The 2004 Frontiers in Addiction Research mini-convention brought together participants from diverse scientific disciplines to share advances and discuss future directions in the neuroscience of drug abuse and addiction. The application of NIDA-supported neuroscience research will enable even greater advances vital to reducing drug abuse, addiction, and their related consequences.
During the Behavioral Neuroscience of Nicotine Addiction section of the program, presenters discussed recent critical findings in the areas of behavioral neuroscience of nicotine addiction, with a particular emphasis on the nicotinic receptor and novel CNS circuitry as they relate to addictive behavior. The topic areas of this symposium were chosen to highlight a range of nicotine research, including the motivationally positive and negative aspects of nicotine. The symposium also provided the additional benefit of an overview of various approaches and models that can be used to study the neuroscience of nicotine, ranging from animal behavioral paradigms to studies with human subjects.
There has been a remarkable evolution of functional biomedical imaging methods into powerful tools in drug abuse research. The Creative Directions in Imaging portion of the program centered on this remarkable evolution. Presenters highlighted state-of-the-science examples of imaging research in animals that are now possible using PET, MRI, and optical methods.
Brain resiliency and repair is an exciting frontier in neuroscience, yet few drug abuse researchers are looking toward clinical interventions that are currently being evaluated for other neurodegenerative diseases. Presentations on Mechanisms of Brain Resiliency and Repair addressed the relation of neural injuries induced by drug abuse to the innate capacity of CNS neurons to repair themselves and to regenerate connections. The speakers were leaders in neural recovery and examined the role of chemical and trophic interventions to enhance repair and connectivity.
Historically, the primary focus of research on the neurochemical mechanisms involved with drug abuse and addiction has been on the central monoaminergic systems. However, recent research has implicated glutamate as an important neurotransmitter associated with the various aspects of drug addiction. In The Role of Glutamate in Drug Addiction session, researchers focused their discussions on recent findings that support the integral role of glutamate in brain plasticity related to certain drugs of abuse.
Behavioral Neuroscience of Nicotine Addiction
- A Dual Role for Nicotine in Reinforcement: The Interaction Between Nicotine and Non-Drug Cues
Nadia Chaudhri’s presentation challenged the widely held belief that smoking is driven solely by the direct, pharmacological actions of nicotine in the brain. Her team, using a standard model of drug self-administration in rats, collected data showing that nicotine reinforcement is strongly influenced by the interaction between nicotine and nonpharmacological environmental stimuli. Furthermore, they determined that this interaction does not require the response-contingent delivery of nicotine, suggesting that primary reinforcement is not the exclusive mechanism underlying nicotine addiction.
The team’s findings indicate that the rapid, robust levels of operant responding observed during the acquisition and maintenance of nicotine self-administration result from a synergistic interaction between nicotine and non-drug stimuli. Furthermore, this synergism appears to reflect two separable actions that Chaudhri termed the “dual-reinforcing effects of nicotine.” The dual-reinforcing hypothesis states that in addition to acting as a primary reinforcer, nicotine can enhance the reinforcing value of other stimuli in a manner that is not dependent on a close temporal association between nicotine and other stimuli, or the behavior controlling their delivery.
- Imaging the Alpha4/Beta2 nAChR in the Human Brain
Yu-Shin Ding, Ph.D.
Nicotinic acetylcholine receptors (nAChR) are involved in various pharmacological effects of nicotine and are altered in various disease states, such as schizophrenia, Parkinson’s, and Alzheimer’s diseases. The goal of Dr. Yu-Shin Ding’s research was to develop suitable PET radioligands to study nAChR in humans. Using F-18-labeled nicotinic agonists, 2–[18F]fluoro-A-85380 (2–[18F]FA) and 6-[18F]fluoro-A-85380 (6-[18F]FA), his research team carried out comparative PET studies in both baboons and humans. They concluded that 6-[18F]FA displays high brain uptake in baboons and that the distribution of radioactivity was consistent with that of nAChR in humans. Their results demonstrated that: (1) 6-[18F]FA is a suitable radioligand for quantitative PET studies of nAChR in the human brain, and that a total 4-hour study with 2 hours of actual scanning time is adequate for kinetic analysis; (2) the binding of 6-[18F]FA is specific to nAChR and can be displaced by the nicotine in cigarettes; and (3) white matter binding of 6-[18F]FA is specific for nAChR. These studies also showed that 6-[18F]FA is a potentially useful in vivo tool to better understand the role of nAChR in various diseases and their relationship with the abnormality in white matter.
- Aversion Versus Reward in Nicotine Reinforcement Circuits
Steven R. Laviolette, Ph.D.
Various neural systems have been implicated in the processing of nicotine’s bivalent motivational properties. However, considerable evidence points to a central role for the ventral tegmental area (VTA) and the functionally associated mesolimbic dopamine (DA) system and brainstem pedunculopontine nucleus as critical players in the central processing of nicotine’s rewarding and aversive psychological properties. Dr. Steven Laviolette’s research suggests that in the acute phase of nicotine exposure, the rewarding effects of nicotine are dependent on the pedunculopontine nucleus, while DA transmission signals the aversive effects of nicotine. However, following chronic exposure to nicotine, the motivational effects of continued nicotine use may depend on transmission through the mesolimbic DA system.
This presentation discussed the emerging characterization of separate dopaminergic and GABAergic neural systems within the VTA that can mediate either the acute rewarding or aversive psychological effects of nicotine versus the motivational effects of nicotine in the dependent and addicted state, providing new insights into (1) how functional interactions between these systems may determine vulnerability to nicotine addiction, and (2) how continued exposure to nicotine may induce plastic molecular alterations within the VTA, triggering an “addiction switch” to a state of nicotine dependence, craving, and compulsive nicotine use.
- GABAergic and Glutamatergic Mechanisms Underlying Nicotine Addiction
Athina Markou, Ph.D.
Two factors that lead to drug dependence, including habitual tobacco smoking, are the rewarding effects of drugs of abuse and the motivation to alleviate the negative affective aspects of drug withdrawal. Dr. Athina Markou suggested in her presentation that cholinergic-glutamatergic-GABAergic interactions in the ventral tegmental area (VTA) and other brain sites may be critically involved in mediating the rewarding effects of nicotine, and may exhibit adaptations with the development of nicotine dependence that contribute to nicotine withdrawal. GABA is the main inhibitory neurotransmitter and glutamate is the main excitatory neurotransmitter in the brain. Dr. Markou discussed the methods and results of her research examining nicotine self-administration in rats, as well as cue-induced reinstatement of nicotine-seeking behavior. Dr. Markou also gave an overview of her research team’s investigations involving the role of mGlu2/3 receptors, possibly mediating the negative affective aspects of nicotine withdrawal.
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- The Dopamine D3 Receptor System: New Possibilities for Dopamine-Based Reward
Christian A. Heidbreder, Ph.D.
The restricted localization of the dopamine (DA) D3 receptor in key elements of the mesolimbic DA system in both the rat and human brain has led to the hypothesis that it may play an important role in drug addiction. This presentation focuses on recent advances in the DA D3 system that may lead to the development of new pharmacotherapies. Virtually all drugs of abuse activate the mesolimbic DA system. The enhanced extra-synaptic release of DA facilitates incentive-learning or the attribution of positive-incentive salience to cues. Thus, any responses to drugs that occur during the period of raised extra-synaptic DA have the potential to produce positive-incentive salience and to contribute to dependence. Dr. Christian Heidbreder introduced evidence suggesting that selective antagonism of DA D3 receptors may represent a unique pharmacotherapeutic strategy for treating drug-seeking and drug-taking behaviors. Specifically, he showed that SB-277011-A has significant efficacy in a wide range of behavioral paradigms assessing the abuse properties of nicotine, alcohol, cocaine, and heroin. His team of researchers used different experimental animal paradigms to assess the effects of SB-277011-A, and each had unique relevance for different aspects of human drug addiction. Altogether, their findings suggest that selective DA D3 receptor antagonism may hold the highest promise for attenuating cue-, drug-, or stress-evoked relapse to addictive drug use.
Creative Directions in Imaging
- Translational Imaging of Methylphenidate in Developing Animals
Susan L. Andersen, Ph.D.
Magnetic resonance imaging (MRI) is a powerful tool that can be used to show functional changes in the brain that may occur as a result of drug exposure. Due to the relatively noninvasive nature of MRI, multiple scans across the course of development can reveal how drug-induced changes in brain function emerge with maturation. Dr. Andersen presented data from rat studies that demonstrated how prepubertal exposure to methylphenidate produces enduring changes in regional cerebral blood volume (rCBV) later in life. Previous behavioral studies suggested that prepubertal methylphenidate exposure reduces the rewarding properties of cocaine in adulthood. For this study, drug-induced changes were detected with monocrystalline iron oxide nanoparticles (MION), which constitute a long-lasting, iron-based contrast agent that has high magnetic susceptibility and increases the contrast-to-noise ratio over blood oxygen level-dependent (BOLD) preparations. Upon challenge with methylphenidate in adulthood, averaged rCBV values were significantly enhanced in the cortical regions associated with executive function in juvenile methylphenidate-treated rats relative to controls. With further maturation, this relative percentage in blood volume increased even more. Rats with juvenile exposure to methylphenidate were also challenged with cocaine in adulthood. The magnitude and time course of rCBV changes in response to cocaine (1 mg/kg IV) in methylphenidate-treated animals were significantly less than in vehicle or naïve animals. Dr. Andersen posited that MRI can be applied to the study of developmental drug exposure and subsequent responsiveness later in life.
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- Multiscale Imaging as an Approach To Elucidate CNS Sites of Stimulus Action
Maryann E. Martone, Ph.D.
Dr. Maryann Martone explained that the ability to combine gross structural imaging with high-resolution microscopy on a large scale will significantly advance the ability to detect and analyze pathology across multiple brain regions. Dr. Martone’s presentation highlighted the work of the newly created Biomedical Informatics Research Network (BIRN) project, a program created to acquire and analyze large-scale multiresolution brain maps. The BIRN project is creating a high-bandwidth and large-capacity network for sharing multimodal and multiresolution data obtained at multiple centers concerned with human disease and associated animal models. As part of the BIRN project, researchers at Duke University and the University of California, San Diego are collaborating on a multiscale investigation of a dopamine transporter knockout mouse. These animals exhibit chronic elevated extracellular dopamine and exhibit motor abnormalities, such as hyperactivity. Dr. Marton’s research team is combining gross structural analysis using structural MRI along with correlated histology and higher resolution light and electron microscopic techniques.
- MRI/PET Imaging of Fetal Development
Helene Benveniste, M.D., Ph.D.
An accurate understanding of maternal-fetal exchange, fetal receptor maturation, and fetal pharmacodynamics is of pivotal importance to understanding how the developing human brain is affected by molecular, genetic, and environmental factors. This has become increasingly important as epidemiological and experimental studies now indicate that diseases such as autism, schizophrenia, depression, addiction, obesity, and certain forms of degeneration may have their origin early in life. One way to capture such complex interactions would be to longitudinally and noninvasively characterize (a) the maturation of fetal brain receptors, (b) the changes that might occur following various stressors, and (c) how such changes link to specific behaviors in the progeny later in life. Dr. Helene Benveniste discussed a noninvasive multimodality PET and MRI imaging approach for visualization of maternal-fetal drug exchange in the macaca radiata species developed by her research team. She described how their method allows them to quantify dopamine transporter uptake binding sites in the maternal and fetal brain. The presentation concluded with a discussion of their current and future studies, which focused on examination of characterizing receptor processes in the 2nd and 3rd trimesters.
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- In Vivo Mammalian Brain Imaging Using One- and Two-Photon Fluorescence Microendoscopy
Mark J. Schnitzer, Ph.D.
One of the major limitations in the current set of techniques available to neuroscientists is the fact that there is a dearth of methods for imaging individual cells deep within the brains of live animals. To overcome this limitation, Dr. Mark Schnitzer and his research team developed two minimally invasive fluorescence microendoscopy techniques and tested their abilities to image cells in vivo. Both one- and two-photon fluorescence microendoscopy are based on compound gradient refractive index lenses that range from 350° to 1,000° microns in diameter and provide micron-scale resolution. One-photon microendoscopy allows full-frame images to be viewed by the eye or with a camera and is well suited to fast frame-rate imaging. Two-photon microendoscopy is a laser-scanning modality that provides optical sectioning deep within tissue. Using in vivo microendoscopy, the research team acquired video-rate movies of thalamic and CA1 hippocampal red blood cell dynamics and still-frame images of CA1 neurons and dendrites in anesthetized rats and mice. Dr. Schnitzer predicted that microendoscopy will help meet the growing demand for in vivo cellular imaging that has been created by the rapid emergence of new synthetic and genetically encoded fluorophores, which can be used to label specific brain areas or cell classes.
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Mechanisms of Brain Resiliency and Repair
- Unraveling Survival Strategies
Valina L. Dawson, Ph.D.
Dr. Dawson presented research results on preconditioning, a phenomenon in which brief episodes of sublethal insults induce robust protection against the deleterious effects of subsequent prolonged toxic challenges. Through differential screening using DAzLE and microarray analysis, Dr. Dawson and her team identified 31 potential preconditioning neuroprotective genes. Using a retrovirus expression cloning strategy, they identified another 29 confirmed neuroprotective and cytoprotective genes. Importantly, several of these genes are protective in neurons and fibroblasts, suggesting that there exist general survival mechanisms that could be exploited to spare cells at risk in the brain. Several genes among these sets of genes have been previously shown to be protective; roughly half are novel genes of unknown function. The characterization of these gene sets was discussed, along with a novel clone (932) that protects against excitotoxicity and oxidative stress in neurons, and PARP-1-dependent cytotoxicity induced by MNNG and oxidative stress in fibroblasts.
- Novel Therapeutic Approaches for Neurodegenerative Disorders: Enhancing Brain Resiliency and Repair
Ole Isacson, M.D.
Dr. Ole Isacson presented his findings related to the search for compounds that can alter the development and health of a neuron. Several neurotrophic factors have shown impressive growth-inducing effects during development and protective effects in animal models of neurodegeneration, including brain-derived neurotrophic factor (BDNF), glial-derived neurotrophic factor (GDNF), transforming growth factor (TGF)-ß, and basic fibroblast growth factor (bFGF). The identification of these factors helped pave the way for the detection and analyses of the effects of other molecules and compounds with specific trophic and tropic effects on subsets of neuronal populations. In Parkinson’s disease (PD), the major degenerative circuitry change resides in the dopaminergic and midbrain to basal ganglia connection and synapses. Dr. Isacson and his research team have demonstrated the molecular signals required for successful differentiation of embryonic stem cells into specific and functional midbrain dopamine (DA) neurons, which in turn can be transplanted in PD models. They found that fluorescence-activated cell sorting or equivalent cell-selection procedures are necessary steps for an adequate and appropriate surgical DA neuronal cell replacement-therapy method. The team also evaluated alternative methods of restoring DA cells to the circuitry by stimulation of the brain’s endogenous stem cells by chemical and trophic factors. Future successful clinical trials using cell therapy for PD will require continued rational development, cell formulation, and the sophisticated use of stem cell technology.
- Brain Repair and Rehabilitation: Behavorial and Pharmacological Treatments
Bryan Kolb, Ph.D.
Evidence is accumulating that at least partial functional restitution is possible after cortical injury. This functional improvement is associated with both compensatory changes in the remaining, intact neural circuitry, as well as with the generation of new circuitry. Such changes can be influenced by various factors, including experience, psychomotor stimulants, and neurotrophic factors. Recent work suggests that there may be a common mechanism that involves the upregulation of neurotrophic factors, such as FGF-2. Thus, stimulants such as nicotine and amphetamine also enhance recovery and again appear to work, at least in part, via their action on trophic factors. Preinjury administration of these compounds appears to be problematic, however, and may retard functional recovery and/or prevent later plastic changes. Finally, preliminary studies have shown that the mobilization of intrinsic stem cells may also be a way to stimulate functional recovery.
- Estrogen Regulates the Structure and Function of Synapses in the Hippocampus
Catherine S. Woolley, Ph.D.
Fluctuating levels of the hormone estrogen produce dramatic plasticity of synaptic connections in the hippocampus, a brain region critical for spatial memory. Dr. Woolley and her laboratory research team have used a combination of anatomical and electrophysiological approaches to define a sequence of synaptic changes in the hippocampus induced by estrogen. They found that estrogen initially and transiently disinhibits hippocampal neurons, as evidenced by a decrease in GABAA receptor-mediated inhibitory postsynaptic currents. The presence of estrogen receptor immunoreactivity at some inhibitory synapses suggests that estrogen may act directly at synapses to regulate synaptic physiology. With longer exposure to estrogen, inhibition is restored to control levels and excitatory synaptic input to hippocampal neurons is enhanced. In addition, dendritic spine and synapse numbers are increased, sensitivity to the NMDA subtype of glutamate receptor-mediated synaptic input is enhanced, and the capacity for long-term potentiation is increased. These estrogen-induced changes in hippocampal synaptic connectivity are paralleled by differences in spatial working memory that may play a role in cognitive functions, which indirectly facilitate reproduction.
The Role of Glutamate in Drug Addiction
- Excitatory Control of Mesolimbic and Mesocortical Dopamine Neurons
Susan R. Sesack, Ph.D.
Dr. Susan Sesack presented research suggesting that excitatory brain stem afferents provide important sources of glutamate and cholinergic synapses onto mesolimbic DA neurons that are likely to contribute to the rewarding properties of drugs and their addictive liability. Her team’s research has focused on the major sources of excitatory drive to the ventral tegmental area (VTA), namely the prefrontal cortex (PFC) and the brain stem laterodorsal tegmentum (LDT), to identify the specific sources of afferents that innervate different populations of VTA DA neurons with discrete forebrain targets, specifically those projecting to the nucleus accumbens (NAc; mesolimbic) or to the PFC (mesocortical). Dr. Sesack presented electron microscopic evidence that VTA afferents from the PFC selectively target DA neurons that project back to the PFC and not those that innervate the NAc.
Dr. Sesack also suggested that LDT, a major source of cholinergic innervation to DA neurons, is a likely anatomical substrate for the reinforcing properties of nicotine. Researchers at her laboratory observed that both LDT afferents and axons labeled for cholinergic markers preferentially synapse onto DA cells with projections to the NAc. In addition, Dr. Sesack presented preliminary data showing that nicotinic alpha-7 receptor subunits in the VTA are localized to excitatory synapses, consistent with their role in the presynaptic regulation of glutamate release.
- Cocaine- and Extinction-Induced Neuroplasticity in AMPA Receptors Regulate Drug-Seeking Behavior
David W. Self, Ph.D.
Dr. David Self presented data suggesting that that behavior-based approaches aimed at modulating neuroplasticity in the mesolimbic dopamine system could be useful in treating cocaine addiction. Using viral-mediated overexpression of GluR1 in vivo to study the role of enhanced AMPA-mediated input to VTA neurons on the motivation for cocaine in self-administration tests, his team found that chronic cocaine self-administration upregulates the phosphorylated form of the GluR1 AMPA receptor subunit in the VTA, an effect that could contribute to enhanced AMPA-mediated excitability of dopamine neurons. Their results suggest that GluR1 upregulation in VTA neurons increases the motivation for cocaine and that protein kinase A-mediated GluR1 phosphorylation may be critical for this effect.
In contrast to the VTA, GluR1 levels in the NAc shell are up-regulated by repeated extinction training experience, an effect that coincides with a reduction in cocaine-seeking behavior. The research team studied viral-mediated overexpression of GluR1 in NAc neurons, which prevents the development of cocaine sensitization and reduces the ability of D2 dopamine receptor stimulation to elicit cocaine-seeking behavior. Although overexpression of a dominant negative GluR1 mutant to weaken AMPA-mediated input to NAc neurons facilitates sensitization of D2 receptor-mediated behavioral responses, their results suggest that extinction-induced increases in GluR1 in the NAc lessen cocaine addiction by reducing dopaminergic stimulation of D2 receptors.
- Role of VTA Glutamate in Contextual Cue-Induced Relapse to Heroin-Seeking
Yavin M. Shaham, Ph.D.
In humans, exposure to stimuli associated with drug intake can provoke relapse to drug use after prolonged abstinence. These stimuli are of two general types: discrete drug cues, which are temporally associated with drug reward, and contextual drug cues, which are not temporally associated with the acute rewarding effects of drugs but predict drug availability. Many laboratories explore the neuronal mechanisms underlying discrete cue-induced drug-seeking in rat models. In contrast, little is known about the mechanisms underlying contextual, cue-induced drug-seeking, despite the important role that these cues play in human drug relapse.
To study the role of the drug context in relapse, Dr. Yavin Shaham and colleagues recently adapted a renewal procedure from the fear-conditioning literature. They found that in rats trained to self-administer cocaine or a heroin-cocaine mixture, exposure to the drug self-administration context, after extinction of the drug-reinforced behavior in a different context, reliably reinstated drug-seeking. Dr. Shaham summarized these findings and presented new data on the effect of systemic and intracranial injections of a group II metabotropic glutamate receptor agonist, LY379268, on context-induced reinstatement of heroin-seeking.
- Phenotype-Driven Approaches to Understanding the Neuronal Plasticity Associated With Drugs of Abuse
Marc G. Caron, Ph.D.
Neurotransmitter transporters play a fundamental role in the regulation of neuronal activity by limiting the diffusion and action of neurotransmitters in extracellular space. Genetic inactivation of plasma membrane and vesicular transporters in the mouse has revealed a key role for these proteins in the maintenance of the homeostasis of presynaptic neurochemistry and physiology. Through reverse genetic approaches, Dr. Marc Caron and colleagues used genetic animal models to identify the underlying molecular components associated with neuronal plasticity and relate them to drugs of abuse. These genetic animal models—which recapitulate the pharmacological models of “behavioral sensitization” associated with exposure to psychostimulants (dopamine transporter knockout [DATKO]), tricyclic antidepressants (norepinephrine transporter knockout), and reserpine (vesicular monoamine transporter knockout)—all demonstrated enhanced behavioral responses to direct or indirect DA receptor agonists. Mice lacking PSD-95 (PSD-95-GK) recapitulated the molecular, cellular, and behavioral phenotypes of the original mouse models, suggesting that modulation of PSD-95 might contribute not only to learning and memory but also to drug-related plasticity. Dr. Caron’s team used the ENU mutagenesis of DATKO mice to identify dominant modifier genes of the hyperactivity phenotype. DATKO mice, unlike their heterozygote littermates, display a novelty-dependent hyperactivity phenotype. Several putative mutants were identified that either enhance or attenuate the DATKO phenotype or provoke a hyperactivity phenotype in the DAT heterozygotes. In the DATKO and amphetamine-treated mice, the team demonstrated that neuronal and behavioral responses to either DA or psychostimulants are mediated, at least in part, via a lithium-sensitive signaling cascade involving concomitant inactivation of Akt/PKB and activation of GSK-3. These biochemical changes are not affected by activation of the cAMP pathway but are effectively reversed by the inhibition of DA synthesis, D2 receptor blockade, or lithium. Pharmacological inhibition or genetic inactivation of GSK-3 significantly reduces the DA-dependent locomotor behaviors in mice. These findings support a role for the Akt/GSK-3 signaling pathway as an important mediator of psychostimulant actions in vivo. Pharmacological modulation of this signaling pathway might be relevant to the management of drugs of abuse.
- Are New Targets for Addiction Pharmacotherapy Hiding in Excitatory Synapses?
Peter W. Kalivas, Ph.D.
Dr. Peter Kalivas discussed recent studies demonstrating that withdrawal from repeated cocaine exposure produces enduring changes in the content and/or function of several proteins in the nucleus accumbens that regulate excitatory synaptic transmission. It was proposed that by reversing these adaptations, it would be possible to block the behavioral plasticity produced by repeated cocaine administration, such as locomotor sensitization and the reinstatement of drug-seeking. Candidate proteins for this process include the catalytic subunit of the cystine/glutamate exchanger (xCT); long-form Homer-1 and Homer-2 gene products; activator of G protein signaling 3 (AGS3); and proteins regulating the disassembly of actin, including Lim kinase (LIMK). These proteins are altered by withdrawal from repeated cocaine exposure, and a variety of strategies were used to restore protein function to normal or to mimic cocaine-induced functional change in drug-naive rats, including systemic drug administration (N-acetylcysteine to restore xCT), adeno-associated viral transfection of Homer cDNA, and Tat-HIV fusion proteins with binding domains of AGS3 or cofilin (LIMK substrate). Dr. Kalivas presented data showing that these proteins offer novel pharmacotherapeutic targets for inhibiting relapse in the reinstatement model of drug-seeking.
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