REVIEwS. Opioid receptors: drivers to addiction?

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1 REVIEwS Opioid receptors: drivers to addiction? Emmanuel Darcq 1 and Brigitte Lina Kieffer 1,2* Abstract Drug addiction is a worldwide societal problem and public health burden, and results from recreational drug use that develops into a complex brain disorder. The opioid system, one of the first discovered neuropeptide systems in the history of neuroscience, is central to addiction. Recently, opioid receptors have been propelled back on stage by the rising opioid epidemics, revolutions in G protein- coupled receptor research and fascinating developments in basic neuroscience. This Review discusses rapidly advancing research into the role of opioid receptors in addiction, and addresses the key questions of whether we can kill pain without addiction using mu- opioid-receptor- targeting opiates, how mu- and kappa- opioid receptors operate within the neurocircuitry of addiction and whether we can bridge human and animal opioid research in the field of drug abuse. Psychotomimetic An effect caused by drugs, mimicking symptoms of psychosis, such as agitation, delusions and delirium. 1 Douglas Mental Health Institute, Department of Psychiatry, McGill University, Montreal, Quebec, Canada. 2 Institut de Génétique et de Biologie Moléculaire et Cellulaire, INSERM, Centre National de la Recherche Scientifique and University of Strasbourg, Strasbourg, France. *e- mail: brigitte.kieffer@ douglas.mcgill.ca s x Opium is extracted from the seeds of poppies (Papaver somniferum) and has been used for more than 4,000 years in medicinal and recreational practices to relieve pain and cause euphoria. Morphine was isolated in 1805 as the most active component of opium 1 and remains the most potent painkiller in modern medicine, despite severe adverse effects and a strong potential for addiction. Heroin, the diacetylated form of morphine, was originally marketed as the first non- addictive opiate to treat cough and asthma in 1898, yet heroin addiction has represented a major societal problem ever since. More recently, an opioid epidemic has emerged in occidental countries, particularly in North America 2. The overprescription of opioids for pain relief in the past 20 years has led to a rapid surge in the non- medical use of prescribed opioids, with deaths by overdose and transition to heroin abuse rising at alarming rates 3 6. The increasing availability of low- cost synthetic opioids, such as non- pharmaceutical fentanyls 7, further feeds the epidemic. This opioid crisis has fostered novel public policies and much interest in developing better opioids to treat pain. For medical purposes 8, the ideal opiate drug would relieve pain with high and sustained efficacy (that is, without tolerance), without the threats of respiratory depression (the main cause of overdose) and without drug dependence (contributing to addiction). The early 1970s saw the game- changing discovery that opiate drugs bind to receptors in the brain (see references cited in ref. 1 ) and hijack a complex endogenous neuromodulatory system. The opioid system comprises three homologous G protein- coupled receptors (GPCRs) known as mu-, delta- and kappa- opioid receptors (MORs, DORs and KORs, respectively). Under physiological conditions, opioid receptors are stimulated by endogenous opioid peptides, forming a peptide family that includes β- endorphin, enkephalins and dynorphins. Distributed throughout the nervous system, opioid peptides and receptors reduce responses to painful stimuli and stress, and influence reward processing and mood. The latter influence represents a fundamental role of the opioid system and will be a main focus of this Review. Note that the activity of the endogenous opioid system is extremely broad and covers many other aspects of physiology and behaviour (reviewed in ref. 9 ), but these are less related to addiction and will not be discussed here. In the late 1980s and early 1990s, the isolation of three genes encoding opioid peptide precursors (namely, POMC, PENK and PDYN, which encode proopio melanocortin, preproenkephalin (also known as proenkephalin) and preprodynorphin (also known as prodynorphin), respectively) and the genes encoding MORs (OPRM1), DORs (OPRD1) and KORs (OPRK1) opened an era of molecular and genetic investigations of the opioid system 10,11. Oprm1 deletion in mice simultaneously eliminated the analgesic, rewarding and dependence- inducing effects of morphine 12, demonstrating that the MOR is the sole responsible receptor for both the therapeutic and the adverse actions of morphine. The MOR is also the key molecular target for biological effects of other clinically useful and/or abused opiates (including heroin, fentanyl, oxycodone and methadone). Given the high risk of adverse effects of MOR agonists, the enthusiasm for drug discovery efforts targeting MORs abated in the late 1990s (although new efforts are starting; see Box 1). On another front, Oprd1-knockout mice revealed that DORs have anxiolytic and antidepressant functions 13, decidedly distinguishing this receptor from MORs. Many pharmacological studies have now implicated DORs in mood disorders and chronic pain By contrast, KOR activation produces both aversive and psychotomimetic effects 17. This peculiar profile has strongly limited the Nature Neuroscience

2 Box 1 can we kill pain without addiction using Mor opiates? Many drug discovery programmes targeting mu- opioid receptors (MOrs) for analgesia without addiction have had limited success. However, recent advances in G protein- coupled receptor (GPCr) biology have revitalized the field. Biased signalling GPCr conformation depends on biological context. the active complex is determined by the drug and neighbouring proteins, including effectors 150. thus, different agonists may engage distinct effectors, leading to biased agonism (see part a of the figure). Biased MOr agonists might reduce pain with minimal adverse effects (part b of the figure). Mice lacking β- arrestin 2 (β- arr) one of the two main MOr effectors showed enhanced morphine analgesia 154 and reduced morphine- induced constipation and respiratory depression 155. G i - biased MOr agonists that minimally recruit β- arr include trv130 (also known as oliceridine; identified through cell- based assays) 156, which is in a phase iii trial for analgesia 157, mitragynine pseudoindoxyl (developed from natural products) 158 and PZM21 (identified using computational docking) 26. these compounds confer strong analgesia with reduced constipation and respiratory depression in rodents. Novel MOr agonists covering the entire G i β- arr bias range were recently designed, with G i bias correlating with the analgesia respiratory depression therapeutic window 159. Biased signalling in cells has not often been correlated with addiction-related behaviours (for example, drug reward), as these in vivo experiments are less amenable to medicinal chemistry efforts. the three compounds in part b do not induce conditioned place preference at analgesic doses 26,158. However, further research should assess the hedonic and motivational properties of G i - biased drugs; a first study has mixed conclusions 160. Moreover, part of the gap between in vitro predictions and in vivo responses depends on location bias 161 and systems bias 162. studies on receptor signalling in different neuronal compartments and at different brain sites will expand in the future. other approaches Other approaches 163 (part c of the figure) include identifying compounds acting at multiple opioid receptors, opioid receptor dimers (MOr delta-opioid receptor (DOr); see supplementary Box 1) or opioid non-opioid receptor dimers (for example, MOr CC- chemokine receptor 5 (CCr5)); compounds with agonist and antagonist activity at MOrs and anti- opioid receptors (such as cholecystokinin, neurokinin 1 or the nociceptin peptide receptor), respectively; and compounds targeting effectors besides arrestins (for example, regulator of G protein signalling (rgs) proteins). in addition, a compound binding a truncated MOr isoform (lacking the first transmembrane domain) had an encouraging pharmacological profile, although with unusual properties 164,165 ; the biological importance of this isoform remains unclear 166. in a novel approach (part c of the figure), the active MOr receptor structure 167 was used to computationally simulate ligand docking at low ph, with the rationale that binding in an acidic environment might limit effects to injured sites. a ph- sensitive fluorinated fentanyl derivative called NFePP reduced inflammatory pain without central or intestinal effects 168. adverse effects might be limited by mimicking or enhancing endogenous opioid signalling (part d of the figure). endogenous opioid analogues with improved stability and bioavailability have been developed 163. recently, MOr positive allosteric modulators were developed to act on allosteric sites (2) to facilitate agonism at the orthosteric site (1), strengthening MOr activity at the optimal times and sites, with fewer adverse effects (part d of the figure) 169. BMs enhances opioid peptide- induced signalling 170 and probably binds to the MOr Na + -binding site 171. In vivo studies will show whether the concept holds to develop safer opioid analgesics. Part a is adapted with permission from ref. 172, elsevier. a Biased signalling and drug design Drug 1 Drug 2 Drug 3 R R R E1 E2 E1 E3 E2 Low efficacy Optimal Adverse effects c Other approaches for better MOR analgesics Multifunctional opioids Dimer-specific opioids R Clinical and abused opioids MOR G i/o -biased opioids Opioid specific to acidic receptor b Biased signalling at MOR HO HO O MOR H Morphine β-arr Constipation Respiratory depression N CH3 G i/o Analgesia Reward? Tolerance and/or dependence? e, effector; r, receptor. HO O H O 3 C N NH S Oliceridine O N N H H 3 C CH 3 PZM21 O NH N CH 3 O N H CH3 CH3 S O CH 3 O H 3 C O Mitragynine pseudoindoxyl Behaviour d Endogenous signalling and allostery at MOR Endogenous opioid analogue Endogenous opioid 1 2 Temporally and spatially controlled enhanced signalling Signalling regulator Anti-opioid system Positive allosteric modulator Cl O N S O S Br O CH 3 BMS

3 Biased agonism A signalling response determined by the conformation of the drug receptor effector complex that engages only a subset of cellular effectors. Some high-throughput screening programmes have aimed to design novel biased drugs with improved therapeutic profiles. Therapeutic window Dose range for a drug that allows therapeutic efficacy with no (or minimal) side effects. Location bias Bias in receptor signalling dictated by the location of the receptor in the cell (for example, at the surface or in endosomes or Golgi) and the availability of effectors at this site. Systems bias Bias in receptor signalling driven by anatomical localization within brain circuits subserving the behavioural response and the effectors available at those sites. Precision medicine Also known as personalized medicine. An innovative approach in medicine in which interindividual variability (in lifestyle, environment and genes) is taken into consideration for disease prevention and/or treatment. Hedonic balance The equilibrium between positive and negative affect. A positive hedonic state is considered a state of well- being, whereas a negative hedonic state is unpleasant. development of KOR agonists for pain control and has given KORs a reputation of being associated with the dark side of emotional and perceptual experience 18,19 (Box 2). Overall, the very distinct profiles of MORs, DORs and KORs have now been clarified, and opportunities in opioid research are rapidly advancing. The past decade has witnessed revolutions in GPCR structure and signalling research 20,21. In 2012, the first atomic structures of the MOR 22, DOR 23, KOR 24 and the structurally related nociceptin/orphanin FQ receptor 25 (Box 3) solved by X- ray crystallography were published (Fig. 1a), opening the way to design opioid drugs with entirely novel chemical scaffolds (for example, see ref. 26 ) and perhaps to get closer to the ideal analgesic (Box 1). Many signalling effectors activated by opioid receptor stimulation have been identified (for example, those in Fig. 1b). Furthermore, new technologies (such as neural tracing, connectome analysis, optogenetics and chemogenetics) enabled the dissection of neural circuit organization and function at microscale or macroscale levels 27 29, and the notion of precision medicine is gradually entering the areas of pain 30 and addiction 31 treatment. Opioid research strongly benefits from advances in these domains, and this Review addresses three questions that arguably foster the strongest interest in current opioid research. First, can we kill pain without addiction using MOR- targeting opiates (addressed in Box 1)? Second, how do MORs and KORs operate within the neurocircuitry of addiction? Third, can we bridge human and animal opioid research in the field of drug abuse? Opioid receptors in addiction circuits Experimental evidence from animal research has positioned the role of the opioid system at the centre of reward and aversion processing, and has highlighted the notion that the dysregulation of opioid neurotransmission is a main driver to drug abuse (Fig. 2). Opioid system and reward aversion in physiology. Searching for reward (such as food, sex and social interactions) and avoiding punishment or discomfort (for example, pain) are two fundamental forces Box 2 The intriguing hallucinogenic properties of Kor agonists Beyond aversion, kappa- opioid receptor (KOr) agonists show hallucinogenic properties a facet of KOr function that is unique among opioid receptors. salvinorin a is a natural product from the sage Salvia divinorum, or magic mint, that was long used by the Mazatecs of Oaxaca, Mexico for spiritual rituals and medicinal practice, and was recently discovered to be a specific KOr agonist 173,174. salvinorin a is a highly potent hallucinogen for which the psychoactive effect is comparable to that of lysergic acid diethylamide (LsD). the use of salvinorin a, which is much less regulated than, for example, LsD use, is currently gaining popularity among young individuals in a recreational context. the finding that this KOr agonist has psychoactive effects definitively establishes a specific role for KOrs in higher cognitive and perceptual functions, the dysregulation of which is associated with psychiatric disorders such as psychosis, bipolar disorders and dementia 175. this particular KOr function remains poorly understood and adds another aspect to the known role of this receptor in stress responses, mood deficits and drug abuse. the circuits underlying this phenomenon are yet to be characterized. Notably, new analogues of salvinorin a with partial agonism or biased ligand properties are now proposed for the treatment of neuropsychiatric diseases, including addiction 176 ; this research is still at an early stage. that drive decision making. Aminergic neurotransmitter systems (such as the dopamine (DA) and serotonin (5-HT) systems) have been extensively studied in this context. Circuit mechanisms encoding reward and/or aversion involve the activity and interaction (sometimes bidirectional) of overlapping networks 32. The opioid system interacts anatomically and functionally with reward aversion networks 33,34, to maintain hedonic balance, regulate mood states and cope with stress (Fig. 2a). Furthermore, the notions that, for example, endogenous opioids contribute to the rewarding effects of pain relief 35 and that physical and social pain (such as that associated with social rejection) share common opioid- mediated mechanisms 36,37 are gaining interest. Overall, the opioid system can be considered a central regulator in basic processes of individual and species survival, geared to enhance reward- based learning and reduce aversive experiences. Opioid receptors are therefore critical in the emergence of neuropsychiatric disorders that manifest when reward and aversion processing are dysfunctional. These pathologies primarily include addiction and depression, and opioid receptor targets have therapeutic potential in both of these disease areas. Opioid receptor function in mood disorders has been reviewed elsewhere 38. Opioid receptors and the multiple faces of addiction. Addiction is a complex, relapsing disorder in which drugs of abuse hijack, overstimulate and compromise reward- processing systems and associated networks. The disease develops from an initiation phase, during which the drug produces pleasurable effects and is consumed recreationally. Upon repeated consumption, control over drug taking is gradually lost, leading to compulsive drug seeking and drug taking 39,40 (Fig. 2b). Whereas recreational drug use is essentially motivated by reward seeking, drug intake in individuals who are addicted is also driven by other factors that arise owing to brain adaptations to chronic drug exposure (Fig. 2b). These include reduced self- control 41, enhanced incentive salience (that is, importance of the context) and habit formation 42, altered reward processing and stress reactivity 43, and the emergence of a negative affective state upon withdrawal 19 or with protracted abstinence (see ref. 44 and references therein). Together, alterations of both positive and negative affect contribute to the development and maintenance of addiction, and relevant transmitter and circuit adaptations are being actively studied 39. All three opioid receptors are involved in the process, although with very different contributions (Fig. 2b). The effects of MORs and KORs in regulating addiction networks and, more importantly, the neuronal populations in which these receptors operate, have been well characterized and are discussed below (Fig. 2c). DOR- mediated circuit mechanisms contributing to addiction have been less well studied using cell- specific genetic approaches (only one study 45 ); thus, the current knowledge of DOR involvement in addiction is reviewed more briefly. Nature Neuroscience

4 Reverse pharmacology An approach in which a receptor or endogenous ligand is discovered first, the physiological function is determined. Conditioned place preference (CPP). A behavioural paradigm in rodents that determines the rewarding or aversive effect of a drug on the basis of time spent in a drug- associated context after conditioning. Tetrahydrocannabinol (THC). The principal psychoactive component of cannabis, which produces central effects by acting at cannabinoid CB1 receptors. Psychostimulants A group of substances (including cocaine and amphetamines) that enhance physical and cognitive performance. Psychostimulants are used to treat attention deficit hyperactivity disorder. MORs in reward, motivation and self- control. MORs mediate the pleasurable properties of therapeutic and/or abused opiates in vivo. Importantly, genetic approaches have demonstrated that these receptors are also necessary for the rewarding effects of other drugs of abuse (reviewed elsewhere 11,46 and in Supplementary Table 1). Briefly, Oprm1-knockout mice show lower levels of voluntary alcohol drinking and self- administration (SA) and fail to express conditioned place preference (CPP) in response to tetrahydrocannabinol (THC) or nicotine. The latter molecules primarily activate non- opioid receptor targets (that is, cannabinoid receptors and nicotinic receptors, respectively), and these activated receptors, in turn, trigger opioid release at appropriate MOR- expressing brain sites to produce reward 11. The data are less clear for cocaine and amphetamine, as Oprm1- knockout mice show decreased SA for these substances, but no change in CPP (see ref. 11 ), consistent with the notion that psychostimulants hijack reward systems via mechanisms that do not necessarily engage MORs, at least for their rewarding effects 47,48. Genetic approaches have also demonstrated the essential role of MORs in mediating natural rewards. The constitutive Oprm1 deletion reduced maternal attachment in 4 8 day old mutant pups 49 ; furthermore, adult knockout mice showed impaired social interactions together with several other signs of autistic- like behaviours 50, confirming the essential function of MORs in social bonding that was previously suggested by the pharmacology 36,38. Oprm1-null mutants also showed reduced motivation for both food and sucrose in an SA paradigm 51. Finally, Oprm1-null mutant mice showed no naloxone aversion in a conditioned place aversion (CPA) paradigm, indicating that MORs are crucial in mediating the positive hedonic tone elicited by endogenous opioids 52. Together, the findings above show that MOR activity both mediates natural rewards and promotes recreational drug use, with the latter in turn favouring the onset of drug abuse. MORs are expressed throughout the addiction circuitry 53 (Fig. 2c). Several brain sites for MOR- mediated reward have been identified by local pharmacology, and these mainly belong to mesocorticolimbic networks 54. Central to this circuitry is the well- characterized ventral tegmental area (VTA) nucleus accumbens (NAc; also known as the ventral striatum) DA pathway. The prevailing disinhibition hypothesis postulates that activation of MORs expressed by VTA GABAergic interneurons relieves the local inhibitory tone and thus disinhibits DA neurons, which release DA to signal drug reward 55,56. This hypothesis is supported by many pharmacology and electrophysiology studies 57. However, this is not the only mechanism through which MORs affect DA signalling, as MORs are also abundant in brain areas that receive DA neuron projections and, notably, among NAc neurons that project back to the VTA 58,59. Furthermore, DA- independent opioid reward is also documented 60 63, but in fact little is known about MOR- expressing cells driving opioid reward outside the VTA. A few studies using genetic approaches in mice have recently interrogated MOR function outside the VTA. In the striatum, MOR expression is robust in DA D 1 receptor (D 1 R)-expressing medium spiny neurons Box 3 The opioid system s cousin: the opioid- like receptor and nociceptin/orphanin FQ the cloning of the opioid- receptor-encoding genes led to the identification of homologous G protein- coupled receptor (GPCr)-encoding genes, some of which were known (such as those encoding somatostatin receptors). However, the closest homologue encoded a GPCr for which the ligand was unknown, thereafter named opioid- like receptor (OrL1). this receptor does not bind opioids with high affinity, and therefore cannot be classified as an opioid receptor. using reverse pharmacology 177, two teams discovered its endogenous ligand, a 17-amino- acid peptide named either orphanin FQ 178 or nociceptin 179 based on its first reported characteristics (its peptide sequence and its effect on pain). this was one of the first GPCr de- orphanizations, and the receptor and peptide are now classically known as the nociceptin opioid peptide receptor (NOP) and N/OFQ, respectively. strikingly, just as NOP shows the highest homology with the kappa- opioid receptor (KOr), the N/OFQ peptide shows sequence similarity with dynorphin (although lacking the amino- terminal tyrosine typical of opioid peptides), suggesting a common ancestor in evolution. similar to the opioid receptors, NOP is an inhibitory G i/o - coupled GPCr that reduces neuronal activity and/or neurotransmitter release. Many synthetic NOP ligands developed in academia and industry 180, as well as genetic mouse mutants, have been used to study the multiple in vivo functions of the NOP system and its potential interactions with the opioid system (reviewed in ref. 181 ). NOP- regulated physiology and pathology cover areas of pain control, reward processing and drug abuse, stress, anxiety and mood disorders, feeding and obesity, motor control and cognition, and operate broadly in the nervous system (reviewed in refs 181,182 ). to the best of our knowledge, no cell- specific genetic studies have yet addressed NOP function in defined brain networks or neuronal populations. Much evidence supports a role for the N/OFQ NOP system in addiction and this has been reviewed extensively in brief, N/OFQ and NOP agonists reduce drug reward and basal or drug- induced (mostly cocaine- induced) dopamine release in the nucleus accumbens, alleviate signs of alcohol withdrawal and diminish stress- primed or drug- primed reinstatement of cocaine place preference, possibly by antagonizing corticotropin releasing factor (CrF) stress systems. Furthermore, the fact that N/OFQ blocks morphine- induced supraspinal analgesia and conditioned place preference suggests that the N/OFQ NOP system acts as an anti- opioid system for some responses. indeed, NOP genetic inactivation or blockade reduces analgesic tolerance to morphine, confirming functional interactions between the two systems. rodent research has therefore positioned the NOP system as a feasible target for the development of addiction treatments. at this stage, however, translation to non- human primates and the clinic may prove challenging for two reasons. First, the anatomical localization of the N/OFQ NOP system seems to be different in rodent, non- human primate and human brains. the recent synthesis of positron emission tomography (Pet) tracers for NOP will help to guide clinical trials 180. second, the effects of N/OFQ on the drug- abuse-associated aspects of physiology (such as stress and anxiety) have proved to be complex; both agonists and antagonists need be examined for their potential therapeutic utility. the current thinking is that NOP blockade has potential for treating depression and obesity, whereas NOP activation may reduce anxiety and help to treat several aspects of addiction, including a reduction of consumption and the prevention of relapse 180,182 ; however, the best valid strategy remains open. as an illustration, a NOP agonist (cebranopadol; phase iii) and a NOP antagonist (JtC-801; phase ii) have both reached clinical trials to treat pain, and the NOP antagonist LY was tested in phase ii trials for major depressive disorders and for alcohol dependence

5 a First solved atomic structure of opioid receptors MOR β-fna DOR naltrindole KOR JDTic NOP C-24 ECL2 ECL2 ECL3 1 ECL1 ECL1 7 7 ECL3 EC membrane 1 S S C-24 ECL NPXXY Helix ICL1 ICL2 DRY ICL3 IC membrane b The active MOR and signalling effectors ECL2 Ca 2+ channel K + channel MOR AC G proteindependent signalling Gα i/o Arr GRK ERK JNK G proteinindependent signalling 10 Å CAMKII STAT3 PKA PKC p38 MAPK Fig. 1 opioid receptor structure and signalling. a First resolved atomic structures of inactive opioid receptors and the nociceptin opioid peptide receptor (NOP); see Box 3). The receptors are bound to their specific antagonists β- funaltrexamine (β- FNA) for the mu- opioid receptor (MOR) 22, naltrindole for the delta- opioid receptor (DOR) 23 and JDTic for the kappa- opioid receptor (KOR) 24 as well as the peptide mimetic antagonist Compound 24 (C-24) for the NOP receptor 25. The receptors each have seven transmembrane domains (numbered). DRY and NPXXY, shown here for the KOR structure, are conserved motifs important for receptor function. b MOR activation and signalling in cells. The left side of the panel shows a comparison of the seven- helical arrangement of active (green) and inactive (blue) forms of the receptor 167. The main modification is a 10 Å outward shift of the intracellular part of transmembrane domain 6, which typically interacts with cellular effectors and notably the Gα subunit of G proteins. The conformation of the extracellular receptor domains shows minimal changes. The structure also revealed a network of polar amino acid residues, which links the binding site to the cytoplasmic face of the receptor and is involved in G protein- coupled receptor (GPCR) signal propagation 167. The switch from inactive to active structures was also probed using solution- state NMR, revealing that the conformational changes of transmembrane domains 5 and 6, the main receptor movements to reach full activation, require engagement of both the ligand and the G protein mimic in the complex 183. The right side of the panel illustrates MOR signalling in cells. All three opioid receptors are inhibitory- type GPCRs, the activation of which reduces postsynaptic neuronal excitability or presynaptic neurotransmitter release 151. At the cellular level, opioid receptors activate G protein- dependent pathways involving both Gα i/o and Gβγ subunits, as well as G protein- independent signalling cascades that involve scaffold proteins such as arrestins (Arr). Altogether, many downstream signalling effectors have been identified for each receptor 8,72,184,185. The scheme illustrates currently known effectors for MOR and indicates which are G protein- dependent (green) or G protein- independent (red) or whether G protein and/or Arr dependency is unclear (orange). The cell adapts to repeated receptor stimulation, leading to desensitization of receptor signalling (via, for example, receptor trafficking and effector uncoupling) and/or compensatory upregulation of related cellular pathways 72. As for most GPCRs, opioid receptor activation is subjected to biased agonism; that is, the cellular and in vivo responses are often agonist- dependent 185. AC, adenylyl cyclase; CAMKII, calmodulin- dependent protein kinase II; EC, extracellular ; ECL, extracellular loop; ERK, extracellular- signal-regulated kinases; GRK, GPCR kinase; IC, intracellular ; ICL, intracellular loop; JNK, Jun N- terminal kinase; MAPK, mitogen- activated protein kinase; PKA, protein kinase A ; PKC, protein kinase C; STAT3, signal transducer and activator of transcription 3. Structures in part a adapted from refs 22 25, Macmillan Publishers Limited. Structure in part b adapted from ref. 167, Macmillan Publishers Limited. (so- called D 1 MSNs) and is barely detectable in DA D 2 receptor (D 2 R)-expressing MSNs (D 2 MSNs) 58,59. Rescuing MOR expression in D 1 MSNs of Oprm1- knockout mice using a bacterial artificial chromosome Pdyn- MOR transgene restores morphine CPP and partially restores remifentanil SA 58, suggesting that MORs expressed in the striatonigral pathway (that is, by D 1 MSNs) are sufficient to mediate opioid reward. However, whether this receptor population is necessary for drug reward is less clear. Conditional Oprm1 Nature Neuroscience

6 a Opioid system physiology MOR Euphoria Hedonic tone DOR Mood Low anxiety Positive affect KOR Dysphoria stress Negative affect b Opioid receptors in the disease process Reward Recreational drug use Adaptions to the drug Lower reward MOR adaption MOR Lower self-control Maladaptive habits Higher context salience MOR/DOR/KOR? Binge Intoxication Preoccupation Anticipation Withdrawal Aversive state Aversion Increased dysphoria Lower mood Higher anxiety Increased stress reactivity KOR Dyn DOR c Opioid receptor function in addiction circuits MOR HIP KOR HIP FC HB FC HB CP NAc BNST VTA DRN CP NAc BNST VTA DRN AMY AMY Preoccupation or anticipation Binge or intoxication Withdrawal or aversive state Low Receptor density Medium High Fig. 2 opioid receptors in physiology and addiction. a Opioid receptors regulate reward and aversion. The opioid system contributes to self and species survival by promoting reward elicited by natural stimuli (such as food, sex and social interaction), regulating mood states and facilitating efficient coping with pain and stress. Mu- opioid receptors (MORs) and kappa- opioid receptors (KORs) oppositely regulate hedonic homeostasis, with MOR agonists and KOR agonists producing euphoria and dysphoria, respectively. Stress and drug abuse both enhance KOR dynorphin (Dyn) signalling, contributing to an increase in dysphoric states 43,83. By contrast, delta- opioid receptor (DOR) activity reduces anxiety and depressive states, and regulates learning and memory 14,105. KOR blockade and DOR activation therefore have the potential to improve emotional responses. Finally, MORs and DORs show contrasting activities on motor impulsivity (see main text). Opioid receptors are therefore prime targets for the treatment of addiction and disorders such as depression 38 that are characterized by low- reward or high- aversion states. b Opioid receptors in the addiction cycle. In highly simplified terms, addiction can be characterized by low- reward, high- aversion functioning, as negative affective states progressively overtake positive affect. A well- accepted view from animal research is that drug abuse unfolds as a three- stage cycling process, involving binge or intoxication episodes, followed by withdrawal and a negative affective state when the drug clears, in turn leading to a preoccupation or craving step, during which desire for the drug intensifies (craving) and triggers the next intoxication episode (inspired by refs 39,40 ). Neuroplastic changes occur at all the stages, contributing to reinforce the addiction process in a vicious cycle; opioid receptors contribute to all facets of the disease. Animal data (see Supplementary Table 1 for behavioural models) indicate that MORs drive rewarding properties of opioid drugs (via direct, on- target effects) and other drugs of abuse (via indirect, opioid peptide- mediated effects) in both recreational consumption and binge or intoxication, and that repeated MOR activation leads to reduced drug reward (tolerance) and compensatory adaptations (dependence or withdrawal symptoms). KOR Dyn activity is important in the negative affect that characterizes withdrawal as well as in short- term or prolonged abstinence, whereas DORs should limit development of this aversive state. All three opioid receptors probably influence the preoccupation anticipation stage, and are implicated in drug- biased motivation, habit formation and loss of control, but these roles are less well understood. In the latter stage, or relapse to drug taking, evidence supports a role for DORs in drug cue or context learning 103. c MOR and KOR function in neurocircuits of addiction. This simplified scheme summarizes animal data involving genetic or cell- specific approaches to study opioid receptors in circuits of addiction- related behaviours. Similar studies for the DOR are only just starting 45. Brain regions involved in drug abuse are represented (circles) with their known connectivities (light grey lines) and include most regions studied in rodent addiction research. Circles are outlined in different colours depending on whether the brain structure has been associated with binge or intoxication (orange), withdrawal or aversion (blue), or preoccupation or anticipation (red) stages and are based on information in refs 39,40, with the addition of the habenula (HB) and dorsal raphe nucleus (DRN), which are the focus of increasing interest in the context of aversive aspects of addiction. Receptor density 36,54 (represented in a naive mouse brain) is indicated for each region, and the opioid- receptor-regulated pathways identified in the studies discussed in this Review are shown by black arrows. In brief, MORs in GABAergic interneurons of the ventral tegmental area (VTA) facilitate dopamine release and drug reward 57, whereas MORs in the striatum (nucleus accumbens (NAc) and caudate putamen (CP)), expressed mainly in dopamine D 1 -type neurons 58,59, regulate both drug reward (for alcohol and morphine) 58,64 and heroin seeking 59. MOR adaptations in the DRN drive depressive- like states and social withdrawal in protracted heroin abstinence 74. The roles of MOR- expressing cell populations in regions that modulate negative affective states, and in regions where the receptor is abundant (HB, DRN and amygdala (AMY)), remain to be studied. KORs in VTA dopamine neurons oppose MOR- stimulating activity at the level of terminals in the NAc and prefrontal cortex 18,89 and disrupt behavioural inhibition 94. KORs in NAc medium spiny neurons differentially gate D 1 and D 2 neuron activities and their AMY inputs to negatively regulate motivational processes 91. KORs in DRN serotonin (5-HT) neurons promote stress- induced reinstatement to cocaine seeking 96,97 ; KORs in excitatory basolateral amygdala (BL A) neurons projecting to the bed nucleus of stria terminalis (BNST) promote anxiety 98 ; and KORs in GABAergic interneurons of the central AMY contribute to excessive alcohol drinking, probably through interactions with corticotropin releasing factor systems 39,78,83. Finally, MORs and KORs are also expressed in brain regions involved in contextual memory and executive functions (including the hippocampus (HIP) and frontal cortex (FC)) 78,103, but the potential implication of these receptors in preoccupation or anticipation has not been studied so far.

7 Inhibitory controls A central component of executive functions, geared to inhibit or delay dominant responses to achieve a goal. deletion from GABAergic forebrain neurons (mostly from D 1 neurons), using a Dlx5/6-Cre driver line in floxed Oprm1 mice, reduced voluntary alcohol drinking and alcohol CPP 64, indicating that this receptor population indeed contributes to alcohol reward. Intriguingly, the latter genetic manipulation did not impair heroin CPP presumably because VTA MORs were intact in these mice but did increase seeking behaviour for heroin and palatable food in SA experiments 59, dissociating MOR- mediated hedonic effects from motivational regulation. Thus, in addition to driving pleasurable drug effects via VTA GABAergic interneurons and, to some degree, striatal MSNs (depending on the drug and paradigm), MORs also regulate motivational aspects of behaviour, specifically at the level of striatal projecting neurons. This notion is consistent with sophisticated pharmacological studies that show that MORs not only assign hedonic values to rewards, but also contribute to decision making through coordinated activity in core and shell compartments of the NAc, which integrate reward- related information and guide goal- directed actions 65. MORs may also contribute to another facet of addiction that is, impaired self- control although this aspect has been less explored. Impulsivity is a critical susceptibility factor for addiction 66, and inhibitory controls decline with disease development 39. Strikingly, Oprm1- knockout mice showed remarkably lower motor impulsivity in a signalled nose- poke task 67. The notion that MORs may facilitate the loss of control that characterizes drug abuse is intriguing. Further studies are required to identify the key MOR- expressing neuron populations responsible for this effect. An important question is whether, in addition to facilitating drug reward and drug seeking in the traditionally studied mesocorticolimbic circuitry, MORs could also regulate aversion processing in other brain networks. The site of the densest MOR expression in the brain is, in fact, the medial habenula (MHb) 68. The MHb and lateral habenula form the habenular complex, which is active in the anticipation of aversive outcomes and has garnered increasing interest in both addiction and depression research 69,70. As the habenular complex is considered to be a centre for aversive processing and acts as an anti- reward system notably, by inhibiting DA neuron activity the hypothesis that MORs may regulate aversion processing, and perhaps limit aversive responses at the level of MHb neurons, is appealing, and investigations along these lines have just begun 71. Finally, an important consideration is that MORs are chronically activated under repeated drug exposure, and even more so under opiates. With chronic activation, receptor signalling adapts throughout the brain, triggering long- term molecular modifications within and outside the opioid system 72 ; such neuroadaptations to chronic opiates are being intensively studied 73. Behavioural consequences of these adaptations include the development of tolerance (weakening drug effects with repeated exposure), and dependence (which manifests in the form of withdrawal symptoms when the drug is no longer present). Acute withdrawal and prolonged abstinence from opiates are both associated with negative emotional states that can be modelled in rodents 44. In this context, MORs in the dorsal raphe nucleus (DRN), the main serotonergic nucleus, were shown to be crucial for the development of despair- like behaviour and deficits in social interaction in heroin- abstinent mice 74. This finding indicates that DRN MORs regulate serotonergic transmission and that adaptations of MOR signalling in the DRN in response to chronic opiate exposure may profoundly alter mood during abstinence. This mechanism is particularly interesting in the area of addiction depression comorbidities 75. KORs in dysphoria, stress and depression. In contrast to MOR agonists, KOR agonists are strongly aversive. In humans, these drugs induce an acute dysphoric state, deteriorate mood and have psychotomimetic effects 17,76 (Box 2). These KOR activities may have strong implications for several psychiatric disorders, including drug abuse, affective disorders and, potentially, psychosis 18. Animal model research has established that the KOR and dynorphin, the preferred endogenous KOR ligand, form an anti- reward brain system 43,77. Notably, in the absence of environmental challenge, the endogenous KOR dynorphin system has only subtly dysphoric activity; however, this system is highly responsive to stress 78, and accumulating evidence has shown that addiction dramatically activates brain stress systems, including endogenous KOR signalling 43, In turn, these events facilitate the emergence of a negative affective state which characterizes withdrawal episodes of the addiction cycle as well as protracted abstinence 82 and promote escalation of drug consumption, stress- induced potentiation of drug reward and stress- induced reinstatement of drug seeking 83,84. KORs are expressed at many sites in the addiction neurocircuitry (Fig. 2c), and a circuit mechanism for KOR- mediated dysphoria has long been proposed. Behavioural analyses have demonstrated CPP and CPA in response to MOR agonists and KOR agonists, respectively, and microdialysis studies have revealed opposing activities for the two types of drugs on DA release, with MOR agonists disinhibiting VTA DA neurons at the level of the cell bodies and KOR agonists inhibiting DA- releasing terminals in the NAc 85,86. These seminal findings may explain why MORs and KORs have such contrasting effects (that is, euphoria and dysphoria, respectively), and have established the notion that endogenous activities of the two receptors fine- tune DA tone to regulate hedonic homeostasis. Accordingly, deletion of Oprk1 from DA neurons reduced CPA to KOR agonists 87, had anxiolytic effects and enhanced cocaine- stimulated locomotor activity 88. Electrophysiology demonstrated that KORs in DA neurons inhibit terminals not only in the NAc, but also in the prefrontal cortex (PFC) 89. As in the NAc, KORs reduced DA release locally in the PFC via a presynaptic mechanism and thus contributed considerably to KOR agonist- induced CPA 90. Therefore, KORs in DA neurons produce dysphoria via presynaptic inhibition at both NAc and PFC sites. Nature Neuroscience

8 Operant photostimulation Instrumental conditioning in which animals learn to self- administer optogenetic stimulation; used to determine whether a neuronal population mediates reward. Escalation In animal research, extended access to the drug leads to a daily increase (or escalation) of drug intake, which is suggested to reflect loss of control. In the NAc, KORs are expressed by D 1 and D 2 MSNs and at the terminals of glutamatergic projections from the basolateral amygdala (BLA), and dynorphin is produced by D 1 MSNs. A recent optogenetic study examined KOR function throughout the entire NAc microcircuitry and demonstrated that two distinct presynaptic KOR- mediated mechanisms regulate D 1 and D 2 MSN activities: one pathway- specific (affecting BLA inputs to the NAc) and the other local (affecting MSN collaterals) 91. This study reveals a mechanism whereby KORs in the NAc dampen excessive MSN activation that is driven by incentive stimuli or stress, thereby limiting reward- seeking behaviour. Thus, in addition to the role of KORs in influencing euphoria dysphoria balance, the notion that KORs negatively regulate motivation is also gaining support. Another optogenetic manipulation revealed functional heterogeneity among Pdyn- positive neurons (D 1 MSNs) in the NAc 92. Photostimulation of these neurons in the dorsal or ventral NAc shell produced approach or avoidance behaviour, respectively, in a real- time place- preference assay. These opposing effects were both mediated by KORs and were confirmed by operant photostimulation. These findings add support to the emerging notion that reward and aversion mechanisms are highly intermingled within the NAc (as was previously shown for the VTA 93 ) and further support the view that dysfunctional KOR dynorphin activity in the NAc affects motivated behaviours. Another study in mice used an operant test of cognition (in which lower rates of response were differentially reinforced) to show that both stress and KOR activation impair behavioural inhibition, an effect prevented by Oprk1 gene deletion in DA neurons 94. Thus, KOR blockade might reduce stress- induced compulsive behaviours that contribute to drug seeking and addiction. KOR activity modulates other brain regions and transmitter systems (in addition to the DA system) that are crucial for negative mood and aversive responses (reviewed in ref. 78 ). First, KORs in the DRN modulate 5-HT transmission, similar to how they modulate DA release from VTA neurons. Local application of a KOR agonist decreased 5-HT release in the DRN 95, suggesting that a low DA tone may not be the sole cause for KOR- mediated dysphoria and that reductions in 5-HT may also contribute. Accordingly, a KOR antagonist infused in the DRN abolished the CPA induced by systemic KOR agonist administration, and prevented stress- induced reinstatement of cocaine CPP, through a mechanism that involved p38 mitogen- activated protein kinase (MAPK) in 5-HT neurons and their projections to the NAc 96,97. Second, amygdala- based mechanisms contribute to KOR- mediated anxiety. Oprk1 deletion in the BLA was anxiolytic in mice, and the anxiolytic effects produced by photostimulation of the BLA bed nucleus of stria terminalis (BNST) pathway were blocked by systemic administration of a KOR agonist 98. The same study also showed that KORs inhibit excitatory BLA terminals in the BNST, and that dynorphin in the BNST thus regulates the activity of BLA BNST projections to control the amygdala- centred anxiety circuit. KOR activity in the central amygdala (CeA) was also studied in the context of alcohol dependence. Infusion of a KOR antagonist into the CeA reduced the increase in alcohol SA elicited by chronic intermittent exposure to alcohol vapours (a procedure designed to trigger escalation of alcohol consumption) 99. Slice electrophysiology showed that KORs tonically inhibit local GABAergic transmission in the CeA via a presynaptic mechanism and has thus been proposed to regulate the effects of ethanol there 100,101. KORs also interact in a complex manner with the stress hormone corticotropin releasing factor (CRF) 102. This KOR CRF signalling interaction is being increasingly studied for a role in the pro- addictive effects of stress, in anxiety disorders and in related psychiatric conditions (reviewed elsewhere 39,78,83 ). DORs in mood and learning. Similar to MORs and KORs, DORs contribute to the development of drug abuse, although very differently from the two other receptors. Oprd1-knockout mice show intact morphine SA, intact THC CPP, decreased nicotine SA and increased alcohol SA (reviewed in refs 11,38 and see Supplementary Table 1). Therefore, contrary to MORs, DORs are not essential for drug reward. Rather, as proposed by pharmacological studies, DORs seem to have a complex regulatory role in motivated behaviours 65,103. Unlike Oprm1-knockout mice and Oprk1-knockout mice, DOR- null mutants show a remarkable anxiogenic and depressive- like phenotype, largely confirmed by pharmacological approaches 14,104, suggesting that DOR activity normally helps to alleviate the negative mood associated with acute withdrawal and prolonged abstinence. In support of this notion, Oprd1-knockout mice undergoing protracted abstinence to heroin show exacerbated sucrose anhedonia 74. Another role of DORs with potential relevance to addiction is the regulation of learning and memory (reviewed elsewhere 103,105 ). DOR- null mice show impaired spatial learning and, consistently, reduced drug context association in CPP paradigms 106. These and other observations suggest that DOR activity in fact enhances drug context memories and facilitates relapse. These findings would suggest that the DOR is pro- addictive; however, by alleviating the low affect associated with withdrawal, DOR activity may also prevent relapse. In addition, opposing the phenotype of MOR- null mutants, Oprd1-knockout mice show increased motor impulsivity in a signalled nose- poke task 67, suggesting that DOR activity increases inhibitory controls. Thus, altogether, the role of DORs in addiction is complex. DORs are expressed in all areas subserving reward and motivation, mood control, and learning and memory 53,54. The identification of circuit mechanisms underlying addiction- related functions of DORs through cell- specific genetic approaches is just starting. Only one conditional Oprd1-mutant mouse has been produced, demonstrating that DORs in GABAergic forebrain neurons mediate the locomotor- stimulant effect of SNC80 (a prototypic DOR agonist), counteract D 1 R agonist- induced hyperactivity and exert an anxiogenic effect 45. Further cell- targeted genetic studies in the future may address, for example, the intriguing proposed role of DORs in stimulus- based decision- making at the level of the BLA NAc shell circuit 65.