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October 13, 2006
Atlanta, Georgia

Introduction

The 2006 Frontiers in Addiction Research mini-convention brought together participants from all over the world, representing a diverse array of scientific disciplines, to share cutting-edge advances in research and to discuss future directions in the neuroscience of drug abuse and addiction. Many of the discoveries and research advances discussed here hold great promise for eventual translation into effective interventions to treat and prevent the serious public health problems of drug abuse and addiction and their related health and social consequences.

Roles of Hypthalamic Peptides in Addiction and Obesity

Dissecting Neural Circuits That Control Feeding
Scott M. Sternson, Ph.D.

In the hypothalamic arcuate nucleus (ARC), pro-opiomelanocortin (POMC) neurons inhibit feeding, and neuropeptide-Y/agouti-related protein (NPY/AgRP) neurons stimulate feeding. These neuron populations are important for the anorexigenic action of leptin. We have used laser-scanning photostimulation to map the functional connectivity between the ventromedial hypothalamic nucleus (VMH) and these molecularly defined neurons in the ARC. We show that POMC and NPY neurons, which are interspersed in the ARC, are nevertheless regulated by anatomically distinct synaptic inputs from the VMH. We are also investigating the functional contribution of these individual cell types to the physiologic effects of leptin. To address this, we have developed a chimeric receptor with variants of the FK506 binding protein (Fv2) fused to the cytoplasmic signaling domain of LepR (Fv2-LepR). A dimeric small molecule ligand that tightly binds Fv is used to activate LepR-like signaling. Using BAC transgenesis, we targeted this receptor specifically to the anorexigenic POMC-expressing neurons and, in a separate mouse line, to the orexigenic AgRP-expressing neurons. When these mice receive the small molecule dimerizer, leptin-receptor signaling is activated only in POMC or AgRP neurons, respectively, allowing the role of these neurons in leptin physiology to be dissected. These mouse lines are currently being characterized in studies of feeding and energy homeostasis. Both of these approaches can be applied to other molecularly defined neuron populations controlling feeding and body weight.

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Stress, Arousal, and Addiction: The Hypocretin Connection
Luis de Lecea, Ph.D.

Hypocretins, also known as orexins, are two neuropeptides now commonly described as critical components to maintain and regulate the stability of arousal. Several lines of evidence have raised the hypothesis that hypocretin-producing neurons are part of the circuitries that mediate the hypothalamic response to acute stress. Intracerebral administration of hypocretin leads to a dose-related reinstatement of drug- and food-seeking behaviors. Furthermore, stress-induced reinstatement can be blocked with hypocretin receptor 1 antagonism. These results, together with recent data showing that hypocretin is critically involved with cocaine sensitization through the recruitment of NMDA receptors in the ventral tegmental area, strongly suggest that the activation of hypocretin neurons play a critical role in the development of the addiction process. The activity of hypocretin neurons may affect addictive behavior by contributing to brain sensitization or by modulating the brain reward system. Hypocretinergic cells, in coordination with brain stress systems, may lead to a vulnerable state that facilitates the resumption of drug-seeking behavior. Hence, the hypocretinergic system is a new drug target that may be used to prevent the relapse of drug seeking.

Orexin Neurons, Reward-Seeking, and Addiction: It All Comes Together in the Lateral Hypothalamus
Gary Aston-Jones, Ph.D.

The lateral hypothalamus (LH) is a brain region historically implicated in reward and motivation, but the neurotransmitters of LH neurons involved with these functions are unknown. The orexins (or hypocretins) are neuropeptides recently identified as neurotransmitters in LH neurons. Although knockout and transgenic overexpression studies have implicated orexin neurons in arousal and sleep, many orexin cells project to reward-associated brain regions, including the nucleus accumbens and ventral tegmental area (VTA). This indicates a possible role for these neurons in reward function and motivation, which is consistent with previous studies implicating these neurons in feeding. We found that the activation of LH orexin neurons is strongly linked to preferences for cues associated with morphine, cocaine, or food reward. In addition, we showed that the chemical activation of LH orexin neurons reinstates an extinguished conditioned place preference (CPP). This reinstatement effect was completely blocked by the prior administration of an orexin A antagonist. Moreover, the administration of the orexin A peptide directly into the VTA also reinstated CPP. Finally, we discovered that specific lesion of the LH orexin projection to the VTA prevents learning a morphine CPP. All these findings were found specifically for LH orexin neurons; orexin neurons in the perifornical or dorsomedial hypothalamus did not exhibit these reward-linked properties. These data reveal a novel role for LH orexin neurons in reward-seeking, drug relapse, and addiction.

Hypocretin Enhances Synaptic Strength in VTA Dopamine Neurons
Stephanie L. Borgland, Ph.D.

Dopamine neurons in the ventral tegmental area (VTA) represent a critical site of synaptic plasticity induced by addictive drugs. Orexin/hypocretin-containing neurons in the lateral hypothalamus project to the VTA, and behavioral studies have suggested that hypocretin neurons play a critical role in motivation, feeding, and adaptive behaviors. However, the role of hypocretin signaling in neural plasticity is poorly understood. We demonstrate that in vitro application of hypocretin 1 induces the potentiation of N-methyl-D-aspartate receptor (NMDAR)-mediated neurotransmission via a PLC/PKC-dependent insertion of NMDARs in VTA dopamine neuron synapses. Interestingly, this hypocretin-mediated acute potentiation of NMDARs resulted in an augmentation of AMPAR-mediated synaptic transmission several hours later. We examined the behavioral relevance of this plasticity using locomotor sensitization. This behavioral paradigm is characterized by an augmented locomotor response to repeated injections of cocaine, and its initiation is largely dependent on NMDAR receptor activation in the VTA. In vivo administration of a hypocretin 1 receptor antagonist systemically or within the VTA blocked locomotor sensitization to cocaine and occluded cocaine-induced potentiation of excitatory currents in VTA dopamine neurons. These results provide in vitro and in vivo evidence of a critical role for hypocretin signaling in the VTA in neural plasticity relevant to addiction. Furthermore, data regarding the role of hypocretin signaling in cocaine self-administering rats will be discussed. Thus, hypocretin receptors may provide novel pharmacotherapeutic targets for motivational disorders, such as drug-craving.

Keynote Speech: Jacob P. Waletzky Award Recipient

Introduction
Terry Robinson, Ph.D., Director, NIDA Training Program in Neuroscience, University of Michigan

Established in 2003, the Society for Neuroscience Jacob. P. Waletzky Memorial Award is given for innovative research in drug addiction and alcoholism. This year's award recipient was Yavin M. Shaham, Ph.D.

Incubation of Cocaine Craving: Behavioral and Neuronal Mechanisms
Yavin M. Shaham, Ph.D.

Using a rat model of drug relapse and craving, we previously found time-dependent increases in cocaine seeking induced by exposure to drug cues after withdrawal from the drug, suggesting that cocaine craving incubates over time. In subsequent studies, we found that the time-dependent increases in cocaine seeking are associated with increases in the peptide levels of the plasticity-related growth factor BDNF in the nucleus accumbens, amygdala and ventral tegmental area (VTA), and that a single VTA infusion of BDNF induces long-lasting increases (up to 30 days) in cocaine seeking after withdrawal. I will discuss these findings and also present results from a series of experiments that led us to conclude that the central, but not the basolateral, amygdala ERK signaling pathway mediates the incubation of cocaine craving.

Social Neuroscience - Applications to Addiction

Social Defeat, Gene Expression, and Vulnerability to Addiction
Huda Akil, Ph.D.

In humans, it is well established that stressful life events play an important role in triggering drug-seeking behavior in vulnerable individuals and in contributing to the maintenance and relapse of substance abuse. Because life events are typically psychosocial in nature, it is important to ask in animal models whether there are distinct neural mechanisms that encode social stress and how these mechanisms affect drug-seeking behavior.

We have chosen territorial social behavior in male rodents as our animal model. This behavior typically results in a clearly dominant and clearly subordinate animal, and the latter is presumed to have undergone "social defeat." We characterized the neural pathways that encode agonistic behavior and distinguished the subordinate from the dominant animal. The pathways associated with social defeat show remarkable parallels to the circuitry implicated in severe depression in humans.

We then used social defeat in animals that have different environmental reactivity, different propensities to seek novelty, and different endocrine responses to stress and, importantly, that exhibit a differential propensity to self-administer drugs of abuse. This is the high-responder versus low-responder trait. We have shown that social defeat stress has a differential impact on the behavior of these two types of animals. Moreover, the neural changes associated with social defeat are unique in these two behavioral phenotypes. Many of the observed changes are relevant to the mechanisms of neuroplasticity that appear to differ between animals and are critical for responsiveness both to social defeat stress and to the development of addiction.

The findings will be discussed in terms of multiple pathways to addiction and their distinctive impact on brain and behavior.

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PET Imaging of Dopamine D2Receptors in Cocaine Abuse: A Social Neuroscience Perspective 
Michael A. Nader, Ph.D.

The primary goal of this research is to achieve a better understanding of the individual differences in the susceptibility and vulnerability to the reinforcing effects of cocaine using a unique nonhuman primate model of drug abuse. To accomplish this, we have combined the study of primate social behavior with intravenous drug self-administration and the noninvasive brain imaging procedure positron emission tomography (PET) to examine how environmental and pharmacological variables influence the behavioral and reinforcing effects of cocaine. These studies use male and female monkeys to examine trait variables that are predictive of eventual social rank, such as locomotor activity, measures of impulsivity, hormone levels, and measures of dopamine (DA) and serotonin function with PET. We also examine how social group formation influences these measures and how cocaine self-administration is affected by social rank. Studies will be described related to the plasticity of the DA system during cocaine abstinence and following social group reorganization as well as to the impact of these manipulations on cocaine intake. The use of these novel and homologous nonhuman primate models of cocaine abuse should aid in understanding how environmental and pharmacological variables contribute to the vulnerability, maintenance, and relapse to drugs of abuse. This information could lead to better treatment and prevention strategies.

Getting To Know You: Reputation and Trust in a Two-Person Economic Exchange
P. Read Montague, Ph.D.

Recent work in neuroimaging has combined computational models with novel imaging methods to begin to probe the neural correlates of social exchange. The importance of these approaches to understanding reward-processing and the nature of addictive (pathological) reward-processing derives in part from the influence of group pressures on perception and action. In the context of a group, perceptions can be dramatically changed, and valuation and decision-making can be perturbed. The growing capacity to make multibrain measurements in the context of live social exchanges offers a new approach to the dynamic brain states that underlie both the normal and pathological processing of social signals and the decisions they engender. In this talk, I will emphasize the need to use quantitative behavioral models to help guide the design and interpretation of social exchange experiments.

Social Cognitive Neuroscience: Exploring the Psychological and Neural Bases of Socioemotional Experience and Behavior
Kevin Ochsner, Ph.D.

The past few years have seen an explosion of interest in using cognitive neuroscience methods to study the mechanisms underlying human emotion and social behavior. In this talk I will give a brief overview of the social cognitive neuroscience approach that motivates this work; illustrate its usefulness for studying emotion regulation, using examples from our own research on cognitive reappraisal; and consider the relevance of this work for understanding the mechanisms underlying substance abuse.

Recent Results From Genome-Wide Scans for Addiction and Other Brain Diseases

Pooled Association Genome-Scanning for Addictions: Convergent Observations
George R. Uhl, M.D., Ph.D.

Classical genetic studies document strong, complex genetic contributions to the abuse of multiple addictive substances and to the ability to quit smoking. Mnemonic processes that are likely to include those involved with substance dependence, and to the volumes of brain gray matter in regions that are likely to contribute to mnemonic/cognitive and addictive processes, also show substantially heritable individual differences. Whole genome association studies have been increasingly recognized as methods of choice for elucidating the genetic underpinnings of complex genetically and environmentally influenced disorders, such as addictions.

We can now identify the results of whole genome association studies—using multiple samples from individuals addicted to drugs and matched controls—that now identify many of the loci and genes that contain allelic variants that are likely to provide the heritable components of human addiction vulnerability. These data identify a surprising number of haplotypes in genes that are involved with cell adhesion processes, whereby neurons recognize each other during development and in the synaptic modifications that occur in adult brains. Data from these addiction-vulnerability genes, as well as data from the genes that distinguish successful versus unsuccessful quitters, are likely to inform our understanding of addictions and also to provide sets of markers that are clinically useful for better assignment of individuals to appropriate treatment and prevention modalities. Medium-sized clinical trials of nicotine cessation, for example, could gain substantial power and reduce costs by using genotypic stratification.

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Understanding the Genetic Architecture of Common Disease: A Comparison of Genome Scans 
Dennis G. Ballinger, Ph.D.

Perlegen Sciences has conducted several genome-wide SNP association studies. The different designs of these studies will be compared and contrasted. In addition, common and distinct features of the genetic architecture of the various phenotypes will be discussed. The studies involved will include predisposition to Parkinson's disease with both sibling and unrelated controls; late onset Alzheimer's disease cases and matched controls; and nicotine-addicted and matched exposed nonaddicted controls.

A Hybrid Approach to Genetic Studies: Genome-Wide Association Study and Comprehensive Candidate Gene Analysis of Nicotine Dependence
Laura J. Bierut, M.D.

Smoking is the leading source of preventable death in the United States, and twin studies consistently demonstrate strong genetic contributions to smoking. The NICSNP Project is a hybrid study of a genome-wide association (GWA) study in tandem with the systematic coverage of biologically relevant candidate genes. The sample consisted of 1,050 nicotine-dependent cases and 879 nondependent smokers as controls. All participants were selected from two community-based studies, the Collaborative Genetics Study of Nicotine Dependence (United States) and the Nicotine Addiction Genetics Project (Australia). The GWA study performed pooled genotyping of 2.4 million single nucleotide polymorphisms (SNPs) followed by individual genotyping of the top 40,000 signals. The second arm of the study was a comprehensive candidate gene study, where individual genotyping was conducted in more than 300 genes chosen for their biological significance by experts in the field of addiction. There were convergent findings in these complementary approaches. Common variants in candidate genes exhibited the highest level of statistical significance and are among the top 25 signals from the GWA study. In addition, the GWA study results identified novel loci not previously associated with the risk for nicotine dependence. These findings are a major advance in the genetics of complex human disease, and data will be available upon publication for further analyses by the scientific community.

Statistical Methodologies for Analyzing Whole Genome Association Data
John P. Rice, Ph.D.

Although it is clear that whole genome association (WGA) studies will be done in the near future on many genetic disorders, it is less clear how to design, analyze, and interpret these studies. We will address several of these issues and will underscore that there are limitations in most current methods. It is essential to understand these limitations and to evaluate the underlying assumptions.

We will cover the distinction between linkage disequilibrium (LD) blocks and bins and the different properties of D-Prime and R-Square in single nucleotide polymorphism (SNP) selection. We will describe analytic issues - problems of multiple testing, use of prior linkage information, use of genomic controls, and the problem of whether to base the analysis on allelic or genotypic differences. We will also discuss the analysis of haplotypes and SNPs in different genes.

Finally, we will consider the implications of WGA studies for our understanding of the genetics of substance use disorders. The public availability of genotypic data and DNA to qualified investigators is a key area of debate.

Molecular Mechanisms of Synapse Formation: Adhesion Molecules

Functional Regulation of Synaptic Adhesion Complexes by Alternative Splicing
Peter Scheiffele, Ph.D.

The formation of synapses during the development of the central nervous system requires the coordinate differentiation of presynaptic and postsynaptic membrane domains. Cell adhesion molecules have been proposed to play important roles in directing this differentiation process as well as the selectivity of synaptic cell-cell interactions. We have explored the role of the heterophilic neuroligin-neurexin adhesion complex in synapse assembly. Neuroligins and neurexins each constitute protein families, with multiple isoforms generated from multiple genes through alternative transcription start sites and alternative splicing. We observed that the alternative splicing of neuroligin and neurexin isoforms underlies their selective function at GABAergic and glutamatergic synapses in hippocampal neurons. Specific classes of neuroligin and neurexin isoforms exclusively promote the assembly of glutamatergic presynaptic and postsynaptic structures, whereas other classes function exclusively at GABAergic synapses. These data suggest that the highly diverse extracellular domains of neuroligin and neurexin isoforms encode selective transsynaptic interactions that contribute to the assembly or modification of different types of central synapses.

SynCAMs: From Synaptic Adhesion to Synapse Formation
Thomas Biederer, Ph.D.

Synaptogenesis is a defining process of neuronal network formation. Synapse formation is most intense in early postnatal development, but synapses continue to turn over and form anew throughout adulthood. Only few interactions in the mammalian central nervous system (CNS) are known to directly induce new synapses. The analysis of these interactions, mediated by SynCAM 1 and the neuroligin/neurexin membrane proteins, has shown that these synaptic adhesion proteins can drive the formation of synaptic specializations through transsynaptic interactions. SynCAM 1 drives neurons to form fully functional presynaptic terminals upon physical contact.

SynCAM 1 is a homophilic cell adhesion molecule widely expressed in the CNS. It is an N-glycosylated, single-spanning membrane protein and is expressed during the peak period of synaptogenesis. Importantly, SynCAM 1 is enriched in synaptic membranes and induces the formation of new, functional presynaptic terminals in a coculture assay of hippocampal neurons. This activity of SynCAM 1 is monitored using live-cell optical imaging, and we employ it to reconstitute synaptic transmission. Using this reconstitution approach, properties of evoked synaptic transmission can be observed and analyzed under defined conditions in vitro. Concurringly, the overexpression of SynCAM 1 in transfected neurons increases the frequency of minis. This activity of SynCAM 1 depends on its cytosolic sequence, and the SynCAM 1-induced facilitation of minifrequencies is specific for excitatory currents.

Do these adhesive interactions at synaptic sites specify key properties of synaptogenesis? To begin to address this question in detail, we study the SynCAM family, founded by SynCAM 1. It consists of four members. All SynCAM proteins have three extracellular immunoglobulin-like domains and a highly conserved cytosolic tail with a PDZ interaction motif. We demonstrate through real-time RT-PCR analysis and in situ hybridization that the four SynCAM proteins are neuronally expressed, present in the developing vertebrate brain, and exhibit distinct regional and developmental expression profiles. Our biochemical studies identify that three of the four SynCAM proteins, including SynCAM 1, exert homophilic adhesive interactions. In addition, specific heterophilic adhesive interactions can occur among particular family members. These studies indicate that particular SynCAM interactions may constitute an adhesive code during synaptogenesis. We currently determine to which extent the four members of the SynCAM family of neuronal adhesion molecules drive the formation of presynaptic terminals. It is our hypothesis that all SynCAM proteins can drive synapse formation and that their homophilic and heterophilic interactions contribute to synapse specification.

These studies aim to provide deeper insight into the molecular mechanisms directing the initial steps of synapse formation in the CNS. Future analyses have a goal to determine the roles of synaptogenesis in the modulation of neuronal circuits.

Regulation of Synapse Formation and Sprouting by Cadherin Adhesion Complexes
Shernaz X. Bamji, Ph.D.

Recent studies suggest commonalities between the development of addictive behaviors and traditional learning models. For example, synaptic plasticity, which has long been the molecular correlate of learning and memory, has been demonstrated in neural reward circuits and is believed to contribute to the learning of addictive behaviors. The rapid formation and elimination of synaptic sites occurs throughout life and represents one aspect of synaptic plasticity in which synaptic communication is modified in the long term. Synaptic adhesion proteins are of particular interest in this context because presynaptic to postsynaptic membrane adhesion is one of the initial events during synapse formation and remains a fundamental component of the maintenance of synapses in maturity. Our studies demonstrate a role for the cadherin adhesion complex in the localization of synaptic vesicles to developing presynaptic compartments. Despite the requirement for cadherin-based adhesion in some aspects of synapse formation, we show that the maintenance of strong cell-cell adhesion is detrimental to the formation of new synapses in the presence of the plasticity factor brain-derived neurotrophic factor (BDNF). We show, using time-lapse confocal analysis, that BDNF mobilizes synaptic vesicles at existing synapses, resulting in small clusters of synaptic vesicles "splitting" away from synaptic sites. BDNF's ability to mobilize synaptic vesicle clusters depends on the dissociation of cadherin/β-catenin adhesion complexes that occurs following tyrosine phosphorylation of β-catenin. Artificially maintaining cadherin/β-catenin complexes in the presence of BDNF abolishes the BDNF-mediated enhancement of synaptic vesicle mobility and also abolishes the longer term BDNF-mediated increase in synapse number. Together these data demonstrate that the disruption of cadherin/β-catenin complexes following BDNF treatment is an important molecular event through which BDNF increases synapse density. We are currently exploring the hypothesis that enhanced synaptic vesicle mobility contributes to the formation of new synapses at adjacent regions of the axon and that the disruption of strong cadherin-based adhesion catalyzes this event. We believe that the molecular adhesive machinery required for synapse assembly in development plays an essential role in modulating synaptic architecture in the context of plasticity-related structural remodeling.

Regulation of Synaptic Connectivity in C. Elegans: From Cell Adhesion to Morphogenetic Gradient
Kang Shen, M.D., Ph.D.

The presynaptic regions of axons accumulate synaptic vesicles and active zone and periactive zone proteins. The rules for the orderly recruitment of presynaptic components are not well understood. We systematically examined the molecular mechanisms of presynaptic development in egg-laying synapses of C. elegans, demonstrating that two scaffolding molecules, SYD-1 and SYD-2, play key roles in presynaptic assembly. SYD-2/liprin was previously shown to regulate the size and shape of active zones. We now show that in syd-1 and syd-2 mutants, synaptic vesicles and numerous other presynaptic proteins fail to accumulate at presynaptic sites. SYD-1 and SYD-2 function cell autonomously at presynaptic terminals, downstream of the synaptic specificity molecule SYG-1. SYD-1 likely acts upstream of SYD-2 to positively regulate its activity. These data suggest a hierarchical organization of presynaptic assembly, in which transmembrane specificity molecules initiate synaptogenesis by recruiting a few key scaffolding proteins, which in turn assemble other presynaptic components.

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