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FOCUS ON ADDICTION

FOREWORD FOCUS CONTENTS 623 Transcriptional and epigenetic mechanisms of addiction !"#$%&'()*+,-.+/&0/%& 1#-2&'()3$.4!$# 638 Common cellular and molecular mechanisms in obesity and drug addiction 506!&'()7$//8 652 Dysfunction of the prefrontal cortex in addiction: !"#$%&'(% ()* +% (,) and clinical implications *-40&9():+!%.4$-/ 0/%& 3+#0&;()<+!=+> 670 Pharmacogenetic approaches to the treatment of alcohol addiction ?0#=6.&@$-!-AB&;0C-%& :+!%D0/B&E0%$&F$##$44-/0/%&GH0#!$.&5()IJF#-$/ 685 OPINION Opiate versus psychostimulant addiction: the +%-!#! .!,)+$)&'//!# !%+&F0%-0/-B&;0C-%&F$!-/B& ;0C-%&1K.4$-/B&;+//0& G0!6&0/%&L0C-/&MH0H0D

Leonie Welberg, Senior Editor, Nature Reviews Neuroscience doi:10.1038/nrn3131

Addiction: from mechanisms to treatment It needs no explanation that addiction is an extremely serious problem, considering its impact on both health and society. Unsurprisingly, addiction is a major focus of neuroscience research, and the molecular, cellular and circuit mechanisms underlying addiction are slowly beginning to be understood. Despite these research efforts, few effective treatments exist, highlighting the need for continuing investigation. This Focus on Addiction brings together five articles that review the current state of the field and that point to potential new treatment opportunities. Much of addiction research focuses on the brain’s reward system. A popular idea of addiction is that drugs of abuse ‘hijack’ this system, disrupting the normal behavioural responses to natural rewards. However, it has been argued that natural rewards can also induce an addiction-like state. For example, the hedonic properties of palatable food can, in some people, lead to dysregulation of food intake, resulting in binge eating and, ultimately, obesity. In his Review, Paul J. Kenny describes how excessive intake of palatable foods and drugs of abuse cause similar molecular, cellular and circuit changes, not only in the reward system but also in the brain stem, hypothalamus and several cortical areas. Studies in animals and humans have shown that the prefrontal cortex (PFC) has a major influence on drugtaking behaviour, owing to its regulation of reward circuits and its role in executive functions such as selfcontrol. Goldstein and Volkow review a decade’s worth of neuroimaging studies on the PFC in addicted individuals. Based on these studies, they present a model of how interactions between dorsal and ventral PFC regions change in the course of the addiction process. Understanding how PFC functioning is altered in addiction may help in the development of new treatments; for example, cognitive–behavioural approaches that target specific PFC functions may prevent relapse. Relapse can occur after weeks, months or even years of abstinence. Drug-induced long-lasting changes in the transcriptional potential of genes in brain regions involved in reward processing may contribute to this phenomenon, and in their Review, Robison and Nestler examine the evidence for this hypothesis. They show that chronic exposure to drugs of abuse alters the expression or activity of several transcription factors, induces changes in the epigenetic status of several genes through histone tail or DNA modification, and alters the

expression of microRNAs in reward regions. Epigenetic changes alter steady-state gene expression but may also influence the inducibility of genes in response to subsequent drug exposure, which — as the authors suggest — could affect the adaptability of the addicted individual. Current theories of addiction assume that different types of addiction have a common psychobiological substrate. However, as argued in a Perspective article by Badiani and colleagues, it is important to acknowledge that several factors distinguish different types of addiction. Focusing on psychostimulant and opiate addictions, they show that there are numerous cognitive, neurobiological and behavioural differences between these conditions. Such differences have important implications for theories of drug addiction as well as for the development of treatments. Indeed, despite decades of research into the neurobiological processes that underlie addiction, very few effective treatments exist. Differences between addictions as well as genetic heterogeneity in addicted individuals suggest that there may not be a ‘magic bullet’. The fact that addiction is partially heritable points to the potential of pharmacogenetic treatment approaches. In their Review, Heilig and colleagues discuss this issue in the context of alcohol addiction, using naltrexone therapy for alcohol addiction as a case in point. Although initial studies indicated that naltrexone had a small effect size, subsequent studies showed that it is in fact effective in individuals with a particular polymorphism of the mu opioid receptor gene. Genetic variations in the corticotropin-releasing factor, serotonin and GABA systems have also been implicated in alcohol addiction and may become new pharmacotherapeutic targets. As the authors point out, more attention needs to be paid to personalizing pharmacotherapy for alcohol — and other — addictions.

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FOCUS ON ADDICTION

REVIEWS Transcriptional and epigenetic mechanisms of addiction Alfred J. Robison and Eric J. Nestler

Abstract | Investigations of long-term changes in brain structure and function that accompany chronic exposure to drugs of abuse suggest that alterations in gene regulation contribute substantially to the addictive phenotype. Here, we review multiple mechanisms by which drugs alter the transcriptional potential of genes. These mechanisms range from the mobilization or repression of the transcriptional machinery — including the transcription factors ΔFOSB, cyclic AMP-responsive element binding protein (CREB) and nuclear factor-κB (NF-κB) — to epigenetics — including alterations in the accessibility of genes within their native chromatin structure induced by histone tail modifications and DNA methylation, and the regulation of gene expression by non-coding RNAs. Increasing evidence implicates these various mechanisms of gene regulation in the lasting changes that drugs of abuse induce in the brain, and offers novel inroads for addiction therapy.

Fishberg Department of Neuroscience and Friedman Brain Institute, Mount Sinai School of Medicine, One Gustave L. Levy Place, BOX 1065, New York, New York 10029, USA. Correspondence to E.J.N.  e-mail: eric.nestler@mssm. edu doi:10.1038/nrn3111 Published online 12 October 2011

Drug addiction exacts an enormous medical, financial and emotional toll on society in the form of overdose and health complications, family disintegration, loss of employment and crime. The National Institute on Drug Abuse (NIDA), part of the US National Institutes of Health, estimates that the total cost of drug abuse in the United States exceeds US$600 billion annually, and it is particularly alarming to note a sharp increase in the abuse of prescription drugs and in drug abuse by teenagers (see the NIDA web site). These data substantiate the need for more research into the neuronal effects of drugs of abuse and the mechanisms of addiction, in the expectation of uncovering novel targets for treating and preventing addictive disorders. Although most individuals are exposed to drugs of abuse, only a subset experience the loss of control over drug use and compulsion for drug seeking and taking that defines the addicted state. Entrance into this state is strongly influenced by both an individual’s genetic constitution and the psychological and social context in which drug exposure occurs1–3. Although the genetic contribution to risk for addiction is roughly 50%1, the specific genes that are involved remain almost completely unknown. The addictive phenotype can persist for the length of an individual’s life, with drug craving and relapse occurring even after decades of abstinence. This persistence suggests that drugs induce longlasting changes in the brain that underlie addiction behaviours. The many cells of an individual organism, although they contain essentially identical complements of DNA,

differentiate to form distinct tissues and organs through regulated changes in the transcriptional potential of each gene, based on environmental cues, cell-to-cell signals and other, probably random factors4. It is becoming clear that many of the same processes of gene regulation that are involved in the normal differentiation of cells and tissues during development are also engaged in the adult organism to mediate cellular adaptation to environmental stimuli5,6. The processes that are involved in the regulation of transcriptional potential are varied and highly complex, and include activation and inhibition of transcription factors, modification of chromatin and DNA structure, and induction of non-coding RNAs. Increasing evidence supports the hypothesis that each of these mechanisms of epigenetic regulation is directly affected by drugs of abuse, and that such adaptations are one of the main processes by which drugs induce highly stable changes in the brain that mediate the addicted phenotype. This Review summarizes the findings that support this hypothesis, and highlights areas in which future research will extend this fundamental knowledge of addiction and exploit it for new therapeutics.

Drug action and gene transcription A seemingly similar syndrome of addiction can result from exposure to a wide variety of chemical substances or even rewarding activities, from cocaine to gambling to sex. One common mechanism in these various forms of addiction is thought to be activation of the brain’s reward circuitry, which centres on dopaminergic neurons in

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REVIEWS Limbic system A collection of cortical and subcortical structures that are important for processing memory and emotional information. Prominent structures include the hippocampus and amygdala.

Sensitization Enhanced drug responsiveness — on the behavioural, cellular and/or molecular levels — with repeated exposure to a constant dose.

the ventral tegmental area (VTA) of the midbrain and their projections to the limbic system — in particular, the nucleus accumbens (NAc; also known as the ventral striatum), dorsal striatum, amygdala, hippocampus and regions of prefrontal cortex 7–9 (FIG. 1). This reward circuitry is activated by stimuli or pursuits that promote evolutionary fitness of the organism, such as nutrientrich foods, sex and social stimulation. As drugs of abuse activate this circuitry far more strongly and persistently than natural rewards, and without being associated with productive behavioural outcomes, chronic exposure to drugs modulates brain reward regions partly through a homeostatic desensitization that renders the individual unable to attain sufficient feelings of reward in the absence of drug. An alternative, but not mutually exclusive, hypothesis of addiction focuses on incentive sensitization, whereby drugs alter the reward circuitry to cause increased assignment of incentive salience to drug cues, effectively making drug-associated environmental stimuli more difficult to ignore and leading to intense drug craving and relapse10. Pathological druginduced changes in the reward circuitry further impair behavioural control over drug taking. Virtually all rewarding drugs or activities increase dopaminergic transmission from the VTA to the NAc and other target limbic regions, although they each employ partly distinct mechanisms and in some cases involve other neurotransmitter systems as well7–9. The actions of drugs on the NAc are further complicated by the cellular heterogeneity of this brain region (BOX 1). Although drugs differ in their acute mechanisms of action, the common syndrome of addiction suggests that chronic activation of these distinct, acute mechanisms induces some shared molecular adaptations in brain reward regions that mediate the lasting nature of the addictive phenotype.

We, and others, have long proposed that changes in the transcriptional potential of genes — through the actions of transcription factors, chromatin modifications and non-coding RNAs — contribute substantially to many of the neuroadaptations that result from chronic exposure to drugs of abuse11 (FIG. 2). We know that many mRNAs display altered expression in brain reward regions after chronic drug exposure, which suggests that transcription of individual genes is differentially regulated under these conditions. Over the past ~5 years, studies at the chromatin level have confirmed the involvement of such transcriptional mechanisms in vivo. Moreover, beyond stable changes in steady-state mRNA levels, this work has shown that the ‘inducibility’ of a gene — its ability to be induced or repressed in response to the next drug exposure or some other environmental stimulus — is also altered by chronic drug exposure, and that such gene ‘priming’ or ‘desensitization’ is mediated by stable drug-induced changes in the chromatin state around individual genes (FIG. 3). This transcriptional and epigenetic model of chronic drug action provides a plausible mechanism for how environmental influences during development can increase (or decrease) the risk for addiction later in life. For example, there is mounting evidence that stress during adolescence increases the risk of addiction, and that exposure to drugs in utero increases the risk in adolescence and adulthood12,13. Long-lasting changes in gene transcription or in the potential for transcription that results from early-life stress or drug exposure — mediated at the chromatin level in the absence of genetic differences in the primary DNA sequence — might render an adult brain more vulnerable to the addictive process. As alterations in transcriptional potential can last for many years, this model also explains how relapse can occur despite decades of abstinence.

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Figure 1 | Brain reward circuitry. The brain on the left depicts dopaminergic afferents that originate in the ventral tegmental area (VTA) and release dopamine in the nucleus accumbens (NAc) and many other limbic targets. Also shown are other monoaminergic nuclei — the noradrenergic locus coeruleus (LC) and serotonergic dorsal raphe (DR) — which modulate drug reward and other actions. The brain on the right highlights glutamatergic regions that are important for reward: medial prefrontal cortex (mPFC), orbitofrontal cortex (OFC), anterior cingulate cortex (ACC), thalamus (Thal), hippocampus and amygdala, all of which send excitatory projections to the NAc. Drugs of abuse alter this reward circuitry in complex ways, and this leads to addiction.

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F O C U S O N A DRDEI C ON V ITEI W S Recent studies of rodent models of addiction have provided considerable support for this hypothesis and have contributed substantially to our understanding of in vivo transcriptional and epigenetic regulation in the

Box 1 | Cellular organization of the nucleus accumbens /0#"!1!"% 3(4(# +,-./,.0/1,2

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The nucleus accumbens (NAc) is composed of multiple neuronal cell types (see the figure), with each cell type seeming to exhibit different transcriptional responses to drugs of abuse and to mediate distinct aspects of drug reward and addiction. Glutamatergic afferents from the hippocampus, prefrontal cortex and amygdala, among other regions, excite all subtypes of NAc neurons36, with such excitation differentially regulating drug reward and motivation, as shown by recent optogenetic experiments130,131. These excitatory inputs are modulated by dopamine afferents from the ventral tegmental area (VTA), and psychostimulant drugs such as cocaine and amphetamine act by directly prolonging the effects of these dopamine signals. Excitatory inputs to the NAc are also modulated by endogenous opioid peptides that are both expressed locally and released by input neurons. Opiate drugs thus act directly on NAc neurons that express opioid receptors, and they also promote dopamine release in the NAc indirectly by inhibiting VTA GABAergic interneurons. Cannabinoids also have a role in regulating NAc neurons — they act primarily by locally repressing the function of glutamatergic synapses. Much work is needed to further understand the cellular specificity of drug action in the the NAc. 95% of NAc neurons are GABAergic medium spiny neurons (MSNs), which can be further differentiated into those that express the D1 dopamine receptor (D1-type MSNs) along with dynorphin and substance P, and those that express the D2 dopamine receptor (D2-type MSNs) along with encephalin132. Drug induction of ΔFOSB14,133,134, and the effects of ΔFOSB and G9a on cell morphology and behaviour, differ between D1-type and D2-type MSNs135, and neuronal activity of these two cell types causes opposing effects on the rewarding properties of cocaine130. In addition, acute cocaine causes extracellular signal-regulated kinase (ERK)-dependent phosphorylation of mitogen- and stress-activated kinase 1 (MSK1; also known as ribosomal protein S6 kinase α5) and of histone 3 specifically in D1-type MSNs75, although the functional consequences of this histone modification are not yet known. By contrast, the effects of cannabinoids seem to predominate at glutamatergic synapses on D2-type MSNs136. About 1–2% of NAc neurons are spiny large cholinergic interneurons, which have been shown to play an important part in cocaine reward131, and a similar number are GABAergic interneurons, the function of which are less well understood. Although these studies are important, so far they have barely scratched the surface of what promises to be an important new focus in addiction research: to overlay the alterations in transcriptional potential of genes induced by chronic exposure to drugs onto the map of cellular subtypes in the NAc. ACh, acetylcholine.

brain. Here, we highlight key examples of transcriptional and epigenetic mechanisms of drug action, and identify some of the novel potential targets for therapeutic intervention during the addiction process.

Transcription factors in addiction The classic mechanism for the regulation of gene expression is through the actions of transcription factors: proteins that, in response to cell signalling pathways, bind to specific sequences of DNA — generally in the promoter or enhancer regions of target genes — and increase or repress the expression of these genes by respectively promoting or blocking the recruitment of the RNA polymerase II transcriptional complex. Transcription factors operate as part of large protein complexes, with their mechanisms of action eventually involving alterations in chromatin structure (see below). Although neurons contain hundreds of transcription factors, studies of adaptations induced by drugs of abuse have focused primarily on a small subset. ΔFOSB. ΔFOSB14 is encoded by the FosB gene and shares homology with other FOS family transcription factors. It heterodimerizes with JUN family proteins to form activator protein 1 (AP1; also known as transcription factor AP1) complexes that bind to AP1 sites in responsive genes to regulate transcription. There is some evidence from in vitro studies that ΔFOSB may also homodimerize15. Although all FOS family proteins are induced transiently by acute drug exposure, chronic administration of virtually any drug of abuse induces the long-lasting expression specifically of ΔFOSB14,16,17, a process that is most robust in the NAc and dorsal striatum, but is also seen in several other reward-related brain regions, including prefrontal cortex 17. ΔFOSB induction in the NAc and dorsal striatum by drugs of abuse, regardless of whether the drug is investigator-administered or self-administered, occurs only in the subtype of medium spiny neuron (MSN) that expresses D1 dopamine receptors (D1-type MSNs)14 (M. K. Lobo, S. Zaman and E. J. N., unpublished observations). ΔFOSB is a carboxy-terminal truncation of full-length FOSB that is generated by alternative splicing; it lacks the two degron domains that are present in the full-length protein and that are conserved among all other FOS family proteins. This absence results in a fourfold increase in protein stability 18. In addition, ΔFOSB is phosphorylated in vivo at serine 27 (as well as at several other sites) and this phosphorylation further stabilizes the protein by roughly tenfold, both in vitro and in vivo19,20. This intrinsic and regulated protein stability is a particularly interesting feature of the molecule, as it provides a molecular mechanism by which drug-induced changes in gene expression can persist for weeks after drug intake stops. ΔFOSB has been linked directly to several addictionrelated behaviours. In adult bi-transgenic mice, in which removal of doxycycline induces ΔFOSB overexpression specifically in D1-type MSNs of the NAc and dorsal striatum, such induction causes increased locomotor sensitivity to cocaine21, increased conditioned placepreference to cocaine and morphine21,22, and increased

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Self-administration A form of operant conditioning using a drug as a reward, generally by administration through an intravenous line that is controlled directly by the animal’s actions.

Medium spiny neurons (MSNs). The main cell population of the ventral and dorsal striatum; these GABAergic projection neurons form the two main outputs of these structures, called the direct pathway (D1-type MSNs) and indirect pathway (D2-type MSNs).

Degron domains A specific amino acid sequence that targets a protein for degradation through proteasomal or other proteolytic processes.

Conditioned place-preference A behavioural test in which animals learn to prefer an environment that is associated with rewarding drug administration. It provides an indirect measure of drug reward.

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Figure 2 | Mechanisms of transcriptional and epigenetic regulation by drugs of abuse. In eukaryotic cells, DNA is organized by wrapping around histone octomers to form nucleosomes, which are then further organized and condensed to form chromosomes (left part). Only by temporarily unravelling compacted chromatin can the DNA of a specific gene be made accessible to the transcriptional machinery. Drugs of abuse act through synaptic targets such as reuptake mechanisms, ion channels and neurotransmitter (NT) receptors to alter intracellular signalling cascades (right part). This leads to the activation or inhibition of transcription factors (TFs) and of many other nuclear targets, including chromatin-regulatory proteins (shown by thick arrows); the detailed mechanisms involved in the synaptic regulation of chromatin-regulatory proteins remain poorly understood. These processes ultimately result in the induction or repression of particular genes, including those for non-coding RNAs such as microRNAs; altered expression of some of these genes can in turn further regulate gene transcription. It is proposed that some of these drug-induced changes at the chromatin level are extremely stable and thereby underlie the long-lasting behaviours that define addiction. CREB, cyclic AMP-responsive element binding protein; DNMTs, DNA methyltransferases; HATs, histone acetyltransferases; HDACs, histone deacetylases; HDMs, histone demethylases; HMTs, histone methyltransferases; MEF2, myocyte-specific enhancer factor 2; NF-κB, nuclear factor-κB; pol II, RNA polymerase II.

cocaine self-administration23. In addition, virus-mediated overexpression studies show that cocaine-mediated induction of ΔFOSB in orbitofrontal cortex, a subregion of prefrontal cortex, mediates the ability of chronic cocaine to induce tolerance to the cognition-disrupting effects of acute drug exposure24. Such overexpression also enhances impulsivity during drug withdrawal, and both of these effects further promote drug self-administration24,25. Importantly, genetic or viral overexpression of ΔJUND — a dominant negative mutant of JUND that antagonizes ΔFOSB and other AP1-mediated transcriptional activity — in the NAc or orbitofrontal cortex blocks these key effects of drug exposure14,22,24. This indicates that ΔFOSB is both necessary and sufficient for many of the changes that are wrought in the brain by chronic drug exposure. ΔFOSB is also induced in

D1-type NAc MSNs by chronic consumption of several natural rewards, including sucrose, high fat food, sex and wheel running, and this promotes the consumption of such rewards14,26–30. This implicates ΔFOSB in the regulation of natural rewards under normal conditions and, perhaps, during pathological addictive-like states. Progress has been made in identifying the broad range of transcriptional targets (some activated and some repressed) through which ΔFOSB produces these various behavioural phenotypes in response to drug exposure31,32. By regulating numerous genes that are related to dendritic spine architecture, including synaptotagmin, microtubule associated proteins, activity-regulated cytoskeleton-associated protein (ARC), actin-related proteins, cyclin-dependent kinase 5 (CDK5) and kinesin31–33, ΔFOSB mediates the structural

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F O C U S O N A DRDEI C ON V ITEI W S !

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calmodulin-dependent kinase II (CaMKII)31,39, which is consistent with the hypothesis that it mediates key aspects of the synaptic plasticity that is exhibited by MSNs after drug exposure34,40. ΔFOSB is far more stable than all other transcription factors that have been linked to addiction so far. Nevertheless, drug relapse can occur after decades of abstinence, a timescale dwarfing even phosphorylated ΔFOSB’s prolonged turnover rate. It is possible that ΔFOSB remains stably linked to individual gene promoters for long periods of time or induces longlasting changes to the chromatin structure of individual genes (see below) to influence relapse behaviour long after total cellular levels of the protein have returned to baseline. These possibilities remain to be investigated in future experiments.

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Figure 3 | Gene priming and desensitization. In addition to regulating the steady-state expression levels of certain genes, cocaine induces latent effects at many other genes, which alter their inducibility in response to a subsequent stimulus. a | Analysis of mRNA expression after acute or chronic cocaine. Heat maps that are !"#$%&'()*&+&,*-'&!..&/$0$,&)*!)&!"$&12"$/1.!)$%&(0&)*$&013.$1,&!331 4$0,&56*-1"&!7)$"& a cocaine challenge in naive animals (acute), in animals treated repeatedly with cocaine 8"$2$!)$%&9&!31)$:&-"&(0&!0( !.,&!7)$"&56'$$#&-7&'()*%"!'!.&8'%:&7"- &"$2$!)$%& cocaine (repeated wd + acute). Associated heat maps show how the same genes were affected under the other two conditions. Examples of desensitized transcriptional responses after repeated cocaine are indicated by ***. b | Early evidence suggests that epigenetic mechanisms are important in mediating such gene priming and desensitization, and that many of these changes are latent, meaning that they are not reflected by stable changes in steady-state mRNA levels. Instead, such changes alter chromatin structure, so that a later drug challenge induces a given gene to a greater (primed) or lesser (desensitized) extent based on the epigenetic modifications that are induced by previous chronic drug exposure. A major goal of current research is to identify the chromatin signatures that underlie gene priming and desensitization. A, acetylation; M, methylation; P, phosphorylation; pol II, RNA polymerase II. Part a is reproduced, with permission, from REF.  37 © (2010) American Association for the Advancement of Science.

Tolerance Reduced drug responsiveness with repeated exposure to a constant dose.

Dominant-negative mutant A mutant molecule that forms heteromeric complexes with the wild-type protein’s targets to yield a non-functional complex. This antagonizes the activity of the endogenous wild-type protein.

plasticity that is induced in NAc by cocaine34–36: it is both necessary and sufficient for cocaine-induced increases in the dendritic spine number of NAc MSNs37 (BOX 2). As discussed below, ΔFOSB controls the activity of several other transcriptional and epigenetic regulatory proteins, which then further influence NAc dendritic arborization. This suggests that ΔFOSB serves as one of the master control proteins that govern this structural plasticity. ΔFOSB also regulates proteins that are important for glutamatergic synaptic function and plasticity, including AMPA receptor subunits21,38 and Ca2+/

CREB. Cyclic AMP (cAMP)-responsive element binding protein (CREB) forms homodimers that can bind to genes at cAMP-responsive elements (CREs). It primarily activates transcription after it has been phosphorylated at serine 133 (by any of several protein kinases), which allows recruitment of CREB-binding protein (CBP) that then promotes transcription (see below)41,42. The mechanism by which CREB activation represses the expression of certain genes is less well understood. Psychostimulants (for example, cocaine and amphetamine) and opiates increase CREB activity, and do so acutely as well as chronically — as measured by increased phospho-CREB (pCREB) or reporter gene activity in CRE–lacZ transgenic mice — and in multiple brain regions, including the NAc and dorsal striatum41–43. Experiments that involve the inducible overexpression of CREB or a dominant-negative mutant form of CREB, either in bi-transgenic mice or using viral vectors, have shown that CREB induction in the NAc, which occurs in both D1- and D2-type MSNs41, decreases the rewarding effects of cocaine and opiates44,45. This promotes drug self-administration, presumably through negative reinforcement 46. CREB shows more complicated and varied responses to rewards or drugs of abuse other than cocaine and opiates. For example, chronic nicotine47 or ethanol48,49 administration reduces pCREB levels in the NAc but CREB activity seems to be necessary for nicotine to establish a place preference50. In addition, exposure to Δ9-tetrahydrocannabinol (THC, the active compound in marijuana) increases pCREB in the prefrontal cortex and hippocampus51, and stimuli that are associated with natural reward increase pCREB in the NAc52. Other CREB family proteins, such as inducible cAMP repressor (ICER; a product of the cAMP-responsive element modulator (CREM) gene) and activating transcription factors (ATFs), have also been implicated in the long-term actions of drugs of abuse and require further study 53. CREB activity has been directly linked to the functional activity of NAc MSNs. The electrical excitability of MSNs is increased by CREB overexpression, whereas dominant-negative CREB decreases it 54. Possible differences between D1- and D2-type MSNs in this regard have not yet been explored. The observation that

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REVIEWS Box 2 | Epigenetic regulation and dendritic spine plasticity

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For changes in gene transcription and chromatin modifications to affect complex behaviours such as addiction, they must result in some functional output, such as a change in neuronal excitability (intrinsic membrane properties) or connectivity (synapse number or strength). Indeed, it is clear that nearly all drugs of abuse alter the structural connectivity of neurons in the reward circuitry, an effect that is most evident in changes in the number, shape and size of dendritic spines on medium spiny neurons (MSNs) in the nucleus accumbens (NAc)34–36 (see the figure, part a, which shows cocaine-induced increases in dendritic spine number that can be blocked by viral overexpression of G9a or ΔJUND, or mimicked by viral overexpression of ΔFOSB). These changes seem to be behaviourally relevant, as they correlate with behavioural sensitization137. However, certain conditions that increase spine density cause opposite behavioural effects60,106. Moreover, the nature of these changes varies with the abused substance, time of withdrawal and method of intake, even within a single brain region. For example, experimenter-administered cocaine increases the number of thin spines on NAc MSNs during and shortly after chronic exposure, but increases mushroom spines and dendritic complexity during withdrawal34,36. Moreover, opiates and psychostimulants both induce locomotor activity acutely, and locomotor and reward sensitization chronically138, whereas morphine consistently reduces NAc MSN spine density and complexity34,35. Resolving this discrepancy is an important future research goal. It is also likely that structural plasticity of the NAc plays a part in volition and decision-making, as self-administered drugs generally cause larger changes in spine density than the same doses administered by experimenters35,36. Although the molecular underpinnings of these structural changes remain incompletely understood, several factors that control gene transcription and chromatin regulation have been implicated (see the figure, part b). These include ΔFOSB37, cyclic AMP-responsive element binding protein (CREB)55, myocyte-specific enhancer factor 2 (MEF2)60, G9a37 and DNA methyltransferase 3A (DNMT3A)106, each of which has been linked directly to cocaine regulation of NAc MSN spine density. A key goal is to now identify how these epigenetic factors control cytoskeletal and cytoskeleton-altering genes to regulate spine morphology and consequently changes in neuronal circuitry and addiction-related behaviours. LIMK, LIM domain kinase; RAC, Ras-related C3 botulinum toxin substrate. Part a, right parts are reproduced, with permission, from REF.  37 © (2010) American Association for the Advancement of Science. Part a, left part is reproduced, with permission, from REF.  34 © (2010) Cell Press.

virus-mediated overexpression of a K+ channel subunit in the NAc, which decreases MSN excitability, enhances locomotor responses to cocaine suggests that CREB might act as a break on behavioural sensitization to cocaine by upregulating MSN excitability 54. Numerous CREB target genes that mediate these and other effects on NAc MSNs have been identified31,32,41,42,44,55. Prominent examples include the opioid peptide dynorphin, which feeds back and suppresses dopaminergic signalling to the NAc41,44, as well as certain ion channels and glutamate receptor subunits that control NAc excitability 54,55. It is interesting to compare these effects of CREB in the NAc to similar data from the locus coeruleus, where CREB has also been found to increase neuronal excitability and thereby mediate aspects of drug tolerance and dependence (BOX 3). NF-κB. Nuclear factor-κB (NF-κB), a transcription factor that is rapidly activated by diverse stimuli, was studied initially for its role in inflammation and immune responses, and more recently has been linked to synaptic plasticity and memory 56. NF-κB has been shown to be induced in the NAc by repeated cocaine administration, where it is required for the cocaine-induced increase in NAc MSN dendritic spine density (BOX 2) and sensitization to the rewarding effects of the drug 57. It has also been associated with nicotine dependence in humans58. A major goal of current research is to identify the target genes through which NF-κB causes cellular and behavioural plasticity. Interestingly, cocaine-induced expression of NF-κB is mediated through ΔFOSB14, illustrating the complex transcriptional cascades that are involved in drug action. The role of NF-κB in MSN spinogenesis has recently been extended to stress and depression models59. This finding is of particular importance considering the co-morbidity of depression and addiction, and the well-studied phenomenon of stress-induced relapse to drug abuse. MEF2. Multiple myocyte-specific enhancer factor 2 (MEF2) proteins are expressed in the brain (including in NAc MSNs), where they form homodimers and heterodimers that can activate or repress gene transcription depending on the nature of the proteins that they recruit (for example, co-activator p300 and co-repressors known as class II histone deacetylases (HDACs) (see below)). Recent work suggests that chronic cocaine exposure suppresses striatal MEF2 activity, partly through D1 receptor–cAMP-dependent inhibition of calcineurin, a Ca2+-dependent protein phosphatase60. Cocaine-mediated induction of Cdk5, which is a target gene for ΔFOSB33, may also be involved. This reduction in MEF2 activity is required for the cocaine-induced increase in MSN dendritic spine numbers, but seems to inhibit behavioural sensitization to cocaine60. Although these data suggest that MEF2 plays an important part in the structural and behavioural changes that result from repeated cocaine administration, they also demonstrate an apparent inconsistency between MSN spine increases and behavioural sensitization to cocaine that merits further study 34. Although ethanol has been shown

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F O C U S O N A DRDEI C ON V ITEI W S Dendritic spine A small protrusion from a dendrite that is typically associated with synaptic input from a glutamatergic axon at its tip but may receive other inputs along its sides or neck.

to decrease MEF2 expression in rat cardiomyocytes61, little is known about the effects of other drugs of abuse on MEF2 function in the brain. Additional transcription factors. The transcription factors that are listed here are the ones that are most extensively studied in addiction models. However, increasing evidence links several other transcription factors to drug exposure. These include the glucocorticoid receptor,

Box 3 | Chronic morphine action in the locus coeruleus !"#$%"&

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The locus coeruleus is the major noradrenergic nucleus in the brain, and it has served as a useful model of opiate action11,139,140. Acute morphine decreases the firing rate of locus coeruleus neurons, whereas chronic exposure to the drug allows the rate to return to baseline (a phenomenon known as tolerance) and withdrawal from morphine causes firing rates to increase dramatically over baseline (a phenomenon that is characteristic of dependence and withdrawal) (see the figure, left part)139,141,142. Chronic morphine exerts these effects on firing rate partly by upregulating the cyclic AMP–cAMP-responsive element binding protein (CREB) pathway, including induction of adenylyl cyclase type 8 (AC8) and CREB itself (see the figure, right part, shown by arrows in red boxes). Indeed, inhibiting or removing components of this pathway prevents the effect of chronic morphine on locus coeruleus neuron firing (see the figure, left part). As this pathway is acutely inhibited by the drug, cAMP–CREB upregulation can be seen as a classic negative feedback mechanism11,139. These cellular and molecular effects of chronic morphine are independent of synaptic inputs and can be induced by direct activation of opioid receptors on locus coeruleus neurons in brain slices142. Moreover, the proposed role for CREB in locus coeruleus, which was based originally on overexpression systems, has been validated more recently by the local knockout of endogenous CREB from locus coeruleus neurons142. The nature of the ion channel (or channels) that mediate the cAMP– CREB-dependent changes in locus coeruleus excitability remains unknown, but activation of the cAMP–CREB pathway in locus coeruleus neurons is behaviourally relevant, in that it contributes to symptoms of physical opiate dependence and withdrawal, which are mediated in part by locus coeruleus activation. These studies establish the molecular details of a transcriptional mechanism of intrinsic homeostatic plasticity that is involved in the development of opiate tolerance and dependence, and have provided key insight into the chronic actions of opiates and of other drugs of abuse in several other CNS regions, including those directly related to reward, such as the nucleus accumbens and ventral tegmental area11. AC8 KO, AC8 knockout mouse; dnCREB, dominant negative CREB; PKA, protein kinase A; Rp–cAMP, (Rp)-adenosine 3ʹ,5ʹ-monophosphorothioate (a competitive inhibitor of cAMP-dependent processes). Left part of figure is reproduced, with permission, from REF. 142 © (2010) National Academy of Sciences.

nucleus accumbens 1 transcription factor (NAC1), early growth response factors (EGRs) and signal transducers and activators of transcription (STATs)11,14. For example, glucocorticoid receptor expression is required in dopamine receptor-expressing neurons to facilitate cocaine seeking 62 but not for molecular and behavioural responses to morphine63, and polymorphisms of this gene may be associated with the initiation of alcohol abuse in teenagers64.

Epigenetics of addiction Over the past decade, research into the regulation of transcriptional potential through modification of DNA and chromatin structure has exploded. As it became clear that epigenetic change underlies adaptations in the adult organism, investigations of epigenetic mechanisms have proven fruitful in numerous fields, including drug addiction65,66. Here, we describe three major mechanisms of epigenetic regulation — histone tail modification, DNA methylation and microRNAs — and summarize the major findings that have linked each of these mechanisms to addiction. Histone tail modification. Most DNA in eukaryotic cells is densely packed into chromatin, where 147 base pairs (bp) are wrapped around a nucleosome core in ~1.7 superhelical turns67. Nucleosomes are composed of octamers that contain four histone homodimers, one each of histones H2A, H2B, H3 and H4, with H1 binding to spans of non-nucleosomal DNA. Numerous types of post-translational modifications of the amino-terminal tails of histones alter chromatin compaction to create more ‘open’ states (euchromatin, which is transcriptionally permissive) versus ‘closed’ states (heterochromatin, which is transcriptionally repressive)68 (FIG. 3). Many residues in the tails of histones are covalently modified in numerous ways, resulting in a complex ‘code’ that is thought to control the accessibility of individual genes to the transcriptional machinery 69. Histone acetylation, which negates the positive charge of lysine residues in the histone tail, is associated with transcriptional activation. This process is controlled by histone acetyltransferases (HATs) and HDACs, each of which comprise multiple enzyme classes whose expression and activity are exquisitely regulated67. Histone methylation has been associated with both transcriptional activation and repression, depending on the particular residue and the extent of methylation70,71: both lysine and arginine residues can be methylated by several families of histone methyltransferases (HMTs), and this reaction can be reversed by equally diverse histone demethylases (HDMs). Histone tail modifications also include phosphorylation, ubiquitylation, sumoylation and ADP ribosylation, among many others67. The prospect of deciphering the histone code is daunting, given the seemingly infinite number of possible patterns of histone modifications, and the possibility that a particular pattern may have various meanings, depending on the individual gene involved. Nevertheless, new tools are accelerating progress in mapping the epigenetic state of individual gene promoters and

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Dependence A physiological state that develops to compensate for persistent drug exposure and that gives rise to a withdrawal syndrome after cessation of drug exposure.

Histone deacetylases Enzymes that catalyse the deacetylation of histone amino-terminal tails.

Nucleosome The basic building block of chromatin in which 147 base pairs of DNA are wrapped (~1.7 turns) around a core histone octamer.

Histone acetyltransferases Enzymes that catalyse the acetylation of histone amino-terminal tails.

Histone methyltransferases Enzymes that catalyse the methylation of histone amino-terminal tails.

Histone demethylases Enzymes that catalyse the demethylation of histone amino-terminal tails.

Sirtuins Proteins that have been categorized as Class III histone deacetylases, but that also serve as protein deacetylases for many non-histone proteins and as part of transcriptionrepressive complexes, seemingly independently of catalytic activity.

the genome as a whole, and future research will determine the feasibility of identifying functionally meaningful chromatin codes72. Multiple drugs of abuse induce changes in histone acetylation in the brain, and evidence has begun to accumulate that these modifications underlie some of the functional abnormalities found in addiction models66,70. First, global (that is, total cellular) levels of H3 and H4 acetylation are increased in the NAc after acute or chronic exposure to cocaine65,73, and gene promoters that show increased H3 or H4 acetylation have been mapped genome-wide32. Despite these global increases, many genes show decreased histone acetylation after chronic cocaine, raising a key question as to what governs gene-specific acetylation changes in the face of global modifications. Another key question concerns the precise intracellular signalling cascades through which cocaine induces changes in histone acetylation — there is some information that such changes may be specific to D1-type MSNs and involve regulation of growth factor-associated kinases74,75. Second, alcohol withdrawal has been shown to increase HDAC activity and reduce histone acetylation in the mouse amygdala76, and in Drosophila melanogaster the commonly abused inhalant benzyl alcohol regulates potassium channels that are tied to alcohol tolerance through H4 acetylation77. Third, exposure to THC increases HDAC3 expression in trophoblast cells78. However, this alteration was absent in a genome-wide screen of brain tissue from Δ9-THCtreated mice79, demonstrating that experiments on cell lines can yield effects that are very different from those found in a complex heterogeneous tissue like the brain. These data highlight the need for further research to define the effects of drugs of abuse on histone acetylation in brain in a region- and cell type-specific manner, and to identify the specific HAT and HDAC subtypes and intracellular signalling pathways that mediate this regulation in vivo. Experimental alterations in histone acetylation potently affect addiction-related behaviours. Shortterm systemic or intra-NAc administration of nonspecific HDAC inhibitors potentiates place conditioning and locomotor responses to psychostimulants and to opiates65,73,80. More prolonged HDAC inhibition has been reported to induce changes in the opposite direction81,82, perhaps through adaptations that oppose initial enzyme inhibition. Studies of specific HDAC isoforms have yielded interesting information: overexpression of HDAC4 or HDAC5 decreases behavioural responses to cocaine73,80, whereas genetic deletion of HDAC5 hypersensitizes mice to the chronic effects (but not to the acute effects) of the drug 80. Similarly, mutant mice with reduced expression of CBP, a major HAT in the brain, exhibit decreased sensitivity to chronic cocaine83. Much additional work is needed to define the influence of specific HAT and HDAC subtypes on addiction-related phenomena. The potential complexity involved in histone acetylation in addiction models is indicated by recent findings on sirtuins, which are considered Class III HDACs but in reality influence many non-histone proteins.

Genome-wide studies of chromatin alterations in the NAc after chronic cocaine revealed an upregulation of two sirtuins, NAD-dependent deacetylase sirtuin 1 (SIRT1) and SIRT2. Pharmacological inhibition of sirtuins decreases cocaine place preference and selfadministration, whereas activation increases rewarding responses to cocaine32. SIRT1 and SIRT2 induction is associated with increased H3 acetylation and increased ΔFOSB binding at their gene promoters32, which suggests that sirtuins are downstream targets of ΔFOSB. Studies are now needed to identify the proteins that are affected by cocaine-induced regulation of these sirtuins. For example, sirtuins deacetylate several transcription factors such as forkhead box O (FOXO) proteins or NF-κB, and serve scaffolding functions by contributing to transcriptional repressive complexes84 — processes that now warrant study in models of cocaine addiction. These findings illustrate the ability of genome-wide efforts to identify previously unknown mechanisms that are involved in drug action. Histone methylation is also directly regulated by drugs of abuse: global levels of histone 3 lysine 9 dimethylation (H3K9me2) are reduced in the NAc after chronic cocaine exposure37, and a genome-wide screen revealed alterations in H3K9me2 binding on the promoters of numerous genes in this brain region32; both increases and decreases were observed, indicating again that epigenetic modifications at individual genes often defy global (that is, cell-wide) changes. The global decrease in H3K9me2 in the NAc is probably mediated by cocaine-induced downregulation of two HMTs, G9a and G9a-like protein (GLP), which catalyse the demethylation of H3K9me2 (REF.  37). These adaptations mediate enhanced responsiveness to cocaine, as selective knockout or pharmacological inhibition of G9a in the NAc promotes cocaine-induced behaviours, whereas G9a overexpression has the opposite effect. Similarly, G9a downregulation mediates the ability of cocaine to increase the spine density of NAc MSNs 37 (BOX  2) . Interestingly, there is a functional feedback loop between G9a and ΔFOSB: ΔFOSB seems to be responsible for cocaine-induced suppression of G9a, and G9a binds to and represses the Fosb promoter, such that G9a downregulation may promote the accumulation of ΔFOSB that is seen after chronic cocaine37. In addition, G9a and ΔFOSB share many of the same target genes. Chronic cocaine also downregulates H3K9me3, a mark of heterochromatin, specifically in the NAc, and this change is associated with a decrease in the total amount of heterochromatin in NAc MSN nuclei and an increase in the volume of these nuclei85. Genome-wide mapping of H3K9me3 after chronic cocaine indicates that most of the cocaine-mediated regulation of this mark occurs at non-genic regions, including at repetitive line elements, which are consequently induced by cocaine85. Although the functional implications of this regulation are not yet known, these findings highlight the profound effects that cocaine exerts on the genome within NAc neurons. Studies are now needed to examine the actions of other drugs of abuse on these histone endpoints, as well

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DNA methyltransferases Enzymes that methylate cytosine nucleotides, in CpG sequences, in DNA.

DNA methylation. Methylation of DNA occurs at the 5′ position of cytosine nucleotides, with the resulting methyl group projecting into the major groove of the DNA double helix 91. In mammals, this occurs almost exclusively in 5′-CpG-3′ sequences, and methylation is common throughout the genome — ~3% of all cytosines in human DNA are methylated92 — with proper cytosine methylation required for normal development, genetic imprinting and X-chromosomal inactivation93. CpG sequences are not evenly dispersed throughout the genome, but are concentrated in regions termed CpG islands. These are CG-rich regions that overlap with the promoters of 50–60% of human genes and are typically methylated to a much lower extent than CpG dinucleotides that are found outside of islands94.

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as the effect of drugs on many other types of histone modifications that are known to regulate eukaryotic gene expression in other systems, in addiction models. Examples include recent, preliminary observations of chronic cocaine-mediated regulation of histone arginine methylation and poly-ADP ribosylation, of several families of chromatin remodelling proteins, and of histone variant subunits in the NAc, all of which illustrate the complexity of epigenetic changes that are associated with drug exposure86–89. Moreover, it will be important to relate drug-induced modifications of histones, which occur at specific drug-regulated genes, with the recruitment of numerous additional proteins that ultimately constitute the transcriptional activation or repression complexes that mediate such regulation. For example, early studies have shown that cocaine-induced expression of CDK5 in the NAc involves a cascade of events, including binding of ΔFOSB to the Cdk5 gene promoter, followed by the recruitment of CBP, increased H3 acetylation and the recruitment of specific chromatin remodelling factors, such as transcription activator BRG1 (REF. 73 ) (FIG. 4). Such activation also involves reduced repressive histone methylation at this promoter, which is mediated through cocaine-induced suppression of G9a. By contrast, a very different cascade mediates chronic amphetamine-induced repression of the Fos gene. Here, ΔFOSB binds to the Fos promoter and recruits HDAC1 and SIRT1, and presumably numerous other proteins90. Also, chronic amphetamine induces increased repressive histone methylation at the Fos promoter, perhaps mediated through increased G9a binding 37. It is interesting that such increased G9a binding occurs despite the global decrease in G9a expression, once again highlighting gene-specific changes that occur on top of global modifications. Understanding the molecular basis of such gene-specific modifications — for example, why ΔFOSB triggers a cascade of transcriptional activation when it binds to one promoter, but a cascade of transcriptional repression when it binds to another — is a crucial goal of current research. So far, these efforts have been pursued on a protein-by-protein basis, which is experimentally painstaking. There is a major need in this field to develop tools to analyse the complete protein complexes that are recruited to individual genes in concert with drug exposure.

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Figure 4 | Epigenetic basis of drug regulation of gene expression. The mechanisms by which chronic cocaine, through ΔFOSB, activates the cyclin-dependent kinase 5 (Cdk5) gene and represses the Fos gene. a | ΔFOSB binds to the Cdk5 gene and recruits several co-activators, including cyclic AMP-responsive element (CREB)-binding protein (CBP; a type of histone acetyltransferase (HAT) leading to increased histone acetylation), transcription factor BRG1 (also known as brahma-related gene 1; a type of chromatin remodelling factor) and proteasome 26S ATPase subunit 5 (SUG1; another type of chromatin-regulatory protein). ΔFOSB also represses G9a expression, leading to reduced repressive histone methylation at the Cdk5 gene. The net result is gene activation and increased CDK5 expression. b | ΔFOSB can also bind to the Fos gene and recruits several co-repressors, including histone deacetylase 1 (HDAC1) and sirtuin 1 (SIRT 1). Additionally, the gene shows increased G9a binding and repressive histone methylation (despite global decreases in these marks). The net result is Fos gene repression. As transcriptional regulatory complexes contain dozens or hundreds of proteins, much further work is needed to further define the activational and repressive complexes that cocaine recruits to particular genes to mediate their transcriptional regulation and to explore the range of distinct activational and repressive complexes that are involved in cocaine action.

CpG methylation is catalysed by a family of enzymes termed DNA methyltransferases (DNMTs), some of which are responsible for the maintenance of DNA methyl states, whereas others perform de novo CpG methylation91,92. The process of demethylation is less well understood, and may involve DNA repair mechanisms, such as growth arrest and DNA damage-inducible protein (GADD45) family members92 and methylcytosine dioxygenase TET1 (REFS  95–97). A variant of DNA methylation, 5-hydroxycytosine methylation, also seems to be important in gene regulation98,99 but has not yet been investigated in addiction models. DNA methylation is generally considered to repress gene transcription through recruitment of co-repressor complexes (for example, HDACs and HMTs) that can

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Hypomorphic A mutation that causes a wild-type gene product to be produced at a reduced level.

sterically hinder the transcriptional machinery or modify nucleosome structure. Such complexes involve several DNA methyl-binding domain proteins (MBDs)92, which are required for normal cell growth and development. Indeed, mutations in methyl CpG binding protein 2 (MeCP2), a prominent MBD, cause the majority of Rett syndrome cases and are found in a small number of patients with other autism spectrum disorders100. There are multiple known links between DNA methylation and addiction. Cocaine self-administration increases MeCP2 expression in the NAc101 and dorsal striatum102, and lentiviral knockdown of MeCP2 in the dorsal striatum (but not the NAc) decreases drug intake under extended but not limited access conditions103. Hypomorphic Mecp2 mutant mice show reduced locomotor sensitization and place conditioning after chronic amphetamine104, however, the same study reported that viral knockdown of MeCP2 in the NAc increases amphetamine-induced place conditioning, whereas local overexpression decreases this behavioural response104. The reasons for this discrepancy are unclear, but it seems likely that developmental abnormalities in the mutant mice, or the effects of reduced Mecp2 expression in other brain regions, explain these differences. These findings therefore emphasize the importance of using inducible and brain region-specific tools. Two possible mechanisms for the actions of MeCP2 in drug reward have been proposed. First, a reduction in MeCP2 prevents amphetamine-mediated increases in NAc dendritic spine density while increasing the number of GABAergic synapses104. This is complemented by an increase in MeCP2 phosphorylation specifically in GABAergic interneurons in the NAc, which regulates its transcriptional activity and correlates strongly with behavioural sensitization to amphetamine 104. An alternative model suggests that MeCP2 represses the transcription of specific microRNAs (see below), resulting in reduced repression of brain-derived neurotrophic factor (BDNF)103, which is also a target for CREB. BDNF has previously been described to promote cocaine self-administration105, consistent with the MeCP2 data. Although these models are not mutually exclusive, further work is necessary to integrate them with our growing understanding of the multiple brain regions and cell types that are involved in reward behaviours. A direct link between CpG methylation and addiction involves DNA (cytosine-5)-methyltransferase 3A (DNMT3A). Repeated cocaine administration dynamically regulates DNMT3A expression in the mouse NAc, with decreases seen during early phases of withdrawal and sustained increases seen at later time points82,106. Experimental reduction of DNMT3A activity in the adult NAc — achieved either through virus-mediated local knockout in floxed Dnmt3a mice or through local infusion of a DNMT inhibitor — increases behavioural responses to cocaine, whereas DNMT3A overexpression in this region decreases these responses but also has the paradoxical effect of increasing NAc MSN spine density 106, similar to the effects of MEF2 manipulation in this brain region60. Future research may identify the

specific genes whose methylation status changes in response to chronic cocaine and consequently regulates cellular and behavioural adaptations to the drug. These observations that chronic cocaine alters DNMT3A and MBDs in the NAc and dorsal striatum raise the possibility that drug-induced changes in DNA methylation might also occur in germ cells and be passed on to subsequent generations to regulate the propensity of the offspring for addictive behaviours. The idea of such trans-generational transmission of DNA methylation changes and the resulting behavioural plasticity remains highly speculative, although recent research has shown robust effects of adult cocaine exposure in rats on cocaine responses in their progeny 107. Gene priming and desensitization. Ongoing studies of chromatin regulation in addiction models support the view that epigenetic modifications at individual genes not only underlie stable changes in the steady-state levels of mRNA expression of certain genes but also alter the inducibility of many additional genes in response to some subsequent stimulus, without affecting baseline expression levels of these genes. Although such studies are still in relatively early stages of development, these types of latent epigenetic changes can be viewed as ‘molecular scars’ that dramatically alter an individual’s adaptability and contribute importantly to the addicted state. Such priming and desensitization of genes is evident in a recently published microarray study 37. Numerous desensitized genes were identified: ~10% of genes whose transcription is induced acutely in the NAc by cocaine are no longer induced by a cocaine challenge after prior chronic exposure to the drug (FIG. 3a). Conversely, numerous genes are primed: genes that are not affected by acute cocaine become inducible after a chronic course of cocaine, with approximately three times more genes being induced in cocaine-experienced animals. The mechanisms that underlie such gene desensitization and priming remain incompletely understood; our hypothesis is that epigenetic mechanisms are crucial (FIG. 3b). A subset of primed genes in the NAc show reduced binding of G9a and H3K9me2 at their promoters, suggesting the involvement of this epigenetic mark37. Desensitization of the Fos gene in the NAc, discussed above and shown in FIG. 4, involves stable increases in the binding of ΔFOSB, G9a and related co-repressors, which — although not affecting steadystate levels of Fos mRNA — dramatically repress its inducibility by subsequent drug exposure90. There is now a major need in this field to investigate the many additional chromatin mechanisms that are recruited by drug exposure to mediate gene priming and desensitization, and to understand the detailed mechanisms that target those particular genes. The goal of such studies would be to identify ‘chromatin signatures’ that underlie such long-lasting regulation. The prominence of gene priming and desensitization indicates that studies of steady-state mRNA levels per se would miss important aspects of drug regulation that

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F O C U S O N A DRDEI C ON V ITEI W S are not captured at the particular time point examined. For example, the aforementioned microarray study 37 measured mRNA levels 1 hour after a cocaine challenge, and preliminary evidence suggests that a partly distinct set of genes show evidence of priming and desensitization at 4 hours. These observations highlight the unique utility of genome-wide assays of chromatin regulation, as such assays would reveal priming and desensitization more globally 32. MicroRNAs. Increasing attention has focused on a variety of non-coding RNAs that are important in biological regulation108. These include microRNAs, which are generally around 22 bp long, are found in all mammalian cells and are post-translational regulators that bind to complementary sequences on target mRNAs to repress translation and thus silence gene expression. Like histone modifications and DNA methylation, expression of microRNAs can alter the transcriptional potential of a gene in the absence of any change to the DNA sequence, and thus can be considered an epigenetic phenomenon. Several recent studies have implicated microRNAs in addiction behaviours, and microRNAs whose expression is altered by drugs of abuse have been shown to regulate the expression of many proteins that are strongly linked to addiction109. Cocaine self-administration in rats reportedly increases expression of the microRNA miR-212 in striatum, and experimentally increasing miR-212 levels in this region decreases cocaine reward110. The actions of miR-212 depend on upregulation of CREB, which is known to decrease the rewarding effects of cocaine (see above), and more recent work shows that MeCP2 may interact homeostatically with miR-212 to control BDNF expression and cocaine intake103. It has been proposed103 that this CREB–miR-212–MeCP2–BDNF mechanism is at least partially responsible for cocaine tolerance and escalating intake. Chronic cocaine also regulates miR-124 and miR-181a in brain, where they are decreased and increased, respectively 111. miR-124 overexpression in the NAc reduces cocaine place conditioning, whereas overexpression of miR-181a has the opposite effect 112, suggesting that drug-induced regulation of these microRNAs may also act as a mechanism of tolerance and escalating intake. Like miR-212, miR-124 and miR-181a may operate through the CREB–BDNF pathway, as miR-124 overexpression downregulates both of these genes111,113. However, these microRNAs have also been shown to affect the expression of the dopamine transporter 112, so their mechanisms of action are likely to be complex. Finally, argonaut 2 protein (AGO2) — which is important in microRNA-mediated gene silencing — has recently been implicated, along with several specific microRNAs, in cocaine-mediated regulation of gene expression selectively in the D2 subclass of striatal MSNs114. Other drugs of abuse have also been linked to microRNAs. Opioid receptor activation downregulates miR-190 in cultured rat hippocampal neurons in a beta arrestin 2-dependent manner 115, and the let-7 family of microRNA precursors is upregulated by

chronic morphine exposure in mice116. Interestingly, the mu opioid receptor is itself a direct target for let-7, and the resulting repression of the receptor has been suggested as a novel mechanism for opiate tolerance116. In zebrafish and in cultured immature rat neurons, morphine decreases miR-133b expression, and this might influence dopamine neuron differentiation117. In addition, both acute and chronic alcohol exposure upregulates miR-9 in cultured striatal neurons, and this may contribute to alcohol tolerance through regulation of large-conductance Ca2+ activated K+ (BK) channels118. miR-9 seems to preferentially downregulate BK channel isoforms that are sensitive to alcohol potentiation, perhaps shifting BK channel expression towards more tolerant subtypes119. miR-9 also targets the D2 dopamine receptor 119 and so probably influences alcohol reward. In the future, next-generation sequencing of microRNAs in several brain regions after exposure to drugs of abuse will be essential to uncover how specific microRNAs (and, eventually, the genes that they control) are regulated. Indeed, this process has already begun, as such screens are revealing that numerous microRNAs are regulated in the NAc by chronic cocaine114,120. For example, cocaine-mediated regulation of the miR-8 family suggests novel mechanisms for drug-induced alterations in the neuronal cytoskeletal and synaptic structure120. Exploring this mechanism in drug-induced regulation of NAc dendritic morphology is an important line of future investigation.

Future directions This Review has summarized the increasing array of findings that support a role for regulation of the transcriptional potential of myriad genes in the brain’s maladaptations to drugs of abuse. The mechanisms of transcriptional and epigenetic regulation are themselves varied and highly complex, and future studies are needed to catalogue the vast number of regulatory events that occur as well as to understand the precise underlying mechanisms that are involved. One key question is what controls the recruitment or expulsion of individual transcriptional regulatory proteins to a particular target gene. Our hypothesis is that the underlying epigenetic state of that gene is a crucial determining factor. However, if this is the case, what controls the formation and maintenance of distinct epigenetic states at particular genes? Also, what are the intracellular signalling cascades that transduce the initial drug action at the neurotransmitterreceptor level to the neuronal nucleus to regulate the epigenetic state of specific subsets of genes? The existing literature on transcriptional and epigenetic mechanisms of addiction is limited in several key ways. So far, most studies have employed conditioned place-preference and locomotor sensitization paradigms. Although these behavioural assays provide useful insight into an animal’s sensitivity to the actions of drugs of abuse on the brain’s reward circuitry, they do not provide direct measures of drug reinforcement or addiction per se. Instead, the field needs to make greater use of drug self-administration and relapse

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REVIEWS Box 4 | Sex differences in drug addiction: epigenetic mechanisms? Addiction research has historically neglected female subjects, particularly in animal studies, although both human and animal studies have found robust sex differences in drug responses143,144. In self-administration studies with various drugs, female rats are more responsive in general and exhibit particularly enhanced responses in the transition phases of acquisition or relapse compared to the maintenance phase145,146. In addition, the locomotor effects of many psychostimulants are greater in female rats147,148. Although, in general, ovariectomy reduces these differences and oestrogen administration increases them, this is not true of all drugs of abuse, and some contradictory results have been reported143. These data suggest that drugs of abuse have differential effects on the two sexes, and that the reward system may be different between men and women; clinical evidence supports these hypotheses. Women usually have a later age of onset for substance abuse, although they progress to addiction more rapidly than men149. In the specific case of cocaine, women report shorter periods of abstinence, have greater drug intake and respond more strongly to cue-induced craving143. These differences may be directly related to the brain’s reward circuitry, as men have been reported to show greater striatal dopamine release than women in response to psychostimulant challenges150. Interestingly, stress upregulates the expression of DNA methyltransferases (DNMTs) and DNA methyl-binding domain proteins (MBDs) in the nucleus accumbens (NAc)106; these effects predominate in females and inhibition of DNMT3A in the NAc of female rats increases natural reward151, suggesting that the sexes may undergo differential epigenetic regulation of the reward circuitry. Furthermore, as activation of the reward circuitry by sexual behaviour induces ΔFOSB27,29,30and other regulators of transcription, there is little doubt that future studies will reveal further sexual dimorphism in the regulation of transcriptional and epigenetic mechanisms by drugs of abuse — findings that may have important consequences for treatment.

assays, which are considered the best available animal models of addiction121–123. Similarly, in most studies the drugs of abuse were experimenter-administered, but we know that drugs exert some distinct actions when selfadministered or given within a particular environmental context. Studies that move beyond the relatively short time frames of most current experiments are also needed to examine transcriptional and epigenetic endpoints after much longer periods of drug exposure and longer periods of withdrawal from drug exposure. Such studies might lead to a molecular hypothesis that explains the phenomenon of relapse in human addicts after years or even decades of abstinence. In addition, studies should be extended from investigating cocaine action in NAc, which has been the main focus so far, to investigating several other drugs and several other reward-related brain regions. Future studies of gene regulation will better inform drug discovery efforts as they increasingly incorporate experimental paradigms that better model human addiction. Another limitation of the existing literature is the reliance of many studies on overexpression systems — viral or transgenic — which often induce levels of expression that are far greater than those seen under normal conditions or even after drug treatment. Such overexpression of transcription factors, chromatin-regulatory proteins or their dominant-negative mutants, can lead to non-physiological changes in gene expression and subsequent alterations in cell morphology, physiology and/or behaviour. It is reassuring that many of the phenomena described above, resulting from studies that utilized overexpression systems, have been validated with other methods. For example, the genes that are regulated by overexpression

of ΔFOSB in the NAc of inducible bi-transgenic mice31 overlap extensively with genes that show enrichment of endogenous ΔFOSB binding after cocaine exposure32. Similar caveats exist for the use of constitutive knockout animals, in which loss of a gene in early development and in all tissues makes it difficult to interpret any changes that are observed in drug regulation involving a single brain region of an adult animal. Ultimately, a truly accurate understanding of the transcriptional and epigenetic regulation of the addiction process will require the generation of novel tools that control protein expression with greater spatial, temporal and accumulation precision. Methodological advances in epigenetics are needed as well. Current levels of experimental proof of epigenetic mechanisms of drug action have so far involved the overexpression or deletion of a given epigenetic protein (for example, an HAT, HDAC, HMT or a DNMT) within a brain region of interest. However, such manipulations affect the epigenetic states of perhaps thousands of genes without targeting those genes that are specifically altered by drug exposure. Being able to experimentally manipulate the epigenetic state of an individual gene within a discrete brain region of an adult animal would represent a major advance for the field. Tools such as artificially designed zinc-finger proteins124 or sequence-specific transcription activator-like effectors (TALEs)125, which are designed to bind specific DNA sequences in vivo, would offer exciting possibilities for future studies. Similarly, all genome-wide studies of drug-induced epigenetic changes in the brain so far have used total extracts of brain regions, even though we know that drugs produce very different effects on distinct neuronal and non-neuronal cell types within a given region. Genome-wide epigenetic analyses in a cell type-specific manner are crucially needed in addiction research126. Advances in bioinformatics are also needed. Genome-wide studies of transcription factor binding and chromatin modifications generate enormous datasets, which require the development of better tools to effectively mine the resulting data. For example, it will be crucial to overlay such epigenetic analyses with genomewide changes in RNA expression and to compare data obtained from animal models with those from human post-mortem brain tissue. On a similar note, the findings from studies on drug regulation of gene expression reviewed here must be integrated with findings obtained at several other levels of analysis. How do individual differences in genome sequences relate to individual differences in epigenetic regulation? Do drug-induced epigenetic modifications occur in peripheral tissues such as blood, and do any such changes reflect addictionrelevant phenomena? Recent studies, for example, have found altered levels of methylation of the monoamine oxidase A (MAOA) and MAOB gene promoters in the blood of smokers127,128. Additionally, altered methylation of MAOA in lymphoblasts is associated with nicotine and alcohol dependence in women but not in men129, emphasizing the need for studies of sex differences in epigenetic regulation in addiction models, which until now have focused almost exclusively on male animals (BOX 4).

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F O C U S O N A DRDEI C ON V ITEI W S As information on transcriptional and epigenetic mechanisms of addiction accumulates, it is essential to integrate it with equally important information regarding post-transcriptional (translational and post-translational) regulation to obtain a complete understanding of how chronic exposure to a drug

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85. Maze, I. et al. Cocaine dynamically regulates heterochromatin and repetitive element unsilencing in nucleus accumbens. Proc. Natl Acad. Sci. USA 108, 3035–3040 (2011). This study used ChIP–Seq to show drug-induced regulation of repetitive genomic sequences in the NAc after repeated cocaine administration. This supports the utility of such genome-wide methods in revealing new mechanisms of drug action. 86. Sun, H. et al. Cocaine and stress regulates ATPasecontaining chromatin remodelers. Soc. Neurosci. Abstr. 909.14 ((Washington DC, 12–16 Nov 2011). 87. Damez-Werno, D. et al. Histone arginine methylation in the nucleus accumbens in response to chronic cocaine and social stress. Soc. Neurosci. Abstr. 909.16 (Washington DC, 12–16 Nov 2011). 88. Kennedy, P. J. et al. Differential histone H2A variant expression in the nucleus accumbens following repeated exposure to cocaine or morphine. Soc. Neurosci. Abstr. 909.15 (Washington DC, 12–16 Nov 2011). 89. Scobie, K., Damez-Werno, D., Sun, H., Kennedy, P. J. & Nestler, E. J. Role of poly(ADP-ribosyl)ation in drugseeking behavior and resiliency to stress. Soc. Neurosci. Abstr. 909.18 (Washington DC, 12–16 Nov 2011). 90. Renthal, W. et al. Delta FosB mediates epigenetic desensitization of the c-fos gene after chronic amphetamine exposure. J. Neurosci. 28, 7344–7349 (2008). 91. Newell-Price, J., Clark, A. J. & King, P. DNA methylation and silencing of gene expression. Trends Endocrinol. Metab. 11, 142–148 (2000). 92. Kim, J. K., Samaranayake, M. & Pradhan, S. Epigenetic mechanisms in mammals. Cell. Mol. Life Sci. 66, 596–612 (2009). 93. Chahrour, M. & Zoghbi, H. Y. The story of Rett syndrome: from clinic to neurobiology. Neuron 56, 422–437 (2007). 94. Wang, Y. & Leung, F. C. An evaluation of new criteria for CpG islands in the human genome as gene markers. Bioinformatics 20, 1170–1177 (2004). 95. Guo, J. U., Su, Y., Zhong, C., Ming, G. L. & Song, H. Hydroxylation of 5-methylcytosine by TET1 promotes active DNA demethylation in the adult brain. Cell 145, 423–434 (2011). This study provides the first demonstration of TET1-mediated demethylation of DNA in the adult brain, laying the groundwork for future in vivo studies that may provide insight into the role of DNA demethylation in addiction-related phenomena. 96. Williams, K. et al. TET1 and hydroxymethylcytosine in transcription and DNA methylation fidelity. Nature 473, 343–348 (2011). 97. Wu, H. et al. Dual functions of Tet1 in transcriptional regulation in mouse embryonic stem cells. Nature 473, 389–393 (2011). 98. Pastor, W. A. et al. Genome-wide mapping of 5-hydroxymethylcytosine in embryonic stem cells. Nature 473, 394–397 (2011). 99. Ficz, G. et al. Dynamic regulation of 5-hydroxymethylcytosine in mouse ES cells and during differentiation. Nature 473, 398–402 (2011). 100. Chahrour, M. & Zoghbi, H. Y. The story of Rett syndrome: from clinic to neurobiology. Neuron 56, 422–437 (2007). 101. Host, L., Dietrich, J. B., Carouge, D., Aunis, D. & Zwiller, J. Cocaine self-administration alters the expression of chromatin-remodelling proteins; modulation by histone deacetylase inhibition. J. Psychopharmacol. 25, 222–229 (2011). 102. Cassel, S. et al. Fluoxetine and cocaine induce the epigenetic factors MeCP2 and MBD1 in adult rat brain. Mol. Pharmacol. 70, 487–492 (2006). 103. Im, H. I., Hollander, J. A., Bali, P. & Kenny, P. J. MeCP2 controls BDNF expression and cocaine intake through homeostatic interactions with microRNA-212. Nature Neurosci. 13, 1120–1127 (2010). This work provided an alternative, but not mutually exclusive, mechanism for MeCP2 action in addiction models to that presented in reference 104. The authors showed that MeCP2 controls cocaine intake through microRNA-mediated regulation of BDNF, demonstrating the complex interactions among the various mechanisms of epigenetic modifications in the drug-exposed brain. 104. Deng, J. V. et al. MeCP2 in the nucleus accumbens contributes to neural and behavioral responses to psychostimulants. Nature Neurosci. 13, 1128–1136 (2010).

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This study provided novel evidence that MeCP2 activity in NAc regulates synaptic responses to psychostimulants, and established a link between MeCP2 and behavioural sensitization (see also REF. 103). 105. Graham, D. L. et al. Dynamic BDNF activity in nucleus accumbens with cocaine use increases self-administration and relapse. Nature Neurosci. 10, 1029–1037 (2007). 106. LaPlant, Q. et al. Dnmt3a regulates emotional behavior and spine plasticity in the nucleus accumbens. Nature Neurosci. 13, 1137–1143 (2010). This study provided crucial evidence of the importance of DNMT3A in the NAc in regulating cellular and behavioural plasticity to repeated cocaine exposure. 107. Vassoler, F. M., White, S. L., Ortinski, P. I., Sadri-Vakili, G. & Pierce, R. C. Paternal transmission of a cocaine resistance phenotype in male offspring. Soc. Neurosci. Abstr. 219.2 (Washington DC, 12–16 Nov 2011). 108. Taft, R. J., Pang, K. C., Mercer, T. R., Dinger, M. & Mattick, J. S. Non-coding RNAs: regulators of disease. J. Pathol. 220, 126–139 (2010). 109. Li, M. D. & van der Vaart, A. D. MicroRNAs in addiction: adaptation’s middlemen? Mol. Psychiatry 24 May 2011 (doi: 10.1038/mp.2011.58). 110. Hollander, J. A. et al. Striatal microRNA controls cocaine intake through CREB signalling. Nature 466, 197–202 (2010). This study provided a detailed mechanism by which miR-212 enhances CREB signalling in dorsal striatum after cocaine, and demonstrated a key role for this adaptation in blunting the sensitivity of animals to the motivational effects of the drug. 111. Chandrasekar, V. & Dreyer, J. L. microRNAs miR-124, let-7d and miR-181a regulate cocaine-induced plasticity. Mol. Cell. Neurosci. 42, 350–362 (2009). 112. Chandrasekar, V. & Dreyer, J. L. Regulation of MiR-124, Let-7d, and MiR-181a in the accumbens affects the expression, extinction, and reinstatement of cocaine-induced conditioned place preference. Neuropsychopharmacology 36, 1149–1164 (2011). 113. Rajasethupathy P. et al. Characterization of small RNAs in Aplysia reveals a role for miR-124 in constraining synaptic plasticity through CREB. Neuron 63, 803–817 (2009). 114. Schaefer, A. et al. Argonaute 2 in dopamine 2 receptor-expressing neurons regulates cocaine addiction. J. Exp. Med. 207, 1843–1851 (2010). 115. Zheng, H. et al. mu-Opioid receptor agonists differentially regulate the expression of miR-190 and NeuroD. Mol. Pharmacol. 77, 102–109 (2010). 116. He, Y., Yang, C., Kirkmire, C. M. & Wang, Z. J. Regulation of opioid tolerance by let-7 family microRNA targeting the mu opioid receptor. J. Neurosci. 30, 10251–10258 (2010). 117. Sanchez-Simon, F. M., Zhang, X. X., Loh, H. H., Law, P. Y. & Rodriguez, R. E. Morphine regulates dopaminergic neuron differentiation via miR-133b. Mol. Pharmacol. 78, 935–942 (2010). 118. Pietrzykowski, A. Z. et al. Posttranscriptional regulation of BK channel splice variant stability by miR-9 underlies neuroadaptation to alcohol. Neuron 59, 274–287 (2008). 119. Pietrzykowski, A. Z. The role of microRNAs in drug addiction: a big lesson from tiny molecules. Int. Rev. Neurobiol. 91, 1–24 (2010). 120. Eipper-Mains, J. E. et al. MicroRNA-Seq reveals cocaine-regulated expression of striatal microRNAs. RNA 17, 1529–1543 (2011). As the role of microRNAs in addiction becomes clearer, the requirement for an unbiased approach to identifying microRNA-regulated genes becomes more pressing. Here, RNA–Seq was used to identify novel messages regulated by cocaine-induced microRNAs in the striatum, with a particular focus on regulation of synaptically located microRNAs. 121. Pelloux, Y., Everitt, B. J. & Dickinson, A. Compulsive drug seeking by rats under punishment: effects of drug taking history. Psychopharmacology 194, 127–137 (2007). 122. Pickens, C. L. et al. Neurobiology of the incubation of drug craving. Trends Neurosci. 34, 411–420 (2011). 123. O’Connor, E. C., Chapman, K., Butler, P. & Mead, A. N. The predictive validity of the rat self-administration model for abuse liability. Neurosci. Biobehav. Rev. 35, 912–938 (2011). 124. Laganiere, J. et al. An engineered zinc finger protein activator of the endogenous glial cell line-derived neurotrophic factor gene provides functional neuroprotection in a rat model of Parkinson’s disease. J. Neurosci. 30, 16469–16474 (2010).

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F O C U S O N A DRDEI C ON V ITEI W S 125. Zhang, F. et al. Efficient construction of sequencespecific TAL effectors for modulating mammalian transcription. Nature Biotech. 29, 149–153 (2011). 126. Cheung, I. et al. Developmental regulation and individual differences of neuronal H3K4me3 epigenomes in the prefrontal cortex. Proc. Natl Acad. Sci. USA 107, 8824–8829 (2010). Understanding the epigenetic regulation of addiction behaviours will require the delineation of individual genes whose chromatin structure is altered by drug exposure in specific regions and cell types within the brain. This study validated a method for cell-type specific ChIP–Seq on brain tissue that makes such studies possible. 127. Philibert, R. A. et al. The effect of smoking on MAOA promoter methylation in DNA prepared from lymphoblasts and whole blood. Am. J. Med. Genet. B Neuropsychiatr. Genet. 153B, 619–628, (2010). 128. Launay, J. M. et al. Smoking induces long-lasting effects through a monoamine-oxidase epigenetic regulation. PLoS ONE 4, e7959 (2009). 129. Philibert, R. A., Gunter, T. D., Beach, S. R., Brody, G. H. & Madan, A. MAOA methylation is associated with nicotine and alcohol dependence in women. Am. J. Med. Genet. B Neuropsychiatr. Genet. 147B, 565–570 (2008). 130. Lobo, M. K. et al. Cell type-specific loss of BDNF signaling mimics optogenetic control of cocaine reward. Science 330, 385–390 (2010). Using cell-type specific expression strategies and optogenetic control of neuronal activity, this study showed that activation of D1-type and D2-type MSNs enhances and suppresses behavioural responses to cocaine, respectively. The authors implicated neurotrophin signalling in mediating these opposite responses (see also REF. 131). 131. Witten, I. B. et al. Cholinergic interneurons control local circuit activity and cocaine conditioning. Science 330, 1677–1681 (2010). This study showed that cholinergic interneurons in the NAc are activated by cocaine, and that optogenetic supression of this activity blocks cocaine conditioning, providing an important, complementary mechanism to those proposed by reference 130. 132. Self, D. W. in The Dopamine Receptors 2nd edn (ed. Neve, K. A.) 479–524 (Humana Press, New York, 2010).

133. Nye, H. E., Hope, B. T., Kelz, M. B., Iadarola, M. & Nestler, E. J. Pharmacological studies of the regulation of chronic FOS-related antigen induction by cocaine in the striatum and nucleus accumbens. J. Pharmacol. Exp. Ther. 275, 1671–1680 (1995). 134. Lee, K. W. et al. Cocaine-induced dendritic spine formation in D1 and D2 dopamine receptorcontaining medium spiny neurons in nucleus accumbens. Proc. Natl Acad. Sci. USA 103, 3399–3404 (2006). 135. Maze, I. et al. G9a regulates cocaine-induced behavioral and transcriptional plasticity in a cell-type specific manner Soc. Neurosci. Abstr. 574.7 (California, 13–17 Nov 2010). 136. Singla, S., Kreitzer, A. C. & Malenka, R. C. Mechanisms for synapse specificity during striatal long-term depression. J. Neurosci. 27, 5260–5264 (2007). 137. Li, Y., Acerbo, M. J. & Robinson, T. E. The induction of behavioural sensitization is associated with cocaineinduced structural plasticity in the core (but not shell) of the nucleus accumbens. Eur. J. Neurosci. 20, 1647–1654 (2004). 138. Russo, S. J., Mazei-Robison, M. S., Ables, J. L. & Nestler, E. J. Neurotrophic factors and structural plasticity in addiction. Neuropharmacology 56, 73–82 (2009). 139. Nestler, E. J. & Aghajanian, G. K. Molecular and cellular basis of addiction. Science 278, 58–63 (1997). 140. Van Bockstaele, E. J., Reyes, B. A. & Valentino, R. J. The locus coeruleus: a key nucleus where stress and opioids intersect to mediate vulnerability to opiate abuse. Brain Res. 1314, 162–174(2010). 141. Han, M. H. et al. Role of cAMP response elementbinding protein in the rat locus ceruleus: regulation of neuronal activity and opiate withdrawal behaviors. J. Neurosci. 26, 4624–4629 (2006). 142. Cao, J. L. et al. Essential role of the cAMP-cAMP response-element binding protein pathway in opiateinduced homeostatic adaptations of locus coeruleus neurons. Proc. Natl Acad. Sci. USA 107, 17011–17016 (2010). This study detailed a signalling mechanism by which long-term exposure to morphine induces homeostatic plasticity intrinsic to locus coeruleus neurons, which involves induction of CREB and its downstream targets, such as adenylyl cyclase 8. These adaptations result in enhanced excitability of the neurons and partly mediate physical opiate withdrawal.

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143. Fattore, L., Altea, S. & Fratta, W. Sex differences in drug addiction: a review of animal and human studies. Womens Health 4, 51–65 (2008). 144. Carroll, M. E. & Anker, J. J. Sex differences and ovarian hormones in animal models of drug dependence. Horm. Behav. 58, 44–56 (2010). 145. Lynch, W. J. & Carroll, M. E. Sex differences in the acquisition of intravenously self-administered cocaine and heroin in rats. Psychopharmacology 144, 77–82 (1999). 146. Roth, M. E. & Carroll, M. E. Sex differences in the escalation of intravenous cocaine intake following long- or short-access to cocaine self-administration. Pharmacol. Biochem. Behav. 78, 199–207 (2004). 147. Cailhol, S. & Mormede, P. Strain and sex differences in the locomotor response and behavioral sensitization to cocaine in hyperactive rats. Brain Res. 842, 200–205 (1999). 148. Robinson, T. E., Becker, J. B. & Presty, S. K. Long-term facilitation of amphetamine-induced rotational behavior and striatal dopamine release produced by a single exposure to amphetamine: sex differences. Brain Res. 253, 231–241 (1982). 149. Hernandez-Avila, C. A., Rounsaville, B. J. & Kranzler, H. R. Opioid-, cannabis- and alcohol-dependent women show more rapid progression to substance abuse treatment. Drug Alcohol Depend. 74, 265–272 (2004). 150. Munro, C. A. et al. Sex differences in striatal dopamine release in healthy adults. Biol. Psychiatry 59, 966–974 (2006). 151. Hodes, G. E., Christoffel, D. J., Golden, S. A., Ahn, H. F. & Russo, S. J. Sex differences in epigenetic regulation of stress-related disorders. Soc. Neurosci. Abstr. 219.01 (Washington DC, 12–16 Nov 2011).

Acknowledgements Preparation of this Review was supported by grants from the US National Institute on Drug Abuse (NIDA).

Competing interests statement The authors declare no competing financial interests.

FURTHER INFORMATION Eric J. Nestler’s homepage: http://neuroscience.mssm.edu/ nestler The NIDA web site: http://www.nida.nih.gov ALL LINKS ARE ACTIVE IN THE ONLINE PDF

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REVIEWS

Common cellular and molecular mechanisms in obesity and drug addiction Paul J. Kenny

Abstract | The hedonic properties of food can stimulate feeding behaviour even when energy requirements have been met, contributing to weight gain and obesity. Similarly, the hedonic effects of drugs of abuse can motivate their excessive intake, culminating in addiction. Common brain substrates regulate the hedonic properties of palatable food and addictive drugs, and recent reports suggest that excessive consumption of food or drugs of abuse induces similar neuroadaptive responses in brain reward circuitries. Here, we review evidence suggesting that obesity and drug addiction may share common molecular, cellular and systems-level mechanisms. Hyperphagia Excessive consumption of food (above caloric requirements), which can reflect increased motivation to consume palatable food and/or deficits in brain circuitries that regulate satiety.

Laboratory of Behavioral and Molecular Neuroscience, Department of Molecular Therapeutics, and Department of Neuroscience, The Scripps Research Institute Florida, 130 Scripps Way, Jupiter, Florida 33458, USA. e-mail: pjkenny@scripps.edu doi:10.1038/nrn3105

One of the primary functions of the brain during periods of negative energy balance is to reprioritize behavioural output to procure and consume food, thereby replenishing energy stores that are depleted by caloric expenditure. Much is known about hypothalamic and hindbrain circuitries that control energy homeostasis and the hormonal regulators of hunger and satiety, such as leptin, ghrelin (also known as appetiteregulating hormone) and insulin, on these circuitries (FIG. 1). In addition to these homeostatic energy systems, reward systems also have key roles in regulating feeding behaviour. In particular, brain reward systems control learning about the hedonic properties of food, shifting attention and effort towards obtaining food rewards and regulating the incentive value of food or environmental stimuli that predict the availability of food rewards. Hormonal regulators of energy homeostasis can also act on brain reward circuits, most notably on the mesoaccumbens dopamine system1, to increase or decrease the incentive value of food depending on energy requirements. However, electrical or chemical stimulation of brain areas that regulate food reward can trigger binge-like overeating even in recently fed animals in which homeostatic satiety signals have been engaged2,3. This suggests that obtaining the pleasurable effects of food is a powerful motivating force that can override homeostatic satiety signals, and in agreement with this, meals that consist of palatable food are generally consumed with greater frequency and in greater portion size than those consisting of less palatable food4. As a single meal of increased portion size can trigger

increased food intake over several days5, such hedonic overeating is likely to be an important contributor to weight gain and the development of obesity. As common brain circuits regulate the hedonic properties of palatable food and drugs of abuse, and as there are striking phenomenological similarities between the overeating in obesity and excessive drug use in addiction, it is perhaps not surprising that these disorders have been proposed to share common underlying neurobiological mechanisms1. Nevertheless, it is important to point out that there is much ongoing debate about the idea that food can be ‘addictive’ in the same sense as drugs of abuse6,7. Here, we provide an overview of the brain systems that process information that is related to the hedonic properties and incentive value of palatable food, and discuss how addictive drugs can ‘hijack’ these systems. In addition, we highlight common cellular and molecular mechanisms in these circuitries that may contribute to both obesity and drug addiction.

Brain systems encoding food palatability Genetic factors play a major part in regulating vulnerability to obesity, and levels of adiposity have been shown to be a highly heritable trait (BOX 1). In many cases, genes that are associated with excessive body weight contribute to obesity by increasing preference for palatable food. It is well established that palatable food that is rich in fat and refined sugars can provoke hyperphagia. Palatable high-fat food promotes larger meal sizes, less postprandial satiety and greater caloric intake than diets that are high in carbohydrates but low

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F O C U S O N A DRDEI C ON V ITEI W S in fat 8. Hence, the perceived palatability of food contributes importantly to overconsumption and weight gain. The sensory characteristics of food, most notably its taste, smell, texture and appearance, have key roles in determining its palatability. The sensory information that is derived from the ingestion of palatable food is integrated in the primary and secondary gustatory cortices (FIG. 2). Chemosensory neurons in the oral cavity that are involved in tastant detection project to the nucleus tractus solitarius (NTS) in the brainstem9. The NTS in turn projects to the gustatory thalamus (ventroposteromedial (VPM) thalamic nucleus)10, which innervates the primary gustatory cortex (PGC) in the insula and operculum10. As the name implies, the PGC is critically involved in processing information related to the taste of food and its hedonic valuation11. Afferents from the PGC

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Figure 1 | Overview of homeostatic feeding circuits. a | Hormonal regulators of hunger, satiety and adiposity are released from the periphery. These include leptin and other adipokines, and also inflammatory cytokines, from adipose tissue. Insulin and pancreatic polypeptide (PP) are secreted from the pancreas. Furthermore, ghrelin (also known as appetite-regulating hormone), pancreatic peptide YY3-36 (PYY3-36), glucagon-like peptide 1 (GLP1, a cleavage product of glucagon) and cholecystokinin (CCK) are released from the gastrointestinal tract. These hormonal regulators of energy balance act on hindbrain and hypothalamic brain sites to influence hunger and satiety. b | Hormonal signals from the viscera that regulate energy balance, and vagal nerve input that is related to stomach distention after meal ingestion, alter neuronal activity in the nucleus tractus solitarius (NTS). The NTS relays information related to energy balance to homeostatic feeding circuits in the hypothalamus. c | In the arcuate nucleus in the mediobasal hypothalamus, so-called first-order neurons that contain agouti-related peptide (AgRP) and neuropeptide Y (NPY) are activated by orexigenic signals and inhibit the so-called second-order neurons that express melanocortin 4 receptor (MC4R), and this tonically inhibits feeding behaviour. Conversely, anorexigenic signals activate first-order neurons containing cocaine- and amphetamine-regulated transcript (CART) and proopiomelanocortin (POMC), which stimulates the release of α-melanocytestimulating hormone (αMSH), a cleavage product of POMC. This results in the activation MC4R neurons and inhibition of feeding behaviour.

project to a region of the the caudolateral orbitofrontal cortex (OFC) termed the secondary gustatory cortex (SGC). In addition to taste, other modalities of sensory input related to food palatability (for example, smell, sight and texture) also converge on the PGC and SGC10. The PGC and SGC project to the striatum, particularly the nucleus accumbens (NAc), thereby modifying neuronal activity in feeding-related striatohypothalamic and striatopallidal circuitries1. These striatal feeding circuits are in turn influenced by mesolimbic and nigrostriatal dopaminergic inputs1. It is well established that the striatum regulates consumption of both palatable food and drugs of abuse1,12. As described in detail below, recent evidence suggests that other components of the brain circuitry that are involved in processing food palatability — particularly the NTS, insula and OFC — also regulate the consumption of addictive drugs.

Nucleus tractus solitarius in food and drug reward Neurons that produce catecholamine neurotransmitters are a major class within the NTS that is involved in regulating feeding behaviour (FIG. 3). The NTS receives information from chemosensory neurons in the oral cavity that process the taste of food, and ascending projections transmit this information to thalamic brain sites. In addition, NTS catecholamine neurons are activated by afferents from the gastrointestinal tract that signal meal ingestion or gastric distension, and by circulating satiety signals such as cholecystokinin (CCK)13. The NTS relays this visceral information to homeostatic feeding centres in the hypothalamus. Intriguingly, rats or mice that are maintained on a high-fat diet or mice that are genetically prone to develop obesity show decreased responsiveness of NTS catecholamine neurons to lipid ingestion14,15. This suggests that the hyperphagia that is associated with consumption of palatable high-fat food may be related to adaptive responses in the NTS, resulting in decreased sensitivity to gut hormones that signal satiety. In addition to thalamic and hypothalamic feeding centres, NTS catecholaminergic neurons — specifically those in the A2 region of the NTS that produce noradrenaline — also project densely to limbic brain regions that are involved in stress and reward processing, including the shell region NAc, the central nucleus of the amygdala (CeA) and the bed nucleus of the stria terminalis (BNST)16 (FIG. 3). These same brain regions, collectively part of a larger contiguous cluster of functionally, structurally and chemically related brain structures termed the extended amygdala, have key roles in regulating the acute reinforcing properties of drugs of abuse and the development of drug dependence during chronic drug exposure17 (see BOX 2 for a discussion of the role of stress in obesity and addiction). Intriguingly, nicotine that is applied to the tongue of rats excites gustatory neurons in the NTS and simultaneously decreases their responsiveness to a broad range of tastants18. This suggests that the actions of nicotine and other drugs of abuse on peripheral sensory systems converge on NTS neurons19,20, or the direct actions of these drugs within the NTS, could contribute to their potential for abuse. Consistent with this possibility, the rewarding

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REVIEWS Protracted drug abstinence This is an aversive state that can persist in drug-dependent subjects long after cessation of drug use. Protracted abstinence is thought to increase vulnerability to relapse to drug-taking behaviour.

Reinforcer This is a stimulus (object or event) that is obtained or that occurs in response to a particular behaviour and that is associated with an increased probability that the behavioural response that resulted in delivery of the stimulus will occur again. In essence, a reinforcer is anything that increases the likelihood that a given behaviour will be repeated.

properties of morphine are completely ablated in dopamine β-hydroxylase (DBH) knockout mice, which cannot synthesize noradrenaline21. However, virus-mediated re-expression of DBH in the NTS of the knockout mice re-established their sensitivity to morphine reward21. In addition to drug reward, the NTS also plays an important part in the development of drug dependence and the aversive consequences of drug withdrawal. NTS activity is increased in rats undergoing opiate withdrawal, resulting in higher levels of noradrenaline transmission in the extended amygdala22, which contributes to the expression of aversive aspects of withdrawal22. Persistent activation of the NTS during periods of protracted drug abstinence in dependent rats also enhances sensitivity to the motivational properties of addictive drugs and increases vulnerability to stress-induced reinstatement of drug seeking behaviours (that is, relapse)16. The increased sensitivity to drug reward in rats undergoing periods of protracted abstinence is associated with decreased sensitivity to food reward23. As such, longterm alterations in NTS function may contribute to the enhanced motivational properties of addictive drugs and the diminished value of food and other natural reinforcers that are evident in drug-addicted individuals23. Insights are beginning to emerge into the molecular signalling events in the NTS that contribute to obesity and drug dependence. For example, the vagus nerve transmits information that is related to gastric distension to the NTS24, and vagal nerve activation suppresses food intake in rats25 and humans26. Human brain imaging studies have shown that an implantable device that triggers stomach expansion in response to vagal nerve stimulation increases metabolism in areas of the brain that are involved in food reward and palatability, including the OFC, striatum and hippocampus27. Intriguingly, bariatric surgery in overweight individuals can increase alcohol use28. These findings support the idea that the NTS influences activity in brain reward circuits and

Box 1 | Genetic and epigenetic factors that contribute to obesity Familial forms of obesity have been identified in which null mutations in single genes implicated in homeostatic regulation of energy balance, such as those encoding leptin or the melanocortin 4 receptor (MC4R), can profoundly increase adiposity independent of the type of diet that is consumed. In addition, genome-wide association studies have identified single nucleotide polymorphisms that increase vulnerability to obesity in a polygenic manner. Polymorphisms in genes that are involved in energy balance often increase adiposity independently of the type of diet available161. However, in many cases genetic loci that are associated with body weight encode transcripts that increase risk of obesity by increasing preference for palatable food. This highlights the importance of hedonic brain systems in influencing propensity to overeat. Epigenetic mechanisms may also influence preference for palatable food and weight gain162,163. For example, consumption of a palatable high-fat diet increases DNA and histone methylation and decreases histone acetylation status in the promoter region of the opioid receptor mu 1 (MOR1) gene, which correlates with decreased MOR expression. Worryingly, chromatin remodelling in response to nutritional status !"#$%&' or during early postnatal development can affect dietary preference and metabolism, and thereby influence vulnerability to obesity later in life. Moreover, epigenetic alterations in gene expression, including genes that are expressed in brain reward circuitries that regulate the motivation to consume palatable food or drugs of abuse, can be transmitted across generations of offspring, resulting in trans-generational vulnerability to obesity and obesity-related diseases162,164.

thereby regulates food and drug intake. In rats, repeated vagal nerve stimulation increases expression of the transcription factor ΔFOSB in NTS29. Similarly, the development of opiate dependence in rats is also associated with increased NTS expression of ΔFOSB30. ΔFOSB is a splice variant of the full-length FOSB gene product 31 and is known to accumulate in the striatum and other rewardrelated brain areas in rats and mice during chronic exposure to various classes of addictive drugs, and it persists long after drug exposure has ceased. Moreover, ΔFOSB increases the motivational properties of addictive drugs, probably by triggering structural and functional alterations in reward circuitries that increase their responsiveness to drugs and drug-associated stimuli32. Hence, it is possible that ΔFOSB signalling in the NTS could contribute to the development of obesity. In addition, ΔFOSB accumulation in the NTS could account for the simultaneous increase in sensitivity to drug reward and decrease in sensitivity to food reward, described above, in animals undergoing protracted abstinence from chronic drug exposure. Nucleus tractus solitarius neuropeptides in drug reward. In addition to catecholaminergic neurons in the NTS, separate neuronal populations produce neuropeptides such as proopiomelanocortin (POMC) or glucagon-like peptide 1 (GLP1, a cleavage product of glucagon). In a similar way to noradrenaline-containing neurons, NTS POMC neurons are activated by vagal afferents from the gastrointestinal tract and circulating satiety signals, and they contribute to limiting food intake33. Enhancing POMC transmission in the NTS can induce weight loss and protect against diet-induced obesity 34. Intriguingly, NTS infusion of opiates, which is known to increase food intake, inhibits POMC neurons33, suggesting that these cells may play a part in opiate reward and dependence. GLP1 is primarily synthesized by intestinal L cells, and it serves to lower blood glucose levels and stimulate insulin secretion35. GLP1 is also produced by a small number of neurons in the NTS that inhibit food intake36, particularly in response to gastric distention37, stress and illness38. Disruption of GLP1 production in the NTS or GLP1 receptor signalling in the brain results in hyperphagia in rats38, suggesting that overeating may induce deficits in central GLP1 receptor signalling that contribute to obesity. Activation of GLP1 receptors in the NTS probably decreases food intake through a mechanism involving protein kinase C (PKC)-mediated concurrent inhibition of AMP-activated protein kinase (AMPK) and stimulation of mitogen-activated protein kinase (MAPK) cascades39. So far, the roles of GLP1 receptors in the brain, and AMPK and MAPK in the NTS, in regulating drug reward and dependence have not been investigated.

Insular cortex in obesity and drug addiction The insula and operculum primarily encode and store information related to the valence (appetitive or noxious) and magnitude of the hedonic properties of palatable food1,10 (FIG. 2). In addition to its role in taste memory, the insula may also regulate the experience

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F O C U S O N A DRDEI C ON V ITEI W S

Figure 2 | The neurocircuitry controlling palatable food and drug consumption. The palatability of food is related to its touch and temperature, and is processed mainly by mechanoreceptors in the oral cavity that project to the gustatory thalamus. Texture also contributes to palatability, and may play an important part in detecting fat content in food. Taste plays a key part in food palatability, with chemoreceptors that detect tastants on the tongue projecting to the nucleus tractus solitarius (NTS). The smell of food is processed through the olfactory bulb (OB) and pyriform cortex. The appearance of palatable food is processed through the visual cortices (V1, V2 and V4) and then through the interior temporal visual cortex (ITVc). Information related to food palatability from these different modalities of sensory input converge on the amygdala, insular cortex and orbitofrontal cortex (OFC), and from there into feeding circuits in the striatum and lateral hypothalamus (LH). The sensory properties of drugs of abuse can activate the same brain systems as palatable food. Furthermore, drugs of abuse penetrate into the CNS and act directly in these brain systems. The sites of action of most major classes of addictive drugs on the neurocircuitory controlling food palatability are indicated (shown by dashed arrows). In addition, the NTS has a prominent role in regulating opiate reward and the development of dependence.

insular activation. Consistent with this interpretation, obese individuals show enhanced insular activation in response to palatable food46. Moreover, young adults who are at risk of developing obesity (both parents had a body mass index (BMI) score of â&#x2030;Ľ27) showed enhanced insula and operculum activation in response to monetary or food rewards compared with adolescents who have a low risk of developing obesity (both parents with a body mass index score of <25)47. This suggests that innately enhanced responsiveness of the insula, which may contribute to increased sensitivity to the taste of palatable food and a shift in dietary preference towards such food, increases vulnerability to obesity 1. In addition to its role in taste memory and food preference, the insula also plays a key part in drug addiction. Abstinence-induced cigarette craving in smokers is highly correlated with activation of the insular cortex 48. More notably, stroke-related damage to the insula in human smokers can result in a disruption of tobacco addiction, characterized by spontaneous cessation of the smoking habit and a low urge to smoke thereafter 49. In rats, chemical inactivation of the insula, or disruption of hypocretin receptor type 1 (also known as orexin receptor type 1) signalling in this structure, decreases intravenous nicotine self-administration behaviour 50 and amphetamine-seeking behaviour 51. Within insular neurons, cocaine treatment 52 or exposure to environmental cues that predict availability of palatable food53 increase expression of the immediate early gene and transcriptional regulator early growth response protein 1 (also known as transcription factor ZIF268), which plays a key part in neuronal plasticity and long-term memory formation. This suggests that palatable food and drugs of abuse can induce similar adaptive responses in the insular cortex. Mice that are permitted to consume highly palatable food show a marked increased in MAPK signalling in the insular cortex 54. Moreover, this increase in insular MAPK signalling, perhaps as a consequence of NMDA and metabotropic glutamate 5 receptor activation55, controls the induction of a long-term taste memory 56. Little is known about the effects of drugs of abuse on MAPK signalling in the insula and its involvement in drug-seeking behaviours.

of conscious urges and cravings40. Humans or rodents with access to palatable food show a marked decrease in consumption when less palatable food than anticipated is made available, a phenomenon termed negative contrast 41,42. This shift in preference towards the most hedonic food available, and the rejection of less palatable options, may play a key part in the development of obesity by contributing to persistent overconsumption of palatable energy-dense food41,42. Importantly, lesions to the insula abolish diet-associated negative contrast effects43. Similarly, a lesion to the gustatory thalamus, which is innervated by the NTS and in turn projects to the insula, also abolishes diet-associated negative contrast44. Obese human subjects show decreased functional connectivity strength in the insular cortex under resting conditions45, perhaps reflecting diminished control over

Orbitofrontal cortex in obesity and addiction In contrast to the insula, which encodes information related to the valence and magnitude of the hedonic properties of food, the OFC seems to continuously update information related to the relative motivational value of palatable food, based on information from metabolic or hedonic circuitries in the brain57. As such, the OFC probably plays a key part in the development of sensory-specific satiety during meals based on the diminished incentive value of any given food item, independent of changes in the perception of its palatability57. In a recent study, volunteers who were asked to imagine repeatedly eating a particular type of desirable food (chocolate or cheese) subsequently consumed far less of that food when it was actually available compared with the amounts eaten by individuals who imagined eating less of the food, those who envisioned eating a different

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REVIEWS type of palatable food or those who did not consider the food at all58. The decreased food consumption was not related to changes in subjective hedonic value, the participants simply desired it less (that is, they experienced sensory-specific satiety following imagined consumption)58. These findings show how readily the incentive value of food can be dissociated from its absolute hedonic properties58, and they show the importance of higher-order cortical brain centres that are involved in mental representations in attributing the relative motivational value of any given food item. Considering the key role of the OFC in attributing value to food59, these and related findings suggest that disruption of OFC function could result in inappropriate attribution of incentive value to food, resulting in weight gain60. Consistent with this possibility, obesity in humans is associated with marked deficits in OFC metabolism60. Furthermore, frontotemporal dementia resulting in atrophy of the OFC and insula triggers the emergence of binge-like overeating of palatable food in humans61. Recently, it was shown that activation of mu opioid receptors in the OFC induces hyperphagia in rats62. This suggests that local opioid receptor transmission in the OFC62, which could influence the activity of downstream feeding circuits in the striatum (see below), controls feeding behaviour. The OFC may also play a key part in attributing motivation value to cocaine and other drugs of abuse. !*+ 0$%1"'-(%)&

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Figure 3 | The nucleus tractus solitarius in food and drug consumption. The nucleus tractus solitarius (NTS) receives input from the gastrointestinal tract from the vagal nerve, and in turn projects to midbrain, thalamic, hypothalamic, limbic and cortical brain regions that are involved in processing food palatability, hedonic aspects of food and drugs of abuse, and the effects of stress on food and drug consumption. The NTS expresses different populations of neurons that are involved in regulating food and drug intake, including catecholaminergic neurons that express the enzyme tyrosine hydroxylase (TH+), those that express proopiomelanocortin (POMC) and those that express glucagon-like peptide 1 (GLP1, a cleavage product of glucagon). BNST, bed nucleus of the stria terminalis.

Chemical inactivation of the OFC rendered rats insensitive to alterations in the relative reinforcing value of different unit doses of cocaine that were available for intravenous self-administration63. Lesions of the OFC also block the ability of drug-paired environmental cues that predict palatable food or drug availability to drive seeking behaviours64,65, perhaps by disrupting the attribution of salience to the food- or drug-paired cues66. A history of intravenous cocaine self-administration behaviour in rats, or repeated exposure to amphetamine, induces structural and functional alterations in the OFC of rats that correlated with deficits in OFC-dependent cognitive performance67,68. Based on these and similar findings, it has been proposed that drug-induced remodelling of the OFC may contribute to the transition from controlled to uncontrolled drug use in addiction 67,69. The underlying molecular mechanisms that contribute to OFC dysfunction are beginning to emerge. In rats, volitional consumption of cocaine or alcohol increases the expression of the transcription factor ΔFOSB in the OFC70. This increase in ΔFOSB expression in OFC exacerbates the increase in impulsive-like behaviour that is observed during withdrawal from chronic cocaine selfadministration71. As increases in impulsive choice are thought to increase vulnerability to addiction, druginduced increases in ΔFOSB in the OFC may drive the development of addiction. It will therefore be important to determine whether overconsumption of palatable food similarly increases ΔFOSB expression in the OFC, and whether this influences vulnerability to obesity.

Mesostriatal system in obesity and addiction Information relating to the sensory properties of palatable food, which is processed in the OFC and other cortical structures, is transmitted to feeding-related circuits in the striatum, particularly to so-called ‘hedonic hot spots’ in the shell region of the NAc. Hedonic hot spots in accumbens project to, and control the activity of, lateral hypothalamic and pallidal brain sites. These striatohypothalamic and striatopallidal systems, which are regulated locally by opioid and endocannabinoid signalling and also by dopamine transmission arising from mesoaccumbens and nigrostriatal input, control responsiveness to environmental stimuli that predict food availability and palatability, approach behaviours and attribution of incentive value to palatable food1. In addition to the sensory properties of palatable food, the striatum also plays an important part in responding to the post-ingestive effects of food metabolism72. Specifically, the release of macronutrients from energydense food can activate metabolic signalling pathways in the viscera and thereby stimulate dopamine inputs onto feeding circuits in the striatum, independently of the sensory properties of the food73,74. The functional transient receptor potential channel subfamily M member 5 (TRPM5) is necessary for detecting sweet, bitter and amino acid (umami) tastants 75. Taste-blind Trpm5 knockout mice do not show a preference for sucrose over water when presented briefly with a choice between both solutions73,74, confirming their inability to detect sweettasting solutions. However, when the Trpm5 knockout

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F O C U S O N A DRDEI C ON V ITEI W S Box 2 | The role of stress in obesity and addiction Stress triggers intense bouts of feeding, particularly of palatable food, in rodents, monkeys and humans, with palatable food consumption thought to attenuate the aversive effects of stress84,165. Obesity is associated with elevated stress-related glucocorticoid secretion, suggesting that stress and obesity are closely intertwined. Indeed, ‘withdrawal’ from the palatable diet increases expression of stress hormone corticotropin-releasing factor (CRF) in the central nucleus of the amygdala of rats and mice, which may drive the emergence of compulsive-like eating in rodents84,166. Amygdalar CRF levels are also increased in rats during withdrawal from all major drugs of abuse, an effect that has been suggested to drive compulsive drug seeking167. Similar to obesity, hunger is a stressor in humans, monkeys and rodents, with food restriction increasing the subjective motivation to eat in response to stress in humans168. Furthermore, rats undergoing cyclic periods of caloric restriction and re-feeding, which sensitizes rats to stress-induced overeating, demonstrate compulsive-like consumption of palatable food169,170. Hence, increased activity of stress pathways in response to overeating and weight gain on the one hand, or food restriction and hunger on the other, may contribute to the development of overeating and weight gain through similar stress-related mechanisms that drive the development of drug addiction.

Direct pathway The direct striatal pathway comprises medium spiny neurons (MSNs) that express dopamine D1 receptors and project directly to the globus pallidus interna (GPi). The indirect pathway comprises MSNs that express dopamine D2 receptors and project to the GPi indirectly through a pathway involving the globus pallidus externa (GPe) and the subthalamic nucleus.

Fixed and progressive ratio schedules A fixed ratio schedule of reinforcement requires an animal to emit a fixed number of responses to earn a reinforcer. A progressive ratio schedule involves the animal emitting progressively greater numbers of responses to earn each subsequent reinforcer.

mice were repeatedly allowed longer access to water or sucrose dilutions at discrete locations in the testing environment, and therefore able to associate postingestive effects of water or sucrose with their consummatory behaviour, they showed a clear preference for the sucrose solutions. Importantly, the Trpm5 knockout mice did not develop a preference for the non-caloric sweetener sucralose under the same test conditions, demonstrating that the post-ingestive caloric effects of sucrose were responsible for the increased preference for sucrose in the knockout mice73,74. Sucrose increased dopamine levels in the NAc and dorsal striatum of the Trpm5 mice73,74, suggesting that non-gustatory metabolic signals in the knockout mice were sufficient to stimulate midbrain dopamine neurons that drive preference for calorically dense solutions. Intriguingly, Trpm5 channels on the tongue also regulate taste responses to nicotine and alcohol, and contribute to their volitional consumption76,77. This suggests that, in addition to their direct actions in the brain, sensory information that is related to inhaled or orally consumed drugs of abuse contributes to their intake. Signalling events downstream of dopamine receptors. Palatable food or drugs of abuse, and environmental cues that predict their delivery, increase dopamine transmission in the striatum, thereby influencing striatohypothalamic and striatopallidal circuitries that control the hedonic and incentive properties of food and abused drugs1. The roles of striatal dopamine transmission in obesity, including the contributions of constitutive and diet-induced alterations in dopamine receptor function, has been reviewed in detail elsewhere1,12,78. Here, the focus will be on emerging evidence suggesting that drugs of abuse and palatable food converge on common intracellular signalling cascades in the striatum and in midbrain dopamine neurons that project to the striatum, which contribute to drug addiction and obesity (FIG. 4). Cocaine and other drugs of abuse increase the expression of ΔFOSB throughout the striatum, particularly in

the D1 dopamine receptor and dynorphin-expressing medium spiny neurons of the direct pathway79. Moreover, gradual accumulation of ΔFOSB in the striatum in response to drug consumption increases their motivation properties, which is thought to contribute to the development of drug addiction80. Interestingly, mice that were exposed to a high-fat diet during early postnatal development (postnatal days 21–28) for 1 week had increased preference for dietary fat intake in adulthood81, and this increased preference for calorically dense food was associated with alterations in intracellular molecular transducers of dopamine receptor signalling 81. In particular, ΔFOSB levels were increased in the NAc of these mice81. Similarly, increased ΔFOSB expression in the striatum was detected in adult mice that were permitted to eat palatable high-fat or sucrose diets82–84, and this effect was associated with enhanced motivation to consume palatable diets. Furthermore, mice with restricted access to food, and that were therefore hungry and highly motivated to consume food, also showed increased striatal ΔFOSB expression85. Transgenic overexpression of ΔFOSB in the striatum, specifically in neurons of the direct pathway, resulted in greater responses for food rewards under fixed and progressive ratio schedules of reinforcement, suggesting that ΔFOSB increases the motivational properties of food86. These findings are strikingly similar to the enhanced responses to cocaine under fixed and progressive ratio reinforcement schedules that are induced by striatal overexpression of ΔFOSB87. Consumption of a palatable high-fat diet can normalize many of the deficits in dopamine receptor-associated signalling cascades in the striatum of ΔFOSB-overexpressing mice88. These deficits include decreases in the transcription factor cyclic AMP-responsive element binding protein (CREB), protein phosphatase 1 regulatory subunit 1B (DARPP32) and brain-derived neurotrophic factor (BDNF)88. In addition, markers of dopamine production and release, particularly tyrosine hydroxylase, the ratelimiting enzyme in the production of dopamine, and the dopamine transporter protein (DAT) were decreased in the ventral tegmental area (VTA)–striatum axis of the ΔFOSB-overexpressing mice88, suggesting that ΔFOSBoverexpressing mice have decreased dopamine production in midbrain systems and decreased dopamine release into the striatum. Evidence of disrupted striatal dopamine transmission in ΔFOSB-overexpressing mice was ameliorated by access to a high-fat diet for 6 weeks88. This suggests that the palatable food may have increased motivational value in these mice because it can normalize deficits in dopamine signalling. Taken together, these data strongly suggest that striatal ΔFOSB signalling controls the motivational properties of food and drugs of abuse. It is important to note, however, that weight gain is similar in wild-type and ΔFOSB-overexpressing mice with access to standard chow or a high-fat diet 88. It is therefore an intriguing possibility that caloric usage or other aspects of metabolism may be increased in ΔFOSB-overexpressing mice to compensate for their increased motivation to seek food, a possibility that has not yet been tested.

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Figure 4 | Intracellular signalling cascades in the striatum and mesoaccumbens dopamine pathway that regulate food intake and drug use. The receptors for leptin, insulin and brain-derived neurotrophic factor (TRKB) are expressed on ventral tegmental area (VTA) dopamine neurons, where they regulate the phosphinositide 3-kinase (PI3K)–serine/threonine kinase AKT–mammalian target of rapamycin (mTOR) signalling cascade. Leptin can also regulate the JAK–STAT (Janus kinase–signal transducer and activator of transcription) signalling pathway. Leptin, insulin and BDNF signalling are necessary to maintain dopamine homeostasis, probably through actions involving the PI3K signalling cascade. Drugs of abuse like cocaine can also potentiate PI3K–AKT–mTOR signalling in midbrain dopamine neurons. Insulin receptors are also probably expressed presynaptically on dopamine terminals in the nucleus accumbens, and postsynaptically on medium spiny neurons, that express either dopamine D1 or D2 receptors, the so-called direct and indirect pathway neurons, respectively. Insulin receptors in the accumbens promote dopamine release and enhance the activity of the dopamine transporter (DAT), and thereby play an important part in accumbal dopamine homeostasis. This action probably contributes to the satiety-related actions of insulin and its ability to decrease palatable food intake. Conversely, all major drugs of abuse stimulate dopamine release into the accumbens, an action that is considered critical to their motivational properties. Dopamine signalling in the accumbens modulates the activity of ΔFOSB, cyclic AMP-responsive element binding protein (CREB), protein phosphatase 1 regulatory subunit 1B (DARPP32) and cyclin-dependent kinase 5 (CDK5) signalling pathways in medium spiny neurons, and thereby influences the motivational properties of food and addictive drugs. Neuropeptides that are produced in the lateral hypothalamus (LH) can also modulate the activity of VTA dopamine and striatal neurons. LH neurons that produce hypocretin (also known as orexin), project to the VTA and regulate VTA dopamine neurons and their responsiveness to palatable food and addictive drugs. LH neurons that produce melaninconcentrating hormone (MCH) project to the accumbens and control the motivational properties of food and addictive drugs, and also the responsiveness of medium spiny neurons, through MCH receptors expressed in this area. The main sites of action of most major classes of addictive drugs are indicated (shown by red boxes). IRS, insulin receptor substrate; HCRTR1, hypocretin receptor type 1; S6K, ribosomal protein S6 kinase β1.

Other components of dopamine receptor signalling in the striatum also regulate the motivational properties of both drugs of abuse and food. For example, expression of cyclin-dependent kinase 5 (CDK5) in the striatum is regulated by ΔFOSB and cocaine89,90. Pharmacological or genetic disruption of CDK5 signalling in striatum increases cocaine reward in mice91,92. This suggests that drug-induced increases in CDK5 expression in striatum may be an adaptive response in brain reward circuits to counter the effects of cocaine and thereby protect against addiction93. Disruption of CDK5 signalling in the brain also increases the incentive motivational properties of food92, suggesting again that common biochemical mechanisms in the striatum regulate the motivational properties of addictive drugs and food. Lastly, activation of D1 dopamine receptor signalling in the striatum

is known to cause the dephosphorylation of DARPP32 at serine residue 97. Replacement of serine 97 with an alanine reside, thereby preventing the phosphorylation-mediated regulation of DARPP32 through this site, results in profound decreases in sensitivity to the motivational properties of cocaine and food rewards94. Taken together, these observations provide compelling evidence that similar dopamine-activated signalling cascades in the striatum control the motivational properties of drugs of abuse and food, and that disruption of these cascades may contribute to the development of obesity or addiction.

Neuropeptide and hormonal signalling In addition to downstream signalling events that are related to dopamine receptor activation, palatable food

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F O C U S O N A DRDEI C ON V ITEI W S and drugs of abuse can trigger neuroplasticity in striatal feeding circuits through hormonal and neuropeptide regulators of energy balance. Two major neuropeptides that are produced in the lateral hypothalamus and that are known to modulate striatal feeding circuits and dopamine input to these pathways, are melanin-concentrating hormone (MCH) and hypocretin (also known as orexin). MCH and hypocretin are produced in the lateral hypothalamus95 — a brain region that is involved in regulating both feeding behaviour and reward processing — and increases in MCH or hypocretin signalling stimulate feeding behaviour 96,97. Interestingly, genetic ablation of hypocretin neurons in the lateral hypothalamus leads to overeating, weight gain and obesity in mice98, suggesting that hypocretin transmission plays a complex part in regulating food intake and weight gain. MCH receptors are expressed in the NAc, with activation of these receptors stimulating feeding behaviour 99 and inhibiting NAc neuronal firing 100. These effects are likely to involve a decrease in adenylyl cyclase activity, and the consequent reductions in CREB activity, and reduced surface expression of the AMPA glutamate receptor subunit 1 (GluR1)100. Disruption of MCH receptor signalling in the NAc blocks the stimulant and conditioned reward effects of cocaine in mice101. Furthermore, ablation of MCH receptor signalling in the NAc also decreases intravenous cocaine self-administration and blocks relapse-like behaviour101. Hypocretin-containing neurons project from the lateral hypothalamus to the VTA, where hypocretin receptor type 1 (HCRTR1; also known as orexin receptor type 1) plays a key part in regulating mesolimbic dopamine transmission and the rewarding properties of various drugs of abuse and food, probably through regulation of PKC-dependent signalling cascades102–104. In summary, feeding-related neuropeptides, like MCH and hypocretin, have key roles in controlling food intake and drug use through modification of reward system activity, and probably contribute to the development of obesity and addiction.

Anorexigenic A stimulus (object or event) that decreases appetite and food consumption.

Leptin signalling in the ventral tegmental area. In addition to hypothalamic neuropeptides, hormonal regulators of appetite that are produced in the viscera can modulate brain reward function. For example, ghrelin, which is produced in the stomach and pancreas, can increase appetite and food intake. Ghrelin acts partly by stimulating midbrain dopamine transmission and thereby increasing motivation for food or drugs of abuse105. Another major hormonal regulator of energy balance that modulates brain reward activity is leptin. Congenital leptin deficiency results in increased striatal activation in response to images of food106, and leptin replacement therapy attenuates striatal activation of selfreported liking of food in these individuals106. Leptin can modulate striatal responses to food by controlling mesolimbic dopamine pathways. Leptin receptors are expressed on midbrain dopamine neurons107–109, and leptin infusion into the VTA inhibits the activity of dopamine neurons109, decreases food intake109–111 and induces generalized decreases in sensitivity to reward in rats111. Conversely, knockdown of leptin receptors in the VTA

in rats increases preference for palatable food109 and enhances the motivational properties of food112. In hypothalamic circuitries, the JAK–STAT (Janus kinase–signal transducer and activator of transcription) cascade is a major pathway through which leptin signals its anorexigenic effects113. Infusion of leptin into the VTA, at doses that decrease feeding behaviour, activates the JAK–STAT cascade109,110, and inhibition of JAK–STAT signalling in the VTA attenuates the anorexigenic effects of leptin110. Chronic cocaine treatment has been shown potentiate JAK–STAT signalling in the VTA114. It has therefore been proposed that cocaine-induced amplification of JAK–STAT signalling in the VTA may contribute to the long-lasting adaptations in brain reward circuitries that underlie cocaine addiction. In addition, by acting in a leptin-like manner, it is possible that cocaine-induced increases in JAK–STAT signalling in the VTA may contribute to the anorexigenic properties of the drug. Insulin signalling in the ventral tegmental area. Insulin is another hormonal regulator of energy balance that can influence food intake by modulating striatal feeding circuits and midbrain dopamine input onto these circuits. Insulin activates the insulin receptor and a signalling cascade that involves insulin receptor substrate (IRS)-mediated activation of phosphoinositide 3-kinase (PI3K). PI3K subsequently activates tyrosine-protein kinase BTK (also known as ATK), which then activates mammalian target of rapamycin (mTOR) and its downstream effector ribosomal protein S6 kinase β1 (S6K1). Insulin receptors are expressed in the striatum115 and on midbrain dopamine neurons107. Infusion of insulin into the VTA decreases food intake in rats111,116, and conversely, selective deletion of insulin receptors in midbrain dopamine neurons in mice results in hyperphagia and increased weight gain compared with control mice117. These effects are related to a loss of insulinstimulated PI3K signalling in dopamine neurons 117. Diabetic rats have greatly diminished levels of dopamine in midbrain and striatal brain sites and are less sensitive to the rewarding properties of methamphetamine than control rats with physiological levels of insulin118,119, demonstrating that insulin signalling is necessary to maintain dopamine transmission. These data suggest that acute activation of insulin receptors in the VTA can decrease the activity of dopamine-containing neurons in this brain site. However, insulin seems to act in a neurotrophic manner in the VTA as disruption of insulin signalling results in deficits in dopamine transmission. Disruption of BDNF expression throughout the forebrain, or specifically in the VTA, results in hyperphagia and weight gain in mice, particularly when permitted access to a palatable high-fat diet 120, similar to the effects of knocking out insulin receptors in the VTA. Moreover, central depletion of BDNF is associated with a profound deficit in dopamine signalling in the NAc, suggesting that, like insulin, BDNF is essential to maintain appropriate levels of mesolimbic dopamine signalling 120. Intriguingly, in addition to the acute inhibitory effects of leptin on VTA dopaminecontaining neurons and the feeding behaviour that

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REVIEWS are described above109,121, hyperphagic ob/ob mice, in which leptin signalling is disrupted, have lower levels of tyrosine hydroxylase in midbrain dopamine neurons, a key enzyme in the biosynthesis of dopamine108. ob/ob mice also have reduced evoked dopamine release into the NAc108 and decreased somatodendritic vesicular stores of dopamine in the VTA122. These deficiencies in dopamine signalling are normalized by treatment with exogenous leptin108. Together, these findings suggest that insulin, BDNF and leptin, which can all signal through the PI3K–serine/threonine kinase AKT–mTOR cascade, are necessary for appropriate dopamine production and signal transmission. Deficits in their actions disrupt the mesoaccumbens dopamine system and increase the animal’s propensity to over-consume palatable highfat food and develop obesity. In contrast to the motivational properties of palatable food and weight gain in mice with disrupted insulin, BDNF or leptin signalling in the VTA, these mice show diminished sensitivity to the motivational and psychomotor stimulant effects of cocaine and amphetamine108,117. Furthermore, disruption of the PI3K–AKT–mTOR signalling cascade in the VTA, achieved through virus-mediated expression of a dominant negative insulin receptor substrate 2 (IRS2) protein, attenuates the rewarding properties of cocaine and morphine in mice123,124. Thus, it is possible that disruption of insulin, BDNF and leptin signalling in the VTA not only increases propensity to become obese, which may reflect hedonic overeating to overcome a negative affective state associated with disrupted midbrain dopamine signalling 1, but also decreases sensitivity to the rewarding properties of addictive drugs like cocaine or morphine. Insulin signalling in the striatum. Insulin increases DAT expression and function in the striatum through the canonical IRS–PI3K pathway 125. Moreover, insulin potentiates the inhibitory effects of cocaine on dopamine release from striatal slices, an effect that is blocked by inhibition of PI3K125. Intriguingly, direct infusion of insulin into the NAc exacerbates the emergence of impulsive-like behaviour in rats that are treated with cocaine125, as measured in a five-choice serial reaction time task. High levels of impulsivity in this task are known to predict vulnerability to develop compulsive-like cocaine seeking behaviours in rats126, and humans with constitutively high levels of impulsivity are at increased risk of developing drug addiction or obesity 127. Hence, insulin signalling locally in the striatum may influence vulnerability to addiction through the IRS–PI3K–AKT–mTOR cascade. The idea that the PI3K-AKT-mTOR cascade has a role in addiction is also supported by the finding that pharmacological inhibition of mTOR signalling using rapamycin, particularly in the NAc, decreases the motivational properties of cocaine in rats and mice128. Lastly, the PI3K–AKT–mTOR pathway is known to play an important part in long-term depression (LTD)129, the process by which synaptic strength between neurons is enduringly decreased. Striatal LTD also depends on endocannabinoid and metabotropic glutamate receptor signalling and the transient receptor potential cation channel subfamily V member 1

(TRPV1) channel, all of which are known to regulate the rewarding properties of addictive drugs and the motivation to consume palatable food. Intriguingly, withdrawal from cocaine self-administration can induce deficits in the induction of LTD in the striatum130 and concomitant decreases in striatal expression of core components of the PI3K–AKT–mTOR signalling cascade131. This deficit in LTD gradually recovers during extended periods of abstinence from cocaine self-administration behaviour in rats130. However, failure to recover striatal LTD after a period of extended access to cocaine is associated with the emergence of addiction-like behaviours130. Finally, so-called western diets, which are rich in refined sugars and fat, are deficient in omega 3 fatty acids, and as a result obese individuals are very often deficient in this essential nutrient 132. Omega 3 deficiency in mice induces a striking deficit in LTD in the striatum132, suggesting that striatal LTD deficits that result from dietary deficiencies may contribute to the development of drug addiction and obesity.

Inflammation in obesity and drug addiction Emerging evidence suggests that induction of PI3K– AKT–mTOR-dependent LTD in brain is critically dependent on caspase 3, a signalling molecule that is involved in inflammation and apoptosis. Specifically, activation of NMDA receptors in response to synaptic activity increases intracellular calcium levels, which activates the calcium-dependent phosphatase calcineurin133. This in turn increases the release of cytochrome c from mitochondria through a mechanism that is dependent on the pro-apoptotic factors BCL-XL (BCL2 antagonist of cell death), XIAP (baculoviral IAP repeat-containing protein 4) and the apoptosis regulator BAX133,134. Cytochrome c in turn activates caspase 3, which then regulates the surface expression of AMPA receptor subunits and induces LTD through the AKT pathway 133,134. Importantly, caspase 3 plays a key part in inflammatory signalling in the brain, including striatal and midbrain dopamine sites135,136, suggesting that inflammatory pathways in the brain could also contribute to drug addiction and obesity. Nuclear factor-κB signalling in obesity and addiction. Initiation of inflammatory signalling cascades triggers activation of nuclear factor-κB (NF-κB), a transcription factor that increases the transcription of proinflammatory cytokines and other genes that are involved in cellular responses to damage, infection and stress (FIG. 5). Adipocytes produce a host of inflammatory cytokines, and obesity is generally associated with a chronic state of inflammation in peripheral tissues137. Inflammation in brain sites that are involved in regulating food intake may play a key part in the development of obesity. In mice that are permitted to consume a high-fat diet and in overweight ob/ob mice, inhibitor of NF-κB kinase subunit-β (IKKB)–NF-κB signalling is abnormally elevated in neurons of the mediobasal hypothalamus (MBH)138. Moreover, genetic disruption of IKKB–NF-κB signalling in the MBH, and specifically in agouti-related peptide (AgRP) neurons in this site (FIG. 1), protects mice

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Figure 5 | Nuclear factor-κB signalling and its regulation by SIRT1. Immune, inflammatory and stress signals in the striatum converge on the inhibitor of Nuclear factor-κB (NF-κB) kinase subunit-β (IKKB). Neuronal activity that is triggered in response to cocaine, neurotrophins or glutamate transmission also activates IKKB. IKKB then phosphorylates IκB. IκB is the major inhibitory factor that retains NF-κB (usually a dimeric complex comprising the p65 and p50 subunits) in the cytoplasm and prevents its activation and translocation to the nucleus. Phosphorylation of IκB by IKKB leads to IκB ubiquitylation and proteolysis, rendering NF-κB free to translocate to the nucleus. IκB can also be phosphorylated by other kinases that are implicated in synaptic plasticity, drug addiction and feeding behaviour, including RAF proto-oncogene serine/threonine protein kinase (RAF1), protein kinase A (PKA), casein kinase 2 (CK2), protein kinase C (PKC) and !" #$%& !"%'($"#)*(+,+)(+)-.,/'-+#).0#)!1+.-2,+344.56!78449:.4).-;+.)$ "+$1<.! -#=!-+(. NF-κB binds to response elements in the promoters of NF-κB-responsive genes such as histone deacetylases (HDACs), CREB-binding protein (CBP) and p300. Peroxisome proliferator-activated receptor-γ (PPARγ) has anti-inflammatory effects through an inhibitory action on NF-κB activity, probably by sequestering key transcriptional co-activators like p300 and CBP. Similarly, NAD-dependent deacetylase sirtuin 1 (SIRT1) has anti-inflammatory actions through its ability to deacetylate the p65 subunit of NF-κB and inhibit its activity. Ac, acetyl; NEMO, NF-κB essential modulator; Ub, ubiquitin.

from obesity when permitted to eat a high-fat diet 138, whereas ectopic activation of IKKB–NF-κB signalling in MBH triggers central insulin and leptin resistance (key physiological features of obesity)138. Brain-specific deletion of MYD88, an important adaptor protein through which toll-like receptors (core components of the innate immune system) activate NF-κB signalling, also protects mice from weight gain and developing leptin resistance when consuming a high-fat diet 139, further supporting a role for inflammatory signalling in the brain in obesity. In addition to overeating, enhanced NF-κB signalling in the hypothalamus, particularly within POMC neurons in the MBH, can trigger other obesity-associated disorders such as hypertension140. Obesity was also associated with inflammation in extrahypothalamic brain sites that are involved in hedonic aspects of feeding behaviour.

Using MRI, obese human subjects were shown to have chronic inflammation of the OFC, an important brain site that is involved in the attribution of incentive value to palatable food (see above)141. Based on this finding, it was proposed that inflammation in cortical brain sites, and perhaps also in limbic, striatal and midbrain sites that are involved in regulating palatable food consumption, may contribute to the development of obesity. Cocaine and other drugs of abuse can also trigger inflammatory responses in brain. In mice, cocaine activates NF-κB signalling in the NAc142,143, leading to an increase in BDNF levels and enhanced sensitivity to cocaine reward142. Cocaine-induced NF-κB signalling also caused structural remodelling in the NAc, resulting in an increased number of dendritic spines on NAc neurons142, which may be an adaptive response that increases vulnerability to addiction142. In addition to cocaine, consumption of alcohol also activates NF-κB signalling in brain, and it has been suggested that this contributes to the development of alcoholism144. SIRT1 in obesity and addiction. Given the importance of NF-κB signalling in weight gain and drug reward, it is perhaps not surprising that proteins that regulate NF-κB signalling — such as the NAD-dependent deacetylase sirtuin 1 (SIRT1) — are also implicated in obesity and drug addiction. SIRT1 has anti-inflammatory actions, primarily through deacetylating and inhibiting the p65 NF-κB subunit 145. Genetic variation in the SIRT1 gene is associated with lower BMI scores in humans145, and genetic ablation of SIRT1 in hypothalamic POMC neurons increases the vulnerably of mice to diet-induced obesity by decreasing energy expenditure146. Cocaine increases expression of SIRT1 in the striatum147 and resveratrol-induced activation of SIRT1 activity enhances the motivational properties of cocaine147. These findings suggest that SIRT1 in hypothalamus and striatum regulates food and drug intake, respectively. It will be interesting to determine whether these actions are related to NF-κB signalling, and whether SIRT1 activity in the striatum also regulates the hedonic properties of palatable food.

New vistas in obesity and addiction research Tantalizing new observations are revealing glimpses of new systems and biological processes that may also be involved in obesity and addiction. For example, circadian rhythms may influence the sensitivity of brain reward circuitries and thereby regulate feeding behaviour and drug use. The transcription factors CLOCK and BMAL1 are core components of circadian master clock, which is located in the suprachiasmatic nucleus (SCN) of the hypothalamus. CLOCK mutant mice are obese148, are more sensitive to cocaine reward than wild-type mice and show enhanced excitability of midbrain dopamine neurons149. It will therefore be interesting to determine how CLOCK–BMAL-regulated genes influence food and drug intake. RNA editing is a post-transcriptional process by which adenosine residues are edited to inosine in the sequence of mature mRNA transcripts, which

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REVIEWS can result in alterations in the amino-acid code of the translated protein150. RNA editing is catalysed by double-stranded RNA-specific adenosine deaminases (ADARs), and perhaps the best-known mRNA transcript that is subjected to RNA editing in the brain is the serotonin 2C (5-HT2C) receptor 151. Disruption of ADAR2 activity in mice (ADAR2 is known to edit AMPA and kainate glutamate receptor subunits) results in hyperphagia and obesity in mice. Furthermore, the small nucleolar RNA HBII 52 controls editing of 5HT2C receptors152, and chromosomal microdeletions of HBII 85 contribute to the features of the neurodevelopmental disorder Prader–Willi syndrome153, a major symptom of which is obesity. MicroRNAs are also involved in post-transcriptional regulation of gene expression and a key role for microRNAs in regulating the motivational properties of cocaine in rats and mice is emerging 154. They have also been heavily implicated in adipogenesis, glucose metabolism and insulin signalling. However, very little is known of the role in feeding behaviour. Agonists of peroxisome proliferator-activated receptor-γ (PPARγ), such as rosiglitazone (Avandia; GlaxoSmithKline plc), are used as insulin-sensitizing agents to treat type 2 diabetes. PPARγ also regulates adipogenesis and one of the major side-effects of PPARγ agonists is weight gain, particularly by targeting PPARγ that is expressed in brain155,156. PPARγ interacts with known regulators of drug intake, including NF-κB (FIG.  5), SIRT1 and CDK5, and PPARγ agonists decrease alcohol consumption and attenuate relapse-like behaviour 157. Hence, it will be important to understand the precise mechanisms through which PPARγ and other nuclear hormone receptors regulate food and drug consumption, and to determine whether they act on the same signalling pathways.

1.

Kenny, P. J. Reward mechanisms in obesity: new insights and future directions. Neuron 69, 664–679 (2011). 2. Wyrwicka, W., Dobrzecka, C. & Tarnecki, R. On the instrumental conditioned reaction evoked by electrical stimulation of the hypothalamus. Science 130, 336–337 (1959). 3. Will, M. J., Pratt, W. E. & Kelley, A. E. Pharmacological characterization of high-fat feeding induced by opioid stimulation of the ventral striatum. Physiol. Behav. 89, 226–234 (2006). 4. McCrory, M. A., Suen, V. M. & Roberts, S. B. Biobehavioral influences on energy intake and adult weight gain. J. Nutr. 132, 3830S–3834S (2002). 5. Kelly, M. T. et al. Increased portion size leads to a sustained increase in energy intake over 4 d in normalweight and overweight men and women. Br. J. Nutr. 102, 470–477 (2009). 6. Benton, D. The plausibility of sugar addiction and its role in obesity and eating disorders. Clin. Nutr. 29, 288–303 (2010). 7. Corsica, J. A. & Pelchat, M. L. Food addiction: true or false? Curr. Opin. Gastroenterol. 26, 165–169 (2010). 8. Warwick, Z. S. Probing the causes of high-fat diet hyperphagia: a mechanistic and behavioral dissection. Neurosci. Biobehav. Rev. 20, 155–161 (1996). 9. Schwartz, G. J. The role of gastrointestinal vagal afferents in the control of food intake: current prospects. Nutrition 16, 866–873 (2000). 10. Rolls, E. T. Brain mechanisms underlying flavour and appetite. Phil. Trans. R Soc. Lond. Series B 361, 1123–1136 (2006).

11.

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Lastly, drugs of abuse decrease neurogenesis, the process by which new neurons are born and mature, in the brains of adult rodents158. Similarly, apoptosis of newly born neurons in the olfactory bulb, a process that may regulate odour-related memory, is increased in mice during the post-prandial period159. This suggests that neurogenesis in the olfactory bulb and perhaps other regions of the brain may contribute to aspects of feeding behaviour and drug use. Hence, it will be important to investigate the contributions of emerging mechanisms of neuroplasticity and gene regulation in the brain to the hedonic aspects of feeding behaviour and the rewarding properties of addictive drugs.

Summary As discussed in this Review, many of the same brain systems regulate food intake and drug use, and similar adaptive responses can be triggered in brain reward systems by drugs of abuse and palatable food. As a result, obesity is now often conceptualized as a form of compulsive consummatory behaviour much like drug addiction. Thus, our understanding of the neurobiological mechanisms of drug addiction may provide a heuristic framework for deciphering the motivational drivers in obesity. Lastly, much emphasis is now being placed on defining the effects of palatable food on brain reward circuits that are implicated in drug addiction. However, it is also worth considering the reverse relationship that exists between the homeostatic feeding circuits in the hypothalamus and the brainstem in regulating consumption of addictive drugs. Nicotine and other drugs of abuse can stimulate hypothalamic feeding circuits and thereby influence weight gain160. It is an intriguing possibility that these hypothalamic feeding circuits may also regulate drug reward and contribute to the loss of control over drug use that characterizes addiction.

An excellent overview of the neurocircuitries that regulate the perception of food palatability. Small, D. M., Zatorre, R. J., Dagher, A., Evans, A. C. & Jones-Gotman, M. Changes in brain activity related to eating chocolate: from pleasure to aversion. Brain 124, 1720–1733 (2001). An important paper that identifies brain systems that are involved in the development of satiety and sites that are recruited to limit further consumption. Volkow, N. D., Wang, G. J. & Baler, R. D. Reward, dopamine and the control of food intake: implications for obesity. Trends Cogn. Sci. 15, 37–46 (2011). Appleyard, S. M. et al. Visceral afferents directly activate catecholamine neurons in the solitary tract nucleus. J. Neurosci. 27, 13292–13302 (2007). Covasa, M. & Ritter, R. C. Reduced sensitivity to the satiation effect of intestinal oleate in rats adapted to high-fat diet. Am. J. Physiol. 277, R279–R285 (1999). Donovan, M. J., Paulino, G. & Raybould, H. E. Activation of hindbrain neurons in response to gastrointestinal lipid is attenuated by high fat, high energy diets in mice prone to diet-induced obesity. Brain Res. 1248, 136–140 (2009). Smith, R. J. & Aston-Jones, G. Noradrenergic transmission in the extended amygdala: role in increased drug-seeking and relapse during protracted drug abstinence. Brain Struct. Funct. 213, 43–61 (2008). Koob, G. & Kreek, M. J. Stress, dysregulation of drug reward pathways, and the transition to drug dependence. Am. J. Psychiatry 164, 1149–1159 (2007). Simons, C. T., Boucher, Y., Carstens, M. I. & Carstens, E. Nicotine suppression of gustatory responses of neurons in the nucleus of the solitary tract. J. Neurophysiol. 96, 1877–1886 (2006).

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19. Wise, R. A. & Kiyatkin, E. A. Differentiating the rapid actions of cocaine. Nature Rev. Neurosci. 12, 479–484 (2011). 20. Lenoir, M. & Kiyatkin, E. A. Critical role of peripheral actions of intravenous nicotine in mediating its central effects. Neuropsychopharmacology 36, 2125–2138 (2011). An important paper demonstrating that non-brain actions of nicotine may contribute to its reinforcing properties. It suggests that addictive drugs may act through peripheral mechanisms to trigger addiction. 21. Olson, V. G. et al. Role of noradrenergic signaling by the nucleus tractus solitarius in mediating opiate reward. Science 311, 1017–1020 (2006). 22. Delfs, J. M., Zhu, Y., Druhan, J. P. & Aston-Jones, G. Noradrenaline in the ventral forebrain is critical for opiate withdrawal-induced aversion. Nature 403, 430–434 (2000). 23. Harris, G. C. & Aston-Jones, G. Activation in extended amygdala corresponds to altered hedonic processing during protracted morphine withdrawal. Behav. Brain Res. 176, 251–258 (2007). 24. Garcia-Diaz, D. E., Jimenez-Montufar, L. L., Guevara-Aguilar, R., Wayner, M. J. & Armstrong, D. L. Olfactory and visceral projections to the nucleus of the solitary tract. Physiol. Behav. 44, 619–624 (1988). 25. Ziomber, A. et al. Magnetically induced vagus nerve stimulation and feeding behavior in rats. J. Physiol. Pharmacol. 60, 71–77 (2009). 26. Burneo, J. G., Faught, E., Knowlton, R., Morawetz, R. & Kuzniecky, R. Weight loss associated with vagus nerve stimulation. Neurology 59, 463–464 (2002).

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F O C U S O N A DRDEI C ON V ITEI W S 27. Wang, G. J. et al. Gastric stimulation in obese subjects activates the hippocampus and other regions involved in brain reward circuitry. Proc. Natl Acad. Sci. USA 103, 15641–15645 (2006). 28. Ertelt, T. W. et al. Alcohol abuse and dependence before and after bariatric surgery: a review of the literature and report of a new data set. Surg. Obes. Relat. Dis. 4, 647–650 (2008). 29. Cunningham, J. T., Mifflin, S. W., Gould, G. G. & Frazer, A. Induction of cFos and ∆FosB immunoreactivity in rat brain by Vagal nerve stimulation. Neuropsychopharmacology 33, 1884–1895 (2008). 30. Nunez, C. et al. Induction of FosB/∆FosB in the brain stress system-related structures during morphine dependence and withdrawal. J. Neurochem. 114, 475–487 (2010). 31. Mumberg, D., Lucibello, F. C., Schuermann, M. & Muller, R. Alternative splicing of fosB transcripts results in differentially expressed mRNAs encoding functionally antagonistic proteins. Genes Dev. 5, 1212–1223 (1991). 32. McClung, C. A. & Nestler, E. J. Regulation of gene expression and cocaine reward by CREB and ∆FosB. Nature Neurosci. 6, 1208–1215 (2003). 33. Appleyard, S. M. et al. Proopiomelanocortin neurons in nucleus tractus solitarius are activated by visceral afferents: regulation by cholecystokinin and opioids. J. Neurosci. 25, 3578–3585 (2005). 34. Zhang, Y. et al. Pro-opiomelanocortin gene transfer to the nucleus of the solitary track but not arcuate nucleus ameliorates chronic diet-induced obesity. Neuroscience 169, 1662–1671 (2010). 35. Holst, J. J. The physiology of glucagon-like peptide 1. Physiol. Rev. 87, 1409–1439 (2007). 36. Turton, M. D. et al. A role for glucagon-like peptide1 in the central regulation of feeding. Nature 379, 69–72 (1996). An important paper showing that GLP1 that is produced in the NTS can control food intake. Further studies will be necessary to determine whether GLP1 also regulates drug intake. 37. Hayes, M. R., Bradley, L. & Grill, H. J. Endogenous hindbrain glucagon-like peptide1 receptor activation contributes to the control of food intake by mediating gastric satiation signaling. Endocrinology 150, 2654–2659 (2009). 38. Barrera, J. G. et al. Hyperphagia and increased fat accumulation in two models of chronic CNS glucagonlike peptide1 loss of function. J. Neurosci. 31, 3904–3913 (2011). 39. Hayes, M. R. et al. Intracellular signals mediating the food intake-suppressive effects of hindbrain glucagonlike peptide1 receptor activation. Cell Metab. 13, 320–330 (2011). 40. Paulus, M. P. Neural basis of reward and craving-a homeostatic point of view. Dialogues Clin. Neurosci. 9, 379–387 (2007). 41. Johnson, P. M. & Kenny, P. J. Dopamine D2 receptors in addiction-like reward dysfunction and compulsive eating in obese rats. Nature Neurosci. 13, 635–641 (2010). This paper shows that consumption of palatable food can become compulsive in much the same way that consumption of addictive drugs can be compulsive. It supports the hypothesis that obesity and addiction share common underlying mechanisms. 42. Cottone, P., Sabino, V., Steardo, L. & Zorrilla, E. P. Opioid-dependent anticipatory negative contrast and binge-like eating in rats with limited access to highly preferred food. Neuropsychopharmacology 33, 524–535 (2008). This paper shows that rats will shift their consummatory preference to the most palatable item available and will reject a less palatable alternative, even one that they previously readily consumed, after a period of exposure to the more palatable item. The authors show that this so-called negative contrast effect is regulated by opioid receptors. 43. Lin, J. Y., Roman, C. & Reilly, S. Insular cortex and consummatory successive negative contrast in the rat. Behav. Neurosci. 123, 810–814 (2009). 44. Reilly, S., Bornovalova, M. & Trifunovic, R. Excitotoxic lesions of the gustatory thalamus spare simultaneous contrast effects but eliminate anticipatory negative contrast: evidence against a memory deficit. Behav. Neurosci. 118, 365–376 (2004). 45. Kullmann, S. et al. The obese brain: association of body mass index and insulin sensitivity with resting state network functional connectivity. Hum. Brain Mapp. 21 Apr 2011 (doi:10.1002/hbm.21268).

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Acknowledgements The author is supported by grants from the US National Institute on Drug Abuse (NIDA). This is manuscript number 21309 from The Scripps Research Institute.

Competing interests statement The author declares no competing financial interests.

FURTHER INFORMATION Paul J. Kenny’s homepage: http://www.scripps.edu/kenny ALL LINKS ARE ACTIVE IN THE ONLINE PDF

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REVIEWS

Dysfunction of the prefrontal cortex in addiction: neuroimaging findings and clinical implications Rita Z. Goldstein* and Nora D. Volkow‡

Abstract | The loss of control over drug intake that occurs in addiction was initially believed to result from disruption of subcortical reward circuits. However, imaging studies in addictive behaviours have identified a key involvement of the prefrontal cortex (PFC) both through its regulation of limbic reward regions and its involvement in higher-order executive function (for example, self-control, salience attribution and awareness). This Review focuses on functional neuroimaging studies conducted in the past decade that have expanded our understanding of the involvement of the PFC in drug addiction. Disruption of the PFC in addiction underlies not only compulsive drug taking but also accounts for the disadvantageous behaviours that are associated with addiction and the erosion of free will.

*Medical Department, Brookhaven National Laboratory, Upton, New York 11973, USA. ‡ National Institute on Alcohol Abuse and Alcoholism, Bethesda, Maryland 20892, USA, and National Institute on Drug Abuse, Bethesda, Maryland 20892, USA. Correspondence to R.Z.G.  e-mail: rgoldstein@bnl.gov doi:10.1038/nrn3119

Drug addiction encompasses a relapsing cycle of intoxication, bingeing, withdrawal and craving that results in excessive drug use despite adverse consequences (FIG. 1). Drugs that are abused by humans increase dopamine in the reward circuit and this is believed to underlie their rewarding effects. Therefore, most clinical studies in addiction have focused on the midbrain dopamine areas (the ventral tegmental area and substantia nigra) and the basal ganglia structures to which they project (the ventral striatum, where the nucleus accumbens is located, and the dorsal striatum), which are known to be involved in reward, conditioning and habit formation1–3. However, preclinical and clinical studies have more recently brought to light and started to clarify the role of the prefrontal cortex (PFC) in addiction4. A number of processes are ascribed to the PFC that are fundamental for healthy neuropsychological function — encompassing emotion, cognition and behaviour — and that help to explain why PFC disruption in addiction could negatively affect a wide range of behaviours (TABLE 1). On the basis of imaging findings and emerging preclinical studies5,6, we proposed 10 years ago that disrupted function of the PFC leads to a syndrome of impaired response inhibition and salience attribution (iRISA) in addiction (FIG. 1) — a syndrome that is characterized by attributing excessive salience to the drug and drug-related cues, decreased sensitivity to non-drug reinforcers and decreased ability to inhibit maladaptive or disadvantageous behaviours7. As a result of these core deficits, drug seeking and taking become a main

motivational drive, occurring at the expense of other activities8 and culminating in extreme behaviours in order to obtain drugs9. Here we review imaging studies into the role of the PFC in addiction from the past decade, integrating them into the iRISA model with the aim to gain a greater understanding of the dysfunction of the PFC in addiction. Specifically, this is the first systematic evaluation of the role of distinct regions within the functionally heterogeneous PFC in the neuropsychological mechanisms that putatively underlie the relapsing cycle of addiction. We review positron emission tomography (PET) and functional MRI (fMRI) studies focusing on regions of the PFC that have been implicated in addiction. These include the orbitofrontal cortex (OFC), anterior cingulate cortex (ACC) and dorsolateral prefrontal cortex (DLPFC) (see TABLE 1 for Brodmann areas; see Supplementary information S1 (table) for Brodmann areas that are not discussed in the main text). We consider the results of these studies (FIG. 2) in the context of the role that the PFC plays in iRISA: first, in the response to direct effects of the drug and drug-related cues; second, in the response to non-drug rewards, such as money; third, in higher-order executive function, including inhibitory control; and fourth, in awareness of the illness. We present a simple model that helps to guide our hypotheses regarding the role of the various PFC subregions in the endophenotype of drug addiction (FIG. 3), as described in more detail below. For preclinical studies on the PFC in addiction or in-depth accounts

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F O C U S O N A DRDEI C ON V ITEI W S into the executive function of the PFC we refer the reader to other reviews10,11. In evaluating this Review, readers need to embrace a myriad of results, which can prove quite confusing as definite conclusions are not always provided. This is particularly true for the localization of functions: for example, are the dorsal ACC and DLPFC involved in the craving response or in control over craving, or in both? Determining which PFC subregion mediates which function can be very difficult, presumably owing to the neuroanatomical and cognitive flexibility of these functions — that is, participants can use multiple strategies when performing neuropsychological tasks, and prefrontal systems seem to have a greater level of functional flexibility than more primary sensorimotor systems. Another decade of research may prove invaluable in our understanding of the PFC’s role in drug addiction. Integrating results from preclinical lesion and pharmacological studies, considering other cortical and subcortical structures in addiction — the PFC is densely interconnected with other brain regions (see BOX 1 for a discussion of early studies examining these networks in the context of addiction) — and using computational modelling may help further in ascribing

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(18F-PET). Positron emission tomography (PET) with a radioligand to image regional glucose uptake, a measure of metabolic activity that can also be used to assess global brain function.

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Methylphenidate (MPH). A mild stimulant (approved for treatment of attention deficit hyperactivity disorder) with similar pharmacological effects to cocaine (it blocks the dopamine transporter) but with lower abuse potential owing to slower rates of clearance from the synapse.

Non-contingent administration Administration of a certain drug that is not dependent on the subject’s behaviour.

Fixed-rate self-administration Self-administration of a certain drug on a ratio between drug delivery and behaviour that is fixed by an experimenter (for example, after emission of a certain number of responses or after a certain time has elapsed following the previous response).

Figure 1 | Behavioural manifestations of the iRISA syndrome of drug addiction. This figure shows the core clinical symptoms of drug addiction — intoxication, bingeing, withdrawal and craving — as behavioural manifestations of the impaired response inhibition and salience attribution (iRISA) syndrome. Drug self-administration may lead to intoxication, depending on the drug, amount and rate of use, and individual variables. Bingeing episodes develop with some drugs, such as crack cocaine, and drug use becomes compulsive — much more of the drug is consumed and for longer periods than intended — indicating reduced self-control. Other drugs (for example, nicotine and heroin) are associated with more regimented drug use. After discontinuation of excessive or repeated drug use, withdrawal symptoms develop, including lack of motivation, anhedonia, negative emotion and enhanced stress reactivity. Excessive craving or drug wanting, or other, more automatic processes such as attention bias and conditioned responses, can then pave the way to additional drug use even when the addicted individual is trying to abstain (see TABLE 1 for clinical characteristics of addiction in the context of iRISA and the role of the PFC in addiction). Figure is modified, with permission, from REF. 7 © (2002) American Psychiatric Association.

probable psychological functions to select PFC regions and in enhancing our understanding of their involvement in drug addiction. Our Review is a step in this direction.

Direct effects of drug exposure Here, we review studies that assessed the effects of stimulant and non-stimulant drugs on PFC activity (Supplementary information S2 (table)). Our model predicts drug-induced enhancements of activity in PFC areas that are involved in drug-related processes — including emotional responses, automatic behaviours and higher-order executive involvement (for example, medial OFC (mOFC) and ventromedial PFC in craving, OFC in drug expectation, ACC in attention bias and DLPFC in forming drug-related working memories). It also predicts drug-induced decreases in nondrug related activity in these same PFC regions, most notably during craving and bingeing in drug-addicted individuals, discussed below (FIG. 3). Consistent with the former prediction, intravenous cocaine administration to overnight-abstinent cocaine-addicted individuals increased self-reports of high and craving, and mainly increased fMRI blood oxygen level-dependent (BOLD) responses in various PFC subregions12,13. Interestingly, activity in the left lateral OFC, frontopolar cortex and ACC was modulated by drug expectation (that is, activity was greater after expected versus unexpected intravenous delivery of cocaine), whereas subcortical regions responded mainly to the pharmacological effects of cocaine (that is, there was no modulation by expectation); the specific direction of the effect differed by region of interest (ROI)13. In an 18Fluorodyoxyglucose PET (PET FDG) study, administration of the stimulant drug methylphenidate (MPH) to active cocaine users increased whole-brain glucose metabolism14. Here, the left lateral OFC showed greater metabolism in response to unexpected than to expected MPH; the opposite pattern to that of the BOLD effect in the above study 13 possibly reflects the different temporal sensitivity of the imaging modalities (see below). Stimulant drugs also increase PFC activity in laboratory animals. For example, regional cerebral blood flow (rCBF) in drug-naive rhesus monkeys increased in DLPFC after non-contingent administration and in ACC during a simple fixed-rate self-administration of cocaine15,16. A PET FDG study in the same animal model showed that cocaine self-administration increased metabolism in OFC and ACC to a greater extent when access to cocaine was extended than when access was limited17 (note that extended access, but not limited or short access, is associated with transition from moderate to excessive drug intake, as occurs in addiction18). Similarly, intracerebroventricular administration of cocaine in rats induced a large fMRI response in selected brain regions, including PFC19. Taken together, the main effect of cocaine (and other stimulants such as MPH) on the PFC is to increase PFC activity, as measured by glucose metabolism, CBF or BOLD (although in a recent study, cocaine reduced PFC cerebral blood volume in macaque monkeys20). As the length of access to the drug and drug expectation

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REVIEWS Arterial spin labelling (Also known as arterial spin tagging). An MRI technique that is capable of measuring cerebral blood flow in vivo. It provides cerebral perfusion maps without requiring the administration of a contrast agent or the use of ionizing radiation, as it uses magnetically labelled endogenous blood water as a freely diffusible tracer.

modulate PFC activity, increases in activity that occur during drug administration may be indicative of the neuroplastic adaptations that ensue in the transition from first or occasional use to regular use, such that drug-related neuropsychological processes, including drug-related anticipation (and other conditioned responses), suppress or eclipse non-drug related processes, such as anticipation of — or the motivation to — pursue non-drug related goals (FIG. 3). In cigarette smokers, rCBF was reduced in the left dorsal ACC (dACC) and this correlated with a decrease in craving after smoking the first cigarette of the day 21. Similar correlations were reported between rCBF in OFC and craving after acute injections of heroin in people who are heroin-dependent 22. The disparity between the effects of cocaine (and other stimulants) and other types of drugs on PFC activity may reflect differences in the direct pharmacological effects of the drugs on the PFC and other brain regions (cannabinoid, mu opioid and nicotine receptors, which are targets for marijuana, heroin and nicotine, respectively, have a distinct regional brain distribution) or on non-CNS targets (cocaine and methamphetamine have peripheral sympathomimetic effects that are distinct from the peripheral effects of marijuana or alcohol), or it may reflect variability in methodological factors (for example, whether studies analysed

absolute or relative (or normalized) values)23. It may also be related to drug-induced craving effects: with drugs like cocaine, craving in addicted individuals increases 10–15 minutes after smoking, whereas the studies discussed above reported decreases in craving immediately after nicotine or heroin administration. Viewed in this light, and consistent with our model, the collective results suggest that when drug intake decreases craving, this is associated with decreases in drug-related PFC activity, and vice versa. Concomitantly with these drug-related decreases, we would expect non-drug related PFC activity to increase, as indeed is the case (see below). Disparities between results in this section, and throughout this Review, could also be attributed to differences between the various imaging modalities — an issue that should be recognized early on in this Review. For example, PET FDG measures glucose metabolic activity averaged over 30 min, whereas fMRI BOLD and PET CBF reflect faster changes in activation patterns. These modalities also differ in their baseline measures: it is not possible to establish an absolute baseline with BOLD fMRI, whereas it is possible with PET and arterial spin labelling MRI. Another common difference between studies is the baseline state of an individual, for example, the duration of abstinence could impact measures of craving and withdrawal.

Table 1 | Processes associated with the prefrontal cortex that are disrupted in addiction Process

Possible disruption in addiction

Probable PFC region

Self-control and behavioural monitoring: response inhibition, behavioural coordination, conflict and error prediction, detection and resolution

Impulsivity, compulsivity, risk taking and impaired self-monitoring (habitual, automatic, stimulus-driven and inflexible behavioural patterns)

DLPFC, dACC, IFG and vlPFC

Emotion regulation: cognitive and affective suppression of emotion

Enhanced stress reactivity and inability to suppress emotional intensity (for example, anxiety and negative affect)

mOFC, vmPFC and subgenual ACC

Motivation: drive, initiative, persistence and effort towards the pursuit of goals

Enhanced motivation to procure drugs but decreased motivation for other goals, and compromised purposefulness and effort

OFC, ACC, vmPFC and DLPFC

Awareness and interoception: feeling one’s own bodily and subjective state, insight

Reduced satiety, ‘denial’ of illness or need for treatment, and externally oriented thinking

rACC and dACC, mPFC, OFC and vlPFC

Attention and flexibility: set formation and maintenance versus set-shifting, and task switching

Attention bias towards drug-related stimuli and away from other stimuli and reinforcers, and inflexibility in goals to procure the drug

DLPFC, ACC, IFG and vlPFC

Working memory: short-term memory enabling the construction of representations and guidance of action

Formation of memory that is biased towards drug-related stimuli and away from alternatives

DLPFC

Learning and memory: stimulus–response associative learning, reversal learning, extinction, reward devaluation, latent inhibition (suppression of information) and long-term memory

Drug conditioning and disrupted ability to update the reward value of non-drug reinforcers

DLPFC, OFC and ACC

Decision making: valuation (coding reinforcers) versus choice, expected outcome, probability estimation, planning and goal formation

Drug-related anticipation, choice of immediate reward over delayed gratification, discounting of future consequences, and inaccurate predictions or action planning

lOFC, mOFC, vmPFC and DLPFC

Salience attribution: affective value appraisal, incentive salience and subjective utility (alternative outcomes)

Drugs and drug cues have a sensitized value, non-drug reinforcers mOFC and vmPFC are devalued and gradients are not perceived, and negative prediction error (actual experience worse than expected)

Orbitofrontal cortex (OFC) includes Brodmann area (BA) 10–14 and 47 (REF. 216), and inferior and subgenual regions of anterior cingulate cortex (ACC) (BA 24, 25 and 32) in the ventromedial prefrontal cortex (vmPFC)217; ACC includes rostral ACC (rACC) and dorsal ACC (dACC) (BA 24 and 32, respectively), which are included within the medial PFC (mPFC). The mPFC also includes BA 6, 8, 9 and 10 (REF. 218); dorsolateral PFC (DLPFC) includes BA 6, 8, 9 and 46 (REF. 219); and the inferior frontal gyrus (IFG) and ventrolateral PFC (vlPFC) encompass inferior portions of BA 8, 44 and 45 (REF. 220). These various processes and regions participate to a different degree in craving, intoxication, bingeing and withdrawal. lOFC, lateral OFC; mOFC, medial OFC; PFC, prefrontal cortex.

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F O C U S O N A DRDEI C ON V ITEI W S !

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Figure 2 | Recent neuroimaging studies of PFC activity in drug-addicted individuals. The areas of activation (measured using MRI, positron emission tomography (PET) or single-photon emission computed tomography (SPECT)) (Supplementary !"#$%&' #!()* (table)) are plotted in stereotaxic space, shown rendered on the dorsal and ventral surfaces (top part) and the lateral and medial surfaces (middle part and bottom part, respectively) of the human brain. a | Activity changes related to neuropsychological features in addiction. Prefrontal cortex (PFC) areas show differences in activity between individuals with addiction and healthy controls during tasks involving attention and working memory (shown in green), decision making (shown in light blue), inhibitory control (shown in yellow), emotion and motivation (shown in red), and cue reactivity and drug administration (shown in orange). In addition, in some PFC areas activity correlates with task performance or drug use (shown in dark blue). b | Activity changes related to clinical features in addiction, !+,-. !/0 !'#1 +&' #!0&!.02 !/3 !/0456#7!0 !0$3.80.$-/5073$30-53.07 '6 !09:(6#-$50#"0'6305'-.;<=0+$&> !/0 456#7!0 !0? !@80.$-/5073$30-53.0*AB(733@5 before the study) and withdrawal (shown in purple; drugs were used more than C(733@s before the study). Areas that showed activation in studies in which the stage of addiction was not specified or could not be determined are also indicated (shown in brown). These are the same studies as those depicted in a. Studies were included only if x, y and z coordinates were provided and if these coordinates were within PFC grey matter; studies in which x, y and z coordinates could not be located or were incorrectly labelled were not included. All x, y and z coordinates were converted to Talairach space (using GingerAle, a Cross-platform Java application for Meta-Analysis) before plotting. The Multi-Level Kernel Density Analysis toolbox213,214 was used (see the University of Colorado CANLab software Web site; see also Supplementary information S8 (figure)).

Responses to drug-related cues At the core of drug addiction are the conditioned responses to stimuli associated with the drug that develop in habitual users — such as objects that are used to administer the drugs, people who procure the drug or emotional states that in the past were either relieved or triggered by the use of the drug — that then drive the desire for drug taking and that are important contributors to relapse. Imaging studies have evaluated these conditioned responses by exposing addicted people to drug-related cues, for example, by showing them

drug-related pictures. Here, we first review studies that compared the PFC response to cue exposure in addicted individuals and controls (Supplementary information S3 (table)), and then we discuss studies that explored the effect of abstinence, expectation and cognitive interventions on the PFC responses to drug-related cues (Supplementary information S4 (table)). We predict that in addicted individuals, PFC responses to drug-related cues mimic the responses to the drug itself, owing to conditioning, and that intervention causes a reduction of the drug-cue conditioned responses in the PFC.

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REVIEWS Effect of cue exposure on PFC activity. Although there are some exceptions24–26, fMRI studies report that compared to controls, drug-addicted individuals show enhanced BOLD responses in PFC to drug-related cues relative to control cues (Supplementary information S3 (table)). ! )*"+,-.'"-/'

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Figure 3 | A model of PFC involvement in iRISA in addiction. A model of how interactions between prefrontal cortex (PFC) subregions may regulate cognitive, emotional and behavioural changes in addiction. The model shows how changes in the activity of PFC subregions in addicted individuals relate to core clinical symptoms of addiction — intoxication and bingeing, and withdrawal and craving — compared to PFC activity in healthy, non-addicted individuals or states. The model focuses particularly on inhibitory control and emotion regulation. The blue ovals represent dorsal PFC subregions (including the dorsolateral PFC (DLPFC), the dorsal anterior cingulate cortex (dACC) and the inferior frontal gyrus; see TABLE 1) that are involved in higher-order control (‘cold’ processes). The red ovals represent ventral PFC subregions (the medial orbitofrontal cortex (mOFC), the ventromedial PFC and rostroventral ACC) that are involved in more automatic, emotion-related processes (‘hot’ processes). Drug-related neuropsychological functions (for example, incentive salience, drug wanting, attention bias and drug seeking) that are regulated by these subregions are represented by darker shades and non-drug related functions (for example, sustained effort) are represented by lighter shades. a | In the healthy state, non-drug related cognitive functions, emotions and behaviours predominate (shown by the large light-coloured ovals) and automatic responses (emotions and action tendencies that could lead to drug taking) are suppressed by input from the dorsal PFC (shown by the thick arrow). Thus, if a person in the healthy state is exposed to drugs, excessive or inappropriate drug-taking behaviour is prevented or stopped (‘Stop!’). b | During craving and withdrawal, drug-related cognitive functions, emotions and behaviours start to eclipse non-drug related functions, creating a conflict regarding drug taking (‘Stop?’). Decreased attention and/or value is assigned to non-drug related stimuli (shown by smaller light-shaded ovals), and this reduction is associated with reduced self-control and with anhedonia, stress reactivity and anxiety. There is also an increase (shown by the larger dark-shaded ovals) in drug-biased cognition and cue-induced craving and drug wanting. c | During intoxication and bingeing, higher-order non-drug related cognitive functions (shown by the small light blue oval) are suppressed by increased input (shown by the thick arrow) from the regions that regulate drug-related, ‘hot’ functions (large dark red oval). That is, there is decreased input from higher-order cognitive control areas (shown by the thin dashed arrow), and the ‘hot’ regions come to dominate the higher-order cognitive input. Thus, attention narrows to focus on drug-related cues over all other reinforcers, impulsivity increases and basic emotions — such as fear, anger or love — are unleashed, depending on the context and individual predispositions. The result is that automatic, stimulus-driven behaviours, such as compulsive drug consumption, aggression and promiscuity, predominate (‘Go!’). This model does not take into account the challenge of localizing PFC functions or the evidence that some addicted individuals use drugs to ‘self-medicate’ in an attempt to normalize PFC functions (although part a could represent an approximation of the normalized PFC functions in these individuals).

These results were reported in the left DLPFC, left medial frontal gyrus and right subcallosal gyrus (Brodmann area 34) in young cigarette smokers27, and in bilateral DLPFC and ACC in short-term 28 and long-term 29 abstinent alcoholics. Similar increases were reported in studies (including PET FDG studies) of cocaineaddicted individuals watching cocaine-related videos30 and of heavy smokers watching cigarette-related videos while handling a cigarette31. Often, there are no differences between addicted and non-addicted individuals in valence or arousal ratings, or even in autonomic reactions (for example, skin conductance responses) to the drug-related cues29, which suggests that neuroimaging measures are more sensitive in detecting group differences in conditioned responses to drug-related cues. Importantly, cue-induced PFC responses were correlated with craving 31 and severity of drug use27, and predicted both subsequent performance on a primed emotion recognition task32 and drug use 3 months later 29, indicating that these measures have clinical relevance. As no PFC activation was elicited by drug-related masked cues33 (which activated subcortical regions instead 34), these effects may only be induced when drug-related cues are consciously perceived, but this needs to be studied further. An interesting line of studies explores cue-related PFC activation during acute pharmacological drug exposure. In heroin-dependent males receiving heroin injections while viewing drug-related videos, CBF in OFC correlated with the urge to use the drug, and CBF in DLPFC (Brodmann area 9) correlated with happiness22 (Supplementary information S2 (table)). In this context, it is interesting to note that the mere taste of alcohol (versus litchi juice) can increase BOLD PFC activity in young drinkers, and this response correlates with alcohol use and craving 35 and is possibly driven by dopamine neurotransmission in the subcortical reward circuit 36. By contrast, in non-dependent alcohol drinkers or cigarette smokers, cue-related OFC activity was reduced by alcohol or nicotine administration, respectively 37. This finding resonates with the finding that in non-addicted subjects, intravenous MPH administration decreased metabolism in ventral PFC regions38 (BOX 2). Future studies could directly compare PFC responses to drug-related cues in non-dependent and dependent individuals and thereby further explore the impact of intoxication on cue-related PFC responses. Modelling of bingeing in drug abusing subjects would be informative for the design of interventions to reduce cue-induced compulsive behaviours. PFC activation to relevant cues has also been reported in behavioural addictions. For example, young males who played internet games for over 30 hours a week showed BOLD activations in OFC, ACC, medial PFC and DLPFC when viewing pictures of the game, and these activations were correlated with the urge to play 39. Similarly, compared to control subjects, pathological gamblers watching gambling videos showed increased activation in right DLPFC and inferior frontal gyrus40, and this activation correlated with the urge to gamble41. By contrast, another study in pathological gamblers showed reduced

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F O C U S O N A DRDEI C ON V ITEI W S Box 1 | Addiction-related changes in PFC connectivity and structure The prefrontal cortex (PFC) is densely interconnected with other cortical and subcortical brain regions and networks, including the ‘default mode network’ (DMN) and the ‘dorsal attention networks’, which are implicated in executive control processes such as attention and inhibition43,155,156. Although the question of how these networks — and other interconnected brain regions — impact drug addiction has only recently begun to be explored, resting-state functional connectivity studies have already shown promise in revealing patterns that predict disease severity and treatment outcomes. For example, in cigarette smokers, dorsal anterior cingulate cortex (dACC)–striatal connectivity is inversely correlated with the severity of nicotine addiction; using a nicotine patch significantly enhanced the coherence strength of several ACC connectivity paths, including those to frontal midline structures157. In addition, in abstinent smokers, withdrawal symptom improvement after nicotine replacement therapy was associated with an increased inverse correlation between the executive control network and the DMN, with altered functional connectivity within the DMN, and with altered functional connectivity between the executive control network and regions implicated in reward158. More recent studies into nicotine addiction adapted an important multi-imaging approach in which connectivity is explored with regard to grey matter integrity and cue reactivity159,160. Network-specific functional connectivity strength is also decreased in other addictions. In cocaine-addicted individuals, the rostroventral ACC (part of the DMN) had lower connectivity with the midbrain, where dopamine neurons are located161, and similar results have been reported in other studies162. Reductions in functional connectivity have also been reported in heroin addiction163, in whom connectivity was modulated by drug-related cues164 and associated with longer duration of heroin use165. Further studies are needed to determine whether resting-state connectivity predicts task performance, and how drugs of abuse or potential medications change these measures — for example, does drug administration increase both resting-brain connectivity and task-induced activations or could an elevated resting or baseline state be associated with reduced task-induced activations? These questions are important because the answers will help to determine individually tailored clinical end-points — for example, medication dose could be tapered based on an individual’s own baseline resting-state functional connectivity. Structural imaging studies have shown reduced PFC grey matter density or thickness across addiction populations (up to 20% loss). For example, grey matter PFC decrements, specifically in the dorsolateral PFC (DLPFC), have been documented in individuals who are addicted to alcohol. These decrements are associated with longer lifetime alcohol use166,167 and worse executive function167, and persist from !"#$%&'()*+,*'%* #-./0)*%0*$%0.*%1*/2)'3&.&4.168–170. Despite some conflicting results171, most studies in individuals who are addicted to cocaine172–174, methamphetamine175, heroin176 (even when on methadone replacement therapy177,178) and nicotine159,160,179,180 report similar PFC grey matter reductions — which are most evident in the DLPFC, ACC and orbitofrontal cortex (OFC) — that are associated with longer duration or increased severity of drug use. The persistence of these structural changes beyond the end of drug use and into long-term abstinence suggests an influence of pre-morbid or stable factors that might predispose individuals to drug use and addiction during development (BOX 3). Nevertheless, such structural abnormalities are not seen in adolescent users of alcohol181 or marijuana182, which suggests these PFC decrements could also be a dose-dependent consequence of drug use. Whether it predisposes to addiction or is a consequence of addiction, such lower PFC grey matter volume, particularly in the medial OFC, is associated with disadvantageous decision making183 that could lead to the catastrophic consequences in the lives of addicted individuals.

Masked cue A cue that is presented below conscious processing level (that is, outside of conscious awareness). This is usually achieved with a very short duration of cue presentation followed by presentation of another cue that is consciously perceived (longer duration).

left ventromedial PFC BOLD responses to winning versus losing in a gambling-like task, and the size of the reduction was correlated with the severity of the gambling addiction, as assessed with a gambling questionnaire42. The opposite directions of the activity changes (hyperactivations versus hypoactivations as compared to controls) may be driven by the ROI (for example, ventromedial PFC task-related deactivations are often seen and have been attributed to the role of the ‘default brain’ network43), differences in craving (craving was reported

in REFS 39–41 but not REF. 42), task differences or methodological factors, which are summarized at the end of this section. Disorders that are characterized by impaired control of food consumption are also associated with abnormal PFC reactivity to cues. This is not unexpected, given that these disorders and addiction involve similar compromises in neuronal circuits44, including decreased striatal dopamine D2 receptor availability 45. For example, women with anorexia or bulimia who are passively viewing pictures of foods (versus non-food related pictures) showed increased fMRI BOLD responses in left ventromedial PFC46. Compared to patients with bulimia, patients with anorexia showed greater right OFC activation in response to food pictures, possibly implicating this region in overly restrictive self-control; by contrast, left DLPFC activity to these pictures was decreased in patients with bulimia when compared to healthy controls, possibly implicating this region in the loss of control over food intake46. In another study, young women with eating disorders, but not control subjects, showed activation of the left ventromedial PFC during the selection of the most negative word from negative body-image related word sets (compared to during the selection of the most neutral word from neutral word sets)47. Such differences were not observed for generally negative words, indicating this region’s activation was driven by words that are most strongly related to the actual concerns of this patient group. Taken together with the results in the pathological gamblers described above42, ventromedial PFC responses may track the emotional relevance of cues of highest concern to the patient population in question (that is, winning or avoiding loss for individuals with pathological gambling, body image for individuals with eating disorders and drugrelated cues for drug-addicted individuals) and could serve as a target for tracking therapeutic interventions in addiction, as was recently suggested48,49. Effect of abstinence, expectation and cognitive interventions. Here, we propose that cognitive intervention and long-term abstinence attenuate cue-induced responses in the PFC, and that drug-related expectation and shortterm abstinence have the opposite effect. The impact of short-term abstinence on PFC cue-related activity has been most extensively studied in nicotine addiction (Supplementary information S4 (table)). In an arterial spin labelling MRI study, 12-hour abstinence in smokers increased craving, global CBF and regional CBF in the OFC, and decreased CBF in the right PFC, with CBF changes in all ROIs correlating with craving and withdrawal symptoms50. Such enhanced cue reactivity was also reported for longer periods of abstinence — up to 8 days in the DLPFC, ACC and inferior frontal gyrus in female smokers51 — and also positively correlated with craving 52. However, some studies report no effect of abstinence on cue-induced PFC activity 53. This could possibly be attributed to other factors that contribute substantial variability to results, such as the expectation to smoke at the end of the study 54. Indeed, as discussed above13, expectation alone may mimic the effects of acute

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REVIEWS Ketamine An NMDA receptor antagonist primarily used for the induction and maintenance of general anaesthesia. In addition, it can induce analgesia, elevated blood pressure and hallucinations, and it has been used as a recreational drug.

[11C]carfentanil A positron emission tomography (PET) receptor radioligand that competes with endogenous opiates for binding to the mu opiate receptor.

drug intake on PFC activation in addicted individuals. Studies in which all three variables — expectation for drug administration, exposure to drug-related cues and abstinence — are explored for main effects and interaction effects on PFC activity would be useful, particularly if they involve large samples. The temporal dynamics of PFC cue reactivity also remain to be explored in longitudinal studies, tracking the same individual throughout longer-term abstinence periods. A promising line of research explores behavioural modulation of cue reactivity. For example, a role for the mOFC in the suppression of craving was suggested by findings from a recent PET study in cocaine users. Craving increased after watching a video of cocainerelated cues, and craving levels correlated with glucose metabolism in the medial PFC55. Importantly, when participants were instructed — before watching the video — to inhibit craving, metabolism in the right mOFC decreased, and this was associated with activation of the

right inferior frontal gyrus (Brodmann area 44), which is a crucial region in inhibitory control. In treatmentseeking cigarette smokers, the instruction to resist craving while viewing smoking-related videos was associated with DLPFC and ACC activation, although unexpectedly, this activation correlated positively with craving 56. A recent study suggests that the direction of the change in activity and correlation with craving may be modulated by the behavioural strategy that is used to suppress craving. In this elegant study, cigarette smokers were instructed to consider the immediate versus long-term consequences of consuming the stimuli depicted in pictures (cigarette-related versus food-related cues) 57. Considering the long-term consequences was associated with increased activity in PFC regions associated with cognitive control (DLPFC and inferior frontal gyrus) and with decreased activity in PFC regions associated with craving (mOFC and ACC). In addition, selfreported craving decreased when subjects considered the

Box 2 | The role of dopamine and other neurotransmitters Dopamine D2 receptors, which are most densely expressed in subcortical regions such as the midbrain and dorsal and ventral striatum, are also distributed throughout the prefrontal cortex (PFC). A series of positron emission tomography (PET) studies reported lower striatal dopamine D2 receptor availability in individuals who are addicted to methamphetamine184, cocaine38 or alcohol185, and in people with morbid obesity186, and these reductions were associated with decreased baseline metabolic activity in the orbitofrontal cortex (OFC) and anterior cingulate cortex (ACC). This suggests that loss of dopamine signalling through D2 receptors may underlie some of the deficits in prefrontal function that are seen in addiction — an idea that is supported by preliminary data showing that striatal dopamine D2 receptor availability was correlated with medial PFC response to money in cocaine-addicted individuals187. Reduced striatal 5%,/$3&.*67*0.4.,'%0*/8/39/2393'-*:/)*/9)%*0.,%0'.5*3&*$/9.*(./8-*)$%;.0)<*2%'(*/1'.0*)$%;3&=*/)*+)+/9*/&5*/1'.0*7>#(%+0)* of abstinence; in the sated condition, the dopamine D2 receptor availability in the bilateral ACC was negatively correlated with the desire to smoke (positive correlations were observed for the striatum and OFC)188. Evidence for dopamine depletion in the dorsolateral PFC (DLPFC) was also reported in young chronic ketamine users, and levels of depletion were correlated with higher weekly drug use189. Other PET studies reported markedly attenuated striatal dopamine release in response to intravenous administration of a stimulant drug (for example, methylphenidate) in cocaine abusers and alcoholics, with a parallel decrease in self-reported experiences of feeling high38,185. Consistent with data from animal studies, these results in addicted individuals point to a blunted striatal dopaminergic function — both at baseline and in response to a direct challenge — that is associated with enhanced craving and severity of use. A blunted striatal dopamine response is predictive of actual choice for cocaine over money in abstinent cocaine-addicted individuals, suggesting that it may predispose subjects to relapse190. The results also suggest that, by regulating the magnitude of dopamine increases in the striatum185, the OFC assumes a crucial role in the modulation of the value of reinforcers; disruption of this regulation may underlie the increased value attributed to a drug reward in addicted subjects. Consistent with this suggestion, metabolism in the medial OFC and ventral ACC in cocaine abusers increased after intravenous stimulant administration, whereas it was reduced in controls; the regional metabolic increases in the abusers were associated with drug craving38. Endogenous opioids also mediate the rewarding responses of many drugs of abuse, particularly heroin, alcohol and nicotine. Repeated drug use has been associated with decreased release of endogenous opioids, an effect that may contribute to withdrawal symptoms, including dysphoria. A study using [11C]carfentanil showed that cocaine abusers had higher PFC mu opiate receptor binding potential (indicative of lower endogenous opioid levels) than healthy &%&?/5534'.5*4%&'0%9)<*/&5*'(/'*'(3)*,.0)3)'.5*3&*'(.*/&'.03%0*10%&'/9*4%0'.@*/&5*ABB*'(0%+=(%+'*C7#:..;)*%1* abstinence191. Elevated mu opiate receptor binding in the DLPFC and ACC before treatment was associated with greater cocaine use and shorter duration of abstinence, and was suggested to be a better predictor of treatment outcome than baseline drug and alcohol use192. Similar results were reported in abstinent alcoholic men193, whereas the level of mu (or kappa) opiate receptor binding is reversed by chronic methadone in heroin-addicted individuals194. Decreased PFC binding potential for a serotonin transporter radioligand has been reported in abstinent methamphetamine abusers195, young recreational MDMA users196 and in recovered alcoholics197. Reduced serotonin transporter availability may reflect neuroadaptations to increased synaptic serotonin, but it could also reflect damage to serotonergic nerve terminals. Other neurotransmitter systems that regulate the PFC and are involved in the neuroadaptations that occur with repeated drug use in laboratory animals include the glutamate198 and the cannabinoid199,200 systems. However, so far there are no published studies with radiotracers to image these systems in human addiction. See D+,,9.$.&'/0-*3&1%0$/'3%&#DE (table) for an overview of studies comparing neurotransmitter systems between addicted individuals and healthy controls.

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F O C U S O N A DRDEI C ON V ITEI W S Box 3 | Vulnerability and predisposition to drug use Studies on how pre-morbid vulnerabilities — such as prenatal exposure to drugs, family history or selected gene polymorphisms and their interactions — impact prefrontal cortex (PFC) function are crucial for the design of future intervention and possibly prevention efforts; these studies highlight the importance of targeting clear biomarkers of vulnerability to drug use and addiction. For example, reduced absolute global cerebral blood flow (CBF) (–10%), and enhanced relative CBF in the dorsolateral PFC (DLPFC) (9%) and anterior cingulate cortex (ACC) (12%) were reported in adolescents with heavy prenatal cocaine exposure201. A hyperactive PFC was also reported in young users of MDMA202, marijuana203 or alcohol204 during the go/no-go task, in which they performed normally (Supplementary information S6 (table)). Similarly, compared to control children and children who had alcoholic parents but were resilient, children who had alcoholic parents and were vulnerable to alcohol drinking (classified based on the level of problem drinking over the course of adolescence) had a hyperactive right dorsomedial PFC, while the bilateral orbitofrontal cortex (OFC) was hypoactive, despite a lack of behavioural differences when silently reading emotional words. Across the entire sample, such dorsomedial PFC hyperactivity was associated with more externalizing symptoms and with aggression205 (Supplementary information S5 (table)). Thus, such changes in PFC activity may be compensatory in the short-term (as evidenced by equal task performance), but in the long-term may promote substance abuse and addiction in these individuals, although this remains to be ascertained. The mechanism that underlies such vulnerability to, or that confers protection against, developing addiction may involve altered dopaminergic neurotransmission. For example, striatal dopamine D2 receptor availability and regional PFC metabolism were higher in young, unaffected members of alcoholic families than in subjects without such family history, which is the opposite to results commonly reported in addicted individuals (BOX 2; see Supplementary information S7 (table))206. The individuals with a family history of alcohol abuse reported lower positive emotionality, and this was associated with both lower striatal dopamine D2 receptor availability and lower OFC metabolism. It is therefore possible that the higher dopamine D2 receptor availability and the enhanced metabolic activity in PFC in individuals with a family history of alcohol abuse increased the level of positive emotionality — although this nonetheless remained below the level in healthy controls — to levels that may have protected these individuals against developing addiction. It is also possible that optimal conditions are needed for the maintenance of such protection, and that suboptimal conditions (for example, chronic stress) could expose these same individuals to addiction later in life, but this remains to be determined in longitudinal studies. Other mechanisms, such as brain dysmorphology207, may also be important in conferring vulnerability to addiction. Genetic contributions to vulnerability to addiction are also important. For example, regular marijuana users with risk alleles of genes that encode the cannabinoid receptor 1 (CB1) or the fatty acid amino hydrolase 1 (FAAH; the enzyme that metabolizes endogenous cannabinoids) had greater drug-related cue reactivity in limbic PFC areas208. Importantly, such gene by environment interactions may be used to predict future disadvantageous behaviour. For example, 1-year increases in body mass of healthy adolescent girls could be predicted by activation of the lateral OFC induced by food-related cues, but only in carriers of the dopaminergic risk alleles dopamine receptor D4 (DRD4) 7-repeat allele or the DRD2 TaqIA A1 allele209. Recent studies also suggest that interactions between certain polymorphisms and familial — including prenatal — drug exposure can influence OFC development210,211. For example, a recent study showed that medial OFC (mOFC) grey matter volume was modulated by the monoamine oxydase A genotype, such that the low-activity variant of this gene drove the mOFC grey matter decreases in cocaine-addicted individuals212, and this was correlated with longer lifetime cocaine use.

long-term consequences, and it was negatively correlated with activity in dACC and DLPFC. A mediation analysis showed that the association between increased activity in DLPFC and regulation-related decreases in craving was no longer significant after including decreased activity in ventral striatum in the model. Nevertheless, preclinical studies using ablation or optogenetic tools are necessary to better understand the interaction of the PFC and the ventral striatum in suppressing craving responses. Taken together, results of studies using

behavioural approaches to suppress craving provide support to our proposed model (FIG. 3), which distinguishes between PFC regions that facilitate non-drug related cognitive effort and inhibitory control (DLPFC, dACC and inferior frontal gyrus) and those that reflect drug-related emotional concern, craving and compulsive behaviours (mOFC and ventral ACC). To summarize, exposure to drug-related cues mimics the effects of direct drug administration on PFC activity in drug-addicted individuals, although the impact of duration of abstinence and expectation of drug use (and related processes such as forming of drugrelated memories), and their unique contributions to PFC function, remain to be assessed in large sample sizes. By expanding studies of cue reactivity to include additional neuropsychological functions, and by exploring the direction of correlations between PFC activity and specific end-points (for example, craving), the functional significance of activations of specific PFC regions in addiction will become clearer. A further recommendation for future studies into cue reactivity is to conduct direct comparisons between sessions (for example, abstinence versus satiety) and task conditions (for example, drug versus neutral cues) and to perform whole-brain correlations with the respective behavioural changes. Future studies could also compare the duration and the pattern of PFC activation following acute drug exposure and following exposure to conditioned cues in the same subjects. Studies in non-addicted individuals could be used to assess the impact of deprivation (for example, of food) and urgent needs (for example, hunger, sexual desire and achievement motivation) on PFC cue reactivity. For example, in young healthy controls, craving of imagined foods — induced by a monotonous diet — was associated with activation in several limbic and paralimbic regions, including ACC (Brodmann area 24)58. It is important to note that as we have not reviewed the ventral striatal literature — and therefore direct comparisons cannot be made between PFC and subcortical responses to these stimuli — we cannot infer, however tempting this may be, that PFC activity itself may contribute to the rewarding effects of drugs and drug cues.

Responses to non-drug rewards We propose that in individuals with drug addiction, PFC activity in response to non-drug related rewards is opposite to PFC activity changes that characterize drug-related processing (FIG. 3). Specifically, in addicted individuals who are in a state of craving, intoxication, withdrawal or early abstinence, sensitivity of the PFC to non-drug related rewards will be markedly attenuated compared with that in healthy non-addicted subjects. Indeed, decreased sensitivity to non-drug related rewards is a challenge in the therapeutic rehabilitation of patients with substance use disorders. Therefore, it is important to study how drug-addicted individuals respond to non-drug related reinforcers. Such decreased sensitivity to non-drug related reward has been explained as an allostatic adaptation59.

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REVIEWS In this interpretation, frequent and high-dose drug use leads to compensatory brain changes that limit appetitive hedonic and motivational processes (‘reward’), instead strengthening aversive (opponent or ‘anti-reward’) systems60. This process is similar to tolerance, in which sensitivity to reward is decreased. It is also captured by the opponent-process hypothesis set forth by Slomon and Corbit 61,62, which describes the temporal dynamics of opposing emotional responses; here, negative reinforcement (for example, withdrawal) prevails over positive reinforcement (for example, drug-induced high) in the transition from occasional drug use to addiction. This process is relevant to emotional reactivity and emotion regulation, which, insofar as emotions are defined as ‘states elicited by reinforcers’63, are bound to be impaired in drug addiction, especially during drug-biased processing such as craving and bingeing. Anhedonia is a defining characteristic of drug dependence64, and criteria for major depressive disorder — which includes anhedonia as a core symptom — are met by many drug-addicted individuals (for example, 50% of cocaine-addicted individuals65). The strong association between mood and substance use disorders is not limited to depression66; for example, emotional distress is a risk factor for drug relapse67. However, research on how altered emotion processing is implicated in substance use disorders is in its infancy 68,69, as discussed below (Supplementary information S5 (table)). Money is an effective abstract, secondary and generalizable reinforcer that acquires its value by social interaction, and it is used in emotional learning in everyday human experience; compromised processing of this reward may therefore point to a socially disadvantageous emotional learning mechanism in addiction. Such a deficit, all the more distinct given the strong motivational and arousal value that is normally associated with this reward, would corroborate the idea that in addiction, brain reward circuits are ‘hijacked’ by drugs, although the possibility for a pre-existing deficit in reward processing also cannot be ruled out. One fMRI study investigated how cocaine-addicted individuals and controls responded to receiving monetary reward for correct performance on a sustained attention and forced-choice task70. In controls, sustained monetary reward (gain that did not vary within task blocks and that was fully predictable) was associated with a trend for the left lateral OFC to respond in a graded fashion (activity monotonically increased with amount: high gain > low gain > no gain), whereas the DLPFC and rostral ACC responded equally to any monetary amount (high or low gain > no gain). This pattern is consistent with the OFC’s role in processing relative reward, as documented in non-human71 and human subjects72–76, and with the DLPFC’s role in attention77. Cocaine-addicted subjects showed reduced fMRI signals in left OFC for high gain compared to controls and were less sensitive to differences between monetary rewards in left OFC and in DLPFC. Remarkably, more than half of the cocaine-addicted subjects rated the value of all monetary amounts equally (that is, US$10 = US$1000)78. Eighty-five percent of the variance in these ratings could

be attributed to the lateral OFC and medial frontal gyrus (and amygdala) responses to monetary reward in the addicted subjects. Although these findings need to be replicated in a larger sample size and with more sensitive tasks, they nonetheless suggest that some cocaineaddicted individuals may have reduced sensitivity to relative differences in the value of rewards. Such ‘flattening’ of the perceived reinforcer gradient may underlie over-valuation or bias towards immediate rewards (such as an available drug)79 and the discounting of greater but delayed rewards80,81, therefore reducing sustained motivational drive. These results may be therapeutically relevant as monetary reinforcement in well-supervised environments has been shown to enhance drug abstinence82, and may also be relevant in predicting clinical outcomes. In line with this idea, in a similar population of subjects, the degree of dACC hypoactivation in a task in which correct performance was monetarily remunerated correlated with frequency of cocaine use, whereas degree of rostroventral ACC (extending to mOFC) hypoactivation correlated with task-induced craving suppression83. There was an inverse association of these PFC ROIs with cue reactivity in the midbrain in cocaineaddicted subjects but not in control subjects, which implicates these ACC subdivisions in the regulation of automatic drug responses84. It should be noted that in the studies described above, subjects were not asked to choose between monetary rewards. We predict that choice would similarly follow a linear function (choice of higher over lower reward) in healthy controls more so than in addicted individuals, who we expect to show less flexibility in choice (choosing drug over other reinforcers), particularly during craving and bingeing. Studies that allow subjects to choose between reinforcers have mostly been conducted in laboratory animals. These studies have shown that, when given the choice, previously drug-exposed animals choose the drug over novelty 85, adequate maternal behaviour 86 and even food87–89, indicating that drug exposure can decrease the perceived value of natural rewards, even those that are needed for survival. In a recent human neuroimaging study in which subjects could win cigarettes or money, occasional smokers were more motivated to obtain money than cigarettes, whereas dependent smokers made similar efforts to win money or cigarettes90. A similar group by reward interaction was observed in the right OFC, bilateral DLPFC and left ACC, such that in the occasional smokers these regions showed higher activity to stimuli predicting an increasing monetary reward than to stimuli predicting a cigarette reward, whereas the dependent smokers showed no significant differences in such anticipatory brain activity. These regions also showed higher activation to money in the occasional than in dependent smokers90. These results, together with behavioural results on neuropsychological tests in cocaine-addicted individuals91,92 (see also BOX 2), contribute to our understanding of how relative reward preferences may change in addiction such that preference for the drug competes with (and sometimes exceeds) preference for other

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F O C U S O N A DRDEI C ON V ITEI W S Affect matching A neuropsychological test in which images of faces are matched based on their emotional facial expressions. This task can be used to assess impairments in emotional (or social) processing.

reinforcers, with a concomitant decrease in the ability to assign relative values to non drug-related rewards. Emotional reactivity. Several studies that are reviewed above compared PFC responses to non-concern-specific yet emotionally arousing stimuli with responses to concern-related (for example, drug-related) cues25,26,28,46,47 (Supplementary information S3 (table)). The PFC was hyperactive in response to images from all emotional categories in alcohol-addicted subjects28, the anterior PFC was hypoactive in response to pleasant pictures in heroin-addicted individuals26, and in patients with eating disorders PFC responses to aversive pictures were normal46,47. Thus, in contrast to our model’s predictions (FIG. 3), there were no differences in the PFC response between drug-related and affective yet non-drug related cues in any of these studies. This result, and the variability in the pattern of results, could be attributed to — among other factors — the small number of studies, differences between studies (such as sample sizes, the primary drug of abuse and duration of abstinence) and sensitivity of the measures used. Future studies would benefit from using event-related potential recordings or electroencephalography, which have much higher temporal resolution than fMRI or PET. A clearer picture emerges when studies incorporate emotional processing into cognitive–behavioural tasks (Supplementary information S5 (table)). For example, when required to empathize with a protagonist in a series of cartoons, each depicting a short story, methamphetamine-addicted individuals provided fewer correct answers than controls to the question “what will make the main character feel better?”93. Compared to control subjects, the addicted individuals also showed hypoactivation in OFC (and hyperactivation in DLPFC) when answering this question. With the exception of one study in abstinent heroin-addicted individuals94, other similar studies also reported differences between addicted and control groups in PFC responses to tasks requiring processing of emotional stimuli such as faces, words or complex scenes. For example, when men with alcohol addiction judged the intensity of five facial expressions, negative expressions were associated with lower activations in the left ACC but higher activations in the left DLPFC and right dACC compared to controls95. In addition, compared to healthy controls, cocaine users showed ACC and dorsomedial PFC hypoactivations while performing a letter discrimination task during the presentation of a set of pleasant (versus neutral) pictures and hyperactivations in the bilateral DLPFC during the presentation of unpleasant (versus pleasant) pictures96. Similarly, compared to healthy controls, marijuana smokers showed left ACC hypoactivations, and right DLPFC and inferior frontal gyrus hyperactivations in response to presentation of masked angry faces (versus neutral faces); right ACC responses positively correlated with frequency of drug use and bilateral ACC responses correlated with urinary cannabinoid levels and alcohol use97. By contrast, the left dACC was hyperactive in methamphetamine-dependent subjects compared to

controls when judging emotional expression on faces in an affect matching task (versus judging the shape of abstract figures) and this was associated with more selfreported hostility and interpersonal sensitivity in the addicted subjects98. Taken together, these studies indicate that the DLPFC is mostly hyperactive during emotion processing in addicted individuals compared to control subjects, especially for negative emotions. The ACC shows mixed results, although with more studies showing hypoactivity than hyperactivity. It is possible that the DLPFC hyperactivity may be compensating for the ACC hypoactivity, which would explain the lack of difference in task performance between drug abusers and healthy controls in most of these studies. Disadvantageous and/ or impulsive behaviours may be observed during greater emotional arousal challenges such as stress, craving or more difficult tasks. Clearly, the roles of these regions in relation to the proposed model (FIG. 3) need to be better understood. It is possible that, by prematurely recruiting higher-order PFC executive function (mediated by the DLPFC), negative emotional arousal enhances risk for drug use in addicted individuals, particularly in situations that place additional strain on the limited cognitive control resources. This interpretation is consistent with the competition between drug and non drug-related processes and between ‘cold’ and ‘hot’ processes in the model (FIG. 3c). Although several of the above studies used negatively valenced stimuli, a lingering question is whether altered sensitivity to non-drug reinforcers in addicted individuals also applies to negative reinforcers such as money loss. Studies in animals show that ‘addicted’ subjects manifest persistent drug seeking even if the drug is associated with receiving an electric shock99. In humans, hypoactivation in the right ventrolateral PFC in smokers during monetary loss, and in gamblers during monetary gain, have been reported 100 (Supplementary information S5 (table)). Although more studies are clearly needed, the implication of reduced sensitivity to negative reinforcers in addiction has practical implications as, in addition to positive reinforcers (such as vouchers and privileges), negative reinforcers (such as incarceration) are increasingly being used in the management of drug abusers. Interventions could be optimized by selecting the most effective type and dose of reinforcer. Future studies could also help to ascertain whether addicted individuals may resort to taking drugs because they are easily bored, frustrated, angry or fearful, perhaps as a result of altered PFC functioning. Low threshold for experiencing any of these emotions, or the inability to sustain goal-directed behaviour (for example, completing a boring task) when experiencing these emotions, may be associated with impaired inhibitory control (that is, enhanced impulsivity) as reviewed below. In cocaine-addicted individuals, PFC activity habituates prematurely to repeated presentation of an incentive sustained attention task101, which could be a measure of compromised sustainability of effort and result in inadequate engagement in treatment activities.

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REVIEWS

Go/no-go task A neuropsychological task that is commonly used to assess inhibitory control. Subjects are required to press a button when one stimulus type appears and withhold a response when another stimulus type appears.

Stop signal reaction time task (SSRT). A neuropsychological test that measures the ability to stop a response that has already been initiated. It is used clinically as an index of inhibitory control. Slower SSRT is associated with disruption of executive functions.

Errors of omission and commission Errors on a go/no-go task: a subject had to go but they did not go (omission of a response) or had to withhold a response but pressed a button instead (commission of an unnecessary response). The former is an index of inattentiveness while the latter is an index of impulsive (premature) responding.

Stroop task A neuropsychological task in which conflict is created between an automatic response (for example, reading) and a slower response (for example, colour naming), with both competing for the same processing resources. Impaired performance on Stroop tasks is associated with prefrontal cortex dysfunction.

Inhibitory control in addiction Drug addiction is marked by mild, yet pervasive, cognitive disruptions102 that may accelerate its course, threaten sustained abstinence103 or increase attrition from treatment 104,105. The PFC is essential for many of these cognitive processes, including attention, working memory, decision making and delay discounting (TABLE 1), all of which are compromised in addicted individuals, as reviewed elsewhere106. Another important cognitive function of the PFC is self-control, and here we focus on the role of the PFC in this process in addiction (Supplementary information S6 (table)). Self control refers, among other operationalizations, to a person’s ability to guide or stop a behaviour, particularly when the behaviour may not be optimal or advantageous, or is perceived as the incorrect thing to do. This is pertinent to addiction as, despite some awareness of the devastating consequences of drugs (see also the section below on disease awareness in addiction), individuals who are addicted to drugs show an impaired ability to inhibit excessive drug taking. Impaired inhibitory control, which is a key operation in self-control, is also likely to contribute to engagement in criminal activities in order to procure the drug, and to underlie the impaired regulation of negative emotions, as suggested above. These impairments could also predispose individuals to addiction. Consistent with previous reports107, children’s self-control during their first decade of life predicts substance dependence in their third decade of life108. Go/no-go and stop signal reaction time tasks. Tasks that are often used to measure inhibitory control are the go/no-go task and the stop signal reaction time task (SSRT). In the go/no-go task, cocaine-addicted individuals showed more errors of omission and commission than controls and this has been attributed to hypoactivation in dACC during stop trials109. In another study, this inhibitory behavioural deficit in cocaine users was exacerbated by a higher working-memory load; again, dACC hypoactivation was associated with deficient task performance110. Similarly, heroin-addicted men showed slower reaction times in the go/no-go task, along with hypoactivation in ACC and medial PFC111. Results from the SSRT are more difficult to interpret. For example, the ACC was hypoactive during successful response inhibitions compared to failed response inhibitions in cocaineaddicted men, and their behavioural performance was similar to that of controls112. The ACC was also hypoactive during both careful behavioural adjustment and risk taking on this task in abstinent alcoholics, particularly in subjects with higher alcohol urge at the time of the fMRI scan113. By contrast, the ACC was hyperactive during inhibition errors113, possibly because the abstinent alcoholics exercised a greater attention in monitoring for the stop signal than controls — a function that is associated with the ACC. Increased activity in other regions of the PFC was also reported in cigarette smokers after a 24-hour abstinence, but (in contrast to expectation for an increased regional activation) accuracy was reduced114 (Supplementary information S4 (table)).

The large variability in results from these studies is possibly caused by differences in the analyses, the type of comparison and by performance differences between the groups, in addition to other variables. Nevertheless, a pattern emerges in which the dACC is hypoactive during these inhibitory control tasks, and this hypoactivity is mostly associated with impaired performance, particularly with shorter abstinence durations. Targeted cognitive–behavioural interventions may alleviate this dysfunction. For example, informative cueing (such as providing a warning of an impending no-go trial) enhanced inhibitory control in a go/no-go task, and this was correlated with enhanced ACC activation in methamphetamine-addicted individuals115. Such cognitive–behavioural interventions could be used as neural rehabilitation exercises and combined with the simultaneous administration of drugs, as discussed below. Stroop tasks. Inhibitory control can also be assessed using the colour–word Stroop task116. Slower performance and more errors during incongruent trials on this task are a hallmark of PFC dysfunction. Neuroimaging research has shown that the dACC and DLPFC are involved in this task117–119, with distinct roles for these regions in conflict detection (dACC) and resolution (DLPFC)120. Studies using the colour–word Stroop task in addicted individuals report results that mostly echo those reported above. For example, cocaine abusers had lower CBF in the left dACC and right DLPFC during incongruent trials compared to congruent trials, whereas the right ACC showed the opposite pattern; moreover, right ACC activation was negatively correlated with cocaine use121 (Supplementary information S6 (table)). In marijuana-using men, lower CBF during this task was reported in several PFC regions, including perigenual ACC, ventromedial PFC and DLPFC122. Methamphetamine-dependent subjects also showed hypoactivations in the inhibitory control network, including dACC and DLPFC while performing this task123. Consistent with the impact of abstinence on the go/no-go task reported above114, cigarette smokers who were tested after a 12-hour abstinence had slowed reaction times, and enhanced dACC and reduced right DLPFC responses to the incongruent trials on the colour–word Stroop task124 (Supplementary information S4 (table)). Importantly, an fMRI study showed that activation of the ventromedial PFC (Brodmann areas 10 and 32) during a colour–word Stroop task performed 8 weeks before treatment onset predicted treatment outcome in cocaine-addicted individuals125. In the emotional variant of this task, colour words are substituted for emotional words or pictures that are related to a particular individual’s area of concern, such as alcohol-related words for alcohol-addicted individuals. Although both the classic and the emotional Stroop tests involve the need to suppress responses to distracting stimulus information while selectively maintaining attention on the stimulus property that is needed to complete the task, only the emotional Stroop task uses emotional relevance as a distractor. Such emotional

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Effects of drug administration during inhibitory control tasks. Deficits in emotion regulation and inhibitory control in addicted individuals and enhancement of PFC activity by direct drug administration (see above and Supplementary information S2 (table)) together could support the self-medication hypothesis131,132. According to this hypothesis, drug self-administration — and the associated increases in PFC activity — ameliorate the emotional and cognitive deficits that are present in drugaddicted individuals. Such a self-medication effect has previously been recognized by the treatment community, as evidenced by using methadone (a synthetic opioid) as a standard agonist substitution therapy for heroin dependence. In an fMRI study, watching heroin-related cues was associated with less craving during a post-dose than during a pre-dose methadone session in heroinaddicted individuals, with concomitant decreases in cuerelated responses in the bilateral OFC133 (Supplementary information S4 (table)). Empirical support is starting to accumulate for a similar effect in cocaine-addicted individuals. For example, intravenous cocaine (which increases extracellular dopamine levels) in cocaine users improved inhibitory control in a go/no-go task, and this was associated with normalization of ACC activity and enhanced right DLPFC activation during the task134. Intravenous MPH (which also increases extracellular dopamine levels) similarly improved performance on the SSRT in cocaine abusers, and this was positively correlated with inhibition-related activation of the left middle frontal cortex and negatively correlated with activity in the ventromedial PFC; after MPH, activity in both regions showed a trend for normalization 135.

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Stroop designs can potentially further demarcate the altered PFC activity in addiction: is it generalizable to any type of conflict or does it occur specifically during conflicts in a drug-related context? An fMRI study in stimulant users showed attention bias to drug-related words: addicted individuals, but not controls, showed more attention bias to drugrelated words (measured as the median response latency of correctly identified colours of drug-related words minus the median response latency of correctly identified colours of matched neutral words), which was correlated with enhanced left ventral PFC responses. Such responses were not observed for the colour–word Stroop task126. Similarly, drug-related pictures amplified dACC responses to task-relevant information in cigarette smokers127. These findings suggest that in addiction, more top-down resources are needed to focus on cognitive tasks when drug-related cues are present as distractors (thus biasing attention) during the task. Conflicting with these and other results128 are studies in current cocaine users, in which drug-related words were not associated with slower performance or more errors83,129. This disparity could be related to task design or the treatment-seeking status of the study participants; we predict that enhanced conflict between drug-related words and neutral words characterizes those individuals who are trying to abstain from drugs. Evidence for such an effect in cigarette smokers was recently published130.

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Figure 4 | The effect of oral methylphenidate on anterior cingulate cortex activity and function in cocaine addiction. Methylphenidate enhances functional MRI cingulate responses and reduces commission errors on a salient (remunerated cue reactivity) cognitive task in individuals with cocaine addiction. a | An axial map of the cortical regions that showed enhanced responses to methylphenidate (MPH) compared to a placebo in cocaine-addicted individuals. These regions are the dorsal anterior cingulate cortex (dACC; Brodmann areas 24 and 32) and the rostroventromedial ACC (rvACC) extending to the medial orbitofrontal cortex (mOFC; Brodmann areas 10 and 32). The significance levels (T scores) of the activations are colour-coded (shown by the colour scale). b | Correlation between BOLD signal (presented as % signal change from placebo) in the rvACC extending to the mOFC (x = –9, y = 42, z = –6; Brodmann areas 10 and 32) during processing of drug-related words and accuracy on the fMRI task (both are delta scores: MPH minus placebo). The subjects are 13 individuals with cocaine use disorders and 14 healthy controls. Figure is reproduced, with permission, from REF. 215 © (2011) Macmillan Publishers Ltd. All rights reserved.

A PET study showed that oral MPH attenuated the reduced metabolism in limbic brain regions — including lateral OFC and DLPFC — that followed exposure to cocaine-related cues in cocaine-addicted individuals136. It also decreased errors of commission, a common measure of impulsivity, during a drug-relevant emotional Stroop task, both in cocaine-addicted individuals and controls, and in the addicted individuals this decrease

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REVIEWS was associated with normalization of activation in the rostroventral ACC (extending to the mOFC) and dACC; dACC task-related activation before MPH administration was correlated with shorter lifetime alcohol use137 (FIG. 4). Although it remains to be studied whether or how the noradrenergic effects of MPH contribute to its ‘normalizing’ effects in cocaine users, taken together these results suggest that the dopamine-enhancing effects of MPH could be used to facilitate changes in behaviour in addicted individuals (for example, improve self-control), particularly if MPH treatment is combined with specific cognitive interventions. It should be noted that the effect of dopamine agonists on normalizing brain–behaviour responses to emotional or cognitive-control challenges may depend on patterns of compulsive drug use126 or other individual differences, such as baseline self-control and lifetime drug use, but these possibilities remain to be studied in larger sample sizes. Also, non-dopaminergic probes (for example, cholinergic or AMPA receptor agonists) may offer additional pharmacological targets for cocaine addiction treatment 138. In summary, results of studies into inhibitory control in drug addiction suggest that there is dACC hypoactivity and deficient inhibitory control in drug-addicted individuals. Enhanced PFC activity has been reported after short-term abstinence, upon exposure to drug-related cues and to the drug itself (or similar pharmacological agents). However, although drug exposure is also associated with better performance in these cognitive tasks, short-term abstinence and exposure to drug-related cues have the opposite result on task performance. Viewed in the context of the proposed model (FIG. 3), although drugs of abuse offer temporary relief, chronic self-medication with these drugs has long-term consequences — reduced inhibitory control mechanisms and associated emotional disruptions — that may not be alleviated with short-term abstinence, and that are prone to be rekindled upon exposure to drug-related cues. Normalizing these functions, using empirically based and targeted pharmacological and cognitive–behavioural interventions — in combination with the relevant reinforcers — should become a goal in the treatment of addiction.

Disease awareness in addiction The capacity for insight into our internal world (encompassing interoception but extending to higher-order emotional, motivational and cognitive self-awareness) is partly dependent on the PFC. Given the impairments in PFC function in people with addiction reviewed above, it is possible that a restricted awareness of the extent of the behavioural impairment or of the need for treatment may underlie what has traditionally been ascribed to ‘denial’ in drug addiction — that is, the assumption that the addicted patient is able to fully grasp his or her deficits but chooses to ignore them may be erroneous. Indeed, studies have recently suggested that addicted individuals are not fully aware of the severity of their illness (that is, their drug seeking and taking behaviour and its consequences) and this may be associated with deficits in the control network139.

Several studies have provided evidence for a dissociation between self-perception and actual behaviour in addiction. For example, in healthy controls the speed and accuracy of responses for a high monetary condition compared to a neutral cue in a monetarily remunerated forced-choice sustained attention task was correlated with self-reported engagement in the task; by contrast, cocaine subjects’ reports of task engagement were disconnected from their actual task performance, indicating discordance between self-reported motivation and goal-driven behaviour 70. Using a recently developed task in which participants selected their preferred pictures from four types of pictures and then reported what they thought was their most selected picture type91, the discordance between self-report and actual choice — indicating impaired insight into one’s own choice behaviour — was most severe in current cocaine users, although it was also discernible in abstinent users, in whom it was correlated with frequency of recent cocaine use92. An underlying mechanism of this dissociation may be an uncoupling of behavioural and autonomic responses during reversal learning, such as has been shown to occur after OFC lesioning in monkeys140. There is some evidence for similar neural–behavioural dissociations also in humans. In an event-related potential study using the task reported above70, control subjects showed altered electrocortical responses and reaction times in the high-money condition compared to the neutral cue condition, and these two measures of motivated attention were intercorrelated. This pattern was not observed in the cocaine-addicted group, in which the ability to respond accurately to money (that is, the more the behavioural flexibility to this reinforcer), negatively correlated with the frequency of recent cocaine use141. Another study showed that, in a gambling task, control subjects’ choices were guided by both actual and fictive errors, whereas cigarette smokers were only guided by the actual errors that they had made, even though the fictive errors induced robust neural responses142, again pointing to neural–behavioural dissociations in addiction. In the proposed model (FIG. 3), this mechanism is represented by a decreased input from higher-order cognitive control regions to regions that are associated with emotional processing and conditioned responses. Importantly, in humans this neural–behavioural dissociation can be validated by comparing patients’ self-reports with those of informants137 such as family members or treatment providers, or with objective measures of performance on neuropsychological tests143. It is important to remember that self-report measures provide an important glimpse into such dissociations, but given the limitations of self-reports, the development of more objective measures of insight and awareness is crucial for both research and clinical purposes. Two promising measures are error awareness and affect matching. Error awareness in a go/no-go task was found to be reduced in young marijuana abusers and this was associated with reductions in bilateral DLPFC and right ACC, and with greater current drug use144. In methamphetamine-dependent subjects, the bilateral ventrolateral PFC was hypoactive during affect matching and this

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F O C U S O N A DRDEI C ON V ITEI W S Alexithymia A state of deficiency in understanding, processing or describing emotions, including the difficulty in identifying and/ or describing one’s own feelings and externally oriented thinking.

was associated with more self-reported alexithymia145. As better awareness of the severity of drug use predicted actual abstinence for up to 1 year after treatment in alcoholics146, this budding line of research could greatly enhance our understanding of relapse in drug addiction, potentially improving currently available intervention approaches, for example, by targeting addicted individuals who have reduced self awareness for tailored interventions.

Study limitations and future directions The main limitation of this Review is our selective focus on the PFC at the expense of excluding all other cortical brain regions and subcortical structures. The architecture supporting higher-order executive function and top-down control is complex and is thought to involve several functional networks that include, in addition to the PFC, other regions such as the superior parietal cortex, insula, thalamus and cerebellum147. Consequently, and also given the inherent limitations of cross-sectional human neuroimaging studies, attribution of causality should be avoided — that is, the PFC may not directly drive the deficits described in this review. Future meta-analyses in which the disruption of these functional networks in addiction is explored should be imbued with results from mechanistic studies in laboratory animals. A notable issue with many of the reviewed studies pertains to their use of functional ROI analyses that sometimes lack the more stringent statistical corrections of whole-brain analyses. For example, to overcome issues of low power, reported results are sometimes restricted to post-hoc analyses in regions that showed significant results across all subjects to all task conditions; wholebrain analyses of the main (for example, group or type of stimulus) or interaction effects, or of correlations with task performance or clinical end-points, are not consistently performed. Therefore, such ROI results could represent a Type I error but they could also miss the key neural substrates that are involved in the phenomenon under investigation, for example, craving or control of craving. A way to circumvent the limitations of post-hoc analyses is to perform both whole-brain analyses and use a priori defined anatomical ROIs148,149, which could also help to standardize the nomenclature of ROIs across studies. Other common issues pertain to incomplete presentation of the actual data (such as not providing both mean and variance, or not providing scatterplots when reporting correlations), which can obscure the direction of an effect (activation versus deactivation), potentially adding to the variability in published results (for example, a hyperactivation could refer to higher activations or lower deactivations from baseline). In summary, this field would benefit from standardization — of procedures related to imaging, tasks, analyses and subject characterization — that would facilitate the interpretability of the findings. Standardization is also crucial for allowing integration of data sets from various laboratories — such data pooling will be particularly important for genetic studies that are aimed at understanding the interplay between genes, brain development, brain

function and the effects of drugs on these processes. For example, the creation of large imaging data sets are going to be important in understanding how genes that are associated with vulnerability for addiction affect the human brain both after acute and repeated drug exposures. Moreover, the ability to integrate large imaging data sets — as has recently been done for MRI images of resting functional connectivity 150 — will allow a better understanding of the neurobiology of addiction that in the future may serve as biomarker to guide treatment. Although there are a few exceptions (implicating the right PFC, particularly the ACC and DLPFC, in compensatory inhibitory processes) the data reviewed here show no clear pattern indicating lateralization of brain changes in addicted individuals. However, lateralization was not the focus of investigation in any of the reviewed studies. Given that there is evidence for disrupted laterality during finger-tapping in cocaine abusers 151, studies that specifically investigate PFC lateralization in iRISA in addiction are needed. Furthermore, there are clear gender differences in responses to drugs and in the transition to addiction, and imaging studies are increasing our understanding of the sexually dimorphic features of the human brain. However, so far, few wellcontrolled studies have focused on sex differences in the role of the PFC in addiction; instead, many studies use either female or male subjects (mostly males). Studies are also needed to explore the potentially modulating effects of other individual characteristics; of particular interest are the impact of co-morbid disorders (for example, depression may exacerbate deficits in addicted individuals152) and of the recency of drug use and duration of abstinence (for example, cocaine may reduce or mask underlying cognitive153 or emotional154 impairments in cocaine-addicted individuals). Longitudinal studies would enable examination of these issues, which are of particular importance to those who abstain from drugs in the hope that PFC functioning will recover. Furthermore, comparison between different types of abused substances would allow differentiation between factors that are specific to certain drugs from factors that could be common across addiction populations. Instead of treating the heterogeneity of neural and behavioural changes in addiction as noise, studies could explore it with the goal of answering key questions: is PFC dysfunction in iRISA more prominent in certain addicted individuals than in others? Does self-medication drive drug taking more in some individuals than in others? How does co-morbid drug use, which is more the rule than the exception (for example, most alcoholics are nicotine-addicted), affect the neurobiology in addiction? What is the implication of this variability to treatment outcome and recovery? Most importantly, how can we use these laboratory results on the PFC functioning in addiction to inform the design of effective treatment interventions?

Summary and conclusions In general, neuroimaging studies have revealed an emerging pattern of generalized PFC dysfunction in drug-addicted individuals that is associated with more

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REVIEWS negative outcomes — more drug use, worse PFC-related task performance and greater likelihood of relapse. In drug-addicted individuals, widespread PFC activation upon taking cocaine or other drugs and upon presentation of drug-related cues is replaced by widespread PFC hypoactivity during exposure to higher-order emotional and cognitive challenges and/or during protracted withdrawal when not stimulated. The PFC roles that are most pertinent to addiction include selfcontrol (that is, emotion regulation and inhibitory control) to terminate actions that are not advantageous to the individual, salience attribution and maintenance of motivational arousal that is necessary to engage in goal-driven behaviours, and self-awareness. Although activity among PFC regions is highly integrated and flexible, so that any one region is involved in multiple functions, the dorsal PFC (including the dACC, DLPFC and inferior frontal gyrus) has been predominantly implicated in top-down control and meta-cognitive functions, the ventromedial PFC (including subgenual

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ACC and mOFC) in emotion regulation (including conditioning and assigning incentive salience to drugs and drug-related cues), and the ventrolateral PFC and lateral OFC in automatic response tendencies and impulsivity (TABLE 1). Dysfunction of these PFC regions may contribute to the development of craving, compulsive use and ‘denial’ of illness and the need for treatment — characteristic symptoms of drug addiction. This PFC dysfunction may in some instances precede drug use and confer vulnerability for developing substance use disorders (BOX 3). Irrespective of the direction of causality, the results of the neuroimaging studies that are reviewed here suggest the possibility that specific biomarkers could be targeted for intervention purposes. For example, perhaps these PFC abnormalities could be used to identify the children and adolescents who would benefit most from intensive drug abuse prevention efforts, and perhaps medications can ameliorate these deficits and help addicted individuals to engage in rehabilitation treatment.

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210. Lotfipour, S. et al. Orbitofrontal cortex and drug use during adolescence: role of prenatal exposure to maternal smoking and BDNF genotype. Arch. Gen. Psychiatry 66, 1244–1252 (2009). 211. Hill, S. Y. et al. Disruption of orbitofrontal cortex laterality in offspring from multiplex alcohol dependence families. Biol. Psychiatry 65, 129–136 (2009). 212. Alia-Klein, N. et al. Gene x disease interaction on orbitofrontal gray matter in cocaine addiction. Arch. Gen. Psychiatry 68, 283–294 (2011). 213. Wager, T. D., Lindquist, M. & Kaplan, L. Meta-analysis of functional neuroimaging data: current and future directions. Soc. Cogn. Affect. Neurosci. 2, 150–158 (2007). 214. Wager, T. D., Lindquist, M. A., Nichols, T. E., Kober, H. & Van Snellenberg, J. X. Evaluating the consistency and specificity of neuroimaging data using metaanalysis. Neuroimage 45, S210–S221 (2009). 215. Goldstein, R. Z. & Volkow, N. D. Oral methylphenidate normalizes cingulate activity and decreases impulsivity in cocaine addiction during an emotionally salient cognitive task. Neuropsychopharmacology 36, 366–367 (2011). 216. Kringelbach, M. L. & Rolls, E. T. The functional neuroanatomy of the human orbitofrontal cortex: evidence from neuroimaging and neuropsychology. Prog. Neurobiol. 72, 341–372 (2004). 217. Blair, R. J. The amygdala and ventromedial prefrontal cortex: functional contributions and dysfunction in psychopathy. Phil. Trans. R. Soc. Lond. B Biol. Sci. 363, 2557–2565 (2008). 218. Ridderinkhof, K. R. et al. Alcohol consumption impairs detection of performance errors in mediofrontal cortex. Science 298, 2209–2211 (2002). 219. Rajkowska, G. & Goldman-Rakic, P. S. Cytoarchitectonic definition of prefrontal areas in the normal human cortex: II. Variability in locations of areas 9 and 46 and relationship to the Talairach Coordinate System. Cereb. Cortex 5, 323–337 (1995). 220. Petrides, M. Lateral prefrontal cortex: architectonic and functional organization. Phil. Trans. R. Soc. Lond. B Biol. Sci. 360, 781–795 (2005).

Acknowledgements This study was supported by grants from the US National Institute on Drug Abuse (R01DA023579 to R.Z.G.), the Intramural NIAAA program and the Department of Energy, Office of Biological and Environmental Research (for infrastructure support). We are grateful for A. B. Konova’s contribution to the design of figure 2. We are indebted to our reviewers whose comments were greatly appreciated and guided our revision of the original manuscript.

Competing interests statement The authors declare no competing financial interests.

FURTHER INFORMATION Rita Z. Goldstein’s homepage: http://www.bnl.gov/medical/ Personnel/Rita-Goldstein/default.asp The Brookhaven National Laboratory Neuropsychoimaging Group homepage: http://www.bnl.gov/neuropsychology National Institute on Drug Abuse homepage: http://www. nida.nih.gov/nidahome.html University of Colorado CANLab Software website: http://wagerlab.colorado.edu/tools

SUPPLEMENTARY INFORMATION See online article: S1 (table) | S2 (table) | S3 (table) | S4 (table) | S5 (table) | S6 (table) | S7 (table) | S8 (figure) ALL LINKS ARE ACTIVE IN THE ONLINE PDF

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Pharmacogenetic approaches to the treatment of alcohol addiction Markus Heilig*, David Goldman‡, Wade Berrettini§ and Charles P. O’Brien§

Abstract | Addictive disorders are partly heritable, chronic, relapsing conditions that account for a tremendous disease burden. Currently available addiction pharmacotherapies are only moderately successful, continue to be viewed with considerable scepticism outside the scientific community and have not become widely adopted as treatments. More effective medical treatments are needed to transform addiction treatment and address currently unmet medical needs. Emerging evidence from alcoholism research suggests that no single advance can be expected to fundamentally change treatment outcomes. Rather, studies of opioid, corticotropin-releasing factor, GABA and serotonin systems suggest that incremental advances in treatment outcomes will result from an improved understanding of the genetic heterogeneity among patients with alcohol addiction, and the development of personalized treatments. Disability-adjusted life years Also known as DALY. A measure of disease burden, expressed as the number of years lost owing to ill-health, disability or early death.

*Laboratory of Clinical and Translational Studies, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, Maryland 20892, USA. ‡ Laboratory of Neurogenetics, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, Maryland 20892, USA. § Department of Psychiatry, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA. Correspondence to M.H.  e-mail: markus.heilig@mail. nih.gov doi:10.1038/nrn3110

Addictive disorders account for an extensive disease burden, and disproportionately affect people in the prime of their lives. In industrialized countries, alcohol use alone causes about 10% of total disability-adjusted life years lost 1, and a recent evaluation in the United Kingdom concluded that in aggregate, the harm to self and others inflicted by alcohol exceeds that caused by heroin or cocaine2. Alcohol consumption in the population is markedly skewed, and a large proportion of alcoholrelated disability is due to alcohol addiction, hereafter equated with alcoholism. This is a condition that in the United States affects more than 12% of the population at some point in their life3. Alcoholism is a chronic, relapsing disorder that shares many characteristics with other complex chronic conditions, such as diabetes or hypertension: it has a considerable component of genetic susceptibility, is under marked influence of environmental factors, and its onset and course are fundamentally shaped by behavioural choices4,5. This prompts the question of whether alcoholism can be tackled with medical treatments. Some efficacy of medications for alcoholism6 as well as opiate7 and nicotine8 addiction has been documented and supports the feasibility of addiction pharmacotherapy. However, with the exception of methadone or buprenorphin maintenance therapy for opioid addictions, the effect sizes of these treatments are small. Despite evidence-based guidelines that pharmacotherapy be considered in all patients with alcoholism, and in particular in those who are not successfully treated with behavioural interventions alone9, only a small minority of patients receive medication for their alcoholism10.

Clearly, extensive unmet medical needs remain in this therapeutic area. In this Review, we first show that there is considerable heterogeneity among people with alcohol addiction, and that this heterogeneity suggests a need for personalized treatment approaches based on, among other factors, genetic variation. We then review evidence for functional genetic variation within biological systems that mediate positive and negative reinforcement from alcohol — including the opioid and corticotropin-releasing factor (CRF; also known as CRH) systems — and summarize evidence that this variation is likely to moderate treatment effects. An overarching objective of this Review is to point the way for translating the considerable advances recently made in the basic neuroscience of alcohol addiction into therapeutic gains for patients.

‘Alcoholics’ differ from each other A clinical diagnosis of alcoholism is currently made on the basis of diagnostic criteria that are standardized across addictive disorders by the Diagnostic and Statistical Manual of Mental Disorders, which is currently in its fourth edition (DSM IV)11. In the absence of reliable biomarkers, this approach eliminates some of the subjective judgement involved in making diagnoses, and has clinical utility. However, there is reason to believe that patients diagnosed using this approach are markedly heterogeneous. In fact, such heterogeneity was already proposed in the 1980s on the basis of clinical characteristics such as age of onset, but also on family history, which is a marker of genetic susceptibility 12. Numerous other

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F O C U S O N A DRDEI C ON V ITEI W S Box 1 | Alcohol addiction is an end-stage disease ,$-$)".'%/0%$1-")/1#$1.(2%.)"33$)*

Patients ultimately diagnosed with the 4/5%3$1$."6%)"*7 clinical condition labelled as ‘alcoholism’ can 8"39%3$1$."6%)"*7 arrive at this phenotype through very different trajectories (see the figure, which indicates two prototypical examples of such trajectories). In the graph, which is based on a model adapted from REF. 16, the x axis represents time and the y axis shows the level of environmental insult — such as exposure to stressors or consumption of :"*$(*$%;/1$ alcohol — that is required for triggering relapse to the next episode of compulsive !"#$%&'$()*+ alcohol seeking and intake, despite the adverse consequences of drinking alcohol. In the example illustrated by the green curve, progression to alcoholism is driven by a high level of exposure to environmental risk factors in the absence of significant genetic susceptibility. In this type of trajectory, a high level of environmental insult is initially required to trigger an episode of compulsive alcohol use. Over time, however, there is a kindling-like process, in which a progressively lower level of environmental insult is required to trigger the next episode of heavy use, and compulsive alcohol use ultimately becomes self-perpetuating. In the example illustrated by the blue dashed curve, genetic susceptibility can instead be thought to have rendered the subject ‘pre-kindled’, or ‘already there’. For these individuals, even small environmental insults will trigger episodes of compulsive alcohol use early on in the process. Each of the lines shown in the graph should be considered as representative of a class of trajectories, because both genetic and environmental risk factors themselves are diverse. On average, studies find that genetic factors account for 50–60% of disease risk in alcoholism, but multiple risk alleles in different combinations contribute to the genetic risk in each individual case5. This results in genetic vulnerability that can be mediated through traits as different as impulsivity and heightened alcohol reward on one hand, and stress sensitivity and anxiety on the other. Likewise, environmental factors that drive progression to the clinical phenotype of alcoholism can vary, and include stress and prolonged consumption of alcohol, which initially may only be due to easy availability or low cost. As a result of this diversity in genetic and environmental risk factors, patient populations that are grouped based on clinical diagnosis alone are likely to be markedly heterogeneous with regard to underlying biology. This in turn implies that subgroups of ‘alcoholics’ defined by their biology will be amenable to different medical treatments. Understanding the diverse genetic and environmental factors that contribute to the pathophysiology of alcoholism in each individual case will be crucial for personalizing treatment approaches. This theoretical analysis applies in principle to most complex disorders, but recent data show that taking it into account will be particularly crucial both for the optimal use of currently available alcoholism treatments, and to enrich chances for success in developing new ones.

Phenocopy An environmentally determined observable trait (phenotype) that mimics one that is genetic in nature. Frequently, the use of intermediate phenotypes can help to distinguish between phenocopies.

Kindling Originally, the act of setting something on fire. In neurology, a process by which repeated electrical or chemical stimulation, initially of insufficient intensity to initiate a seizure, ultimately leads to a lowering of the seizure threshold and spontaneous seizures.

attempts at clinical subtyping of people with alcoholism have since followed. The use of genetic markers offers the possibility of more reliably and consistently capturing the heterogeneity of people with alcoholism, in ways that are closer to its biological underpinnings. Among individuals in the general population who fulfil diagnostic criteria for alcoholism, the majority — about three-quarters — never receive treatment 3. Available data indicate that those people who go on to enter treatment and those who do not are fundamentally different with regard to personality traits, alcohol use patterns and long-term outcomes13–15. Furthermore, classic longitudinal studies show that long-term outcomes and alcohol-related harm vary markedly between individuals in ways that do not seem to have a simple correlation with participation in treatment or the level of alcohol use13,14.

A clinical diagnosis of alcoholism is probably best viewed as an ‘end-stage disease’, similar to congestive heart failure. In this view, the diagnostic category of alcoholism consists of conditions that are phenotypically similar (or constitute ‘phenocopies’), but patients arrive at the disease state through fundamentally different trajectories. This is captured by a conceptualization that was first put forward for major depression16, but is also likely to apply to addiction (BOX 1). In a kindling-like process, brain exposure to cycles of intoxication and withdrawal induces progressive neuroadaptations that ultimately result in escalation of alcohol intake17,18. In the absence of significant genetic susceptibility, escalation will only result following prolonged exposure to alcohol and the environmental factors with which it interacts, such as stress. By contrast, when genetic risk factors are present, progression can be fast. These individuals can be viewed, in terminology borrowed from the depression literature16, as ‘pre-kindled’, or ‘already there’. Emerging evidence indicates that individuals with alcohol addiction who are on trajectories that are driven by different biological mechanisms or who are in different stages of addiction can be expected to respond to different treatments. Fundamentally, treatments for alcohol addiction must intervene with biological mechanisms that provide motivation for alcohol seeking and consumption19. These mechanisms largely fall into two main categories. First, in a similar way to other drugs of abuse, alcohol can activate brain reward pathways, leading to positively reinforced alcohol seeking and use. Secondly, alcohol can acutely suppress negative emotions that result from stress or withdrawal from alcohol itself, such as anxiety and dysphoria, thus setting the scene for negatively reinforced alcohol use18,20. To highlight the distinction between these two incentives for alcohol use, the terms ‘reward drinking’ and ‘relief drinking’ have been introduced21. It is reasonable to expect that these different types of excessive alcohol use will require different treatments. Alcoholism has a moderate to high heritability, and in part shares genetic susceptibility factors with other addictions5. Genetic and environmental factors in alcoholism can result in very different types of vulnerability, ranging from heightened impulsivity and reward from alcohol to enhanced stress responses and anxious personality traits12. Genetic variants that alter alcohol reward- or stress-related emotional processing are therefore probable modifiers of disease trajectories and of responses to treatments that target reward and stress systems.

Targeting opioid-mediated alcohol reward Alcohol reward is in part mediated by endogenous opioids. Although the exact role of mesolimbic dopamine in addiction remains controversial, activation of this pathway is thought to confer incentive salience to addictive drugs, to ‘reward’ their pursuit or consumption, or to be otherwise related to their addictive properties20,22–24. Accordingly, studies in experimental animals25,26 and humans27,28 have demonstrated that alcohol activates the mesolimbic dopamine circuitry.

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REVIEWS *5+"7"5

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Figure 1 | An alcohol–endogenous opioid–dopamine cascade is the target of naltrexone. Schematic of the alcohol–opioid–dopamine cascade that is thought to be the target of naltrexone, based on integration of circuitries originally proposed in REFS 29,156. Dopaminergic ventral tegmental area (VTA) neurons that project to the nucleus accumbens (NAc) are under tonic inhibition by GABAergic interneurons within the VTA. GABA release from these neurons is in turn under negative regulation by the mu-opioid receptor (MOR). When alcohol is ingested, endogenous opioids such as β-endorphins (β-EPs) are released, resulting in inhibition of GABA release in the VTA and removal of the inhibitory tone from the dopamine cells. This cascade ultimately results in increased dopamine release in the terminal areas in the NAc.

Withdrawal Sudden and complete cessation of drug taking. The term is also used to denote the syndrome that results when drug is withdrawn after dependence, including tolerance to drug effects, has developed.

Cohen’s D A measure of standardized effect size, most commonly used in treatment studies, and defined as the difference between group means divided by the pooled variance. By convention, 0.2, 0.4 and 0.8 or greater are considered to be small, medium and large effect sizes, respectively.

Pharmacogenetics The study of inherited variation in the pharmacokinetic or pharmacodynamic effects of drugs. In addictive disorders, the term is used both for the genetic modulation of psychotropic effects produced by the addictive substance and the modulation of therapeutic effects produced by medications used for treatment.

Non-synonymous A non-synonymous polymorphism is a coding DNA variation that results in altered amino acid sequence.

Single nucleotide polymorphism (SNP). A one-letter exchange of the genetic code, the most common class of genetic polymorphism between individuals.

Dopamine neurotransmission in the corticomesolimbic system is modulated by the mu-opioid receptor (MOR; also known as MOR1). Inhibitory tone from GABAergic interneurons onto dopamine cell bodies in the ventral tegmental area (VTA) is removed through MOR activation on GABA neurons by endogenous opioids, which ultimately results in increased dopamine release in terminal areas in the ventral striatum29,30. The exact mechanism by which alcohol interacts with this circuitry remains unknown. However, studies in experimental animals show that MOR blockade in the VTA largely prevents accumbal dopamine release induced by alcohol intake, indirectly showing that alcohol leads to release of endogenous opioids within this structure and thereby drives dopamine release31 (FIG. 1). Another, independent line of research led to development and approval of the opioid receptor antagonist naltrexone as a medication for alcoholism6 (BOX 2). A synthesis of these two research lines leads to the hypothesis that the mechanism through which naltrexone exerts its therapeutic action is by disrupting the cascade that leads to striatal dopamine release following alcohol intake. However, although a meta-analysis6 supports the efficacy of naltrexone treatment in alcoholism, the average effect size is small, with a Cohen’s D of approximately 0.2. One possible conclusion is that endogenous opioids only play a minor part in alcohol reward and excessive alcohol use, limiting the utility of treatments that target this mechanism. In fact, despite solid evidence for its efficacy, naltrexone has not come into widespread clinical use, and scepticism about its efficacy is one of the reasons given by clinicians10. However, an alternative interpretation of the limited overall effect size of naltrexone is that it reflects heterogeneity of response among patients. In fact, both clinical experience and meta-analyses have long indicated a heterogeneity of naltrexone responses in people with alcoholism, and have implied a possible role of genetic factors in this heterogeneity. For instance, a meta-analysis of available clinical trials suggests that a family history

of alcoholism is associated with clinical improvement in response to naltrexone treatment 32. Support for a role of family history in the clinical response to naltrexone has also been found in laboratory studies; family history influenced both the effect of naltrexone on subjective feelings of a ‘high’ from a standard alcohol dose33 and the level of alcohol self-administration34. Although a role of family history could reflect genetic or environment factors (or both), emerging evidence strongly suggests a major role of pharmacogenetics in the clinical response to naltrexone, as discussed below. Functional variation at the OPRM1 locus as a pharmacogenetic determinant. The possibility of pharmacogenetic heterogeneity in the response to naltrexone is particularly important to consider, because more than a decade ago a common functional variant was discovered in the OPRM1 gene, which encodes the MOR, the target for naltrexone35,36. This non-synonymous 118A→G single nucleotide polymorphism (SNP), rs1799971, encodes an asparagine (N) → aspartate (D) substitution in position 40 of the receptor protein (N40D). The exchange occurs in the amino-terminal extracellular loop of the receptor, and results in the loss of a putative glycosylation site (BOX 2). The frequency of the less common (minor) 118G allele at this locus varies between populations of different ancestry (see below). The precise functional consequences of the N40D substitution for MOR function remain unclear, and its role as a genetic risk factor in addictive disorders is controversial36–41. However, based on a secondary analysis of three clinical trials, it was suggested that this polymorphism might moderate the therapeutic efficacy of naltrexone, and that beneficial effects of naltrexone might be largely restricted to OPRM1 118G carriers42. This finding was subsequently replicated in a secondary analysis of the large, US National Institute on Alcohol Abuse and Alcoholism (NIAAA)-sponsored COMBINE trial, in which naltrexone almost doubled the proportion of patients with a ‘good clinical outcome’ in the group of 118G carriers (from ~50% to ~90%), but had no effect on outcome in 118A homozygous patients43. Although one clinical study failed to replicate this finding 44, a role of OPRM1 variation as a moderator of alcohol reward and naltrexone effects was also supported by results of elegant human laboratory studies45,46. The evaluation of pharmacogenetic factors poses considerable challenges. Unless subjects in clinical trials are a priori recruited and randomization is stratified by genotype, undetected sources of bias may obscure true findings. Drug effects that are restricted to carriers of a minor allele are difficult to detect, because the sample size may simply be too small. Rodent models cannot easily be used to address the role of specific human genetic variants in drug responses, because variants that are functionally equivalent to those found in humans are rarely if ever found in rodents owing to the large phylogenetic distance between these species. Studies in nonhuman primates can be helpful in this regard, because functional equivalents of behaviourally important human variants have frequently arisen in non-human primates47. This is of evolutionary interest in its own

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F O C U S O N A DRDEI C ON V ITEI W S Box 2 | The development of naltrexone as a medication for alcohol addiction Naltrexone was the first centrally acting medication approved for the treatment of alcohol addiction. Its development provides several useful lessons with regard to the type of mechanism that can be targeted for treatment of addictive disorders, the importance of clinical observation and translational research for advancing development of medications, and the role of pharmacogenetics in optimally targeting patient populations. The endogenous opioid systems and their receptors were discovered in the 1970s, and functional analysis of their role was facilitated by the development of two small-molecule competitive antagonists derived from the analgesic opioid oxymorphone: naloxone, which is only bioavailable upon parenteral administration, and naltrexone, which is bioavailable upon oral administration146,147. Naltrexone given to rhesus monkeys suppressed alcohol drinking at doses that did not significantly affect the drinking of water148. Similar findings were subsequently obtained in numerous animal studies149. Influenced by these data, in 1983, researchers at the Philadelphia VA Medical Center obtained approval from the US Food and Drug Administration (FDA) to use naltrexone as an ‘Investigational New Drug’ in actively drinking individuals with alcohol addiction. During dose-ranging, unblinded trials, several patients reported a lack of enjoyment from drinking alcohol while taking naltrexone. In a subsequent placebo-controlled trial150, male veterans in an outpatient treatment programme received counselling and group therapy (using the 12-step methods of Alcoholics Anonymous) and were randomized to receive either 50 mg naltrexone daily or placebo. The dose was selected because it had been used in treatment of heroin addiction, and had been observed to block the high from heroin. Despite the intensive behavioural treatment they received, !"#$%#&'()*+(,#-*.*)/)+0#&1'.*2$#-*1'&,*3#($#4*'/5#3-)+6)+0#7)(4)+#89:$+(4,;#'#-*,<1(# that is fairly typical151. By contrast, relapse occurred in only 23% of patients receiving naltrexone. In addition to the lower relapse rate, patients receiving naltrexone reported less alcohol craving and less reward from alcohol if they did drink. The results from this study were not widely accepted until they were replicated at Yale University by O’Malley and colleagues, who conducted a study in male and female outpatients and obtained very similar results152. Through a series of lucky coincidences, the data from these two academic studies were eventually presented to the FDA, and as a result alcoholism was added to opioid addiction as an indication for the use of naltrexone. Subsequent clinical trials in the United States and other countries found mostly positive results of naltrexone treatment for reductions in heavy drinking, but not necessarily for total abstinence6. Clinical observations indicated that some individuals with alcohol addiction showed no response, whereas others improved dramatically. An effort to identify the characteristics of a naltrexone responder revealed the following factors: a strong family history of alcoholism and self-report of strong alcohol craving153. Meanwhile, it had been reported that subjects who are at genetic risk for alcoholism (that is, ‘non-alcoholics’ with a positive family history of alcoholism) showed a significantly greater plasma endorphin response to alcohol in the laboratory154. The working hypothesis that emerged from these findings was that alcohol can activate endogenous opioid transmission — producing reward via some of the same pathways as heroin — and that the strength of this activation might in part be genetically determined. Recent neuropharmacological and genetic studies have provided support for both parts of this hypothesis.

Allele A specific sequence variant encountered at a given position within the genome.

Polymorphism A common genetic variation (typically considered to be with a frequency >1.0%) within a species.

right, but it also offers a resource for addressing questions of addiction vulnerability and pharmacogenetics in humans (BOX 3). Accordingly, an OPRM1 SNP that is functionally equivalent to the human A118G polymorphism, namely C77G (resulting in a proline (P) → arginine (R) exchange in position 26 of the receptor protein, or P26R amino acid exchange) was identified in the rhesus macaque48. Male carriers of the rhesus OPRM1 77G allele showed increased psychomotor stimulation in response to alcohol, increased alcohol preference and increased frequency of alcohol consumption to intoxication49. Because psychomotor stimulation is a proxy marker of mesolimbic dopamine activity, these findings suggested

that activation of the mesolimbic circuitry in response to alcohol primarily occurs in OPRM1 77G carriers. This prompted the hypothesis that OPRM1 77G carriers would also be preferentially sensitive to suppression of alcohol preference by naltrexone. When this was tested, naltrexone indeed only suppressed alcohol preference in carriers of the 77G variant50, a finding that has been independently corroborated51. Both the rhesus and the human data may have limitations when considered separately, but their convergence supports a role of OPRM1 variation as a moderator of naltrexone effects, in a manner that is very similar for the rhesus and human variants (FIG. 2a,b). Interestingly, in monkeys that were homozygous for the major (OPRM1 77C) allele, naltrexone tended to increase alcohol preference, an effect opposite to that observed in the 77G carriers. This pattern parallels that of a human laboratory study in which naltrexone suppressed alcohol self-administration in individuals with a positive family history of alcoholism, but increased it in people without such a family history 34. These observations highlight that treatments may need to be personalized not only to achieve therapeutic benefits but perhaps also to avoid worsening outcomes in other patients. OPRM1 118G: correlation or causation? Establishing whether the OPRM1 A118G SNP is causal for the functional phenotypes described above is challenging. Because a high degree of linkage disequilibrium is present between numerous SNPs across the OPRM1 locus, their genotypes are highly correlated, and their respective contribution to phenotypic outcomes cannot be easily disentangled in association studies. For instance, one human study found that polymorphisms other than A118G within the same haplotype block were associated with diagnoses of alcohol and drug dependence52. By contrast, a haplotype-based reanalysis of the COMBINE study found naltrexone responses to be specifically attributable to OPRM1 118G53. Furthermore, evidence was recently reported for a functional role of another OPRM1 SNP, rs563649, for pain sensitivity and MOR expression54. This SNP is located in the 5′ untranslated region of the OPRM1 gene, and is strongly associated with the expression of a novel MOR isoform, MOR1K. Although consequences of this variant for alcohol or naltrexone effects have, to our knowledge, not yet been examined, modulation of other opioid-mediated phenotypes by rs563649 suggests that such effects are possible. Because the rs563649 SNP is in strong linkage disequilibrium with other SNPs within the OPRM1 locus, an association between any of those SNPs and clinical naltrexone response could be indirect and be caused by differential expression of the MOR1K isoform. Against this background, combining the non-human primate and human alcohol and naltrexone data reviewed above helps to isolate the influence of OPRM1 77G (in rhesus macaques) and OPRM1 118G (in humans) from that of other functional polymorphisms with which the respective variants might be in linkage disequilibrium. The findings show that the OPRM1 C77G SNP in rhesus macaques and the OPRM1 A118G SNP in humans are directly linked to alcohol reward and the response to naltrexone.

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REVIEWS Box 3 | OPRM1 variation in non-human primates: a model for studying alcohol and naltrexone responses The target for the FDA-approved alcoholism medication naltrexone is the mu-opioid receptor (MOR), which is a seven-transmembrane domain Gi/Go-protein coupled receptor. Its activation by endogenous opioid peptides, such as enkephalins or β-endorphin, results in inhibition of cyclic AMP formation, suppression of intracellular Ca2+ levels and, ultimately, reduced cellular excitability. The dimerization and trafficking of this receptor are not fully understood but seem to be of major importance for the regulation of MOR function, and are in part thought to be related to glycosylation of the receptor protein155. The MOR is highly conserved between humans and non-human primates, and comprises 400 amino acids in both humans and rhesus macaques. In humans, an OPRM1 gene 118A→G mutation encodes an N→D amino acid substitution in position 40 of the receptor protein, resulting in the loss of a putative glycosylation site. A functionally equivalent 77C→G mutation exists in rhesus macaques and encodes an R26P exchange, offering a model system in which effects on alcohol and naltrexone responses have been possible to study47.

Linkage disequilibrium The degree with which a certain combination of alleles at different chromosomal locations is encountered together in a population, in excess of what would be expected by chance alone.

Haplotype block A block or stretch of DNA that encompasses polymorphisms that are in linkage disequilibrium.

Haplotype A combination of alleles at different loci on the same chromosome.

Isoform In relation to proteins, isoforms are different forms of a protein that arise from the same gene.

Reverse translational strategy Applying findings from humans to model organisms. For example, human genetic variants are inserted into a model organism, allowing their functional role to be studied under better controlled conditions.

These links are, however, still correlational. Subsequent studies have obtained direct evidence for a causal role of the human 118G variant in alcohol reward using a translational strategy — perhaps more appropriately termed a reverse translational strategy — in humans and genetically modified mice. First, a positron emission tomography (PET) study was carried out to determine whether alcohol-induced dopamine release in the striatum varies as a function of the OPRM1 A118G genotype in humans55. Displacement of the dopamine-D2 receptor ligand [11C]-raclopride was used to determine endogenous dopamine release. In this approach, a high level of displacement — that is, reduction in [11C]-raclopride binding potential — reflects high dopamine release. In response to an alcohol challenge in social drinkers, evidence for alcohol-induced dopamine release in the ventral striatum (which encompasses the human equivalent of the rodent nucleus accumbens (NAc)) was only detected in 118G carriers, whereas in subjects who were homozygous for the more common (major) 118A allele, the data suggested reduced dopamine release following the alcohol challenge55 (FIG. 3). Paralleling the human PET study, the consequences of A118G variation for alcohol-induced dopamine-release were investigated in two humanized mouse lines, in which the mouse Oprm1 gene was replaced with the human sequence. These two mouse lines carried two identical copies of the human OPRM1 sequence either with an A (OPRM1 118AA) or a G (OPRM1 118GG) in position 118, but were otherwise identical. Following administration of alcohol, brain microdialysis experiments showed a fourfold higher dopamine release in the NAc of the 118GG line compared to the 118AA line, indicating that the OPRM1 118A→G substitution is sufficient to cause elevated alcohol-induced dopamine release in this area55. Using a different targeting strategy, the functional role of the human OPRM1 118A→G SNP was independently studied in another pair of mouse lines. In these experiments, a 112A→G mutation was introduced directly into the genetic background of C57/BL6 mice, resulting in an N→D substitution in amino acid position 38 (N38D) of the mouse MOR that is thought to be functionally equivalent to the human N40D substitution56. No alcohol data are to our knowledge yet available from the N38D model, but functional equivalence of the two mouse models is suggested by other observations. In both cases, introduction of the A→G mutation

in position 118 or 112 resulted in decreased sensitivity to morphine56,57, a seemingly paradoxical phenotype that is also found in human OPRM1 118G carriers58. As already mentioned, it is currently unclear how the N40D substitution that is encoded by the human OPRM1 118G variant modifies MOR function. The mutation seems to be a loss-of-function mutation in terms of its effects on morphine sensitivity 56, but a gain-of-function mutation in terms of its effects on alcohol-induced dopamine release55. The reason for this discrepancy is a most striking issue that awaits resolution. Nevertheless, in both cases, introducing the human MOR variant into mice consistently reproduces the human phenotype — that is, enhanced alcohol-induced dopamine release55 and attenuated sensitivity to morphine58. This suggests that the human OPRM1 118G allele is not only correlated with these effects, but in fact causes them. The human PET data combine with the microdialysis findings from the humanized mouse lines to form a consistent pattern with regard to the effect of OPRM1 118G on alcohol-induced dopamine release. It seems that 118G carriers activate dopaminergic reward circuitry in response to alcohol, and that this activation is mediated through actions of endogenous opioids. Activation of this cascade offers a target for naltrexone on the basis of the idea that naltrexone can inhibit alcohol-induced dopamine release by blocking the MOR upstream of the dopamine neurons. Conversely, the data indicate that administration of alcohol is largely without influence on dopaminergic reward circuitry in 118A homozygotes, and that there is therefore nothing for naltrexone to block in these subjects. Importantly, OPRM1 118G carrier frequencies vary across populations of different ancestry, with evidence for recent positive selection. The frequency of 118G (40D) carriers is less than 1 in 10 among African Americans, about 1 in 3 among most white populations, and about 1 in 2 among individuals of Asian descent59. Some observations of population-specific effects suggest that an individual’s genetic background can modify the effects of the OPRM1 A118G variation. Thus, similarly to subjects with a family history of alcoholism60, white OPRM1 118G carriers showed elevated adrenocorticotropic hormone (ACTH) and cortisol responses to a challenge with the injectable naltrexone analogue naloxone compared to white individuals who are homozygous for 118A. By contrast, no such difference was found among individuals of Asian descent 61,62. It is unclear

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Figure 2 | Efficacy of naltrexone is moderated by OPRM1 variation in rhesus macaques and humans. Results from studies indicating that carriers of the minor 77G (rhesus) or 118G (human) alleles of OPRM1 (which encodes the mu-opioid receptor (MOR)) are more sensitive to effects of naltrexone on alcohol preference and consumption than non-carriers. a | Set-up of an alcohol-preference test in monkeys. Each monkey is tagged by a microchip in its collar. Alcohol is made available for 1 hour daily, 5 days a week. During this time, monkeys can walk up, place their head into one of the several ‘bar’ booths, be identified through the chip being read, and choose between an aspartame-sweetened alcohol solution or a solution of aspartame alone. b | Suppression of alcohol preference by naltrexone as a function of OPRM1 genotype. In rhesus 77G carriers (CG), which have a greater baseline alcohol preference, naltrexone suppressed alcohol preference, whereas in rhesus subjects that are homozygous for the more common 77C allele (CC), naltrexone lacked effect. Data from REF. 50. c | Selective increase in ‘good clinical outcome’ after naltrexone treatment compared to placebo in individuals with alcohol addiction carrying the 118G allele (Asp40), and lack of efficacy in subjects who are homozygous for the more common 118A allele (Asn40). ‘Good clinical outcome’ is a dichotomous composite measure of clinical efficacy that includes abstinence or absence of heavy drinking and improvement with regard to negative consequences of drinking. Figure is modified, with permission, from REF. 43  (2009) American Medical Association.

whether opioid antagonist effects on ACTH and cortisol responses are mechanistically related to therapeutic efficacy in alcoholism, but they have been shown to be biomarkers of clinical naltrexone response63. The differential effects of OPRM1 A118G genotype on naloxoneinduced ACTH and cortisol responses in populations of different ancestry therefore suggests the possibility that variation at this locus may not be equally predictive of

clinical naltrexone efficacy in all populations. However, at least one study in patients of Asian ancestry did find that OPRM1 118G carriers took longer to relapse when treated with naltrexone, whereas no such effect was seen in 118A homozygous participants64. In summary, it seems that the small mean effect size of naltrexone in a mixed patient population is likely to represent a robust effect in the minority of patients who are 118G carriers, and that this effect is diluted by the absence of effects in the remaining patient population43. Expressed differently, a biologically defined population of individuals with alcohol addiction — namely, those individuals who are 118G carriers and therefore have what could be termed ‘endorphin-dependent alcoholism’ (approximately one-third of alcohol-addicted individuals of European ancestry) — stands to robustly benefit from naltrexone, and should receive this treatment. Even before pharmacogenetic tests become widely available in clinical practice, behavioural phenotypes that are characteristic of ‘reward drinking’, such as pronounced psychomotor stimulation by alcohol21, may help to identify patients with a high probability of being responsive to naltrexone. Furthermore, disease progression is likely to be as important to consider as genetic factors in personalized treatments, in that reward drinking is likely to have a greater role in relatively early stages of the addictive process. Patients with the right genetic make-up who, in addition, are in these early stages may therefore be particularly good candidates for naltrexone treatment. Other medications will be needed for individuals with alcohol addiction who are unlikely to respond to naltrexone.

Targeting brain stress systems Brain stress systems and ‘relief-drinking’. The use of alcohol to alleviate social anxiety at a party illustrates the well-known ability of this drug to suppress negative emotional states, such as anxiety or dysphoria. In a clinical context, this ability sets the scene for negatively reinforced alcohol use, an incentive that is clearly distinct from that which is driven by activation of brain reward circuitry. Recent preclinical evidence has pointed to the potential for a vicious circle — or rather a spiral — in which negative reinforcement drives the progressive escalation of alcohol consumption over time. In this process, withdrawal from an episode of heavy intoxication leads to symptoms of anxiety. With repeated cycles of heavy intoxication and withdrawal, this negative emotional state sensitizes — or increases in strength — ultimately resulting in negative emotionality that persists and provides a powerful incentive for resumption of alcohol intake (relapse)65. The dynamics of this process closely parallel the ‘opponent process theory of affective regulation’, which was originally proposed more than three decades ago66 and has subsequently been applied to drug addiction67,68. In this conceptualization, drug use initially engages a group of processes that mediate pleasurable emotional states and therefore drive positively reinforced drug seeking and taking. This triggers an activation of opponent processes in the CNS that mediate negative

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Figure 3 | Dopamine release in the ventral striatum in response to alcohol is restricted to OPRM1 118G carriers. The effect of alcohol on activation of the dopaminergic brain reward circuitry in carriers of the OPRM1 118G allele, as assessed using positron emission tomography (PET) and [11C]-raclopride displacement. Alcohol given to male social drinkers under closely controlled conditions induced a robust dopamine release (detected as reduced binding potential of the radioligand) in minor 118G allele carriers (AG), whereas no measurable release was observed in subjects homozygous for the major 118A allele (AA). The units in the PET scan represent the change in binding potential (nCi ml–1). AVS, anterior ventral striatum; PVS, posterior ventral striatum. Figure is modified, with permission, from REF. 55  (2011) Macmillan Publishers Ltd. All rights reserved.

emotional states, such as dysphoria and anxiety, in an attempt to bring emotional homeostasis back to its normal level. With repeated drug use, the opponent processes increase in strength and duration, and ultimately remain activated. This in turn results in an emotional setpoint shift, or ‘allostasis’, such that negative emotionality is experienced in the absence of the drug and drives negatively reinforced drug seeking and use. A progressive increase in the activity of brain systems that mediate behavioural stress responses is a crucial process behind this allostatic setpoint shift. The transition to negatively reinforced drug use is what has become known as “the dark side of addiction”69. Corticotropin-releasing factor and relief drinking. Extrahypothalamic CRF systems are crucial for the process described above. CRF is a 41 amino acid peptide that is highly expressed within neurons of the hypothalamic paraventricular nucleus (PVN). These neurons release CRF into the portal vein system, which runs along the pituitary stalk and delivers CRF to the anterior pituitary. In the pituitary, CRF acts as the releasing factor for ACTH, which in turn stimulates the release of cortisol from the adrenal glands70. CRF-positive cells are, however, also present in extrahypothalamic structures, including the central nucleus of the amygdala (CeA), the bed nucleus of stria terminalis (BNST) and the brainstem71. These extrahypothalamic CRF cells also release CRF in response to stress and mediate a broad range of behavioural (rather than endocrine) stress responses, primarily through actions at CRF receptor 1 (CRF1; also known as CRFR1 and CRHR1), a seven transmembrane domain G s-coupled receptor of the secretin receptor family 72,73. CRF1 is an attractive therapeutic target because its endogenous ligand CRF is not typically released in extrahypothalamic areas under basal, unstressed conditions. Instead, extrahypothalamic CRF systems become activated in response to sustained, high-intensity, uncontrollable stress74,75, illustrating the

principle that neuropeptides are commonly released at high neuronal firing frequencies and act as ‘alarm systems’76. This suggests that CRF1 antagonists may have little if any in vivo activity unless central stress systems are activated, and would therefore have an advantageous safety and tolerability profile. Animal experiments support this notion74,77. Alcohol withdrawal is a highly stressful state. Rats do not typically self-administer alcohol in amounts that result in physical dependence and in withdrawal symptoms upon cessation of alcohol intake unless they have been genetically selected for high alcohol preference. However, physical alcohol dependence can be induced in rats, for example, by allowing them to consume a liquid alcohol diet or breathe alcohol vapour. Studies in rats made dependent on alcohol in this manner have shown that CRF is released in the CeA during acute alcohol withdrawal and mediates anxiety-like behaviour 78–80. Behavioural withdrawal signs in rats peak after about 12 hours, and are gone within 48 to 72 hours into withdrawal. However, if the alcohol-dependent state is maintained for a month or more (before withdrawal), and in particular if the exposure to alcohol is in the form of repeated intoxication and withdrawal cycles, more persistent behavioural consequences of withdrawal are observed. Under these conditions, behavioural stress sensitivity remains upregulated long after acute withdrawal signs have subsided, and this emotional dysregulation is accompanied by escalated voluntary intake of alcohol18,65. At this stage, the increased behavioural stress sensitivity reflects a shift in responsiveness rather than an increase in baseline anxiety. For example, during protracted abstinence from alcohol, rats with a history of dependence showed no increase in baseline anxietylike behaviour in the elevated plus-maze, a pharmacologically validated animal model of anxiety. However, when these animals were challenged with a stressor before testing, anxiety-like behaviour was markedly accentuated compared to animals without a history of

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F O C U S O N A DRDEI C ON V ITEI W S alcohol dependence. This anxiety-like response to stress was blocked by intracerebroventricular administration of the non-selective CRF receptor antagonist D-Phe CRF12–41 (REF. 81). Similar findings of increased behavioural sensitivity to stress in rats with a prolonged history of alcohol dependence have been obtained using several other models82–86. A parallel of these findings in humans is suggested by the observation that brain responses to aversive visual stimuli, as measured by the blood oxygen level-dependent (BOLD) response, were elevated in patients with alcohol addiction during protracted abstinence. Here, the greatest differences were found in a network of cortical structures, presumably reflecting the different nature of the stressor, species differences, or both87. Based on these and other observations, it has been proposed that repeated cycles of intoxication and withdrawal drive a progression to sensitized stress responses and escalation of alcohol intake, and that upregulated expression of Crfr1 (also known as Crhr1), the gene that encodes the CRF1 receptor, has a major role in this process18,82,88. The CRF system plays a part in relapse. Relapse is a key element of addictive disorders and can be modelled in laboratory animals89. When alcohol self-administration is established in rats and then extinguished over the course of several weeks, relapse to alcohol seeking is reliably triggered by footshock stress, even if there was no prior physical dependence and the animal did not show any withdrawal signs90. Blockade of CRF1 in the brain blocks this stress-induced relapse to alcohol seeking 77,91–93, but the exact neurocircuitry that mediates this activity is not clear. Many anti-stress actions of CRF1 antagonists have been mapped to the amygdala, and this structure, along with the BNST, is also implicated in relapse to drug-seeking 94. In addition, however, blockade of stress-induced relapse is in part mediated by CRF1 blockade in the median raphe nucleus (MRN)95. Projections from the MRN are largely restricted to midline subcortical structures and do not include the amygdala or the BNST96. This suggests that CRF activity may contribute to stress-induced relapse at multiple sites along the complex neurocircuitry that drives this behaviour 94. In addition to stress, relapse can be triggered by exposure to alcohol-associated cues or alcohol itself (‘priming’)89. CRF1 antagonism does not block cue- or priming-induced relapse to alcohol seeking. Conversely, naltrexone blocks relapse induced by both alcoholassociated cues and alcohol priming, but leaves stressinduced relapse unaffected92,97. This suggests that it may be possible to target these two mechanisms in an additive manner for clinical treatment. CRF1 blockade also blocks relapse to alcohol seeking induced by a pharmacological stressor, the α2-adrenergic antagonist yohimbine98. Furthermore, non-selective CRF receptor antagonists or CRF1-selective antagonists decrease the escalated levels of alcohol self-administration that are observed in rats following a prolonged period (a month or longer) of alcohol dependence, and this decrease is mediated through actions in the amygdala77,99,100. The same effect of CRF1 antagonism is seen when escalation

of alcohol self-administration is induced by yohimbine98. Finally, the CRF system may become engaged in earlier stages of the addictive process than previously thought if alcohol consumption occurs in a binge-like pattern101,102. In summary, work in animal models shows that increased activity of the CRF system is associated with both escalated voluntary alcohol intake and increased sensitivity to stress-induced relapse. It can be speculated that different populations of CRF neurons differentially contribute to these behaviours, with the amygdala driving escalated consumption and the BNST and MRN being involved in relapse. However, the precise contribution of these brain structures to the respective effects remains to be determined. Nevertheless, the findings with CRF1 antagonists together suggest that CRF1 is an attractive treatment target for alcohol addiction. Genetic variation within the CRF system and alcoholrelated behaviours in animal studies. We have so far described findings that establish a role for central CRF signalling in escalated alcohol intake and stress-induced relapse once this system has been sensitized through a prolonged history of exposure to cycles of alcohol intoxication and withdrawal. These findings also predict that an individual with innate or stress-induced upregulation of central CRF activity would be at higher risk for escalated alcohol use and stress-induced relapse than an individual who is genetically protected against such an upregulation. By the same token, it would be expected that individuals with upregulated CRF function would be more responsive to CRF1 antagonism as a therapy for alcohol addiction. Data are beginning to emerge in support of these predictions. The first indication that genetic variation within the CRF system might moderate alcohol intake and stressinduced relapse came from experiments with genetically selected Marchigian-Sardinian alcohol-preferring (msP) rats93. These rats have an innate behavioural sensitivity to stress and high voluntary alcohol intake in the absence of prior exposure to alcohol. They are thus partial phenocopies of rats in which the same traits emerge and persist following a prolonged period of alcohol dependence, as described above. A differential gene expression screen revealed that expression of Crfr1 was increased in several brain regions, including the amygdala, of alcohol-naive msP rats compared to alcohol-naive animals of the parental Wistar strain93. The increased expression was accompanied by increased receptor density and signalling, and may be due to a Crfr1 promoter variant that is unique to the msP line. Rats with a history of dependence show similarly increased Crfr1 expression in the amygdala82 (FIG. 4). When msP rats are given free access to alcohol, Crfr1 expression in the amygdala is downregulated to the level of non-dependent, non-selected animals, suggesting the possibility that msP rats drink alcohol to normalize their CRF function103. In addition, both in rats with a history of alcohol dependence and in msP rats, stressinduced relapse is blocked by systemic administration of the prototypical CRF1 antagonist antalarmin in doses that are insufficient to block this behaviour in non-selected animals without a history of dependence93 (FIG. 5).

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REVIEWS " 5%).$%/ )%)2&"/"'."3 $-.

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Figure 4 | Innate or acquired hyperactivity of extrahypothalamic CRF systems is associated with high alcohol preference. a | Schematic localization on a coronal section of the rat brain. The red box indicates the area that approximately corresponds to the subsequent !"# $%&expression panels. b | Low expression of '()(*&in a control rat (not genetically selected for high alcohol preference and without a history of alcohol exposure). c | Markedly upregulated '()(*&expression (darkened areas) in the medial nucleus of the amygdala (MeA) and basolateral nucleus of the amygdala (BLA) of an !"#$#%&#'()*&(&+*&(,*"(#-./"#'(0/)(12,##3"(&/(4!&/-4%*&4!5(6$//'(*$%/+/$($#7#$"8(92,##3"( after exposure to alcohol was terminated. d | Similarly upregulated '()(*&expression in the BLA of a Marchigian-Sardinian alcohol-preferring (msP) rat, observed in the absence of any alcohol exposure. These findings show that increased expression of '()(*&in the BLA can result from a ‘kindling’ process induced by exposure to cycles of alcohol intake and withdrawal, but it can also be an innate trait that is present in the absence of any alcohol exposure, such as in the msP rat line, which has been genetically selected for high alcohol preference. Part a reproduced, with permission, from REF. 157  (2005) Elsevier. Parts b and d reproduced, with permission, from REF. 93  (2006) National Academy of Sciences. Part c reproduced, with permission, from REF. 82  (2008) Elsevier.

Haplotype tagging The concept that most of the alleles and haplotypes (allele combinations) in a particular chromosomal region can be captured by genotyping a small number of markers.

Chromosomal inversion A chromosome rearrangement in which a segment of a chromosome is reversed from end to end. An inversion occurs when a single chromosome undergoes breakage and rearrangement within itself.

These data are consistent with subsequent findings in non-human primates. A screen of genetic variation within the CRF (also known as CRH) gene in rhesus macaques identified a CRF –284C→T SNP. This variant renders hypothalamic CRF expression insensitive to end-product feedback inhibition by cortisol acting at glucocorticoid response elements in the CRF promoter. This means that under stress conditions, when cortisol levels are high, CRF levels remain elevated in 284T carriers and continue to drive a hypercortisolemic state. In contrast to CRF expression in the PVN, CRF expression in the amygdala and the BNST is thought to be under positive regulation by cortisol104, and so the unrestrained activity of the hypothalamus–pituitary–adrenal (HPA) axis in 284T carriers would be expected to result in further upregulation of CRF activity in these structures. As a functional consequence of the continuing high levels of CRF, –284T carriers are predicted to have increased alcohol intake (compared to subjects that are homozygous for the more common allele) following sensitization of the stress systems by sustained and uncontrollable stress,

but to show normal levels of alcohol consumption otherwise. In agreement with this prediction, alcohol consumption in late adolescence and young adulthood in –284T carriers reared under normal conditions did not differ from that of –284C homozygotes with the same rearing history. By contrast, –284T carriers that had been reared under conditions of high adversity had markedly elevated HPA axis responses to stress and increased voluntary alcohol consumption compared to –284C carriers also reared under high adversity 105. Thus, an early life exposure to a sustained stressor seems to set the gain for acute stress responses later in life as a function of CRF –284C/T genotype, and this in turn is linked to the level of voluntary alcohol intake. Human genetic variation within the CRF system and alcohol-related phenotypes. An association between genetic variation at the human CRFR1 gene locus and alcohol-related phenotypes was first reported in the Mannheim Study of Risk Children (MARC), a cohort enriched for individuals who had been exposed to adversity early in life106. At the time of the last assessment, the subjects in this cohort were still in adolescence. After determining haplotype structure based on 14 markers within the CRFR1 gene, the authors selected haplotype tagging SNPs (htSNPs) — rs1876831 and rs242938 — that tag two separate blocks of CRFR1 and examined their association with drinking phenotypes. Both htSNPs were independently associated with binge drinking and lifetime prevalence of intoxication, indicating that variation affecting either haplotype block could influence these behaviours. No evidence for an interaction of the two markers was found106. In the same study, an association of rs1876831 with high alcohol consumption was found in an independent sample of adult individuals with alcohol addiction. Most importantly, a follow-up analysis from the MARC cohort showed that an interaction between adverse life events and rs1876831 influences alcohol-related phenotypes, with the minor (less common) allele being protective107. The latter finding was subsequently replicated and extended in a large independent sample108. Together with several other genes, CRFR1 is located in a large haplotype block on chromosome 17 that may have resulted from a local chromosomal inversion. The minor allele of rs1876831 is within the H2 haplotype at this locus. An Australian–American study examined the possible interaction between genotype at this locus and the effects on alcohol-related behaviour of childhood sexual abuse — a type of adversity known to constitute a risk factor for alcohol use disorders109. More than 1,100 participants in the Australian Nicotine Addiction Genetics project were assessed for alcohol dependence, lifetime alcohol consumption and exposure to childhood sexual abuse. A history of childhood sexual abuse was associated with significantly higher lifetime alcohol consumption and increased risk for alcohol dependence108. Furthermore, childhood sexual abuse was found to interact with the rs1876831 genotype both for measures of alcohol consumption and for a diagnosis of alcohol dependence. Specifically, the presence of the H2 haplotype, which is

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Figure 5 | CRF1 antagonism suppresses stress-induced relapse-like behaviour in msP rats. In the stress-induced relapse model, animals are first trained to establish operant self-administration of alcohol. Once stable self-administration rates are achieved, this behaviour is extinguished by removing alcohol as reinforcer, after which $#7#):.)#""4!5()*&#"('#%$4!#(&/($/,($#7#$"(/7#)(&+#(%/ )"#(/0(*6/ &(;2,##3"(<=-&>?(=-./" )#( &/(*("&)#""/)(@(*(AB2C4! &#(0//&"+/%3(@()#4!"&*&#"()#"./!"#()*&#"(/!(&+#(.)#74/ "$D( alcohol-reinforced lever, even though alcohol continues to be absent. Antalarmin, a corticotropin-releasing factor receptor 1 (CRF1) antagonist, blocks stress-induced relapse-like behaviour in Marchigian-Sardinian alcohol-preferring (msP) rats at doses that are ineffective in rats that are not selected for high alcohol preference. This shows that the CRF1 receptor is crucial for stress-induced relapse, and that the activity of the CRF system is higher in msP rats compared to non-preferring rats. Figure is reproduced, with permission, from REF. 93  (2006) National Academy of Sciences.

Intronic Located in a stretch of DNA between exons; although regulatory elements can reside within introns, genetic variation within introns is often without functional consequences.

Intermediate phenotypes A genetically influenced trait that is less complex and more proximal to the genetic information than the actual behavioural trait of interest, and is informative of the more distal complex trait while being possible to measure with less variance.

selecting patients for CRF1 antagonist treatment. Along the way, this will also be important during clinical development of CRF1 antagonists, because demonstrating their efficacy will be difficult if an effect in a genetically defined subpopulation of subjects is diluted by a lack of effect in other study participants110.

tagged by the minor allele of rs1876831, was protective. In these subjects, childhood sexual abuse exposure was not associated with increase in risk for any of the outcome measures108. An attractive interpretation of these data is that H2 carrier status is protective because it prevents a functional upregulation of CRF system activity that would be caused by exposure to sustained, uncontrollable stress such as childhood sexual abuse, prolonged heavy alcohol use, or both. In studies that have examined populations of European ancestry, about one-third of subjects are carriers of the minor rs1876831 allele that tags the H2 haplotype. A challenge posed by these findings is that none of the markers within the H2 haplotype examined so far seems to be positioned to change the function of CRF1. For instance, rs1876831 is intronic. In fact, because the extended linkage disequilibrium block at this locus encompasses additional genes, it cannot currently be excluded that genes other than CRFR1 account for or contribute to the observed effects. Nevertheless, the hypothesis that patients with alcohol addiction who are not H2 carriers engage the central CRF system under conditions of stress or heavy alcohol use — and would therefore be predicted to respond to CRF1 antagonist therapy — is an attractive one. The animal and human genetic data reviewed above suggest the possibility that pharmacogenetic variation will be found in the response to treatments that target the CRF system. This hypothesis has not yet been addressed in clinical trials, but if it is confirmed, it will be crucial to take CRFR1 genotype into account when

The prospects of CRF1 antagonists as therapeutics for alcohol addiction. CRF1 antagonists were originally developed for the treatment of depression and anxiety. For a long time, the discovery of safe, orally available and brain-penetrant CRF1 antagonists proved challenging. The first such molecule — R121919 — given to humans in an open label, uncontrolled trial in patients with depression seemed promising 111, but the trial was terminated owing to evidence of treatment-emergent hepatotoxicity. Compounds with better properties have since been developed, but trials that tested these compounds for use in the treatment of anxiety and depression have been disappointing 112,113. Failure to take into account the genotype of patients may have contributed to these negative results. As indicated above, the central CRF system is quiescent under non-stressed conditions, and pathological activation of this system may be a feature in some, but not in other cases of depression and anxiety. By contrast, as reviewed above, given sufficient duration of alcohol exposure, the brain CRF system does seem to be consistently activated in animal models. This may make alcohol addiction the most promising indication for CRF1 antagonists. Functional loci at genes other than CRFR1 have also been implicated in the modulation of stress responses and resilience, and could therefore interact with, or act in parallel with, the CRF system to modulate alcoholinduced plasticity of brain stress systems. Putative stress-modulatory functional polymorphisms have been found in the genes that encode FK506 binding protein 5 (FKBP5)114, neuropeptide Y (NPY)115–117, the serotonin transporter (SLC6A4)118 and catechol-O-methyltransferase (COMT)119. It is outside the scope of this Review to describe in detail the role of genetic factors in stress resilience in general. It is, however, noteworthy that although several lines of evidence exist for a stress-modulatory role of these functional loci, findings in traditional case– control association studies have been controversial — this is perhaps best illustrated by the contradictory data regarding the possible interaction between SLC6A4 variation and stress to produce depression120. As pointed out recently 121, this may reflect limitations to approaching hypotheses of gene × environment interactions using association studies alone, and highlights the complementary value of experimental approaches that utilize intermediate phenotypes and animal modelling. This is echoed by the progress made in the case of OPRM1 and CRFR1 using the approaches reviewed above. CRF1 antagonists may become clinically available for the treatment of alcoholism before pharmacogenetic tests become widely available in clinical practice. In a parallel to what has already been stated for the opioid antagonist naltrexone, careful clinical assessment may go a long way towards identifying patients with phenotypes

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REVIEWS conveniently grouped under the ‘relief drinking’ label, who might be particularly good candidates for CRF1 antagonist treatment. Much of the literature reviewed by us also suggests that an important role for relief drinking will be particularly likely in later stages of the addictive process. By extension, it can be expected that patients in those stages of the addictive process will be the most likely to benefit from CRF1 antagonist treatment.

Other neurotransmitter systems With the decreasing cost of genotyping, it will become progressively easier to conduct unbiased genome-wide searches for pharmacogenetic predictors of alcoholism treatment responses. It is, however, important to recognize that this will require stringent statistical thresholds, and this necessitates the collection of very large samples of participants that are consistently recruited, treated and evaluated. In addition, the cost of the clinical studies will remain a challenge. Meanwhile, studies that are designed to target particular genes based on strong biological hypotheses are statistically advisable and potentially fruitful. Two additional neurotransmitter genes that have been implicated in alcohol addiction based on function are 5HT3A (also known as HTR3A), which encodes the ionotropic 5-HT3 receptor for serotonin, and GABRA2, which encodes the α2-subunit of the GABAA receptor. GABRA2 has been implicated on the basis of both its function and previous alcoholism linkage studies. The findings reviewed below are largely exploratory at this point, but are presented to illustrate the general approach and opportunities for translational and experimental medicine for alcohol addiction.

Exon A stretch of DNA that will be represented in the mature, spliced messenger RNA (mRNA).

Synonymous A coding sequence variant that, owing to the redundancy of the genetic code, does not result in an amino acid substitution.

GABAergic transmission and the GABRA2 gene. Among its wide range of CNS effects, alcohol potently influences GABAergic transmission in multiple ways, including modulation of presynaptic GABA release as well as postsynaptic chloride flux. These actions are thought to contribute to some of the subjective effects of alcohol, such as behavioural disinhibition at lower doses and sedation and ataxia at higher doses26. It is therefore perhaps not surprising that the first robust finding of a nervous system-related genetic susceptibility factor in alcoholism was GABRA2, the gene that encodes the α2-subunit of the ionotropic GABAA receptor. Several markers within a haplotype of this gene have in multiple studies been associated with attenuated P300 event-related potentials, an established marker of familial risk for alcoholism. Associations have also been found directly with a diagnosis of alcoholism and with various drinking variables122–127. Experiments in animal models suggest that the effects of alcohol on GABAergic transmission are in part mediated by neuroactive steroids128. An elegant laboratory study set out to examine whether this translates to the human situation129. This study used finasteride, a 5α-steroid reductase inhibitor that blocks the synthesis of several neuroactive steroids. Pretreatment with highdose finasteride potently interacted with the participants’ genotype at markers within the GABRA2 gene to moderate subjective alcohol responses such as stimulation, sedation and desire to obtain more alcohol. Specifically,

subjects were genotyped for rs279858, a SNP marker informative of the GABRA2 haplotype that had been identified as a susceptibility factor in association studies122–127. Subjects who were homozygous for the major A allele at this locus reported markedly higher psychomotor stimulant-like effects on the ascending limb of the blood alcohol concentration curve compared to AG or GG subjects, and this was largely blocked by finasteride. By contrast, finasteride had no effect on the psychomotor response to alcohol in AG or GG subjects129. The moderating effects of this GABRA2 haplotype on subjective alcohol effects have been replicated in an independent sample130. Furthermore, a recent imaging genetics study 131 showed that the same GABRA2 haplotype moderates insula activity during outcome anticipation in a monetary incentive delay task, an established imaging-based measure of brain reward system activation132,133. Thus, genetic variation in GABRA2 seems to influence alcohol reward. The molecular mechanism for the interaction between GABRA2 genotype and alcohol effects is not clear, because none of the markers in the susceptibility haplotype of GABRA2 examined so far seems to be functional. For instance, although rs279858 is located within exon 4 of GABRA2, it is synonymous. If functional polymorphisms in significant linkage disequilibrium with rs279858 can be identified, however, an appealing hypothesis emerges. Psychomotor stimulant effects are highly correlated with activation of mesolimbic dopamine transmission, and dopamine neurons originating in the VTA are under tonic GABAergic inhibition. It can therefore be speculated that GABRA2 variation — or variations that alter the function of one of the other GABAA subunit genes found nearby in the gene cluster in which GABRA2 is located — moderates the ability of alcohol to disinhibit these dopamine cells through effects on GABAergic transmission, ultimately resulting in altered alcohol reward and psychomotor effects. In summary, the work reviewed above provides some additional support for a role of neurosteroids in alcohol responses, a role that has been proposed on the basis of animal studies128. However, these data suggest that if drugs targeting the neurosteroid response to alcohol are developed for therapeutic use in alcoholism, subjects will need to be selected for treatment on the basis of their GABRA2 genotype. In fact, this may also apply more broadly to therapeutics that target dopamine-mediated alcohol reward, as dopamine-mediated alcohol reward is influenced by GABA transmission at VTA synapses. Serotonergic transmission, serotonin transporter gene variation and ondansetron. The development of selective serotonin reuptake inhibitors (SSRIs), such as fluoxetine (Prozac; Eli Lilly), has made the role of serotonergic transmission in psychiatric disorders the subject of great interest, not only among scientists but also among the general public. Because SSRIs have been effective in a surprisingly wide range of conditions, they were evaluated as a potential treatment for alcoholism in multiple trials, but overall were not found to be effective134. Another serotonergic medication, however, yielded promising

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F O C U S O N A DRDEI C ON V ITEI W S results. Ondansetron, an antagonist of the ionotropic 5-HT3 receptor, was reported to reduce heavy drinking in individuals with early-onset alcoholism, a clinical subtype characterized by the onset of the disorder before the age of 25, and often during the teenage years. Secondary analyses indicated that reductions in craving and improvement of mood disturbances might contribute to the reduction in heavy drinking 135–137. The therapeutic actions of 5-HT3-receptor antagonists in people with alcoholism might be due to the presence of 5-HT3 receptors on dopamine terminals in the NAc and their ability to modulate dopamine release138. The serotonin transporter, which is encoded by SLC6A4, is a key element of serotonergic transmission. A variable-length polymorphism in the promoter region of this gene known as 5-HTTLPR (serotonin-transporterlinked polymorphic region) results in differential transcriptional activity 139 and has been extensively studied for association with a wide range of behavioural and clinical phenotypes that are beyond the scope of this Review 121. A recent randomized controlled treatment study showed that individuals who are homozygous for the higher expression (LL) allele of the 5-HTTLPR had a better treatment response to the 5-HT3-receptor antagonist ondansetron, measured both as the mean number of drinks per drinking day and as the percentage of days of total abstinence. Combining the analysis of 5-HTTLPR genotype with a SNP located in the 3′ untranslated region of the serotonin transporter transcript, rs1042173, further strengthened this pharmacogenetic effect140. Additional support for the reduction of drinking by ondansetron among alcohol-dependent individuals with the 5-HTTLPR LL genotype comes from a study that was carried out in non-treatment-seeking volunteers, and used alcohol self-administration under laboratory conditions as a measure of outcome141. Although the exact mechanism mediating these effects remains to be determined, 5-HTTLPR is clearly functional in that it regulates transcriptional activity of the serotonin transporter, whereas rs1042173 might be related to microRNA-mediated regulation of transcript stability 142. Both are therefore well positioned to moderate the effects of a therapeutic acting on serotonergic transmission. Thus, if ondansetron or other 5-HT3-receptor antagonists are developed for the treatment of alcohol addiction, their efficacy should be tested in patients who have been selected on the basis of their genotype at the SLC6A4 locus.

Conclusions Addressing the extensive unmet medical needs related to alcohol addiction will require that novel pharmacotherapies be developed. Numerous mechanisms that could potentially be targeted have been discovered by basic addiction neuroscience, but clinical translation remains a challenge19. Developing therapeutics that target these mechanisms will require a considerable investment, at a time when the willingness of the pharmaceutical industry to invest in drug development for behavioural disorders has diminished143. When searching for ‘blockbuster drugs’ has become the dominant

strategy, tailored treatments that target subpopulations of patients with addictive disorders are particularly endangered. We believe that these challenges should prompt some rethinking in industry, academic institutions and government of the approach to the development of medications for addictive disorders. Small, mechanistic, experimental medicine studies that use intermediate phenotypes as surrogate markers of clinical efficacy have the potential to help guide development efforts, and to make these efforts more cost effective. These experimental medicine studies can use insights from preclinical research to guide their selection of subjects and outcome measures, increasing the probability of detecting a drug effect in limited-size studies. For instance, when developing CRF1 antagonists, it might be beneficial to recruit genetically susceptible, anxious individuals with alcohol addiction, and to measure stress-induced alcohol craving. This type of approach can be adapted to a range of diverse mechanisms, in what has been called a ‘Rosetta Stone’ approach144. We also think that there is reason to rethink the clinical outcomes that are pursued. The search for novel treatments has largely been focused on finding medications that would be effective as measured by their ability to lead to complete abstinence. This is, for instance, the position currently held by the US Food and Drug Administration when evaluating novel addiction therapeutics for approval. However, the science clearly shows that complete abstinence, although desirable, is not the only worthwhile outcome. Even in the absence of complete abstinence, reductions in heavy drinking can have substantial clinical benefits145. We have largely structured our presentation of the available empirical data by neurobiological system. This is convenient for the purpose of a scientific review, but clinical realities are clearly more complex. The pathophysiology of addiction may engage shifting combinations of mechanisms, not only in different individuals but also in different stages of the disease process. In some stages, reward- and relief-drinking-related mechanisms may combine, calling for combination treatment. At other times, one type of mechanism may dominate. In this sense, optimally personalized treatment will always remain a moving target. This prompts the need for developing clinical assessments — ideally based on the use of biomarkers — that will allow treatment to be tailored on an ongoing basis. In conclusion, treatments that on average seem to produce only small improvements in ‘alcoholics’ may result in considerable clinical benefits in subpopulations of patients that are better defined with regard to their biology. We predict that genetic variation will emerge as one of the most important categories of biological factors that will need to be considered in this context, but that awareness of disease progression will also be crucial for improving treatments. Rather than ‘finding a cure’, we look forward to the addition of multiple, appropriately targeted novel treatments that will incrementally improve outcomes and help to reduce the devastating consequences of alcohol addiction.

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137. Johnson, B. A. et al. Ondansetron for reduction of drinking among biologically predisposed alcoholic patients - a randomized controlled trial. JAMA 284, 963–971 (2000). 138. Dremencov, E., Weizmann, Y., Kinor, N., GispanHerman, I. & Yadid, G. Modulation of dopamine transmission by 5HT2C and 5HT3 receptors: a role in the antidepressant response. Curr. Drug Targets 7, 165–175 (2006). 139. Lesch, K. P. et al. Association of anxiety-related traits with a polymorphism in the serotonin transporter gene regulatory region. Science 274, 1527–1531 (1996). 140. Johnson, B. A. et al. Pharmacogenetic approach at the serotonin transporter gene as a method of reducing the severity of alcohol drinking. Am. J. Psychiatry 168, 265–275 (2011). 141. Kenna, G. A. et al. A within group design of nontreatment seeking 5-HTTLPR genotyped alcohol dependent subjects receiving ondansetron and sertraline. Alcohol. Clin. Exp. Res. 33, 315–323 (2009). 142. Seneviratne, C., Huang, W., Ait-Daoud, N., Li, M. D. & Johnson, B. A. Characterization of a functional polymorphism in the 3′ UTR of SLC6A4 and its association with drinking intensity. Alcohol. Clin. Exp. Res. 33, 332–339 (2009). 143. Miller, G. Is pharma running out of brainy ideas? Science 329, 502–504 (2010). 144. Koob, G. F., Lloyd, G. K. & Mason, B. J. Development of pharmacotherapies for drug addiction: a Rosetta Stone approach. Nature Rev. Drug Discov. 8, 500–515 (2009). 145. Johnson, B. A., Ait-Daoud, N., Akhtar, F. Z. & Ma, J. Z. Oral topiramate reduces the consequences of drinking and improves the quality of life of alcohol-dependent individuals: a randomized controlled trial. Arch. Gen. Psychiatry 61, 905–912 (2004). 146. Akil, H. et al. Endogenous opioids — biology and function. Annu. Rev. Neurosci. 7, 223–255 (1984). 147. Snyder, S. H. & Pasternak, G. W. Historical review: opioid receptors. Trends Pharmacol. Sci. 24, 198–205 (2003). 148. Altshuler, H. L., Phillips, P. E. & Feinhandler, D. A. Alteration of ethanol self-administration by naltrexone. Life Sci. 26, 679–688 (1980). 149. Egli, M. Can. experimental paradigms and animal models be used to discover clinically effective medications for alcoholism? Addict. Biology 10, 309–319 (2005). 150. Volpicelli, J. R., Alterman, A. I., Hayashida, M. & O’Brien, C. P. Naltrexone in the treatment of alcohol dependence. Arch. Gen. Psychiatry 49, 876–880 (1992).

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Acknowledgements The authors want to acknowledge many co-workers in their respective laboratories who over the years have contributed to work reviewed here; C.P.O. particularly wishes to acknowledge contributions by D. Oslin. The laboratories of M.H. and D.G. are supported by the intramural programme of the US National Institute on Alcohol Abuse and Alcoholism. W.H.B. is supported by US National Institutes of Health (NIH) grants P20-DA-025995, R01-DA-025201 and P60-DA 05186. C.P.O. is supported by NIH grants P60-DA-005186-23, 5 - P 5 0 - D A - 0 1 2 7 5 6 - 11 , R 0 1 - D A - 0 2 4 5 5 3 a n d R01-AA017164-2.

Competing interests statement C.P.O. declares competing financial interests; see Web version for details. The remaining authors declare no competing financial interests.

FURTHER INFORMATION Markus Heilig’s homepage: http://www.niaaa.nih.gov/ ResearchInformation/IntramuralResearch/AboutDICBR/ LCTS/Pages/default.aspx David Goldman’s homepage: http://www.niaaa.nih.gov/ ResearchInformation/IntramuralResearch/AboutDICBR/LNG/ Pages/default.aspx Wade Berrettini’s homepage: http://www.med.upenn.edu/ ins/faculty/berrett.htm Charles P. O’Brien’s homepage: http://www.med.upenn.edu/ ins/faculty/obrien.htm ALL LINKS ARE ACTIVE IN THE ONLINE PDF

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FOCUS ON ADDICTION

PERSPECTIVES

Abstract | The publication of the psychomotor stimulant theory of addiction in 1987 and the finding that addictive drugs increase dopamine concentrations in the rat mesolimbic system in 1988 have led to a predominance of psychobiological theories that consider addiction to opiates and addiction to psychostimulants as essentially identical phenomena. Indeed, current theories of addiction — hedonic allostasis, incentive sensitization, aberrant learning and frontostriatal !"#$%&'()%*+*,--*,./$0*#).*,*$%(',.!*,&&)$%'*)#* .$/*, (&'()%1234("*5(06*("* challenged by behavioural, cognitive and neurobiological findings in laboratory ,%(7,-"*,% *4$7,%"1280.09*60*,./$0*'4,'*):(,'0*, (&'()%*,% *:"!&4)"'(7$-,%'* addiction are behaviourally and neurobiologically distinct and that the differences have important implications for addiction treatment, addiction theories and future research.

cognitive and neurobiological data from laboratory animals and humans. We first discuss differences in the cognitive and neurobiological effects of opiate and psychostimulant administration. We then review data from animal models of addiction showing behavioural and neurobiological differences between opiates and psychostimulants. Next, we consider selected studies in humans that also point to differences between opiate and psychostimulant addiction. We conclude by discussing how behavioural and neurobiological differences between opiates and psychostimulants may have implications for addiction treatment, addiction theories and future research on drug addiction. We restrict the discussion in this Perspective to differences between opiates and psychostimulants, but our argument that there are substantial differences in the neurobiological mechanisms of these two classes of drugs is also likely to apply to other classes of drugs of abuse, including nicotine, alcohol, cannabis, benzodiazepine and barbiturates.

psychobiological substrates for addiction, across drug classes: incentive sensitization11, aberrant learning 12–14, frontostriatal dysfunction15–17 and hedonic allostasis18 (BOX 1). A unified view is at the core of current clinical definitions of drug addiction19. Unified theories of drug addiction have led to many important discoveries, some of which are described below, but they have also diverted investigators’ attention away from psychological and neurobiological processes that distinguish opiate addiction from psychostimulant addiction. For example, in the mid 1980s, studies using the intravenous drug self-administration (BOX 2) procedure in rats showed that dopamine-receptor blockade or lesions of the mesotelencephalic dopamine system decrease cocaine or amphetamine reward but not heroin or morphine reward20,21 (BOX 3; FIG. 1). The controversy that was stirred by these findings was quickly swept away by the tide of evidence (some of which is discussed below) that was used to support a unitary account of addiction. In this Perspective, our goal is to highlight differences between opiate and psychostimulant addictions, using behavioural,

Cognitive and neurobiological effects Cognitive effects. Addiction is associated with impairments in prefrontal cortex (PFC)dependent cognitive functions; it is thought that these impairments promote compulsive drug use and relapse15,17. Opiate addicts and psychostimulant addicts share some deficits in memory, cognitive flexibility and decision making 22–25. Studies using laboratory animals have shown that repeated exposure to cocaine or heroin impairs spatial memory 26,27 (however, see REF.  28 for different results) and causes transient deficits in attention29,30. These data suggest common neurobiological substrates for opiate- and psychostimulantinduced cognitive impairment. However, there is evidence that indicates that for some cognitive functions, particularly those related to impulsivity (a personality trait that is associated with drug addiction31,32), there are some fundamental differences between opiates and psychostimulants. For example, cocaine and amphetamine addicts are more impulsive and show more pronounced deficits in attention and cognitive flexibility than heroin addicts33–37. These behavioural differences resonate with observations that functional and structural abnormalities in

OPINION

Opiate versus psychostimulant addiction: the differences do matter Aldo Badiani, David Belin, David Epstein, Donna Calu and Yavin Shaham

In 1950, addiction experts in the World Health Organization proposed that drug addiction is fundamentally characterized by psychic dependence, independent of drug class1. Subsequently, early psychobiological theories identified common denominators of addiction in phenomena such as psychic tolerance (the presumed cause of escalating drug intake) and psychic withdrawal or abstinence agony (the presumed main obstacle to abstinence)2–4. In the 1970s and 1980s, building on the discovery that electrical stimulation of specific brain areas can induce reward5, investigators proposed that the mesotelencephalic dopamine system is the neurobiological substrate for the rewarding effects of both opiates (for example, heroin and morphine) and psychostimulants (for example, cocaine, amphetamine and metamphetamine)6,7. The same system was also implicated in the motivational effects of drug-associated cues8 and in the development of psychomotor sensitization to addictive drugs9. These neuropharmacological developments were the basis for the influential 1987 psychomotor stimulant theory of addiction10 (BOX 1), as well as for subsequent theories that emphasize shared NATURE REVIEWS | NEUROSCIENCE

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PERSPECTIVES the prefrontal cortex are less pronounced in heroin addicts than in cocaine addicts38. It is unclear whether differences between heroin and cocaine addicts are due to drug use or pre-existing differences. Studies in laboratory animals, however, support the former possibility, and they specifically indicate that opiates and psychostimulants have different effects on impulsivity. Withdrawal from cocaine self-administration impaired inhibitory control in both rats39 and non-

human primates40, whereas increased impulsivity was not observed in rats after withdrawal from heroin29. Furthermore, non-contingent experimenter-administered cocaine and amphetamine41 increased impulsivity in rats, whereas heroin did not 42. In conclusion, in both humans and laboratory animals, chronic exposure to psychostimulants seems to cause more pronounced deficits in impulse control and cognitive flexibility than chronic exposure to opiates.

Box 1 | Theories of addiction Over the past decades, several classes of addiction theories have been proposed by animal behaviour researchers and clinical researchers. Below, we describe some of these theories, which over the past two decades have had a substantial influence on the direction that drug addiction research has taken.

Aberrant-learning theories of addiction These theories propose that repeated exposure to addictive drugs heightens Pavlovian and instrumental responsiveness to drug-associated cues through actions on neurons that control normal responses to non-drug conditioned cues; these actions may occur in the ventral striatum12, dorsal striatum14 or both13. A main theme of these theories is that the heightened responsiveness to drug cues is insensitive to outcome devaluation (for example, punishment), leading to continued drug use even when it has adverse consequences227,228. This aberrant learning process has been suggested to be mediated by a progressive dopamine-dependent ventral-to-dorsal striatal shift in control over drug seeking and drug taking227,228. Frontostriatal-dysfunction theories of addiction These theories propose that repeated exposure to addictive drugs causes deficits in top-down executive control over behaviour15–17, leading to loss of impulse control, impaired decision-making processes, exaggerated responsiveness to drug-associated cues and compulsive drug use despite adverse consequences. This idea was first advanced by Jentsch and Taylor15, who proposed that compulsive drug use is due to drug-induced alterations in cortical and limbic circuits, leading to exaggerated responses to drugs and drug-associated cues (owing to nucleus accumbens and amygdala dysfunction) and impaired inhibitory control (owing to medial prefrontal and orbitofrontal cortex dysfunction). Hedonic-allostasis theory of addiction A theory that is based on the opponent-process theory of motivation4; it proposes that although initial drug use is primarily controlled by the drug’s rewarding effects, chronic drug use leads to decreases in its rewarding effects and to recruitment of stress-related systems. This leads to a new emotional state, termed the ‘hedonic allostatic’ state, which represents a chronic change in the normal reward setpoint18. According to this theory, hedonic allostasis causes loss of control over drug use through cortico–striatal–thalamic circuits that are involved in compulsive behaviour. Incentive-sensitization theory of addiction This theory has three main components: first, the idea that addictive drugs increase mesocorticolimbic dopamine neurotransmission; second, the idea that one psychological function of this brain system is to attribute incentive salience to contexts, cues and other events that are associated with activation of this dopaminergic system; and third, the idea that repeated exposure to addictive drugs produces long-lasting adaptations in this neural system, rendering it hypersensitive to drugs and drug-associated cues11. Incentive salience is defined as a psychological process that increases the valence of reward-associated cues and makes them attractive incentive cues. Robinson and Berridge11 also argued that sensitization of neural systems that mediate incentive salience (drug ‘wanting’) occurs independently of changes in neural systems that control pleasurable effects of drugs (drug ‘liking’). They also suggested that motivation systems that control incentive salience are independent of those controlling drug withdrawal states. Pschomotor-stimulant theory of addiction This theory proposes that a common denominator of addictive drugs is their ability to cause psychomotor activation10. The theory is rooted in an earlier theory that all positive reinforcers activate a common biological mechanism that is associated with approach behaviours229. Wise and Bozarth10 put forward the idea that the major substrate of the approach system (and of psychomotor sensitization) is the mesocorticolimbic dopamine system. They also argued that drug withdrawal symptoms, which are drug-class dependent, do not play a major part in controlling compulsive drug use.

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Neurochemical and neurophysiological effects. Opiates and psychostimulants have very different pharmacodynamic profiles10,12,43 but they share the ability to increase dopamine levels in the nucleus accumbens (NAc)7 — one of the terminal regions of the mesocorticolimbic dopamine system — and this increase plays an important part in the rewarding effects of drugs and non-drug stimuli44,45. Psychostimulants do so by blocking dopamine reuptake or inverting dopamine transport 43, whereas opiates indirectly activate dopaminergic neurons in the ventral tegmental area (VTA) — the cell-body region of the mesocorticolimbic dopamine system — through inhibition of GABAergic interneurons46,47. Such similarities in the neurochemical effects of opiates and psychostimulants help to explain why these drugs produce similar effects on neuronal activity in the NAc and in the medial PFC (mPFC) — another terminal region of the mesocorticolimbic dopamine system that has been implicated in the behavioural and cognitive effects of addictive drugs31,48 and in relapse to drug use (see below). In vivo extracellular recording has been used to show that small populations of neurons in these regions are in fact either excited or inhibited during heroin or cocaine self-administration in the rat 49,50. However, when neural activity was assessed using multiple-channel single-unit recordings in rats that consecutively self-administered heroin and cocaine (in the same session), only a small number of drug-responsive neurons (~20%) in the mPFC and NAc showed similar responses to both drugs51. Thus, the rewarding effects of heroin and cocaine seem to be encoded by distinct neuronal subpopulations. Neuroadaptations. Since the early 1990s52,53, a central neurobiological framework for addiction research has been that compulsive drug use and relapse are due to drug-induced neuroadaptations in the mesocorticolimbic dopamine system and in the glutamatergic corticolimbic circuitry in which the dopamine projections are embedded54–56. An implicit assumption has been that the neuroadaptations are independent of drug class11,54, and indeed, some are. For example, both opiates and psychostimulants induce changes in intracellular signal transduction pathways in the mesocorticolimbic dopamine system54,57, induce long-term potentiation (LTP) of glutamatergic synapses in the VTA58–60 impair LTP in the bed nucleus of stria terminalis (BNST)61, and cause sensitization of dopamine and glutamate transmission in the

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F O C U S OPNE RASDPD E ICCTTI IVOEN S terminal regions of the mesotelencephalic dopamine system52,62. In addition, withdrawal from both opiates and psychostimulants is associated with short-term decreases (a few days) in NAc dopamine levels63. However, there are also notable neurobiological differences, which are discussed below. One difference concerns drug-induced synaptic plasticity. Studies using ex vivo whole-cell electrophysiology have shown that morphine and cocaine differ in their ability to induce LTP and long-term depression (LTD) at GABAergic synapses (LTPGABA and LTDGABA) on VTA dopamine neurons. Morphine exerts bidirectional control on such synapses: a single non-contingent injection of morphine in rats abolished both LTPGABA and LTDGABA64,65 in brain slices. By contrast, cocaine seems to downregulate the strength of such synapses: in rats, a single injection of cocaine had only modestly attenuating effects on LTPGABA65, and repeated injections occluded endocannabinoiddependent LTD66, suggesting an induction of an LTD-like state by cocaine in vivo. Additional differences in synaptic plasticity between opiates and psychostimulants are seen in the consequences of drug withdrawal on LTP in the mPFC. Facilitation of LTP in the mPFC of rats occurred after withdrawal from repeated cocaine exposure67,68. By contrast, withdrawal from heroin selfadministration in rats had no effect on LTP as measured by the AMPA:NMDA ratio in the mPFC69. Furthermore, exposure to cues that had previously been associated with heroin intake reduced the AMPA:NMDA ratio in this area, suggesting decreased LTP69. These discrepancies should be interpreted with caution, however, as there were several important experimental differences between the studies, including the age of the rats, the route and type of drug administration (self-administration versus experimenter-delivered), the length of the withdrawal period, the electrophysiological end-points and the mPFC subregions. Nevertheless, the LTP results suggest that exposure to opiates and exposure to psychostimulants can cause qualitatively different changes in mPFC synaptic plasticity. A second notable difference between opiates and psychostimulants concerns their effects on structural plasticity. In 1997, Robinson and Kolb70 found that repeated non-contingent injections of amphetamine in rats induce persistent increases in dendrite branching and spine density in NAc medium spiny neurons and mPFC layer III pyramidal neurons. These findings were extended to cocaine and amphetamine selfadministration71,72. By contrast, morphine

Box 2 | Animal models of drug reward, subjective effects and relapse For many decades, investigators have used animal models to assess the behavioural effects of abused drugs that are potentially related to their effects in humans186,230–233. Below, we describe the main animal models that are currently used by addiction researchers to study the positive reinforcing (or rewarding) effects of drugs, the subjective effects of drugs and relapse to drug seeking.

Conditioned place preference (CCP) model A Pavlovian (classical) conditioning model in which during the training phase one distinct context is paired with drug injections and another context is paired with vehicle injections. During the subsequent testing phase (which is drug-free), the animal’s preference for either context is determined by allowing the animal to move between the two contexts. An increase in preference for the drug-associated context serves as a measure of the drug’s Pavlovian reinforcing (or rewarding) effects. Intravenous drug self-administration model In this model, animals typically make a lever press or nose poke to receive contingent drug injections. The premise of this procedure is that drugs of abuse control behaviour by functioning as operant positive reinforcers. Reinstatement model An animal model of relapse to drug seeking. In the operant-conditioning version of this model, laboratory animals are first trained to self-administer drugs by making a lever press or nose poke, with drug injections typically paired with discrete cues (for example, a tone or a light). Subsequently, the animals undergo extinction training, during which lever presses (or nose pokes) are not reinforced with drug. Reinstatement of lever pressing (or nose pokes) under extinction conditions is then determined after manipulations such as non-contingent priming injections of the drug, exposure to discrete or contextual cues that are associated with drug intake, or exposure to stressors. In the classical-conditioning version of the model, CPP is induced by a drug, extinguished and then induced again by drug priming injections or stressors. Runway model In this operant-conditioning model, the speed with which a laboratory animal traverses a long, straight alley for a positive reinforcer (for example, food or a drug) provides an index of the animal’s motivation to seek the reinforcer. The dependent measure in this model is the run-time from a start box to the goal box in which the positive reinforcer (or the reward) is earned. Drug-discrimination model An animal model of the subjective effects of drugs. In this model, laboratory rodents or monkeys are trained to discriminate between a drug state and a non-drug state, or between different drug states. In a typical experiment, a food-restricted animal is trained in a two-lever operant chamber in which the food-reinforced lever differs as a function of whether drug or saline was administered before the session. After achieving a training criterion of correct responding, subjects are typically injected with various doses of the training drug to generate dose-response curves or injected with other drugs from the same or different drug classes to test for stimulus generalization.

self-administration had the opposite effect; it causes long-lasting decreases in the complexity of dendritic branching and in the number of dendritic spines in NAc and mPFC71 (FIG. 2). These opposing effects might be explained by the differential engagement of the direct and indirect striatal pathways73, as indicated by changes in expression of immediate early genes such as FBJ osteosarcoma oncogene (Fos)74–76. In neurons of the direct pathway, both opiates and psychostimulants increase the expression of Fos 74–76. In neurons of the indirect pathway, only psychostimulants increase Fos expression, whereas opiates (in this case, morphine) reduce it 74–76. This differential regulation of Fos expression is potentially important because repeated drug-induced FOS (the protein product of the Fos gene) induction in the NAc leads to the formation of a more stable

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form of the FOS protein called ΔFOSB77, which plays a major part in drug-induced neuroadaptations in the striatum54, including the regulation of dendritic branches and spines78,79. Lastly, a post-mortem study in cocaine and heroin addicts supports the idea that chronic exposure to these drugs leads to dissociable neuroadaptations: out of approximately 39,000 gene transcripts that were investigated in the NAc, only 25 genes showed changed expression in both cocaine and heroin abusers and in nearly half of these cases, the drugs had opposite effects on expression80. A question for future research is whether opposing cocaine- and heroin-induced changes in neuronal morphology and gene expression help to explain the drugs’ differing behavioural effects in animal models, which are described below. VOLUME 12 | NOVEMBER 2011 | 687

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PERSPECTIVES Animal models Drug addiction is not an automatic outcome of drug use. Only approximately 20% of people who use addictive drugs will switch from controlled to compulsive use81. Thus, one of the aims of modelling drug addiction in the laboratory is to identify the mechanisms that are responsible for the transition from one stage of the disorder to the next: from initial drug use to chronic drug use and then to compulsive, relapsing drug abuse. Vulnerable individuals often exhibit distinct personality traits or psychiatric profiles that are thought to facilitate this transition82. Not surprisingly, this vulnerability seems to be influenced not only by genes but also by environmental factors83, including adverse life experiences (especially in childhood), acute exposure to stressors, drug-associated contextual and discrete cues (acting as conditioned stimuli), and other, more subtle aspects of the environment 84. Indeed, the behavioural and subjective effects of addictive drugs should be seen as the result of complex interactions among the drug, the user’s physiological and mental state (also referred to as ‘set’), and the circumstances of drug taking (referred to as ‘setting’)84–86. Current animal models of drug addiction can help to clarify how drug, set and setting interact to produce the transition from controlled to compulsive drug use84. Under certain conditions, these models reveal

important differences between opiates and psychostimulants. Vulnerability to initial drug use. There are several similarities between the initiation of opiate self-administration and the initiation of psychostimulant self-administration. At the most fundamental level, many studies that use intravenous drug self-administration (the gold-standard procedure for assessment of abuse liability) show that agonists of both drug classes are self-administered by rodents and monkeys87–89. In addition, food restriction strongly facilitates the acquisition of self-administration of both opiates and psychostimulants90, as do other environmental stressors, under certain conditions91,92. Furthermore, repeated non-contingent administration (which causes psychomotor sensitization) facilitates the acquisition of self-administration of both opiates and psychostimulants, as well as inducing conditioned place preference (CPP)93–95(BOX 2). Moreover, for both drug classes, the speed with which self-administration is acquired can be predicted by certain behavioural traits, including high preference for sweet solutions96 and high locomotor response to a novel environment (which is thought to model novelty seeking; a psychological trait that is associated with the initiation of drug use in humans)97–99. Furthermore, for both drugs, inter-individual differences in the

Box 3 | Does dopamine mediate opiate reward? It is well established that the rewarding effects of psychostimulants are mediated by dopamine projections from the ventral tegmental area (VTA) to the nucleus accumbens (NAc); evidence for this is seen in both the self-administration and the conditioned place preference (CPP) models234–237. However, although addiction and neuroscience experts, the popular press and the public commonly assume that this dopamine projection is also crucial for the rewarding effects of opiates, the literature does not support this general conclusion. On the one hand, there is evidence that opiate drugs activate VTA dopamine neurons47 and increase NAc dopamine release7. There is also evidence that fluctuations in NAc dopamine levels correlate with heroin self-administration behaviour238. In addition, morphine and other opiate agonists are self-administered directly into the VTA or the NAc and produce CPP following intracranial injections into either site239–243. Lastly, there is some evidence that morphine or heroin CPP is blocked by systemic or NAc injections of dopamine receptor antagonists236,244,245. On the other hand, the CPP findings are not consistent across studies: 6-hydroxydopamine (6-OHDA) or excitotoxic lesions of the NAc seem to have no effect on CPP for morphine246,247, and dopamine receptor blockade decreases heroin CPP in heroin-dependent rats but not in non-dependent rats248,249. Self-administration data pose an even greater challenge to the theory of a unified, dopamine-based mechanism of drug reward. There is surprisingly little empirical evidence that dopamine transmission is crucial for self-administration of opiates. For example, systemic injections of dopamine receptor antagonists have minimal effect on self-administration of opiate agonists in rats and monkeys, unless the agonists are administered at doses that are high enough to be sedating20,213,250,251 (FIG. 1). In addition, self-administration of heroin or morphine is only minimally reduced by NAc disruptions such as 6-OHDA lesions or local injections of dopamine receptor antagonists250,252–254 (FIG. 1). Finally, chronic blockade of dopamine receptors with a-flupenthixol strongly potentiates, rather than inhibits, the rewarding effect of low heroin doses255. !"#$!#%&'($!)"*'"'+*+,-"./01,*2'"*3$"41"5,%%$"*!-"6,3&'213, these data and related results “argue against a prominent role for dopamine in opioid self-administration.” Research that has been performed since then has not led to new empirical evidence that can be used to refute this conclusion.

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propensity to acquire self-administration are associated with inter-individual differences in the extent to which drug-induced dopamine release in the NAc is modulated by the stress hormone corticosterone (through its actions on glucocorticoid receptors in the VTA and the NAc for opiates and psychostimulants, respectively)100,101. However, there are several fundamental differences in the behavioural effects of opiates and psychostimulants. For example, in rats, exposure to cocaine causes an approach–avoidance conflict towards places that are associated with injection of the drug, whereas exposure to heroin does not. Studies that use a runway model (BOX 2) suggest that intravenous heroin induces an appetitive incentive motivational state that causes an approach behaviour, similar to that induced by palatable food in hungry rats102,103. By contrast, intravenous cocaine induces a motivational state with both an appetitive and an aversive component, leading to approach–avoidance behaviour similar to that caused by simultaneous exposure to food and shock in hungry rats104,105 (FIG. 3a). The mixed motivational state that is induced by cocaine is also observed in the CPP procedure: immediate intravenous cocaine administration (within the 5 min preceding a CPP training session) causes place preference, but delayed cocaine administration (15 min before a training session) causes place aversion106. These data from rats seem consistent with human epidemiological data that show an association between cocaine use and anxiety disorders107,108, and with human laboratory studies in which immediate self-reports of a cocaine high were followed by delayed (~8 min) negative affect-related self-reports of anxiety, paranoia, dysphoria or anhedonia109. A second example is that sex hormones seem to have different effects on the initiation of cocaine self-administration and the initiation of heroin self-administration. Female rats acquire cocaine self-administration faster than males; this sex difference is mediated by ovarian hormones110,111. For heroin, evidence for a sex difference is mixed. Two studies have shown that intact female rats acquire heroin self-administration faster than males96,112. By contrast, a study in which male rats were compared with intact females and with ovariectomized females that were given hormonal replacement found neither sex differences nor a role of ovarian hormones in the acquisition of heroin self-administration113. What might account for the mixed results? One issue to consider is that the rats in the ‘positive outcome’ studies received non-contingent

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F O C U S OPNE RASDPD E ICCTTI IVOEN S

Transition to compulsive drug use. Researchers have attempted to model in animals the loss of control over drug intake that characterizes addiction in humans19. One approach is to give rats prolonged (for example, 6 h per day)115,116 or unlimited daily access to the drug 117–119. Under such conditions, most rats progressively escalate their drug intake, a phenomenon that is not observed in rats with limited drug access (for example, 1 h per day)115,116. Compulsive drug use has also been modelled in laboratory animals by imposing negative consequences on drug seeking and drug taking 120. These include adding quinine (a bitter, aversive substance) to rewarding alcohol solutions121, administering shock to punish drug-taking or drug-seeking responses122–124, or exposing rats to fear-inducing conditioned cues that would normally inhibit operant responding 125. Under these circumstances, some of the rats persist in drug seeking or drug taking. These models show several similarities between heroin and cocaine, at least at the behavioural level29,115,121,126. There are, however, important differences. For example, in rats that are given prolonged drug access, escalation of heroin self-administration does not predict escalation of cocaine self-administration, and vice versa127. In addition, extended access to heroin selfadministration is associated with increased resistance to extinction (that is, after extended access, animals show more persistent attempts to obtain the drug when it is no longer available), whereas this is not the case for cocaine self-administration115. Another well-established difference is that rats that are given unlimited access to opiates

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injections of heroin before the daily sessions96,112, whereas the rats in the ‘negative outcome’ study did not113. Repeated non-contingent drug exposure is known to produce psychomotor sensitization and to facilitate the acquisition of drug self-administration94,95. As female rats develop faster morphine-induced psychomotor sensitization114, the observed sex differences in heroin intake in the ‘positive’ studies may simply be attributable to the experimental procedures. Taken together, results from studies on the initiation of cocaine and heroin self-administration in rats suggest that initial cocaine exposure induces a mixed approach–avoidance motivational state that is not observed with heroin, and that sex differences and ovarian hormones may play a more prominent part in the initiation of cocaine self-administration than in the initiation of heroin self-administration.

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gradually increase drug intake, whereas rats that are given unlimited access to psychostimulants cycle between days of binge intake and days of markedly reduced intake118,128. The consequences of this difference were shown in a study in which cocaine- and heroin-trained rats were given unlimited access to their drug: the heroin-trained rats gradually increased their intake over days

NATURE REVIEWS | NEUROSCIENCE

and then maintained stable intake, whereas the cocaine-trained rats rapidly lost control over intake and, within 12 days, died of overdose117 (FIG. 3b). A third difference is that social defeat promotes escalation of psychostimulant self-administration but not opiate selfadministration. Social-defeat stress (intermittent exposure to a dominant male) VOLUME 12 | NOVEMBER 2011 | 689

© 2011 Macmillan Publishers Limited. All rights reserved


PERSPECTIVES promotes the development of psychomotor sensitization to both opiates and psychostimulants129; this effect is mediated by VTA NMDA receptors130, which control mesolimbic dopamine activity 131. However, social-defeat stress facilitated the progression to binge-like drug intake in rats that had unlimited access to cocaine but not in those that had unlimited access to heroin132. This finding suggests a dissociation between psychomotor sensitization and drug-taking behaviour for opiates but not for psychostimulants. The difference between cocaine intake and heroin intake in the social-defeat paradigm might reflect the differential roles of the mesolimbic dopamine system in heroin self-administration versus cocaine self-administration20,21 (BOX 3). Lastly, escalation of cocaine self-administration is predicted by high trait impulsivity, whereas escalation of heroin self-administration is not. As described earlier, cocaine and heroin have different effects on the expression of impulsive behaviours in rats. There is also evidence that trait impulsivity, which can be assessed before drug self-administration, predicts escalation of cocaine but not heroin intake. Thus, high trait impulsivity,

reflected in premature responses in a fivechoice serial reaction-time test, predicted escalation of cocaine self-administration but not heroin self-administration29,133 (FIG. 3c). In the cocaine study, trait impulsivity was also associated with low expression of D2 dopamine receptors in the NAc133. This indicates that the observed differences between the effects of cocaine and heroin may be yet another illustration of the differential role of the mesolimbic dopamine system in psychostimulant but not opiate self-administration (BOX 3). Taken together, data from animal models indicate that when rats are given unlimited access to psychostimulants, they develop uncontrolled binge intake behaviour that is not seen in rats that are given unlimited access to opiates. In addition, prior exposure to social stress promotes escalation of cocaine self-administration but not heroin self-administration. Lastly, trait impulsivity predicts escalation of cocaine intake but not heroin intake. Drug seeking and relapse. In humans, drug craving and relapse during abstinence are often triggered by acute re-exposure to the

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Figure 2 | Morphine and cocaine have opposite effects on structural neuroplasticity in the NAc and mPFC. a$Y$R */-#$*.$ !"#$%& &$" !'(&)$"*$#&+.=!)5'('#"& $5* -0'(&$* $9*9!'(&$'(" !,&(*/#+E$ .* $#&,& !+$%&&7#3$Q0&$9*(" *+$6 */-#$%& &$6',&($)!'+E$'(" !,&(*/#$'(./#'*(#$*.$,&0'9+&$.* $"0&$#!5&$ -& '*)$*.$"'5&3$A."& $>J5*("0$*.$%'"0) !%!+$. *5$"0&$) /6#C$"0&$ !"#Z$B !'(#$%& &$- *9&##&)$/#'(6$"0&$ R*+6'$#"!'('(6$- *9&)/ &3$@!"#$"0!"$%& &$&<-*#&)$"*$9*9!'(&$#0*%&)$'(9 &!#&)$)&() '"'9$B !(90'(6$ !()$'(9 &!#&)$#-'(&$)&(#'"E$'($B*"0$(/9+&/#$!99/5B&(#$1LA98$5&)'/5$#-'(E$(&/ *(#$!()$5&)'!+$ - &. *("!+$9* "&<$15P?[8$-E !5')!+$(&/ *(#3$SE$9*(" !#"C$ !"#$"0!"$%& &$&<-*#&)$"*$5* -0'(&$0!)$B*"0$ reduced dendritic branching and reduced spine density in these brain regions. b | A summary of changes in spine density and dendritic branching that occur after exposure to cocaine or morphine &+!"',&$"*$9*(" *+#3$A$)'##*9'!"'*($B&"%&&($"0&$&..&9"#$*.$9*9!'(&$!()$5* -0'(&$%!#$!+#*$*B#& ,&)$'($ "0&$* B'"!+$- &. *("!+$9* "&<$1*P?[8$!()$'($"0&$- '5! E$#*5!"*#&(#* E$9* "&<$1X>83$O!"!$. *5$REF. 71.

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self-administered drug 134, drug-associated cues135 or stress136,137. This clinical scenario can be modelled using a reinstatement procedure (BOX 2) in which laboratory animals are exposed to non-contingent injections of the self-administered drug or related drugs (a drug priming manipulation)138, drug cues139 or stress140. Cue-induced drug seeking and drug craving can also be modelled using second-order schedules of reinforcement141 and extinction142 procedures. The use of reinstatement and extinction procedures has led to the discovery of incubation of drug craving143, which seems relevant to human addiction144. These animal models have not provided much evidence for a difference between cocaine and heroin relapse at the behavioural level, except for a finding that is discussed in the next section. Studies in rats have shown that after extinction, seeking of cocaine and heroin is reliably reinstated by acute injections of the drug, by different types of cues (discrete, discriminative or contextual) that are associated with the drug or by stressors such as intermittent footshock or yohimbine (a drug that induces stress-like responses in humans and nonhuman animals)145–148. Discrete cues that are paired with either cocaine or heroin injections also maintain robust drug seeking in second-order schedules of reinforcement 141,149, and incubation of drug craving is equally robust for cocaine, methamphetamine and heroin150–152. There is also evidence of similarities at the neurobiological level (FIG. 4b). Reinstatement of both heroin and cocaine seeking induced by drug priming and different cue types requires dopaminergic projections from the VTA to the NAc and mPFC147,153,154. Cue-induced seeking of both heroin and cocaine also seem to require the dorsolateral striatum155–157. In addition, drug priming-induced and discrete cueinduced reinstatement of both cocaine and heroin seeking require glutamatergic projections from the dorsal mPFC to the NAc core147,158,159. Lastly, reinstatement of both cocaine seeking and heroin seeking induced by intermittent-footshock stress requires activity in extrahypothalamic corticotropin releasing factor (CRF) and central noradrenaline systems160,161. However, there are also several differences (FIG. 4c). First, reinstatement of heroin seeking seems to involve more brain sites compared to reinstatement of cocaine seeking. Cocaine priming-induced reinstatement is attenuated by reversible inactivation of the VTA, dorsal mPFC, NAc core or ventral pallidum, but not of the ventral mPFC, NAc

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F O C U S OPNE RASDPD E ICCTTI IVOEN S cue-induced heroin seeking in the secondorder schedule, as it did with cue-induced heroin seeking in the reinstatement model165. Third, context-induced reinstatement of cocaine seeking seems to involve subregions of mPFC and NAc that are functionally dissociable from those involved in contextinduced reinstatement of heroin seeking. For cocaine, context-induced reinstatement is attenuated by reversible inactivation of the dorsal but not the ventral mPFC167, whereas the opposite is the case for heroin168. In addition, context-induced reinstatement of cocaine seeking is attenuated by reversible inactivation of either the NAc ! 4%516!30).)+"$!"),.&,1&2%$,).&!.3&-,-!).%

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although both second-order schedule and cue-induced reinstatement procedures assess the conditioned reinforcing effects of reward cues142, cue-induced reinstatement does not include drug delivery. Another possible explanation for the discrepancy is the use of different lesion and inactivation methods, and differences in the timing of their application (permanent cell-specific excitotoxic lesions before training 163,164 versus acute reversible inactivation of both cell bodies and fibres of passage by tetrodotoxin165,166). A question for future research is whether reversible inactivation of the basolateral amygdala after training would decrease

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shell, substantia nigra, central and basolateral amygdala or mediodorsal thalamus159. By contrast, heroin priming-induced reinstatement is attenuated by reversible inactivation of any of the above brain areas, as well as the BNST162. Second, pre-training excitotoxic (that is, permanent) lesions of the basolateral amygdala attenuated discrete cue-induced cocaine seeking but not discrete cue-induced heroin seeking in a second-order schedule of reinforcement 163,164. No such dissociation was found after local reversible inactivation165,166. The discrepant results may reflect differences in the experimental procedures:

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PERSPECTIVES ! /$!).&+)"%+&).(,0(%1&).&$%).+"!"%2%."&,3&4,"5&

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E30&%, H ! Similarities and differences in the brain sites controlling reinstatement of cocaine seeking and heroin seeking. a | I*%3T*#/$6 1,+/3*# 15*'3#0 /5, =,1*+*%/3+*63=73+ -*)$=3#, )%*8,+/3*#1 915*'# 7( )&%)6, 63#,1? 2%*= /5, 4,#/%$6 /,0=,#/$6 $%,$ 9UMK? $#- #30%*1/%3$/$6 -*)$=3#, )%*8,+/3*#1 915*'# 7( -$15,- )&%)6, 63#,1? 2%*= /5, 1&71/$#/3$ #30%$ 9SV? /* 4$%3*&1 7%$3#1 $%,$1. $#/5, 06&/$=$/,%03+ )%*8,+/3*#1 915*'# 7( 76&, 63#,1? /* /5, #&+6,&1 $++&=7,#1 9VK+?; b | Several brain sites are implicated in the reinstatement of both heroin seeking and cocaine seeking. The brain areas /5$/ $%, 3#4*64,- -,),#- *# /5, '$( 3# '53+5 %,3#1/$/,=,#/ 31 3#-&+,- L 7( ,C)*13#0 $#3=$61 /* #*#B+*#/3#0,#/ 3#8,+/3*#1 *2 $ -%&0 9W-%&0 )%3=3#0X?. /* -%&0B$11*+3$/,- -31+%,/, *% +*#/,C/&$6 +&,1. or to stress. c ! S*=, 7%$3# 13/,1 $%, -322,%,#/3$66( 3=)63+$/,- 3# 5,%*3# %,3#1/$/,=,#/ 915*'# 3# *%$#0,? $#- +*+$3#, %,3#1/$/,=,#/ 915*'# 3# )&%)6,?. -,),#-3#0 *# 5*' %,3#1/$/,=,#/ '$1 3#-&+,-; M5, 7$1*6$/,%$6 $#- +,#/%$6 #&+6,3 *2 /5, $=(0-$6$ 9YZK $#- [,K. %,1),+/34,6(?. /5, 7,- #&+6,&1 *2 /5, 1/%3$ /,%=3#$631 9YVSM? $#- /5, 4,#/%*=,-3$6 )%,2%*#/$6 +*%/,C 94=OE[? $%, -322,%,#/3$66( 3=)63+$/,- 3# the reinstatement of heroin seeking induced by heroin priming. The vmPFC is also differentially involved in the reinstatement of heroin seeking that is induced by exposure to heroin-paired discrete +&,1 *% +*#/,C/&$6 +&,1; Y( +*#/%$1/. /5, -*%1*=,-3$6 OE[ 9-=OE[? $#- /5, VK+ +*%, $%, -322,%,#tially implicated in the reinstatement of cocaine seeking induced by exposure to cocaine-associated +*#/,C/&$6 +&,1; \I. -*%1$6 53))*+$=)&1] \ZS. -*%1*6$/,%$6 1/%3$/&=] UI. 4,#/%$6 53))*+$=)&1] UO. 4,#/%$6 )$663-&=; Y%$3# 1,+/3*#1 $%, =*-323,-. '3/5 ),%=3113*#. 2%*= FRE; GA< © 9G::A? R61,43,%;

core or shell169, whereas context-induced reinstatement of heroin seeking is attenuated by manipulations of the NAcc shell and not by manipulations of the NAc core170,171. This possible difference between heroin and cocaine reinstatement should be interpreted with caution, because the manipulations that were used to test heroin reinstatement (dopamine-receptor blockade and inhibition of glutamate transmission) differed from those used to test cocaine reinstatement (muscimol in combination with baclofen), and these manipulations can have different effects on behaviour 172. Furthermore, studies by Bossert et al.146,168, described above, indicate that reinstatement of heroin seeking requires activity in the ventral mPFC and NAc shell. By contrast, reinstatement of cocaine seeking is induced by reversible

inactivation of the ventral mPFC (infralimbic area) or NAc shell169,173 and is attenuated by ventral mPFC AMPA receptor activation173. Functionally disconnecting these two brain regions by a unilateral inhibition of ventral mPFC and simultaneous unilateral inactivation of the NAc shell mimics the reinstatement of cocaine seeking induced by bilateral inactivation of either brain area173, suggesting that activation of projections from the ventral mPFC to the NAc shell inhibits cocaine seeking after extinction174. Lastly, recent data suggest that incubation of psychostimulant craving and incubation of opiate craving have different underlying mechanisms143. In this regard, glial cell linederived neurotrophic factor (GDNF) activity in the VTA is crucial for incubation of cocaine but not heroin craving 175,176.

692 | NOVEMBER 2011 | VOLUME 12

What might account for a divergence between the circuits that mediate the reinstatement of cocaine seeking versus heroin seeking? A possible explanation is the differences in the psychological states that are induced by cocaine versus heroin, and by extension, in the psychological states that are induced by cues associated with them. As mentioned above, runway studies by Ettenberg and colleagues102,177 (FIG. 3a) suggest that heroin induces seemingly pure approach behaviour, whereas cocaine induces approach–avoidance conflict behaviour. We speculate that the partial dissociation of brain circuits that control cocaine and heroin seeking (FIG. 4) may mirror this difference in motivational states. Given the evidence for a substantial aversive component in the response to cocaine177, it is perhaps not surprising that the circuits that control inhibition of cocaine seeking after extinction are more similar to the circuits that control fear inhibition174 than are those that control inhibition of heroin seeking. In conclusion, although there are similarities between the brain circuits that control opiate and psychostimulant seeking in animal models of relapse, there are some notable differences with regard to drug-priming and context-induced reinstatement of drug seeking, and incubation of drug craving. The setting of drug taking. It has been known for many years that environmental contexts or places in which drugs are taken play an important part in human addiction84–86,178. So far, animal research has focused mostly on the ability of the environmental context to alter drug seeking or drug taking by inducing stress or conditioned responses. In the previous section, we provided several examples of this. However, setting can also affect drug taking in ways that are not easily attributable to stress or conditioning. For example, the presence of novel objects can reduce intake of amphetamine179,180, and high temperatures can increase intake of 3,4-methylenedioxymethamphetamine181. Even non-physical, seemingly negligible differences in setting can powerfully alter drug-taking behaviour, as indicated by a series of studies in which rats were trained to self-administer heroin or cocaine under two deceptively similar environmental conditions. Some rats were transferred to self-administration chambers immediately before experimental sessions (non-resident rats), a procedure commonly used in most self-administration studies. Other rats were kept in the self-administration chambers at all times (resident rats). Thus, the physical

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F O C U S OPNE RASDPD E ICCTTI IVOEN S characteristics of the self-administration environment for resident versus nonresident rats were virtually identical, with all differences being purely a function of familiarity 84 (FIG. 5). These studies yielded three major findings that challenge the prevailing view that environmental contexts influence opiate and psychostimulant drug taking and drug seeking in a similar way. First, cocaine and amphetamine selfadministrations were greater and seemed to be more rewarding (when tested in a progressive-ratio schedule) in non-resident rats than in resident rats (FIG. 5). By contrast, heroin self-administration was greater and more rewarding in resident rats than in non-resident rats182,183. Second, when rats with double-lumen catheters were permitted to self-administer either cocaine or heroin within the same session, most resident rats preferred heroin over cocaine, whereas most non-resident rats preferred cocaine over heroin184 (FIG. 5). Third, preliminary data suggest that resident and non-resident rats show differential reinstatement of drug seeking after cocaine or heroin priming185. Resident and non-resident rats were first trained to self-administer heroin and cocaine on alternate days. After extinction, heroin priming had a stronger effect on reinstatement in the resident rats than in the non-resident rats. By contrast, cocaine priming had a stronger effect on reinstatement in the non-resident rats than in the resident rats. Additional experiments using the drugdiscrimination procedure186 suggest that opiates and psychostimulants produce interoceptive cues of different strength in resident rats and non-resident rats. Non-resident rats discriminated amphetamine or cocaine from saline more readily than resident rats, whereas resident rats discriminated heroin from saline more readily than non-resident rats84,187. Thus, the setting of drug exposure seems to modulate the effects of opiates and psychostimulants in opposite directions with regard to two major attributes of addictive drugs: the degree of reward and the strength of subjective effects186. Why do the environmental settings differentially affect heroin and cocaine taking, as well as the propensity to relapse to drug seeking? At a proximal, neurobiological level, there is evidence that the initial exposure to low doses of intravenous heroin and cocaine (such as those used in self-administration experiments) differentially activate dorsal striatum neurons in resident and nonresident rats, respectively 188 (FIG. 5). The functional significance of this differential neuronal activation is a subject for future research.

At a more distal level, it is tempting to view the setting as an ecological backdrop against which drug effects are appraised as adaptive or maladaptive184. Thus, the sedative effects of heroin may facilitate introspection in a safe, non-challenging home environment, but may be appraised as performance impairing in a potentially unsafe non-home environment. By contrast, the arousing and activating effects of cocaine may be appraised as performance enhancing in a challenging non-home environment, whereas the same state may be appraised as mainly anxiogenic in the home environment. This hypothesis requires rigorous testing but initial evidence in support of this idea comes from studies showing that ketamine — which, like cocaine, has activating and sympathomimetic effects — is more readily self-administered by rats in the nonresident environment 189. By contrast, alcohol — which, like heroin, initially causes drowsiness and sedation — is more readily selfadministered in the resident environment 190. The ability of setting to differentially affect heroin and cocaine reward may have important implications for the incentive sensitization theory of addiction. Earlier studies have shown in fact that repeated administrations of heroin or morphine produce greater psychomotor sensitization in non-resident than in resident rats191,192. Thus, it appears that psychomotor sensitization and drug reward can be modulated in opposite directions, at least under certain circumstances. This discrepancy goes beyond the reports of mere dissociation between psychomotor sensitization and drug reward193,194. In conclusion, in laboratory rats, the setting of drug availability affects heroin taking and seeking, and cocaine taking and seeking in a different way. Below, we discuss results that suggest that this is also the case in humans. Epidemiological and clinical aspects At the epidemiological level, heroin and smoked cocaine seem similar in terms of the severity and type of harm that they are likely to cause to drug users195, although the proportion of users who become addicted is somewhat higher for heroin (~23%) than for cocaine (~17%)196. Users of these drugs also show similar likelihoods of relapse in the year following treatment 197. Non-pharmacological treatments, such as contingency management or cognitive behavioural therapy, are moderately effective in both types of addiction198,199. Lastly, attempts to identify trait determinants of the ‘drug of choice’, in terms of self-medication

NATURE REVIEWS | NEUROSCIENCE

hypotheses200, have not fared well201–203, and it has even been suggested that an addicted person’s choice of drug is largely determined by chance204. Nevertheless, there is ample evidence that psychostimulant addiction and opiate addiction have distinct profiles, and in the following sections we briefly discuss selected studies that point to fundamental differences between these two drug classes in human addicts. Genetic and environmental factors in the vulnerability to opiate and psychostimulant use. There is some evidence that shared genetic and environmental influences contribute to a generalized vulnerability to drug abuse and dependence83,205. However, there is also evidence that unique genetic and environmental factors underlie a differential vulnerability to heroin versus cocaine use. Data from the Vietnam Era Twin Registry, for example, suggest that vulnerability to heroin use is more strongly influenced by unique genetic factors compared to vulnerability to other drugs, including cocaine83. This finding was not replicated in a cohort study based on the Virginia Twin Registry 205, suggesting that it may have been specific to the all-military Vietnam-era cohort. However, small-scale linkage studies and genome-wide association studies also support the idea that specific genetic variants are differentially associated with opiate and psychostimulant use206. Lastly, both the Vietnam Era Twin Registry and the Virginia Twin Registry cohort studies indicate that a sizeable portion of the variability in the susceptibility to drug use is due to environmental influences that are unique to opiates versus psychostimulants83,205. A host of environmental factors are thought to influence the initiation and maintenance of drug addiction, including price and availability of drug and non-drug rewards, peer pressure, aversive life experiences and occurrence of negative consequences. What is not clear is which of these environmental factors can differentially influence opiate and psychostimulant use. In the next section, we examine at least one way in which environmental context has been shown to interact differently with heroin and cocaine taking in humans. Patterns of heroin or cocaine use in drug addicts. As discussed above, studies conducted using rats have shown differential preferences for heroin and cocaine as a function of the environmental setting or context 184,188. These preclinical findings VOLUME 12 | NOVEMBER 2011 | 693

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PERSPECTIVES in drug availability, peer influence or other socio-demographic factors. Other studies conducted with heroin and cocaine co-abusers208, using real-time electronic diary reports, have examined the predictive value of potential triggers of craving and relapse, such as negative moods, positive moods and exposure to drug-associated cues209,210. Episodes of cocaine use, but not craving, were reliably predicted by any of these triggers on a timescale of 5 hours. For

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!"#$%&'&(&Setting differentially affects heroin and cocaine use in rats and humans. a | In a study that examined drug taking as a function of )%**!+",&)*-+.-$.&*/012%3%$&)%241-.5!+!)*$-*!0+& 67-58%$)&/%$%&#)%.&90+%&2%3%$&/-)&:-!$%.&/!*7& .$#"&!+4#)!0+)&-+.&*7%&0*7%$&2%3%$&/-)&!+-6*!3%;<& =05%&$-*)&/%$%&*$-+)4%$$%.&*0&*7%&67-58%$)& immediately before the start of the sessions 9$%4%$$%.&*0&-)&+0+1$%)!.%+*&$-*);,&/7%$%-)&0*7%$& $-*)&/%$%&>%:*&!+&*7%)%&67-58%$)&-*&-22&*!5%)& 9$%4%$$%.&*0&-)&$%)!.%+*&$-*);<&?%$0!+&/-)&50$%& $%/-$.!+"&!+&*7%&$%)!.%+*&$-*)&*7-+&!+&*7%&+0+1 $%)!.%+*)&$-*)&9!+.!6-*%.&8@&-+&#:/-$.&)7!4*&!+& the dose-response curve). By contrast, cocaine /-)&50$%&$%/-$.!+"&!+&*7%&+0+1$%)!.%+*&$-*)& *7-+&!+&*7%&$%)!.%+*)&$-*)&9!+.!6-*%.&8@&-&2%4*&)7!4*& in the dose-response curve of non-resident rats). b | In a study that examined drug-induced neural activity in the caudate nucleus as a function of setting, drug-naive resident and non-resident $-*)&/!*7&!+*$-3%+0#)&6-*7%*%$)&$%6%!3%.&-&)!+"2%& A)%241-.5!+!)*$-*!0+&.0)%B&04&%!*7%$&7%$0!+&9C'&μg kg–1;&0$&606-!+%&9DEE&μg kg–1), and their brains /%$%&:$06%))%.&FE&5!+&2-*%$&40$& !"# $%&hybridization of '(#&5GHI&%J:$%))!0+<&K06-!+%&%J:0)#$%& induced greater increases in '(# expression in the dorsal and ventral parts of the caudate nucleus of non-resident rats than in that of resi.%+*&$-*),&/7%$%-)&7%$0!+&%J:0)#$%&!+.#6%.& greater increases in '(# expression in resident rats than non-resident rats. c | In a study that examined drug preference as a function of set*!+",&$%)!.%+*&-+.&+0+1$%)!.%+*&$-*)&/!*7&.0#82%1 2#5%+&6-*7%*%$)&/%$%&4!$)*&*$-!+%.&*0&)%241 administer heroin and cocaine on alternate days, -+.&/%$%&*7%+&"!3%+&*7%&0::0$*#+!*@&*0&6700)%& 8%*/%%+&606-!+%&-+.&7%$0!+&/!*7!+&*7%&)-5%& )%))!0+<&L0)*&$%)!.%+*&$-*)&:$%4%$$%.&7%$0!+&03%$& 606-!+%,&/7%$%-)&50)*&+0+1$%)!.%+*&$-*)&:$%ferred cocaine over heroin. d | In a study that examined setting preferences as a function of drug in humans, most addicts reported using heroin at home and cocaine outside the home, $%"-$.2%))&04&/7%*7%$&*7%&*/0&.$#")&/%$%& !+M%6*%.&9*0:&:-+%2;&0$&)+0$*%.&980**05&:-+%2;< N+2@&-&5!+0$!*@&04&-..!6*)&9OPEQ;&!+.!6-*%.&+0& clear preference for the setting of drug taking. Part a,&2%4*&"$-:7&!)&50.!4!%.,&/!*7&:%$5!))!0+,& from REF. 183 ©&9CEER;&=:$!+"%$<&S-$*&a, right graph, data from REF. 182 . Part b is modified, /!*7&:%$5!))!0+,&4$05&REF. 188 ©&9CEET;&=:$!+"%$<& Part c&!)&50.!4!%.,&/!*7&:%$5!))!0+,&4$05&REF. 184. ©&9CEET;&U2)%3!%$<&S-$*&d, data from REFS. 184,207.

constraints. The route of drug taking did not play a major part in these findings, as comparable results were obtained when the analysis was limited to subgroups of respondents who injected or snorted heroin and cocaine separately, often on the same day (FIG. 5). The within-subject design of this study makes the findings especially compelling, because the difference in preferred settings for heroin use compared to cocaine use cannot readily be attributed to differences

=."+-3.!>&"!33$%(. 4"2(.&"!'!"!%*!

are supported by a study in human addicts (outpatients at an addiction clinic), who were heroin and cocaine co-abusers. Retrospective self-reports184 and written diaries207 showed that the respondents used cocaine in different settings from those in which they used heroin. In most cases, heroin was used at home, whereas cocaine was used outside the home; respondents said that these choices of location reflected real preferences rather than social or practical

'(%)*!"#$!%&"

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F O C U S OPNE RASDPD E ICCTTI IVOEN S heroin, the results were nearly the opposite: episodes of craving, but not use, were reliably predicted by increases in triggers that induced negative mood. These findings suggest that heroin and cocaine differ in terms of the factors that induce craving and in terms of their use in daily life. A caveat to this interpretation is that the participants were all in methadone maintenance therapy, which may have partly decoupled heroin craving from heroin use. In summary, the human data reviewed here suggest that there are differences between aspects of heroin and cocaine use, abuse and craving. Indeed, there are no pharmacological treatments that are similarly effective for heroin addiction and cocaine addiction. For example, the effectiveness of agonist maintenance therapy (for example, methadone treatment) for heroin addiction211 has no clear parallel in cocaine addiction, although efforts to find such a therapy continue212. Conclusions and future directions Opiate addiction, psychostimulant addiction and other types of addiction are often seen as mere variants of the same disorder. Indeed, current diagnostic criteria for addiction cut

across drug classes19, and influential theories of addiction emphasize the shared psychological processes and neurobiological substrates of different types of drug addiction (BOX 1). Here, we have attempted to offer a different perspective. We argue that although there are commonalities in the ways in which opiates and psychostimulants affect brain and behaviour, much can be learned from considering the distinctive features of each type of addiction. The human and animal studies reviewed here suggest that there are substantial differences in the neurobiological and behavioural mechanisms underlying opiate addiction and psychostimulant addiction (Supplementary information S1 (box)). At the neurobiological level, the most fundamental difference is that mesocorticolimbic dopamine transmission seems to be crucial for psychostimulant selfadministration but not opiate self-administration (BOX 3). By contrast, the mu opioid receptor is crucial in mediating the effects of intravenous opiate self-administration but plays only a minor part in psychostimulant self-administration20,213. Other notable differences include the distinct populations of mPFC and NAc neurons that are associated

with heroin self-administration compared to cocaine self-administration51, the opposite synaptic and structural plasticity changes in the PFC after withdrawal from opiates and psychostimulants67–69,71,72, the opposite regulation of immediate-early gene expression in the striatal neurons of the indirect pathway by opiates and psychostimulants74–76, and the differential roles of mPFC subregions in context-induced relapse to heroin seeking and cocaine seeking 167,168. Lastly, human studies suggest that there is minimal overlap between the genes that are associated with opiate and psychostimulant addiction83,206,214, as well as minimal overlap between the profiles of post-mortem gene-expression changes in the striatum of opiate and psychostimulant users80. At the behavioural and psychological levels, perhaps the two most fundamental differences between cocaine and heroin (and by implication other psychostimulants and opiates) were revealed in rat models: cocaine exposure leads to a mixed motivational state characterized by approach–avoidance conflict towards drug-associated places, and unlimited cocaine access leads to complete loss of control over drug intake. By contrast, heroin exposure leads to a classical

Glossary AMPA:NMDA ratio

In vivo extracellular recording

Psychic (or psychological) dependence

A measure of postsynaptic changes in synaptic strength. It is defined as the peak synaptic AMPA receptor current relative to the peak synaptic NMDA receptor current.

A set of methods in which bundles of microwires are targeted to specific areas of the brain to allow measurement of extracellular currents (action potentials) of single cells.

Long-term depression

A concept, established early in the addiction field, that refers to a compulsion that requires periodic or continuous intake of an abused drug to produce psychological pleasure or to avoid psychological distress, regardless of whether physical dependence is also present.

(LTD). A form of synaptic plasticity that is defined by a persistent weakening of synaptic strength.

Psychomotor sensitization

Long-term potentiation

A progressive increase in locomotor activity or stereotypy with repeated drug (for example, cocaine) administration.

(LTP). A form of synaptic plasticity that is defined by a persistent increase in synaptic strength.

Relapse

Craving An affective state that can be induced in human drug users by exposure to the drug itself, drug-associated cues or stress. In laboratory animals, craving is often inferred from the subjects’ behavioural response (for example, lever-pressing) to drugs, drug-associated cues or stress.

Direct and indirect striatal pathways The two efferent pathways in the basal ganglia. The direct pathway connects the striatum with the substantia nigra pars reticulata and entopeduncular nucleus. The indirect pathway connects the striatum with the globus pallidus and ventral pallidum.

Extinction The decrease in the frequency or intensity of learned responses after the removal of the unconditioned stimulus (for example, food or a drug) that has reinforced the learning.

Incentive motivational state A motivational state that is induced by exposure to unconditioned aversive or appetitive stimuli or cues that become associated with these stimuli.

Incubation of drug craving A hypothetical motivational process that is inferred from findings of time-dependent increases in cue-induced drug seeking after withdrawal from drug self-administration in rats.

LTDGABA A form of long-term depression (LTD) that is observed in dopaminergic neurons in the ventral tegmental area and that reduces synaptic efficacy between presynaptic GABAergic neurons and postsynaptic dopaminergic neurons.

LTPGABA A form of long-term potentiation (LTP) that is observed in dopaminergic neurons in the ventral tegmental area. It results in increased GABA release and in the strengthening of inhibitory synapses.

Mesotelencephalic dopamine system Also known as the mesocorticolimbic dopamine system. A major ascending dopaminergic pathway that originates in the ventral tegmental area and projects to, among other regions, the nucleus accumbens, the bed nucleus of the stria terminalis, the amygdala, the olfactory tubercle and the medial prefrontal cortex.

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The resumption of drug-taking behaviour after self-imposed or forced abstinence in humans with a history of abuse or dependence.

Second-order schedule of reinforcement A complex reinforcement schedule in which the completion of the response requirement of one schedule is treated as a unitary response that is reinforced according to another schedule.

Stress In animal models, stress typically refers to forced exposure to events or conditions that the animal would normally avoid. In humans, stress often refers to a condition in which the environmental demands exceed the coping abilities of the individual.

Synaptic plasticity Activity-dependent direct or indirect modifications of the strength of synaptic transmission at pre-existing synapses.

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PERSPECTIVES approach behaviour similar to that observed in hungry rats seeking food, and unlimited heroin access does not lead to loss of control over drug intake102,117 (FIG. 3). There are also fundamental differences in the way in which the environment interacts with opiate and psychostimulant reward: in both rats and humans, the preferred setting for opiate use is the home environment, whereas the preferred setting for psychostimulant use is outside of the home environment182–184 (FIG. 5). These findings may help to account for data from population studies that suggest that there are unique environmental influences on opiate addiction and psychostimulant addiction83,214. Furthermore, in rats, escalation of cocaine self-administration is predicted by high trait impulsivity, whereas escalation of heroin self-administration is not29,133. Lastly, in humans, psychostimulants cause more pronounced deficits in impulse control and cognitive flexibility than do opiates33–37. The neurobiological, behavioural and psychological differences between opiates and psychostimulants have implications for addiction treatment. These differences may account for the fact that no known medication effectively treats both opiate and psychostimulant addiction. For example, approved treatments for opiate addiction, such as methadone and buprenorphine, have shown limited efficacy in decreasing cocaine use in concurrent users of heroin and cocaine215–218 (however, see REF. 219 for different results). In addition, the realization that drug choice is crucially dependent on setting has implications for cognitive behavioural therapies in which addicts learn to identify and respond appropriately to use-provoking risk factors. The data reviewed here also have implications for addiction theories (BOX 1), which, as mentioned above, have attempted to provide a unitary account of addiction across drug classes. It is beyond the scope of this Perspective to analyse how each of the neurobiological or behavioural differences discussed above can or cannot be accounted for by current theories. However, we argue that these theories would struggle to explain the opposite modulatory role of the environment on opiate and psychostimulant reward and choice, the finding that escalation of cocaine intake does not predict escalation of heroin use (and vice versa), the finding that opiate self-administration is mostly independent of mesocorticolimbic dopamine transmission, and the opposite structural and synaptic changes in PFC that are induced by exposure to opiates and psychostimulants.

Lastly, the data reviewed here have implications for future neuroscience research on drug addiction. Since the late 1980s and the early 1990s52,53,220, such research has focused on a search for drug-induced neuroadaptations that can account for compulsive drug use and relapse across drug classes. In the vast majority of these studies, cocaine has been used as the prototypical, presumably representative drug of abuse221,222. Based on the differences across drug classes that we have discussed here, we believe that generalizations from cocaine to other drugs of abuse should be made with extreme caution, and that the field would benefit from more systematic comparisons of the roles of different signalling molecules and synaptic-plasticity mechanisms in reward and relapse across drug classes. It is beyond the scope of this Perspective to compare and contrast similarities and differences in the behavioural and neurobiological mechanisms of addiction across all drug classes. However, our concerns about differences between opiates and psychostimulants probably also apply to other addictive drugs. For example, to our knowledge, it has not been established in animal models or human studies that the mesocorticolimbic dopamine system plays a part in addiction to benzodiazepines or barbiturates. Even for nicotine and alcohol, empirical data raise questions about the centrality of the mesocorticolimbic dopamine system in the rewarding effects of these drugs223–226. We hope that this Perspective will serve as a starting point for more balanced future research that will avoid the Scylla of rigidly unified models and the Charybdis of excessively compartmentalized ones. In particular, we believe that it is crucial for models of drug addiction to be formulated and validated on the basis of empirical results from comparative studies that include several classes of addictive drugs. Aldo Badiani is at the Department of Physiology and Pharmacology Vittorio Erspamer, Sapienza University of Rome, Rome, Italy, and at the Drug Addiction and Clinical Pharmacology Unit, University Hospital Umberto I, Sapienza University of Rome, 00185 Rome, Italy. David Belin is in the AVENIR team ‘Psychobiology of Compulsive Disorders’ within the Institut National de la Santé et de la Recherche Médicale (INSERM) Experimental and Clinical Neurosciences Laboratory, University of Poitiers, 86000 Poitiers, France. David Epstein, Donna Calu and Yavin Shaham are at the Intramural Research Program, National Institute on Drug Abuse (NIDA), National Institutes of Health (NIH), 251 Bayview Boulevard, Baltimore, Maryland 21224, USA.

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Correspondence to A.B. and Y.S.  e-mails: aldo.badiani@uniroma1.it; yshaham@intra.nida.nih.gov doi:10.1038/nrn3104 Published online 5 October 2011 1. 2. 3. 4.

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Acknowledgements This Perspective was written with financial support from the Ricerche di Università Program of the Sapienza University of Rome, Italy (A.B.), the Institut National de la Santé et de la Recherche Médicale (INSERM) (D.B.) and the Intramural Research Program of the US National Institutes of Health (NIH) National Institute on Drug Abuse (NIDA) (D.C., D.E. and Y.S.). We thank R. See for sharing with us unpublished data that are included in the summary diagram in FIG. 4 , A. Ettenberg for sharing historical data with us, and M. Heilig and E. Koya for very helpful comments.

Competing interests statement The authors declare no competing financial interests.

FURTHER INFORMATION Yavin Shaham’s homepage: 7**:VWW!$:<.$#"-8#)%<"03WXHGX<:7:

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The regional specificity of rapid actions of cocaine Brandon J. Aragona doi:10.1038/nrn3043-c1

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Differentiating the rapid actions of cocaine Roy A. Wise and Eugene A. Kiyatkin Nature Rev. Neurosci. 12, 479–484 (2011)

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focus on addiction