium channel HEK human embryonic kidney MAPK mitogen-activated protein kinase MOR mu opioid receptor VTA ventral tegmental area
Introduction The mu opioid receptor: a key molecular player in drug addiction
Drug addiction is a chronic relapsing disorder that results from gradual adaptations of the brain to repeated drug exposure. The current understanding of this complex phenomenon is that neurons responding to natural reinforcers such as food, sex, and social interactions are abnormally stimulated, leading to strong deregulations of brain reward pathways [1] and aberrant learning processes [2]. Addiction has many faces, including initiation and maintenance of drug consumption, withdrawal episodes, protracted abstinence, and relapse. Animal models Current Opinion in Neurobiology 2004, 14:370–378
for different aspects of drug-seeking behaviors have been successfully developed, and efforts are made to comprehend the transition between drug use and drug abuse [3]. The brain circuits of addiction involve reward pathways, in association with stress, obsessive-compulsive, and habit-forming systems [4], and molecular adaptations to chronic drug use are being actively examined in this broad neural network. Many neurotransmitter systems are recruited during these processes, with dopaminergic systems being important and the most frequently studied (see Bonci et al. [5] and references therein). The endogenous opioid system is also a major player in addiction. Because the opioid system plays a central part in modulating mood and well being, it is believed that modifications of endogenous opioids participate in the development of drug abuse. In addition, the opioid system could also influence drug craving and relapse by altering stress physiology. Both pharmacological and genetic experimental manipulations of the opioid system support these views and demonstrate that endogenous opioids influence reinforcement and adaptations to many drugs of abuse [4]. The opioid system consists of three G protein-coupled receptors, mu, delta, and kappa, which are stimulated by a family of endogenous opioid peptides [6]. Opioid receptors can also be activated exogenously by alkaloid opiates, the prototype of which is morphine. The finding that morphine’s analgesic and addictive properties are abolished in mice lacking the mu opioid receptor has unambiguously demonstrated that mu receptors mediate both the therapeutic and the adverse activities of this compound [7]. Importantly, a series of studies has shown that the reinforcing properties of alcohol, cannabinoids, and nicotine — each of which acts at a different receptor — are also strongly diminished in these mutant mice [8]. The genetic approach therefore highlights mu receptors as convergent molecular switches, which mediate reinforcement following direct (morphine) or indirect activation (non-opioid drugs of abuse; see Figure 1). Beyond the rewarding aspect of drug consumption, pharmacological studies have also suggested a role for this receptor in the maintenance of drug use, as well as craving and relapse [4]. As a consequence, expanding our understanding of mu receptor function should greatly help to further our knowledge of the general mechanisms that underlie addiction. This review focuses on recent findings regarding cellular regulation of mu receptors in the neuron and molecular mechanisms of responses to mu receptor activation in vivo. The latter part describes several genes involved in mu receptor signaling, highlights the implicated brain www.sciencedirect.com
Mu opioid receptor Contet, Kieffer and Befort 371
Figure 1
Reward circuits
GABAA/NMDA CB1 nACh
Alcohol THC Nicotine
Endogenous opioids? MOR
Natural reinforcers
Morphine MOR signaling Cellular regulation In vivo associated genes In vivo adaptations Addiction Current Opinion in Neurobiology
Mu opioid receptors (MOR) are largely distributed along reward circuits where they mediate the reinforcing activities of morphine and several non-opioid drugs. This was suggested by pharmacological studies and was recently demonstrated genetically by the analysis of mu receptor knockout mice in conditioned place preference or self-administration paradigms. In these mutant mice the rewarding properties of alcohol, D9-tetrahydrocannabinol (THC) and nicotine are abolished (reviewed by Kieffer and Gave´ riaux-Ruff [8]). These non-opioid drugs act at their own receptors (GABAA and NMDA receptors for alcohol, CB1 receptor for THC and nicotinic acetylcholine receptor for nicotine) and are likely to induce the release of endogenous opioid peptides that, in turn, activate mu receptors. Mu receptors, therefore, represent a convergent molecular gate in the initiation of addictive behaviors. Inadequate mu receptor activation might be one of the mechanisms underlying deregulation of reward pathways, which characterizes the addicted state. Whether or not mu receptors also mediate natural rewarding stimuli is presently being investigated in genetically modified mice.
structures and summarizes initial results from genomic approaches.
Regulation of mu receptor signaling in neurons: an agonist-dependent process Opioid agonists binding at mu receptors modulate intracellular effectors through inhibitory Go/Gi proteins. Receptor signaling, in turn, is readily terminated by several cellular regulatory processes that include phosphorylation, desensitization, endocytosis, and downregulation. An important observation from signaling and trafficking studies in transfected cells is that mu receptor activation and subsequent regulations are strongly agonist-dependent. Therefore, the agonist–receptor complex, rather than the receptor itself, determines the ultimate physiological effects of the ligand. Responses to peptidic opioids (DAMGO [(D-Ala2, N-Me-Phe4, Gly5ol)-enkephalin], enkephalins) and alkaloid-type compounds (morphine, fentanyl, methadone) have been examined to find a relationship between agonist efficacy and agonist-induced regulatory processes (see von Zastrow et al. [9] and references therein; [10]). Although correlations do not appear straightforward, it is currently accepted that opioid peptide signaling is efficiently blunted by the different regulatory mechanisms, whereas morphine signaling induces low levels of mu receptor regulation. The impact of agonist-induced mu receptor regulation on long-term in vivo adaptations is a matter of debate [11,12]. www.sciencedirect.com
A recent hypothesis is that mu receptor regulation could protect neurons from overstimulation and prevent counter adaptations that eventually lead to dependence. One approach used to explore this hypothesis in vivo was the simultaneous administration of mu agonists with distinct efficacy and regulatory profiles. The co-administration of a subeffective dose of DAMGO together with morphine reduced tolerance to morphine analgesia, and was accompanied by enhanced receptor internalization in spinal cord neurons [13]. Two recent studies have examined whether or not this agonist combination would similarly enhance mu receptor desensitization in the locus coeruleus. In these neurons, however, morphine did not efficiently desensitize G protein-coupled inwardly rectifying potassium channel (GIRK) currents either with or without the co-application of the low dose of DAMGO [10,14]. In addition, in Bailey’s study [14] subeffective DAMGO could neither facilitate desensitization of the morphine-induced guinea pig ileum twitch inhibition nor trigger morphine-induced internalization of the mu receptor in transfected human embryonic kidney (HEK) cells [14]. Thus, the benefit of mu agonist cocktails in minimizing long-term adaptations remains controversial. At present, the question of whether or not regulatory properties of mu agonists are predictive of abuse liability is a growing topic of attention. The lack of morphine-induced mu receptor internalization, proposed to be responsible for cellular adaptations relevant to addiction, has been widely observed in Current Opinion in Neurobiology 2004, 14:370–378
372 Signalling mechanisms
heterologous systems. It is crucial to examine whether or not morphine also poorly induces endocytosis in brain neurons expressing endogenous mu receptors. In many mu-expressing brain regions [15], including the locus coeruleus [16], treatment of animals with morphine failed to induce mu receptor endocytosis, which is consistent with in vitro studies. In rat nucleus accumbens neurons, however, acute morphine administration unexpectedly produced an intracellular redistribution of mu receptors in dendrites. In the same neurons, morphine was ineffective in cell bodies as previously observed in other systems [17]. This study shows that trafficking of mu receptors following morphine binding depends on which subcellular compartment within the neuron is involved, and is therefore more complex than was anticipated from studies in transfected cells. Finally, intriguing cellular consequences of chronic morphine treatment have been described recently. In one study, prolonged morphine treatment produced recruitment of intracellular delta receptors to the cell surface in dendrites of rat spinal cord neurons, an effect mediated by mu receptors and whose mechanism remains to be elucidated [18,19]. In another study, basal signaling of mu receptors was enhanced in the brain of mice chronically treated with morphine, a phenomenon that might contribute to the establishment of dependence in these animals [20].
Molecular adaptations to mu receptor activation in vivo: genetic approaches Among mu agonists, morphine is most relevant both in terms of clinical use (pain treatment) and in abuse potential (heroin addiction). Morphine essentially activates mu receptors in vivo, and the effects of chronic morphine exposure in whole animals reflect the consequences of repeated mu receptor activation in the nervous system. The analysis of morphine responses in about 30 genetically modified animals has recently revealed many partners of mu receptor signaling in vivo. In Table 1, we have selected data from experimental paradigms reflecting the initial (reward) and maintenance (revealed by precipitated withdrawal) phases of morphine addiction. The inactivation of many genes encoding neurotransmitters and/or their receptors disrupts morphine conditioned place preference or self-administration, two paradigms testing reinforcing properties of morphine. These systems therefore exhibit ‘pro-opioid’ activity, possibly acting in conjunction with mu receptor signaling to produce morphine reinforcement. Interestingly, several of these pro-opioid genes (D2 [dopamine D2 receptor], NK1 [neurokinin-1 receptor], CB1 [cannabinoid 1 receptor], M5 [muscarinic M5 acetylcholine receptor]) also participate in the development of morphine dependence, suggesting a broad recruitment of these transmitter systems in several aspects of addictive behaviors. Some pro-opioid genes (aCGRP [calcitonin gene-related peptide], KOR Current Opinion in Neurobiology 2004, 14:370–378
[kappa opioid receptor]) contribute to physical dependence but not to reward, as a result of temporal and/or spatial dissociation of the two phenomena. The inactivation of other genes results in enhanced morphine effects, and thus, these genes display an ‘anti-opioid’ activity which normally opposes morphine action in wild type mice (see Table 1). Consistent with the pharmacology, CCK2 (cholecystokinin 2 receptor) and OFQ/N (orphanin FQ/nociceptin) systems belong to this category. Gene products known to desensitize G protein signaling in vitro (RGS9 [regulator of G protein signaling 9], b-arrestin 2) also counteract some morphine responses in vivo. Interestingly, the dopamine transporter DAT differentially modulates reward and withdrawal. Signaling effectors otherwise display pro-opioid (PKCg [protein kinase C g isoform], CREB [cAMP responsive element binding protein]), anti-opioid (ERK1; extracellular signal regulated kinase 1), or no (nNOS; neuronal nitric oxide synthase) activity. At present, data from genetically modified mice have essentially addressed the role of genes already known to influence addictive behaviors from the pharmacological perspective. It is likely that a broad ‘morphine screen’ of available mouse mutants would reveal unexpected genes that contribute to adaptations to drugs of abuse, and be informative in terms of gene discovery for addiction research. Identifying neural sites of gene activity is crucial in our understanding of addiction (Figure 2). The constitutive knockout approach provides no clues as to regional gene activity, and, furthermore, compensations could take place during development and account for the observed phenotypes. While strategies for the site-specific knockout of candidate genes are being developed, alternative approaches have provided fascinating results. Recent examples are data showing the modulation of morphine reward and withdrawal following either neurotoxic methods or local gene expression. The selective ablation of cholinergic neurons in the nucleus accumbens of mice, using immunotoxin-mediated cell targeting, led to increased morphine reinforcement as well as increased aversion to morphine withdrawal. Combined with experiments using acetylcholinesterase inhibitors, the data suggest that enhancement of acetylcholine in this brain region hampers the development of addiction [21]. Neurons expressing NK1 receptors were selectively destroyed by local treatments with the neurotoxic substance P–saporin conjugate in several mouse brain areas. Morphine conditioned place preference was reduced when amygdala, but not striatal neurons, were lesioned, which suggests that NK1-bearing neurons of the amygdala are involved in morphine reward [22]. Viral-mediated overexpression of PLCg1 (phospholipase C g 1) in the rat ventral tegmental area (VTA) increased morphine reward when gene transfer was made in the rostral VTA, and produced morphine aversion when the injection site was the caudal VTA. These findings define two distinct www.sciencedirect.com
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Table 1 Genes promoting or counteracting addictive properties of morphine. Morphine reward Pro-opioid
Morphine withdrawal Anti-opioid
Neurotransmitters
Receptors
MOR D2, D2L NK1 CB1 a1b M5
D3
Effectors
PKCg PLCg1 (rVTA)
ERK1 PLCg1 (cVTA) DAT GAT1 RGS9 b-arrestin 2
Regulation
Neutral
Pro-opioid
Anti-opioid
Neutral
aCGRP
aCGRP NT-3 BDNF Orexin MOR KOR ORL-1 D2, D2L NK1 CB1 M5 GluR-A GluRe1 CREB a/D
ppEnk OFQ/N Galanin
NT-4
CCK2 A2a PAC1
DOR
DAT
GAT1 RGS9
KOR CCK2 PAC1
nNOS GRK3 b-arrestin 2
This table summarizes data from gene overexpression (PLCg1, GAT1), gene knockout (others), or a combination of both (galanin, GluRe1, RGS9) in mice. Animals have been examined in paradigms testing either the reinforcing properties of morphine using conditioned place preference and/or self-administration (Reward), or the dependence assessed by naloxone-precipitated withdrawal signs and/or aversion in chronically morphine treated animals (Withdrawal). ‘Pro-opioid’ genes are defined as genes that contribute to morphine reward and withdrawal. Behavioral responses to morphine are decreased when the pro-opioid gene is inactivated, and increased when the gene is overexpressed. In contrast, ‘anti-opioid’ genes are defined as genes whose activity develops during drug exposure to counteract morphine effects. Behavioral responses to morphine are therefore enhanced when the anti-opioid gene is deleted, or decreased when the gene is overexpressed. Genes are classified as ‘neutral’ when their manipulation in mice has no measurable consequence on the behavioral response. Beyond reward and withdrawal, other aspects of chronic morphine adaptations (tolerance, sensitization) have been tested in some genetically modified mice (not displayed in this table). Several mutants show modifications in responses to cocaine also [46], which suggests common molecular mechanisms possibly in the adaptations of dopaminergic circuits. Abbreviations and references: aCGRP, calcitonin gene-related peptide [47]; NT-3, neurotrophin 3 [48]; BDNF, brain-derived neurotrophic factor [49]; orexin [50]; ppENK, preproenkephalin [51]; OFQ/N, orphanin FQ/nociceptin [52]; galanin [53]; NT-4, neurotrophin 4 [54]; MOR, mu opioid receptor [7,55–57]; D2, dopamine D2 receptor [58–60]; D2L, long isoform of dopamine D2 receptor [61]; NK1, neurokinin-1 receptor [62,63]; CB1, cannabinoid 1 receptor [64–69]; a1B, adrenergic a1B receptor [70]; M5, muscarinic M5 acetylcholine receptor [71]; D3, dopamine D3 receptor [72]; KOR, kappa opioid receptor [73]; CCK2, cholecystokinin 2 receptor [74,75]; PAC1, pituitary adenylate cyclase-activating polypeptide type I-receptor [76]; ORL-1, nociceptin receptor [77,78]; GluR-A, AMPA-type glutamate receptor-A subunit [79]; GluRe1, NMDA receptor e1 subunit [25]; A2a, A2a adenosine receptor [80,81]; DOR, delta opioid receptor [51]; PKCg, protein kinase C g isoform [82]; PLCg1, phospholipase C g 1 [23] (rVTA: rostral VTA, cVTA: caudal VTA); ERK1, extracellular signal-regulated kinase 1 [83]; CREB a/D, a and D isoforms of the cAMP-responsive element-binding protein [84]; nNOS, neuronal nitric oxide synthase [85]; DAT, dopamine transporter [86]; GAT1, g-aminobutyric acid transporter I [87]; RGS9, regulator of G-protein signaling 9 [24]; GRK3, G-protein receptor kinase 3 [88]; b-arrestin 2 [89,90].
regions of the VTA that regulate the motivational properties of morphine in opposite directions [23]. Viral gene transfer was also used to rescue RGS9 gene expression in selected brain areas of RGS9 knockout mice. The increased morphine conditioned place preference of the knockout animals was reversed by RGS9 expression in nucleus accumbens, but not caudate putamen, highlighting the contribution of a limited population of RGS9 proteins in the regulation of morphine reward [24]. Gene rescue on a knockout background was also achieved by electroporation in a mouse brain in vivo. The local re-expression of the GluRe1 (NMDA receptor e1 subunit) subunit of the N-methyl-D-aspartate (NMDA) receptor in the nucleus accumbens partially reversed the attenuated morphine withdrawal syndrome of the GluRe1 knockout mice. Morphine analgesic tolerance was otherwise www.sciencedirect.com
rescued in other brain areas, demonstrating spatial dissociation of distinct long-term adaptations to morphine [25].
Mu receptor-activated neural circuits: functional mapping Mu receptors are broadly expressed in all brain areas belonging to the circuits of addiction (Figure 2). These include mesolimbic dopaminergic neurons, which originate from the VTA and project to the nucleus accumbens. These neurons have been widely studied in the past few years. The nucleus accumbens itself is part of a complex network that includes prefrontal cortical areas, hippocampus, and basal forebrain structures known as the extended amygdala. The extended amygdala is formed by a continuum between the shell of the nucleus accumbens, the bed nucleus of the stria terminalis and the central nucleus Current Opinion in Neurobiology 2004, 14:370–378
374 Signalling mechanisms
Figure 2
Hipp PfCx Mesolimbic DA pathway CPu Str NAc
induced MAPK phosphorylation were attenuated along the two chronic morphine regimes (tolerance) in most brain areas, except in the central nucleus in the amygdala [30]. CRE-LacZ reporter mice, in which the LacZ gene encoding b-galactosidase is under the control of cAMP response element-consensus sequence, represent another useful approach for the functional mapping of morphineinduced neuronal activation [31] and it is likely that similar strategies will be developed in the future.
VTA BST
Novel approaches to study mu receptor signaling and molecular adaptations to morphine in vivo
Amg
Extended amygdala Current Opinion in Neurobiology
Schematic representation of brain areas under study in addiction research. The mesolimbic dopamine (DA) pathway has been the main focus of interest during the past few decades. This system includes dopaminergic neurons (black arrows) projecting from the ventral tegmental area (VTA) to striatal structures (Str) (nucleus accumbens, NAc; and caudate-putamen, CPu), the prefrontal cortex (PfCx) and the amygdala (Amg). The role of cortical afferents to the nucleus accumbens originating from the prefrontal cortex and the hippocampus (Hipp) is also investigated (dashed arrows). Recently, several studies have highlighted the involvement of the extended amygdala particularly in the negative emotional aspects of addiction. This structure includes part of the shell of the nucleus accumbens, the bed nucleus of the stria terminalis (BST) and some nuclei of the amygdala (grey area). More detailed functional anatomy of addiction circuits can be found, for example, in the review by Nestler [91].
of the amygdala, and is becoming the focus of much interest in drug abuse research [26]. Other brain areas important in adaptations to drugs of abuse include the locus coeruleus, the hypothalamus, and some brainstem nuclei. Many functional mapping studies conducted following acute or chronic morphine administration have shown neural activation of all these structures. The use of refined behavioral paradigms has recently strengthened our understanding of the importance of the extended amygdala in the negative motivational state of addiction. Low doses of naloxone administered to morphinedependent rats produced place aversion without somatic signs of withdrawal, and this treatment specifically increased c-fos activity in areas of the extended amygdala [27,28]. In a sensitization study, morphine-abstinent rats were re-exposed to the drug in a place conditioning paradigm. The animals showed potentiated morphine reinforcement that was associated with enhanced c-fos expression in the extended amygdala as well as the cingulate cortex, the core of the nucleus accumbens, and the basolateral nucleus of the amygdala [29]. In another functional mapping study, constant or escalating doses of morphine led to the development of either sensitization or tolerance, two conditions in which mitogenactivated protein kinase (MAPK) activation patterns differed markedly, particularly in the basolateral amygdala. Interestingly, also in this study, changes in morphineCurrent Opinion in Neurobiology 2004, 14:370–378
To complement candidate gene approaches several screening methods are now being developed to better understand the consequences of mu receptor activation. The yeast two-hybrid system has identified proteins that physically interact with the carboxy-termini of mu receptors and regulate distinct aspects of mu receptor activity at the cellular level. Phospholipase D2 was found to associate with the mu receptor in the plasma membrane of transfected HEK cells, and accelerated agonist-induced internalization [32]. Filamin A, a cytoskeletal protein known to bind to several membrane proteins, appeared necessary for normal mu receptor trafficking in a study comparing filamin-expressing versus filamin-deficient melanoma cells [33]. By contrast, periplakin, another cytolinker protein, did not alter mu receptor internalization in transfected HEK cells but reduced coupling to the G protein through interaction with helix VIII of the receptor [34]. Proteins that interact with intracellular domains of other G protein-coupled receptors are being discovered, such as GASP (G protein-coupled receptorassociated sorting protein), which interacts with delta rather than mu receptors [35]. Whether or not GASP homologs [36] regulate mu receptor signaling in vivo is an open question. At present, only heterologous systems have been used to study the putative mu receptor partners, and the biological relevance of these interactions remains to be established. Differential gene expression experiments have identified several gene families whose transcription is regulated following treatment with drugs of abuse [37]. The drugs that have been used in these studies are mainly psychostimulants, studies with morphine are still limited. Acute morphine administration modified transcription of 45 genes (down-regulation) and nine genes (up-regulation) belonging mostly to mitochondrial and cytoskeletal gene families in the medial striatum of mice [38]. In the rat frontal cortex, chronic morphine exposure upregulated a set of 14 genes including many heat-shock proteins, whereas naloxone-precipitated withdrawal induced 18 genes that essentially encoded transcription factors [39]. In a series of interesting studies that compared self- versus forced-heroin administration in rats [40], 25 genes were downregulated in the shell of the www.sciencedirect.com
Mu opioid receptor Contet, Kieffer and Befort 375
nucleus accumbens in active but not passive drug consumption [41]. Among these, 18 genes were also up-regulated in the core of the nucleus accumbens irrespective of the administration mode [42]. Together therefore, the two studies reveal genes involved either in the cognitive aspect of voluntary drug use (shell) or in the general plasticity consecutive to heroin pharmacological effects (core). In fact, surprisingly few novel genes have emerged from these initial studies, and these approaches await further refinement of brain dissection methods, combined with parallel proteomic screens and extensive target validation.
Conclusions Recent basic research has first, explored cellular mechanisms that regulate mu receptor activity in neurons, second, identified several genes associated with mu receptor signaling in vivo and third, revealed the crucial role of neuronal activation in the extended amygdala following mu receptor activation. Mu receptor signaling is activated by several drugs of abuse (opioid and non-opioid) and therefore represents a potential target for the therapeutics of addiction. In the clinic, strategies have been developed to either block (naloxone, naltrexone) or partially activate (methadone, buprenorphine) the mu receptor. These therapeutic agents are mainly used in the context of opioid addiction and have had some success in the treatment of alcoholism [43–45]. Although this work is still speculative, it is possible that the clarification of downstream mu receptor effectors in the various recruited brain areas and the discovery of novel proteins that regulate mu-mediated responses will broaden the panel of possible molecular targets useful in the treatment of addictive disorders.
Acknowledgements The authors wish to thank the Human Frontier Science Program, the Mission Interministe´ rielle de Lutte contre la Drogue et la Toxicomanie and the National Institute of Health (NIH-NIDA #DA05010; NIAAA#AA13481) for financial support. They also thank LA Karchewski for helpful advice.
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Current Opinion in Neurobiology 2004, 14:370–378 This review comes from a themed issue on Signalling mechanisms Edited by Richard L Huganir and S Lawrence Zipursky Available online 19th May 2004 0959-4388/$ – see front matter ß 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.conb.2004.05.005 Abbreviations DAMGO [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin GIRK G protein-coupled inwardly rectifying potassium channel HEK human embryonic kidney MAPK mitogen-activated protein kinase MOR mu opioid receptor VTA ventral tegmental area
Introduction The mu opioid receptor: a key molecular player in drug addiction
Drug addiction is a chronic relapsing disorder that results from gradual adaptations of the brain to repeated drug exposure. The current understanding of this complex phenomenon is that neurons responding to natural reinforcers such as food, sex, and social interactions are abnormally stimulated, leading to strong deregulations of brain reward pathways [1] and aberrant learning processes [2]. Addiction has many faces, including initiation and maintenance of drug consumption, withdrawal episodes, protracted abstinence, and relapse. Animal models Current Opinion in Neurobiology 2004, 14:370–378
for different aspects of drug-seeking behaviors have been successfully developed, and efforts are made to comprehend the transition between drug use and drug abuse [3]. The brain circuits of addiction involve reward pathways, in association with stress, obsessive-compulsive, and habit-forming systems [4], and molecular adaptations to chronic drug use are being actively examined in this broad neural network. Many neurotransmitter systems are recruited during these processes, with dopaminergic systems being important and the most frequently studied (see Bonci et al. [5] and references therein). The endogenous opioid system is also a major player in addiction. Because the opioid system plays a central part in modulating mood and well being, it is believed that modifications of endogenous opioids participate in the development of drug abuse. In addition, the opioid system could also influence drug craving and relapse by altering stress physiology. Both pharmacological and genetic experimental manipulations of the opioid system support these views and demonstrate that endogenous opioids influence reinforcement and adaptations to many drugs of abuse [4]. The opioid system consists of three G protein-coupled receptors, mu, delta, and kappa, which are stimulated by a family of endogenous opioid peptides [6]. Opioid receptors can also be activated exogenously by alkaloid opiates, the prototype of which is morphine. The finding that morphine’s analgesic and addictive properties are abolished in mice lacking the mu opioid receptor has unambiguously demonstrated that mu receptors mediate both the therapeutic and the adverse activities of this compound [7]. Importantly, a series of studies has shown that the reinforcing properties of alcohol, cannabinoids, and nicotine — each of which acts at a different receptor — are also strongly diminished in these mutant mice [8]. The genetic approach therefore highlights mu receptors as convergent molecular switches, which mediate reinforcement following direct (morphine) or indirect activation (non-opioid drugs of abuse; see Figure 1). Beyond the rewarding aspect of drug consumption, pharmacological studies have also suggested a role for this receptor in the maintenance of drug use, as well as craving and relapse [4]. As a consequence, expanding our understanding of mu receptor function should greatly help to further our knowledge of the general mechanisms that underlie addiction. This review focuses on recent findings regarding cellular regulation of mu receptors in the neuron and molecular mechanisms of responses to mu receptor activation in vivo. The latter part describes several genes involved in mu receptor signaling, highlights the implicated brain www.sciencedirect.com
Mu opioid receptor Contet, Kieffer and Befort 371
Figure 1
Reward circuits
GABAA/NMDA CB1 nACh
Alcohol THC Nicotine
Endogenous opioids? MOR
Natural reinforcers
Morphine MOR signaling Cellular regulation In vivo associated genes In vivo adaptations Addiction Current Opinion in Neurobiology
Mu opioid receptors (MOR) are largely distributed along reward circuits where they mediate the reinforcing activities of morphine and several non-opioid drugs. This was suggested by pharmacological studies and was recently demonstrated genetically by the analysis of mu receptor knockout mice in conditioned place preference or self-administration paradigms. In these mutant mice the rewarding properties of alcohol, D9-tetrahydrocannabinol (THC) and nicotine are abolished (reviewed by Kieffer and Gave´ riaux-Ruff [8]). These non-opioid drugs act at their own receptors (GABAA and NMDA receptors for alcohol, CB1 receptor for THC and nicotinic acetylcholine receptor for nicotine) and are likely to induce the release of endogenous opioid peptides that, in turn, activate mu receptors. Mu receptors, therefore, represent a convergent molecular gate in the initiation of addictive behaviors. Inadequate mu receptor activation might be one of the mechanisms underlying deregulation of reward pathways, which characterizes the addicted state. Whether or not mu receptors also mediate natural rewarding stimuli is presently being investigated in genetically modified mice.
structures and summarizes initial results from genomic approaches.
Regulation of mu receptor signaling in neurons: an agonist-dependent process Opioid agonists binding at mu receptors modulate intracellular effectors through inhibitory Go/Gi proteins. Receptor signaling, in turn, is readily terminated by several cellular regulatory processes that include phosphorylation, desensitization, endocytosis, and downregulation. An important observation from signaling and trafficking studies in transfected cells is that mu receptor activation and subsequent regulations are strongly agonist-dependent. Therefore, the agonist–receptor complex, rather than the receptor itself, determines the ultimate physiological effects of the ligand. Responses to peptidic opioids (DAMGO [(D-Ala2, N-Me-Phe4, Gly5ol)-enkephalin], enkephalins) and alkaloid-type compounds (morphine, fentanyl, methadone) have been examined to find a relationship between agonist efficacy and agonist-induced regulatory processes (see von Zastrow et al. [9] and references therein; [10]). Although correlations do not appear straightforward, it is currently accepted that opioid peptide signaling is efficiently blunted by the different regulatory mechanisms, whereas morphine signaling induces low levels of mu receptor regulation. The impact of agonist-induced mu receptor regulation on long-term in vivo adaptations is a matter of debate [11,12]. www.sciencedirect.com
A recent hypothesis is that mu receptor regulation could protect neurons from overstimulation and prevent counter adaptations that eventually lead to dependence. One approach used to explore this hypothesis in vivo was the simultaneous administration of mu agonists with distinct efficacy and regulatory profiles. The co-administration of a subeffective dose of DAMGO together with morphine reduced tolerance to morphine analgesia, and was accompanied by enhanced receptor internalization in spinal cord neurons [13]. Two recent studies have examined whether or not this agonist combination would similarly enhance mu receptor desensitization in the locus coeruleus. In these neurons, however, morphine did not efficiently desensitize G protein-coupled inwardly rectifying potassium channel (GIRK) currents either with or without the co-application of the low dose of DAMGO [10,14]. In addition, in Bailey’s study [14] subeffective DAMGO could neither facilitate desensitization of the morphine-induced guinea pig ileum twitch inhibition nor trigger morphine-induced internalization of the mu receptor in transfected human embryonic kidney (HEK) cells [14]. Thus, the benefit of mu agonist cocktails in minimizing long-term adaptations remains controversial. At present, the question of whether or not regulatory properties of mu agonists are predictive of abuse liability is a growing topic of attention. The lack of morphine-induced mu receptor internalization, proposed to be responsible for cellular adaptations relevant to addiction, has been widely observed in Current Opinion in Neurobiology 2004, 14:370–378
372 Signalling mechanisms
heterologous systems. It is crucial to examine whether or not morphine also poorly induces endocytosis in brain neurons expressing endogenous mu receptors. In many mu-expressing brain regions [15], including the locus coeruleus [16], treatment of animals with morphine failed to induce mu receptor endocytosis, which is consistent with in vitro studies. In rat nucleus accumbens neurons, however, acute morphine administration unexpectedly produced an intracellular redistribution of mu receptors in dendrites. In the same neurons, morphine was ineffective in cell bodies as previously observed in other systems [17]. This study shows that trafficking of mu receptors following morphine binding depends on which subcellular compartment within the neuron is involved, and is therefore more complex than was anticipated from studies in transfected cells. Finally, intriguing cellular consequences of chronic morphine treatment have been described recently. In one study, prolonged morphine treatment produced recruitment of intracellular delta receptors to the cell surface in dendrites of rat spinal cord neurons, an effect mediated by mu receptors and whose mechanism remains to be elucidated [18,19]. In another study, basal signaling of mu receptors was enhanced in the brain of mice chronically treated with morphine, a phenomenon that might contribute to the establishment of dependence in these animals [20].
Molecular adaptations to mu receptor activation in vivo: genetic approaches Among mu agonists, morphine is most relevant both in terms of clinical use (pain treatment) and in abuse potential (heroin addiction). Morphine essentially activates mu receptors in vivo, and the effects of chronic morphine exposure in whole animals reflect the consequences of repeated mu receptor activation in the nervous system. The analysis of morphine responses in about 30 genetically modified animals has recently revealed many partners of mu receptor signaling in vivo. In Table 1, we have selected data from experimental paradigms reflecting the initial (reward) and maintenance (revealed by precipitated withdrawal) phases of morphine addiction. The inactivation of many genes encoding neurotransmitters and/or their receptors disrupts morphine conditioned place preference or self-administration, two paradigms testing reinforcing properties of morphine. These systems therefore exhibit ‘pro-opioid’ activity, possibly acting in conjunction with mu receptor signaling to produce morphine reinforcement. Interestingly, several of these pro-opioid genes (D2 [dopamine D2 receptor], NK1 [neurokinin-1 receptor], CB1 [cannabinoid 1 receptor], M5 [muscarinic M5 acetylcholine receptor]) also participate in the development of morphine dependence, suggesting a broad recruitment of these transmitter systems in several aspects of addictive behaviors. Some pro-opioid genes (aCGRP [calcitonin gene-related peptide], KOR Current Opinion in Neurobiology 2004, 14:370–378
[kappa opioid receptor]) contribute to physical dependence but not to reward, as a result of temporal and/or spatial dissociation of the two phenomena. The inactivation of other genes results in enhanced morphine effects, and thus, these genes display an ‘anti-opioid’ activity which normally opposes morphine action in wild type mice (see Table 1). Consistent with the pharmacology, CCK2 (cholecystokinin 2 receptor) and OFQ/N (orphanin FQ/nociceptin) systems belong to this category. Gene products known to desensitize G protein signaling in vitro (RGS9 [regulator of G protein signaling 9], b-arrestin 2) also counteract some morphine responses in vivo. Interestingly, the dopamine transporter DAT differentially modulates reward and withdrawal. Signaling effectors otherwise display pro-opioid (PKCg [protein kinase C g isoform], CREB [cAMP responsive element binding protein]), anti-opioid (ERK1; extracellular signal regulated kinase 1), or no (nNOS; neuronal nitric oxide synthase) activity. At present, data from genetically modified mice have essentially addressed the role of genes already known to influence addictive behaviors from the pharmacological perspective. It is likely that a broad ‘morphine screen’ of available mouse mutants would reveal unexpected genes that contribute to adaptations to drugs of abuse, and be informative in terms of gene discovery for addiction research. Identifying neural sites of gene activity is crucial in our understanding of addiction (Figure 2). The constitutive knockout approach provides no clues as to regional gene activity, and, furthermore, compensations could take place during development and account for the observed phenotypes. While strategies for the site-specific knockout of candidate genes are being developed, alternative approaches have provided fascinating results. Recent examples are data showing the modulation of morphine reward and withdrawal following either neurotoxic methods or local gene expression. The selective ablation of cholinergic neurons in the nucleus accumbens of mice, using immunotoxin-mediated cell targeting, led to increased morphine reinforcement as well as increased aversion to morphine withdrawal. Combined with experiments using acetylcholinesterase inhibitors, the data suggest that enhancement of acetylcholine in this brain region hampers the development of addiction [21]. Neurons expressing NK1 receptors were selectively destroyed by local treatments with the neurotoxic substance P–saporin conjugate in several mouse brain areas. Morphine conditioned place preference was reduced when amygdala, but not striatal neurons, were lesioned, which suggests that NK1-bearing neurons of the amygdala are involved in morphine reward [22]. Viral-mediated overexpression of PLCg1 (phospholipase C g 1) in the rat ventral tegmental area (VTA) increased morphine reward when gene transfer was made in the rostral VTA, and produced morphine aversion when the injection site was the caudal VTA. These findings define two distinct www.sciencedirect.com
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Table 1 Genes promoting or counteracting addictive properties of morphine. Morphine reward Pro-opioid
Morphine withdrawal Anti-opioid
Neurotransmitters
Receptors
MOR D2, D2L NK1 CB1 a1b M5
D3
Effectors
PKCg PLCg1 (rVTA)
ERK1 PLCg1 (cVTA) DAT GAT1 RGS9 b-arrestin 2
Regulation
Neutral
Pro-opioid
Anti-opioid
Neutral
aCGRP
aCGRP NT-3 BDNF Orexin MOR KOR ORL-1 D2, D2L NK1 CB1 M5 GluR-A GluRe1 CREB a/D
ppEnk OFQ/N Galanin
NT-4
CCK2 A2a PAC1
DOR
DAT
GAT1 RGS9
KOR CCK2 PAC1
nNOS GRK3 b-arrestin 2
This table summarizes data from gene overexpression (PLCg1, GAT1), gene knockout (others), or a combination of both (galanin, GluRe1, RGS9) in mice. Animals have been examined in paradigms testing either the reinforcing properties of morphine using conditioned place preference and/or self-administration (Reward), or the dependence assessed by naloxone-precipitated withdrawal signs and/or aversion in chronically morphine treated animals (Withdrawal). ‘Pro-opioid’ genes are defined as genes that contribute to morphine reward and withdrawal. Behavioral responses to morphine are decreased when the pro-opioid gene is inactivated, and increased when the gene is overexpressed. In contrast, ‘anti-opioid’ genes are defined as genes whose activity develops during drug exposure to counteract morphine effects. Behavioral responses to morphine are therefore enhanced when the anti-opioid gene is deleted, or decreased when the gene is overexpressed. Genes are classified as ‘neutral’ when their manipulation in mice has no measurable consequence on the behavioral response. Beyond reward and withdrawal, other aspects of chronic morphine adaptations (tolerance, sensitization) have been tested in some genetically modified mice (not displayed in this table). Several mutants show modifications in responses to cocaine also [46], which suggests common molecular mechanisms possibly in the adaptations of dopaminergic circuits. Abbreviations and references: aCGRP, calcitonin gene-related peptide [47]; NT-3, neurotrophin 3 [48]; BDNF, brain-derived neurotrophic factor [49]; orexin [50]; ppENK, preproenkephalin [51]; OFQ/N, orphanin FQ/nociceptin [52]; galanin [53]; NT-4, neurotrophin 4 [54]; MOR, mu opioid receptor [7,55–57]; D2, dopamine D2 receptor [58–60]; D2L, long isoform of dopamine D2 receptor [61]; NK1, neurokinin-1 receptor [62,63]; CB1, cannabinoid 1 receptor [64–69]; a1B, adrenergic a1B receptor [70]; M5, muscarinic M5 acetylcholine receptor [71]; D3, dopamine D3 receptor [72]; KOR, kappa opioid receptor [73]; CCK2, cholecystokinin 2 receptor [74,75]; PAC1, pituitary adenylate cyclase-activating polypeptide type I-receptor [76]; ORL-1, nociceptin receptor [77,78]; GluR-A, AMPA-type glutamate receptor-A subunit [79]; GluRe1, NMDA receptor e1 subunit [25]; A2a, A2a adenosine receptor [80,81]; DOR, delta opioid receptor [51]; PKCg, protein kinase C g isoform [82]; PLCg1, phospholipase C g 1 [23] (rVTA: rostral VTA, cVTA: caudal VTA); ERK1, extracellular signal-regulated kinase 1 [83]; CREB a/D, a and D isoforms of the cAMP-responsive element-binding protein [84]; nNOS, neuronal nitric oxide synthase [85]; DAT, dopamine transporter [86]; GAT1, g-aminobutyric acid transporter I [87]; RGS9, regulator of G-protein signaling 9 [24]; GRK3, G-protein receptor kinase 3 [88]; b-arrestin 2 [89,90].
regions of the VTA that regulate the motivational properties of morphine in opposite directions [23]. Viral gene transfer was also used to rescue RGS9 gene expression in selected brain areas of RGS9 knockout mice. The increased morphine conditioned place preference of the knockout animals was reversed by RGS9 expression in nucleus accumbens, but not caudate putamen, highlighting the contribution of a limited population of RGS9 proteins in the regulation of morphine reward [24]. Gene rescue on a knockout background was also achieved by electroporation in a mouse brain in vivo. The local re-expression of the GluRe1 (NMDA receptor e1 subunit) subunit of the N-methyl-D-aspartate (NMDA) receptor in the nucleus accumbens partially reversed the attenuated morphine withdrawal syndrome of the GluRe1 knockout mice. Morphine analgesic tolerance was otherwise www.sciencedirect.com
rescued in other brain areas, demonstrating spatial dissociation of distinct long-term adaptations to morphine [25].
Mu receptor-activated neural circuits: functional mapping Mu receptors are broadly expressed in all brain areas belonging to the circuits of addiction (Figure 2). These include mesolimbic dopaminergic neurons, which originate from the VTA and project to the nucleus accumbens. These neurons have been widely studied in the past few years. The nucleus accumbens itself is part of a complex network that includes prefrontal cortical areas, hippocampus, and basal forebrain structures known as the extended amygdala. The extended amygdala is formed by a continuum between the shell of the nucleus accumbens, the bed nucleus of the stria terminalis and the central nucleus Current Opinion in Neurobiology 2004, 14:370–378
374 Signalling mechanisms
Figure 2
Hipp PfCx Mesolimbic DA pathway CPu Str NAc
induced MAPK phosphorylation were attenuated along the two chronic morphine regimes (tolerance) in most brain areas, except in the central nucleus in the amygdala [30]. CRE-LacZ reporter mice, in which the LacZ gene encoding b-galactosidase is under the control of cAMP response element-consensus sequence, represent another useful approach for the functional mapping of morphineinduced neuronal activation [31] and it is likely that similar strategies will be developed in the future.
VTA BST
Novel approaches to study mu receptor signaling and molecular adaptations to morphine in vivo
Amg
Extended amygdala Current Opinion in Neurobiology
Schematic representation of brain areas under study in addiction research. The mesolimbic dopamine (DA) pathway has been the main focus of interest during the past few decades. This system includes dopaminergic neurons (black arrows) projecting from the ventral tegmental area (VTA) to striatal structures (Str) (nucleus accumbens, NAc; and caudate-putamen, CPu), the prefrontal cortex (PfCx) and the amygdala (Amg). The role of cortical afferents to the nucleus accumbens originating from the prefrontal cortex and the hippocampus (Hipp) is also investigated (dashed arrows). Recently, several studies have highlighted the involvement of the extended amygdala particularly in the negative emotional aspects of addiction. This structure includes part of the shell of the nucleus accumbens, the bed nucleus of the stria terminalis (BST) and some nuclei of the amygdala (grey area). More detailed functional anatomy of addiction circuits can be found, for example, in the review by Nestler [91].
of the amygdala, and is becoming the focus of much interest in drug abuse research [26]. Other brain areas important in adaptations to drugs of abuse include the locus coeruleus, the hypothalamus, and some brainstem nuclei. Many functional mapping studies conducted following acute or chronic morphine administration have shown neural activation of all these structures. The use of refined behavioral paradigms has recently strengthened our understanding of the importance of the extended amygdala in the negative motivational state of addiction. Low doses of naloxone administered to morphinedependent rats produced place aversion without somatic signs of withdrawal, and this treatment specifically increased c-fos activity in areas of the extended amygdala [27,28]. In a sensitization study, morphine-abstinent rats were re-exposed to the drug in a place conditioning paradigm. The animals showed potentiated morphine reinforcement that was associated with enhanced c-fos expression in the extended amygdala as well as the cingulate cortex, the core of the nucleus accumbens, and the basolateral nucleus of the amygdala [29]. In another functional mapping study, constant or escalating doses of morphine led to the development of either sensitization or tolerance, two conditions in which mitogenactivated protein kinase (MAPK) activation patterns differed markedly, particularly in the basolateral amygdala. Interestingly, also in this study, changes in morphineCurrent Opinion in Neurobiology 2004, 14:370–378
To complement candidate gene approaches several screening methods are now being developed to better understand the consequences of mu receptor activation. The yeast two-hybrid system has identified proteins that physically interact with the carboxy-termini of mu receptors and regulate distinct aspects of mu receptor activity at the cellular level. Phospholipase D2 was found to associate with the mu receptor in the plasma membrane of transfected HEK cells, and accelerated agonist-induced internalization [32]. Filamin A, a cytoskeletal protein known to bind to several membrane proteins, appeared necessary for normal mu receptor trafficking in a study comparing filamin-expressing versus filamin-deficient melanoma cells [33]. By contrast, periplakin, another cytolinker protein, did not alter mu receptor internalization in transfected HEK cells but reduced coupling to the G protein through interaction with helix VIII of the receptor [34]. Proteins that interact with intracellular domains of other G protein-coupled receptors are being discovered, such as GASP (G protein-coupled receptorassociated sorting protein), which interacts with delta rather than mu receptors [35]. Whether or not GASP homologs [36] regulate mu receptor signaling in vivo is an open question. At present, only heterologous systems have been used to study the putative mu receptor partners, and the biological relevance of these interactions remains to be established. Differential gene expression experiments have identified several gene families whose transcription is regulated following treatment with drugs of abuse [37]. The drugs that have been used in these studies are mainly psychostimulants, studies with morphine are still limited. Acute morphine administration modified transcription of 45 genes (down-regulation) and nine genes (up-regulation) belonging mostly to mitochondrial and cytoskeletal gene families in the medial striatum of mice [38]. In the rat frontal cortex, chronic morphine exposure upregulated a set of 14 genes including many heat-shock proteins, whereas naloxone-precipitated withdrawal induced 18 genes that essentially encoded transcription factors [39]. In a series of interesting studies that compared self- versus forced-heroin administration in rats [40], 25 genes were downregulated in the shell of the www.sciencedirect.com
Mu opioid receptor Contet, Kieffer and Befort 375
nucleus accumbens in active but not passive drug consumption [41]. Among these, 18 genes were also up-regulated in the core of the nucleus accumbens irrespective of the administration mode [42]. Together therefore, the two studies reveal genes involved either in the cognitive aspect of voluntary drug use (shell) or in the general plasticity consecutive to heroin pharmacological effects (core). In fact, surprisingly few novel genes have emerged from these initial studies, and these approaches await further refinement of brain dissection methods, combined with parallel proteomic screens and extensive target validation.
Conclusions Recent basic research has first, explored cellular mechanisms that regulate mu receptor activity in neurons, second, identified several genes associated with mu receptor signaling in vivo and third, revealed the crucial role of neuronal activation in the extended amygdala following mu receptor activation. Mu receptor signaling is activated by several drugs of abuse (opioid and non-opioid) and therefore represents a potential target for the therapeutics of addiction. In the clinic, strategies have been developed to either block (naloxone, naltrexone) or partially activate (methadone, buprenorphine) the mu receptor. These therapeutic agents are mainly used in the context of opioid addiction and have had some success in the treatment of alcoholism [43–45]. Although this work is still speculative, it is possible that the clarification of downstream mu receptor effectors in the various recruited brain areas and the discovery of novel proteins that regulate mu-mediated responses will broaden the panel of possible molecular targets useful in the treatment of addictive disorders.
Acknowledgements The authors wish to thank the Human Frontier Science Program, the Mission Interministe´ rielle de Lutte contre la Drogue et la Toxicomanie and the National Institute of Health (NIH-NIDA #DA05010; NIAAA#AA13481) for financial support. They also thank LA Karchewski for helpful advice.
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