Peter W. Kalivas - US grants

A dysfunction of the brain circuitry mediating the translation of relevant environmental stimuli into adaptive motor responses is thought to have an etiologic or permissive role in many psychiatric disorders. Classic limbic structures provide emotional and cognitive context to environmental stimuli. The circuitry whereby this information is transferred to motor systems and the appropriate behavioral response initiated involves a series of nuclei possessing extensive reciprocal interconnectivity. This circuit is referred to as the 'motive circuit' and includes the ventral tegmental area, nucleus accumbens, ventral pallidum, mediodorsal thalamus, prefrontal cortex and pedunculopontine motor area. The goal of this continuation proposal is to functionally map the flow of information from limbic to motor nuclei through the motive circuit. This will be accomplished using three convergent technologies, 1) retrograde labeling of neurons combined with in situ hybridization for mRNA of relevant transmitter-related proteins, 2) intracranial infusions of transmitter analogues into the circuit to map the connections mediating behavioral activation, and 3) microinjecting transmitter analogues into the circuit and measuring changes in extracellular transmitter concentrations with microdialysis. The proposal is based upon three general hypotheses. 1) A detailed topography exists which permits the transfer of information into discrete compartments within nuclei comprising the motive circuit. 2) The selective transfer of information is, to some extent, chemically coded such that different neurotransmitters route information through different efferent projections. 3) The routing of information is under environmental/genetic control. This latter hypothesis relies on recent observations that the intensity of the motor response elicited by rats in a novel environment is correlated with neurochemical alterations in subnuclei of the motive circuit. Thus, prior to beginning all experiments, rats will be behaviorally screened in a novel open field to permit correlations between their motor response to novelty and other subsequent measures. Many behavioral symptoms defining psychiatric illnesses involve inappropriate behavioral responses to environmental stimuli. The motive circuit is responsible for translating environmental stimuli into adaptive motor responses. By understanding the anatomical and chemical organization of the motive circuit it will be possible to pharmacologically modulate inappropriate behaviors with greater accuracy. Furthermore, the anatomical and functional mapping of the motive circuit will provide a basis for interpreting future imaging studies in humans where attempts are made to correlate changes in spatially discrete nuclei.

A considerable research effort has been made to characterize the central effects of the opioids in terms of brain catecholamine neurotransmission. However, the mesocorticolimbic dopamine system, which has neuronal perikarya in the ventral tegmental area and axonal terminals in the nucleus accumbens, has not been studied to a great extent in this regard. The first objective of this proposal is to characterize the effects of opioids on mesocorticolimbic dopamine function. This will be performe by acute microinjection of opioid into the ventral tegmental area or nucleus accumbens of the rat. After microinjection, I will assess effects on the mesocorticolimbic dopamine system by neurochemical measurements of dopamine release, synthesis and turnover, and by behavioral measurements of motor activity. The effects of peripherally administered opioids will then be compared with the effects of intracranially administered opioids. In addition to the opioids, the indirect sympathomimetics, cocaine and amphetamine, have been shown to affect mesocorticolimbic dopamine function. Therefore, a second objective of this proposal is to evaluate an interaction between the opioids and cocaine and amphetamine. Again, microinjection of drugs into the ventral tegmental area or nucleus accumbens, followed by neurochemical and behavioral measurements shall be used to evaluate a potential interaction. Daily administration with amphetamine or cocaine causes a progressive increase in the mesocorticolimic-mediated behavioral and neurohemical effects produced by these drugs. It has also been found that daily injection with opioid into the ventral tegmental area produces a progressive increase in mesocorticolimbic-mediated responses. Thus, as a third objective of this proposal, I will evaluate an interaction between the opioids, amphetamine and cocaine in this behavioral and neurochemical "sensitization" phenomenon. These studies will provide insight into how the opioids affect the mesocorticolimbic dopamine system. It also evaluates an interaction between the acute and chronic administration of the opioids and the commonly abused sympathomimetics, cocaine and amphetamine. Not only have some studies implicated the involvement of the mesocorticolimbic dopamine system in the reinforcing properties of the opioids, cocaine and amphetamine, but the use of these drugs in combination in our society is becoming an increasingly severe social problem.

A considerable research effort has been made to characterize the central effects of daily administration of drugs of abuse. It has been shown that the motor stimulant effect of amphetamine, cocaine and opioids is progressively enhanced after daily administration. With the exception of amphetamine, relatively little experimentation has been conducted to characterize behavioral "sensitization" to cocaine or opioids in terms of alterations in dopaminergic function. Evidence is presented to suggest that a change in the dopamine (DA) neurons in the A10 region may underlie some aspects of behavioral sensitization. Therefore, neurochemical studies will focus on this brain region, as well as the nucleus accumbens, prefrontal cortex, striatum and A9 DA region. This proposed research will characterize the effects of daily administration of cocaine, ip, morphine, ip, enkephalin analogues, intra-A10, and stress on central DA systems. Rats will receive daily administration of these drugs or stress, and at various times afterward in vitro and postmortem studies will be performed, including measurement of basal and induced DA released from tissue slices, and measurement of DA metabolism, dopa accumulation and DA depletion after tyrosine hydroxylase inhibition. In addition, in vivo measurement of DA release will be assessed using voltammetery and intracranial dialysis. Once changes in dopaminergic function produce by daily exposure to drugs of abuse and stress have been defined, the capacity of daily treatment with one drug to alter the behavioral and neurochemical effects of another drug or stress (i.e. cross-sensitization) will be examined. These studies are designed to determine if a common dopaminergic mechanism may be mediating behavioral sensitization to the daily treatment of cocaine, opioids and stress. Cocaine abuse has markedly increased in recent years, and simultaneous opioid and cocaine abuse appears to be on the rise. Thus, the proposed studies will contribute not only to our understanding of the neurochemical consequence of cocaine and opioid abuse, but how parallel abuse of the drugs may synergize. While a role by stress in promoting drug abuse remains ill- defined, an interaction with DA systems in common with that of drugs of abuse would support such a role.

The mesocorticolimbic dopamine (DA) system arises from perikarya in the ventromedial mesencephalon and innervates a number of cortical and limbic regions. It has become clear that this system is in many ways distinct from the relatively well- characterized nigrostriatal DA system. This is especially true with regard to its postulated physiological functions, and role in the pathophysiology of certain neuropsychiatric disorders. The proposed conference, sponsored by the New York Academy of Sciences, will address the molecular, cellular and functional properties of the mesocorticolimbic DA system as they are distinct from those of the nigrostriatal DA system. In particular, the conference will evaluate data originating in basic research laboratories as they apply to the proposed involvement of the mesocorticolimbic DA system in certain neuropsychiatric disorders. The primary importance of this conference is to provide a forum that will allow basic and clinical research scientists to share their most recent data and hypotheses regarding the physiological and pathological role(s) of the mesocorticolimbic DA system. An entire conference devoted specifically to the mesocorticolimbic DA system has never been organized, and the recent proliferation of studies focused on this system would seem to indicate that such a conference would be useful for defining future research directions. This is particularly crucial considering the number of recent clinical studies attempting to critically evaluate the role of this DA system in certain psychiatric disorders, notably schizophrenia. This conference brings clinical and basic scientists together from divergent backgrounds, including anatomy, electrophysiology, neurochemistry, clinical neuropsychopharmacology and behavior. Another major goal of this conference is to provide an exchange of ideas that will: 1) summarize the state of knowledge in a rapidly evolving field, 2) collectively analyze these data in a multidisciplinary fashion and update old hypotheses or create new ones, and 3) identify productive and creative future research directions, with special emphasis on those that remain at the interface of basic and clinical research efforts.

A considerable research effort has been made to characterize the central effects of daily administration of drugs of abuse. It has been shown that the motor stimulant effect of amphetamine, cocaine and opioids is progressively enhanced after daily administration. With the exception of amphetamine, relatively little experimentation has been conducted to characterize behavioral "sensitization" to cocaine or opioids in terms of alterations in dopaminergic function. Evidence is presented to suggest that a change in the dopamine (DA) neurons in the A10 region may underlie some aspects of behavioral sensitization. Therefore, neurochemical studies will focus on this brain region, as well as the nucleus accumbens, prefrontal cortex, striatum and A9 DA region. This proposed research will characterize the effects of daily administration of cocaine, ip, morphine, ip, enkephalin analogues, intra-A10, and stress on central DA systems. Rats will receive daily administration of these drugs or stress, and at various times afterward in vitro and postmortem studies will be performed, including measurement of basal and induced DA released from tissue slices, and measurement of DA metabolism, dopa accumulation and DA depletion after tyrosine hydroxylase inhibition. In addition, in vivo measurement of DA release will be assessed using voltammetery and intracranial dialysis. Once changes in dopaminergic function produce by daily exposure to drugs of abuse and stress have been defined, the capacity of daily treatment with one drug to alter the behavioral and neurochemical effects of another drug or stress (i.e. cross-sensitization) will be examined. These studies are designed to determine if a common dopaminergic mechanism may be mediating behavioral sensitization to the daily treatment of cocaine, opioids and stress. Cocaine abuse has markedly increased in recent years, and simultaneous opioid and cocaine abuse appears to be on the rise. Thus, the proposed studies will contribute not only to our understanding of the neurochemical consequence of cocaine and opioid abuse, but how parallel abuse of the drugs may synergize. While a role by stress in promoting drug abuse remains ill- defined, an interaction with DA systems in common with that of drugs of abuse would support such a role.

The proposed research will investigate the anatomical and cellular substrates mediating behavioral sensitization to the daily administration of two drugs of abuse, cocaine and morphine. The search for the site of action and neuronal correlates of the enduring behavioral changes that occur in the brain as a result of chronic cocaine or morphine administration constitutes my major career goal. The ultimate progression of which is to develop biological interventions to assist in alleviating the behavioral problems associated with long term drug abuse in humans. Within this context, the immediate goal is to evaluate the role of the A10 dopamine neurons in behavioral sensitization, using in vivo neurochemical measures of dopamine and GABA release. Also, the density of dopamine, GABA and opioid receptors, and the levels of mRNA for some proteins relevant to dopamine and GABA neurotransmission will be measured with light microscopic autoradiography in behaviorally sensitized rats. In addition, a role for GTP binding proteins and K+ conductance will be determined.

The proposed research will evaluate the effects of acute and daily peripheral administration of morphine and cocaine, and intra-A10 injection of enkephalin on the mesocorticolimbic dopamine and ventral striatopallidal GABA systems in rats. In vivo behavioral and neurochemical techniques will be used, and in vitro receptor density and mRNA levels will be measured. All three compounds produce an acute motor stimulant response, and following daily administration the motor stimulant response is augmented. It is hypothesized that daily administration of morphine, cocaine and enkephalin produces behavioral sensitization by modifying the response of A10 dopamine neurons to afferent input, and that this will be reflected in alterations in dopamine release in the nucleus accumbens and A10 region, and in GABA release in the ventral pallidum and A10 region. Release will be estimated by measuring extracellular dopamine and GABA levels with intracranial dialysis. It is also proposed that behavioral sensitization may be reflected in changes in dopamine D1, dopamine D2, mu opioid, delta opioid or GABAA receptors in discrete brain nuclei as measured by receptor autoradiography. Also, in situ hybridization will be used to determine the effects of acute and daily drug treatment on mRNA for tyrosine hydroxylase, glutamic acid decarboxylase, Gi2 subunit or D2 receptors. Finally, the effect of manipulating: 1) D1, D2 or GABAB receptors, 2) potassium channels, or 3) the Gi/Go subunits of GTP binding protein, in the A10 region on the acute and daily effect of cocaine, morphine or enkephalin will be determined. Behavioral sensitization produced by the long-term use of cocaine is manifested clinically as an augmentation of paranoid behaviors. By determining the underlying anatomical substrates and cellular mechanisms mediated sensitization to cocaine and opioids in rats, it is possible that more rational therapy can be designed for intervention in human drug abuse where sensitization is thought to be a factor.

The proposed research will evaluate the effects of acute and daily peripheral administration of morphine and cocaine, and intra-A10 injection of enkephalin on the mesocorticolimbic dopamine and ventral striatopallidal GABA systems in rats. In vivo behavioral and neurochemical techniques will be used, and in vitro receptor density and mRNA levels will be measured. All three compounds produce an acute motor stimulant response, and following daily administration the motor stimulant response is augmented. It is hypothesized that daily administration of morphine, cocaine and enkephalin produces behavioral sensitization by modifying the response of A10 dopamine neurons to afferent input, and that this will be reflected in alterations in dopamine release in the nucleus accumbens and A10 region, and in GABA release in the ventral pallidum and A10 region. Release will be estimated by measuring extracellular dopamine and GABA levels with intracranial dialysis. It is also proposed that behavioral sensitization may be reflected in changes in dopamine D1, dopamine D2, mu opioid, delta opioid or GABAA receptors in discrete brain nuclei as measured by receptor autoradiography. Also, in situ hybridization will be used to determine the effects of acute and daily drug treatment on mRNA for tyrosine hydroxylase, glutamic acid decarboxylase, Gi2 subunit or D2 receptors. Finally, the effect of manipulating: 1) D1, D2 or GABAB receptors, 2) potassium channels, or 3) the Gi/Go subunits of GTP binding protein, in the A10 region on the acute and daily effect of cocaine, morphine or enkephalin will be determined. Behavioral sensitization produced by the long-term use of cocaine is manifested clinically as an augmentation of paranoid behaviors. By determining the underlying anatomical substrates and cellular mechanisms mediated sensitization to cocaine and opioids in rats, it is possible that more rational therapy can be designed for intervention in human drug abuse where sensitization is thought to be a factor.

DESCRIPTION (applicant's abstract): Repeated administration of cocaine produces enduring changes in behavior, including a progressive increase in cocaine-induced motor stimulation. The underlying neuroadaptations mediating motor sensitization in rodents have been proposed to contribute to behaviors associated with cocaine addiction, such as paranoia and craving. Knowledge that monoamine transporters constitute the molecular binding site for cocaine has focused investigation of the cellular underpinnings of behavioral sensitization on dopamine transmission in the nucleus accumbens. Although this research endeavor has revealed many long-lasting neuroadaptations in dopamine transmission that contribute to behavioral sensitization, it has become clear that the focus on neither dopamine nor the nucleus accumbens provides a complete profile of cocaine-induced neuroadaptations mediating addiction. To some extent exploration beyond dopamine is impelled by the realization that behavioral sensitization is potently regulated by learned associations made between the pharmacological actions of cocaine and environmental stimuli. Thus, glutamatergic cortical brain regions involved in learning and memory have become another focus in the search for substrates mediating behavioral sensitization to cocaine. This proposal will evaluate an overarching hypothesis that the enduring changes in pre- and post-synaptic glutamate transmission in the nucleus accumbens and adjacent circuitry mediate behavioral sensitization to cocaine. Three general experiments will be performed. 1) The mechanisms mediating cocaine-induced changes in vesicular- and transporter-mediated glutamate release will be examined. 2) The effect of repeated cocaine on proteins involved in glutamate receptor signaling will be examined and adenovirus gene transfer will be employed to evaluate the role of these proteins in behavioral sensitization. 3) The role that glutamate transmission plays in the conditioned motor responses associated with repeated cocaine administration will be examined.

PROJECT SUMMARY: This is a 5 year competitive renewal that builds upon nearly a decade of previously funded research that has identified the projection from the prefrontal cortex to the nucleus accumbens, and then to the ventral pallidum as a key brain circuit underlying relapse to cocaine use. Relapse is modeled in rats trained to self-administer cocaine and then undergone extinction training, and is defined as the reinstatement of cocaine seeking in response to a conditioned cue, a noncontingent injection of cocaine, or after inhibition of the infralimbic cortex that is hypothesized to disinhibit extinguished responding. In the first aim of the present proposal we will use the recent development of genetically targeted light- activated proteins to validate the role of this circuitry in the reinstatement of cocaine seeking. Previous pharmacological or electrical stimulation techniques used to define brain circuitry suffer caveats such affecting fibers of passage and/or multiple projections from a given nucleus, and these weaknesses are largely circumvented using in vivo light activation of virally transfected channels or transporters. Thus, the first aim is devoted to refining this new technology and validating, or not, previous circuitry studies by our group and others identifying the PFC-accumbens-pallidal circuit as important in relapse. The remaining 3 aims focus on our recent finding that dendritic spine morphology in the nucleus accumbens undergoes rapid and profound changes during a cocaine-primed reinstatement of drug seeking. Firstly, in Aim 2 we will fully characterize these morphological changes in both the core and shell subcompartments of the accumbens in response to different modalities of inducing cocaine seeking (e.g. cue, cocaine and inhibition of infralimbic cortex). Then in Aim 3 we will determine which afferents to the nucleus accumbens are mediating the reinstatement-associated morphological changes, with a focus on glutamatergic inputs from the prefrontal cortex and amygdala, and dopaminergic inputs from the ventral tegmental area. Finally in Aim 4 we conduct experiments to determine if the changes in spine morphology are necessary for reinstating cocaine seeking, or if they are protective and inhibit reinstatement. Accordingly, we will test the capacity of 3 different inhibitors of actin dynamics to simultaneously inhibit primed reinstatement and reinstatement-associated spine morphology. Interestingly, preliminary data presented in the proposal indicates that the rapid morphological changes may be protective rather than causal in reinstating cocaine seeking.

The research experiences for undergraduates (REU) program at the Medical University of South Carolina is designed to provide both didactic and laboratory research experience in cutting-edge neuroscience research. The successful applicants will receive overview training in the neurosciences, exposure to one or more laboratories where they will have hands on experience in a specific research project, as well as an appreciation for the ethical responsibilities faced by researchers responsible for bringing new knowledge into society and training new neuroscientists. Students will have the opportunity to use a variety of biological, chemical and biophysical approaches to understanding cell biology as it pertains to the functioning of neurons. Research will be focussed on the cellular neuroplastic responses of in vitro and in vivo models of central nervous system functions, including studies of receptors, signal transduction pathways, signaling in development and cell differentiation, as well as in memory formation. The research period will be for a period of ten weeks with an immediate goal of, 1) giving the students hands on experience in the excitement of scientific discovery in the neurosciences, and 2) an appreciation for what a career in research in the neurosciences would be like. The overarching goal and significance of this proposal is that through exposure to the REU program we can attract an important human resource into the field of neuroscience research. However, even if a career in science is not chosen, the REU program will give future decision makers in our society a strong appreciation for the discovery process.

DESCRIPTION (provided by applicant): The personal, social and criminal ramifications of psychostimulant abuse is an enormous problem in North America. Clarifying the neural substrates that underlie addiction to drugs of abuse is critical for designing rational pharmacological interventions with the potential to cure addicts. A point in the cycle of addiction where pharmacological intervention can be particularly beneficial is to interfere with the overwhelming desire by addicts to use drugs. The Neurobiology of Addiction Research Center (NARC) uses a rat model of cocaine-seeking and unites an assemblage of investigators with expertise in behavior, neurochemistry, electrophysiology and cell biology. An Animal Core will generate rats with a history of stable cocaine self-administration that have been withdrawn from drug in an extinction or abstinence paradigm. After withdrawal, cocaine-seeking will be induced by presentation of conditioned cues, cocaine or drug context. The rats are then dispersed to the various projects for neurobiological evaluations. This core facility will insure consistency in experimental subjects, thereby permitting more accurate associations to be made between neuroadaptations associated with cocaine-seeking. Another aspect of the NARC that will be held constant is examining the same limbic-motor circuit. Project 1 endeavors to link changes in synaptic proteins, dendritic morphology and electrophysiology in excitatory accumbens synapses that are associated with cocaine-seeking. As well, project 1 will evaluate behaviorally effective medications for effects on the cocaine associated neuroplasticity. Project 2 will explore the cellular basis underlying the capacity of prefrontal BDNF administration to ameliorate cocaine-seeking, with a focus on prefrontal output to the nucleus accumbens and ventral tegmental area. Project 3 explores the neurobiological mechanisms whereby orexin antagonists inhibit cocaine-seeking, focusing on projections to the ventral tegmental area and prefrontal cortex. Finally, Project 4 will examine novel treatments derived from the first 3 projects that possess potential for inhibiting drug-seeking behavior. Thus, this project acts as a translational program to identify systemically active compounds with the potential for entering clinical trials. In addition to this highly integrated, multi-disciplinary approach to the neurobiology and treatment of cocaine-seeking, the NARC will facilitate scientific interactions, provide oversight and mentor junior faculty, pre- and postdoctoral trainees and undergraduates. In part this will be accomplished through a Pilot Core that will directly involve young and established investigators outside the field of addiction in evaluating novel hypotheses related to the neurobiology of relapse. In summary, the NARC provides a multidisciplinary research assault on the neurobiology and medications development for cocaine-seeking and is a mechanism for actively mentoring faculty and trainees. CENTER CHARACTERISITICS

DESCRIPTION (provided by applicant): One of the biggest impediments to the successful treatment of cocaine addiction is the high rate of relapse, following even extended periods of drug abstinence. Exposures to stress, drug-paired cues, and cocaine itself have all been shown to increase reports of craving in human drug users and to elicit a reinstatement of drug-seeking behavior in animal models of relapse. However, there is a lacuna in our understanding of the neural circuitry and neuropharmacology via which these stimuli produce their behavioral effects. Without such information, adequate pharmacotherapies for the treatment of cocaine addiction are unlikely. It is the goal of the proposed project to identify the neural circuitry and examine the neurochemistry underlying stress-induced relapse in cocaine self-administration, particularly within the prefrontal cortex, an area important for behavioral responses to stress. In order to accomplish this goal, rats trained to self-administer cocaine will undergo behavioral extinction and then will be tested for their propensity to reinstate drug-taking behavior following exposure to foot shock stress. In some experiments, rats will have one of several target structures reversibly inactivated by GABA agonist treatment prior to reinstatement testing in order to identify the structures that form the requisite circuitry for stress-induced reinstatement. Following identification of the circuit, the relative importance of prefrontal cortex (PFC) glutamate (GLU) afferents to the nucleus accumbens (NA) in stress-induced reinstatement will be assessed by 1) antagonism of NA GLU or dopamine (DA) receptors during reinstatement; or 2) microdialysis for DA and GLU in the NA combined with antagonism of DA, norepinephrine (NE) or corticotropin-releasing factor (CRF) receptors in the PFC. Finally the source of PFC activation will be determined by combining microdialysis for GABA, NE, GLU or DA in the PFC with inactivation techniques. The hypotheses are that 1) limbic structures (including the central extended amygdala and ventral tegmental area) are important for processing stressful stimuli and transmitting information to motor structures (PFC, NA, ventral pallidum) responsible for activating behavioral output; 2) a rise in accumbal GLU mediates behavioral reinstatement; and dopaminergic activation of the PFC from the ventral tegmental afferents is responsible for activating the glutamatergic projection to the NA.

Determining the neurobiological basis of relapse to cocaine use is a primary mission of the Neurobiology of Addiction Research Center (NARC), with the goal to identify novel drug targets. The Animal Core will provide animals to all projects that have been trained and extinguished or are abstinent from cocaine selfadministration. The goal of Project 1 is to evaluate neuroplasticity in subcompartments of the nucleus accumbens induced by extinction training or homecage abstinence after cocaine self-administration. The three primary measures of neuroplasticity include, 1) levels of proteins related to glutamate homeostasis, actin cycling and postsynaptic glutamate signaling, 2) density of dendritic spines, and 3) presence or absence of longterm potentiation and longterm depression measured as field potential amplitude in the accumbens after stimulating the prefrontal cortex. It is proposed that by comparing multiple modalities, a cause and effect relationship will become apparent that will help other NARC projects focus on the most important aspects of neuroplasticity to use as drug development targets and/or as endpoints to screen for potential therapies. In addition to estimates of basal neuroplasticity associated with withdrawal from cocaine self-administration, cocaine-seeking will be induced by the drug context or cocaine itself, and associated neuroadaptations quantified. Preliminary data are presented to suggest that not only are there marked neuroplastic changes in protein content, dendritic spine morphology and LTP/LTD after withdrawal from cocaine self-administration, but rapid neuroplastic changes are also induced during cocaine-seeking. Another important goal is to integrate Project 1 with the other projects of the NARC by serving to screen new drugs emerging from these projects against the profile of protein, morphological and electrophysiological plasticity. It is proposed that neuroadaptations reversed by drugs shown behaviorally effective at blocking cocaine-seeking will be likely targets for future drug development. This reverse translation from behavior to molecules is a novel aspect of the NARC and should focus our ability to identify neuroplasticity relevant to cocaine-seeking versus other neuroadaptations related to separate aspects of chronic cocaine administration.

instrucfions); The Administrative Core oversees day to day functioning ofthe Neurobiology of Addiction Research Center (NARC). This core provides an administrative structure, an organizational framework, and a plan to insure integration ofthe various components ofthe Center. The primary goal ofthe Administrative Core is to orchestrate the projects to maximize efficiency and savings, provide a forum for the exchange and mutual comparison of data, as well as orchestrate mentoring activities in the field of addiction at the Medical University of South Carolina. Specific goals are outlined below. 1. Provide a forum for integrating ideas, experimental designs and research findings between the projects. 2. Working with the Pilot Core to mentor junior faculty by expanding opportunities in addiction research. 3. Contribute to seminars and journal clubs for pre- and postdoctoral fellows, as well as to promote community outreach to disseminate the latest findings in addiction research and to provide research experience to university undergraduates. 4. Provide access to assistance in statistical evaluation of data generated by the NARC. 5. Provide the financial Infrastructure to assist in hiring personnel, purchasing supplies and equipment, and to meet publication costs. 6. Provide a mechanism for constant internal evaluation of NARC progress, and to establish and arrange for visits from an External Advisory Board. 7. Contribute monies for Principal Investigators to present data generated by the NARC at national meetings. 8. Provide for data sharing, including maintaining NARC server for data storage and NARC website for public dissemination of NARC discoveries 9. Provide an outreach infrastructure, notably to the College of Charieston neuroscience undergraduates and to high school seniors. RELEVANCE (See instrucfions): The Administrative Core has successfully helped the NARC function efficiently, and maintain scientific synergy and rigor. It has also developed significant outreach programs that involve partnering with nearby undergraduate programs and high schools. Coordination of all these efforts in the Administrative Core provides a vital function to promote NARC excellence and to interface between the NARC, the field of addiction research, and the public.

DESCRIPTION (provided by applicant): This application is in response to RFA-OD-09-005, title 'Recovery act limited competition: Supporting new faculty recruitment to enhance research resources through biomedical research core centers (P30). The Neurobiology of Addiction research Center (NARC) unites an assemblage of investigators with expertise in behavior, neurochemistry, electrophysiology, cell biology, neuroimaging and clinical trials. It is proposed to recruit a tenure-track Assistant Professor into the NARC with a primary faculty appointment in the Department of Neurosciences at the Medical University of South Carolina, Charleston. The applicant, Dr. Peter Kalivas, is both director of the NARC and chair of the department of Neurosciences. Although the NARC is administered through the Department of Neurosciences, the 14 primary tenure-track faculty are in both departments of Neurosciences and Psychiatry. The proposed recruit, Dr. Arthur Riegel, is an accomplished neurophysiologist currently funded by a KOI award from NIDA in a position of non-tenure track Assistant Professor in the department of Neurosciences. The NARC presently lacks or has minimal research capacity in two areas of Dr. Riegel's expertise, whole cell patch membrane physiology and live cell multiphoton imaging and recording. In particular, the latter expertise is entirely lacking from the NARC portfolio. Thus, Dr. Riegel's recruitment will establish a facility to conduct live cell imaging that can be shared in part with other members of the NARC. Dr. Riegel will conduct research into the enduring neuroadaptations produced in the regulation of ion channels in dopamine neurons by metabotropic glutamate receptors and corticotropin releasing factor in rats trained to self-administer cocaine. The animals will be derived largely from the animal core maintained by the NARC that trains animals to self-administer cocaine and heroin. The department of Neurosciences guarantees that if appointed, Dr. Riegel will retain his appointment, base salary and laboratory space for a minimum of 5 years, including the 2 years of the proposed P30 funding.

DESCRIPTION (provided by applicant): This grant proposal is being submitted in response to RFA-OD-09-005 that uses the P30 Biomedical Core Center funding mechanism to support the hiring of a newly-recruited faculty member whose research will augment and expand the research capacity of the institution's existing research community. The proposal seeks support to hire an identified Faculty Candidate who will join an interactive and multidisciplinary group of researchers at the Medical University of South Carolina (MUSC) who are currently investigating the plasticity of alcohol addiction and dependence. MUSC has had a long-standing commitment to medical education about the broad negative consequences of alcohol/drug addiction, and a rich and diverse history of clinical as well as preclinical research in alcohol and substance abuse related problems. Alcoholism is a chronic relapsing disorder. Prolonged exposure to alcohol is associated with the development of tolerance and dependence, and alcohol addiction is characterized by craving for alcohol and compulsive alcohol-seeking behavior that often leads to excessive and harmful levels of drinking. Accumulating evidence suggests that these behavioral manifestations of the addiction process likely reflect neuroadaptive changes that are rooted in the plasticity of learning and memory. The proposed new faculty hire will augment and expand the research capabilities of the neuroplasticity Alcohol Core Center (ACC) group at MUSC. The ACC group of investigators at MUSC is actively engaged in numerous multidisciplinary and collaborative research projects. Thus, the new faculty member will be immersed in a highly supportive and productive academic environment with many prominent researchers in the addiction neuroscience field. The specific aims of this proposal are as follows. Aim 1 is to successfully recruit a highly promising and productive junior alcohol researcher into a tenure-track Assistant Professor position. Aim 2 is to provide an appropriate mentoring and research environment for the successful career development of the Faculty Candidate as an alcohol researcher. Aim 3 is to augment and expand the capability of the ACC in the neuroplasticity of alcohol dependence and addiction. PUBLIC HEALTH RELEVANCE: Alcohol is a widely abused substance with tremendous personal, financial and social costs. The hiring of a new research faculty to augment the development of an Alcohol Core Center to study how alcohol exposure leads to changes in brain function that underlies this disorder may lead to the development of effective therapeutic treatments.

DESCRIPTION (provided by applicant): This a 10-year competitive renewal of a Merit Award that builds upon a total of 25 years of previously funded research. Over this period we have identified the role of glutamate transmission in the nucleus accumbens as critical for behavioral sensitization and the reinstatement of cocaine seeking in an animal model of relapse. Both pre- and postsynaptic cocaine-induced neuroadaptations have been discovered. Recently, the diverse array of cocaine induced changes discovered in our laboratory and by others have been amalgamated into the glutamate homeostasis hypothesis of addiction wherein dysregulation in the balance between synaptic and nonsynaptic glutamate release and uptake is proposed to be critical in the vulnerability to relapse. Supporting this hypothesis, restoring glutamate homeostasis with N-acetylcysteine (NAC) has been shown to produce enduring protection against reinstated cocaine seeking, and appears to normalize many cocaine-induced changes in synaptic plasticity. In the aim #1 of the present proposal we will further characterize the behavioral protection by NAC, evaluating dose-response curves and duration of action after discontinuing NAC. The remaining 3 aims focus on our recent findings that NAC produces an enduring restoration in measures of glutamate homeostasis and metabotropic glutamate receptor (mGluR) dependent neuroplasticity in the nucleus accumbens. Accordingly, aim #2 evaluates the enduring restoration by NAC in the level and function of nonsynaptic glutamate release via the cystine-glutamate exchanger and glial glutamate uptake. Aim #3 determines if daily NAC restores classic measures of neuroplasticity, including longterm potentiation, longterm depression and dendritic spine morphology. Finally, aim #4 examines the effects of daily NAC on signaling via presynaptic mGluR2/3 and postsynaptic signaling on mGluR5. Moreover, in this aim it will be determined if mGluR agonists and antagonists can promote or inhibit the capacity of daily NAC to normalize glutamate homeostasis and synaptic plasticity in the nucleus accumbens. PUBLIC HEALTH RELEVANCE: Cocaine addiction is associated with impaired neuroplasticity in the prefrontal cortico-striatal brain circuitry that is important for guiding adaptive behavior. The premise of this proposal is that it is possible to repair this corticostriatal deficit and thereby ameliorate the vulnerability to relapse. Accordingly, we propose to characterize the ability of daily N-acetylcysteine treatment to produce enduring protection from the reinstatement of cocaine seeking, and associate this protection with restoring neuroplasticity.

DESCRIPTION (provided by applicant): The personal, social and criminal ramifications of cocaine abuse and dependence are enormous problems in North America, and yet no proven pharmacotherapies are available. A point in the cycle of addiction where pharmacological intervention can be particularly beneficial is to interfere with the overwhelming motivation by addicts to relapse to drug use. The primary goal of the Neurobiology of Addiction Research Center (NARC) for the last 9 years has been to identify the neuropathology that underpins the drive to engage in cocaine seeking. In this renewal application the NARC has 3 Cores (Administrative, Animal & Pilot), and 4 Scientific Projects. A primary unifying scientific theme of the NARC is that a key neurological consequence of chronic cocaine use is an impairment in 'top-down' control of drug seeking and relapse that resides in pathological changes in the projection from the prefrontal cortex (PFC) to the nucleus accumbens. All projects evaluate this general hypothesis using a variety of approaches, ranging from animal models of relapse, to in vivo and in vitro electrophysiological measurements of pathway function, to morphological changes in neuron structure and biochemical changes in cell signaling. These techniques are combined with state-of-the-art optogenetic and transgenic approaches to validate involvement of the PFC to accumbens projection and dopaminergic afferents to the projection. In addition to circuitry, a key mechanism of NARC synergy is that all projects use rats generated by the animal core, providing consistency in animal models and in the use of optogenetic reagents among the projects. Other important points of synergy within the NARC are through well-established outreach programs to undergraduate and high school students, and through its Pilot Core which involves all faculty in mentoring activity, as well as allows some findings to move into pilot clinical trials. In summary, the renewal of the NARC contains experienced investigators who will bring to bear and integrate new technologies towards understanding how cocaine affects PFC regulation of cocaine seeking, and who will actively mentor new investigators and students towards quality research in the field of addiction neurobiology.

Addiction to drugs of abuse produces pathological changes in synaptic physiology that impair the capacity of the prefrontal cortex (PFC) to communicate with the basal ganglia and impedes the successful regulation of compulsive drug-seeking. The primary portal of entry by the PFC into the basal ganglia is through the nucleus accumbens, and withdrawal from self-administered cocaine is associated with enduring adaptations at PFC synapses in the accumbens. As part of the current Project 1 (2008-13), we characterized differences in accumbens adaptations depending on whether or not rats were withdrawn with or without extinction training. We recently discovered a rapid, transient synaptic potentiation in the accumbens core (NAcore) but not in the shell (NAshell) that correlated with the intensity of cue-reinstated cocaine seeking. Conversely, a similar synaptic potentiation occurs in the NAshell, not NAcore, when rats are placed into an extinguished context. Aim 1 will characterize this rapid accumbens synaptic plasticity using different behavioral protocols. Aim 2 uses optogenetics to test the hypotheses that giutamatergic afferents from the PFC and dopaminergic inputs from the ventral tegmental area (VTA) are necessary forthe rapid, transient synaptic potentiation in excitatory transmission to be characterized in Aim 1. A variety of proteins and signaling mechanisms are purported to account for the cocaine-induced changes in synaptic plasticity at PFC-accumbens synapses. We present preliminary data showing a potential role for matrix metalloproteases (MMPs) in reinstated cocaine seeking and the plasticity being characterized in Aims 1 & 2. MMP activity known to be is necessary for shaping the extracellular matrix and for expressing many forms of plasticity, and in Aim 3 we propose to determine: 1) if inhibiting MMP signaling reduces cocaine seeking and extinction responding, and 2) if this is accomplished by preventing the rapid, transient synaptic potentiation being characterized in Aims 1 & 2. Project 1 synergizes with other NARC projects and cores by sharing animal and optogenetic use through the Animal Core, and through scientific synergisms based on all projects examining different aspects of PFC and VTA regulation ofthe nucleus accumbens.

Relapse is a persistent problem in cocaine addiction, and many important aspects ofthe brain mechanisms involved remain unknown. One key area for cocaine relapse in the rat model is the medial prefrontal cortex (mPFC); in particular, mPFC interactions with the nucleus accumbens (NA) and ventral tegmental area (VTA) are critical for reinstatement of extinguished cocaine seeking. Projects 1 and 2 in this center focus on molecular and cellular mechanisms involved in the mPFC-to-NA pathway during extinction and reinstatement. Little is known about the activities of neurons in mPFC that project to NA, or that receive dopamine (DA) from VTA, during these cocaine behaviors. Here, we will use Fos labeling and unit recording during extinction and reinstatement to measure impulse activity of mPFC neurons identified as projecting to NA core or shell. We also will capitalize on TH::Cre rats and optogenetics methods recently implemented in our lab to determine the influence of endogenous DA release on impulse activity of prelimbic cortex neurons that project to NA core during extinction and reinstatement. Together, these studies will provide an overall map of NA-projecting mPFC neurons that are activated during cocaine behaviors, and also measure their impulse activities with respect to specific task stimuli and behaviors during exinction or reinstatement of cocaine seeking. These findings will provide a detailed circuit analysis of behavior-related activities in these key mPFC neurons that will be important information to extend results of molecular- and cellular-level studies in other projects of this center.

? DESCRIPTION (provided by applicant): Nicotine addiction is a manifold process that involves dysregulated executive control, reward processing and negative affect. Preclinical models reveal that self-administration of nicotine produces an imbalance in corticostriatal glutamate transmission that mediates cue-induced reinstatement to nicotine seeking. Prior research suggests that N-acetylcysteine (NAC), a modified cysteine chain that regulates glial glutamate and normalizes cocaine-induced synaptic plasticity, may attenuate perceived smoking reward and smoking behavior. Crucially however, our preclinical data suggests NAC may be most effective under conditions of abstinence as a relapse prevention aid. Thus, pairing a smoking cessation medication (Varenicline: VRN) that promotes abstinence, with a relapse prevention aid (NAC), may produce greater cessation outcomes than either treatment alone. The overarching goal of this proposal is to use clinical and preclinical neuroscience to investigate limbic-striatal and corticostriatal circuits involved in nicotine addiction and determine the valueof VRN+NAC for normalizing circuitry function and treating nicotine addiction. Smokers interested in quitting will be randomized to one of four placebo (PBO) controlled conditions to examine the individual and combined effects of VRN and NAC over 4-weeks. Following 1-week of treatment, participants will be contingently reinforced for 3 days of smoking abstinence and undergo an fMRI scan. Participants will be followed over the next 3-weeks of treatment and clinically relevant behaviors will be assessed. Relations between fMRI measures and clinical variable will be explored. In our analog preclinical studies that use the same treatment groups as the clinical studies, we will employ an optogenetic strategy to examine the efficacy of VRN+NAC at inhibiting synaptic plasticity in prelimbic (PL) and basolateral amygdala (BLA) inputs to the nucleus accumbens produced during cue-induced nicotine seeking.

Non-technical description
This Research Infrastructure Improvement Track-2 Focused EPSCoR* Collaboration (RII Track-2 FEC) project involves the Medical University of South Carolina as the lead and the University of Alabama at Birmingham, Furman University, and University of South Carolina, Beaufort Campus as collaborative partners. Much of our knowledge of brain function comes from experiments using functional magnetic resonance imaging (fMRI), which directly measures blood flow to regions of the brain, but remains an indirect measure of neural activity. The precise relationship between neural events and hemodynamic response (increased blood flow) is unclear. This project will test the hypothesis that universal rules relate these two brain activities, using direct measurements in the brain and retina of mice and macaques.

Technical Description
The research will develop new instrumentation for in vivo imaging and cell subtype-specific stimulation in both the brain and the retina necessary for interpreting functional Magnetic Resonance Imaging (fMRI) measurements. These activities will help determine the extent of neurovascular coupling and provide a description of the micro-circuitry, which represents a critical and necessary step in understanding the full complexity of the brain. The consortium will track neural and vascular activity with micron-scale resolution, using two-photon microscopy and adaptive optics to measure the presence of synthetic dyes interacting with genetically encoded sensors. Cell subtype-specific stimulation will be monitored using optogenetic techniques. A computational team will analyze the data obtained to tease out hemodynamic signals. Experiments will be performed on macaques, selected because of the similarity in size and functional repertoire with the human brain. Studies in mice, where genetic and molecular tools are more readily available, will also be performed.

*Experimental Program to Stimulate Competitive Research

The purpose of the Animal Core is to provide a consistent, uniform, and reliable source of animals instrumented and trained in cocaine self-administration for use in all of the projects of the NARC. Rats wlll be catheterized and trained to self-administer intravenous cocaine using a standardized and clinically relevant experimental paradigm. Subjects will then be directed towards individual NARC projects for brain tissue extraction, electrophysiology, or assessment of behavioral responding to systemic or site directed manipulations in accordance with the specific aims of each NARC project. The animal core in this renewal will expand to incorporate a transgenic TH::Cre rat colony that will provide transgenic rats to NARC projects with opsins expressed selectively in dopamine neurons. As part of providing optogenetic support for projects using TH:Cre rats, the Animal Core supports an optogenetics core containing the necessary equipment and expertise to allow all NARC optogenetic studies to proceed in a synergistic and competent manner. The primary objectives of the Animal Core core are: 1. Provide rats to all projects that satisfy specific criteria regarding experience in drug self-administration, and in the degree of withdrawal, extinction, and/or abstinence, as well as regarding intracranial injections of viral vectors. 2. Manage the distribution of rats to projects to insure that statistically relevant group sizes and group replications are made available to each project in a timely manner. 3. Provide trained technicians to efficiently generate experimental subjects using a balanced group design. 4. Maintain a colony of TH::Cre rats individually validated for Cre expression and provide the numbers of identified Cre+ and Cre- rats required across NARC projects. 5. Serve as a NARC training center for MUSC and offsite investigators who desire training in self- administration, jugular catheterization, operant conditioning methods, viral vector design, viral vector microinjections, optogenetic practices, and other related experimental procedures. 6. Provide the equipment, resources, and personnel for using optogenetics to manipulate specific neurons as appropriate across NARC projects. This includes maintaining a supply of virus expressing the various opsin transgenes needed by the NARC projects.

? DESCRIPTION: Although research advances in neural circuitry and behavior have improved our understanding of the processes underlying addiction, gaps remain in the development of new biologic or pharmacologic therapies, neuroscience-based behavioral treatment and monitoring methods, and access-to-care strategies. To address these unmet needs, the New York Academy of Sciences, together with the Aspen Brain Forum Foundation, and the journal Science Translational Medicine will present a 2.5-day conference titled, Sixth Annual Aspen Brain Forum: The Addicted Brain and New Treatment Frontiers, on May 18 - 20, 2016, in New York City. This conference will convene approximately 250 attendees - including basic scientists and clinical investigators in academia, industry, and government; physicians and other addiction treatment specialists; pharmaceutical strategists; and lawmakers - in an effort to better understand the neurobiology of addiction. The event will include discussions on neuroplasticity, neurodevelopment, and socio-political issues, with the goals of developing new targets and improving current behavioral and pharmacological treatments. The three central aims of this conference are to: (i) provide a neutral forum through lectures, a keynote presentation, interactive debates, and networking activities for discussing the neurobiological landscape of addiction, therapeutic prospects, and the socio-political environment within which addiction persists; (ii) showcase and encourage the participation of early career, female, and underrepresented minority investigators via short talks, poster presentations, travel fellowships, career mentoring activities, and discounted registration; and (iii) foster collaboration among international investigators from all sectors to promote the translation of research into more effective addiction therapeutics. The conference goals align well with the mission of the National Institute on Drug Abuse (NIDA) to support the advancement of new knowledge on the neurobiological and behavioral processes underlying addictive behaviors, and the translation of information into interventions that may ultimately reduce substance abuse. In addition, NIDA's specific goal of disseminating information about addiction and advances in research will be met through the publication of a post-conference, enduring, open-access, Section 508- compliant Meeting Report in Annals of the New York Academy of Sciences and an online, multimedia report known as an eBriefing, which will distribute the scientific knowledge and ideas exchanged at the meeting to the global research and medical communities. This conference is especially timely given recent advances in neurobiology research and changes to substance laws (e.g., expanded legalization of marijuana usage within many U.S. states and efforts to reform drug criminalization policies). A collaborative examination of this new information among all addiction stakeholders is of significant value for guiding research forward in the field - particularly with respect to potential innovative therapeutic approaches.

PROJECT SUMMARY ? Administrative Core A Administrative Core A oversees day to day functioning of the Center for Opioid and Cocaine Addiction (COCA). Core A provides an administrative structure, an organizational framework, and a plan to insure integration of the various components of the Center. The primary goal of Core A is to orchestrate the projects to maximize efficiency and savings, provide a forum for the exchange and mutual comparison of data, as well as orchestrate mentoring and outreach activities in the field of addiction at the Medical University of South Carolina. Specific goals are outlined below. Promote Scientific Rigor and Synergy 1. Provide a forum for integrating ideas, experimental designs and research findings between the projects. 2. Provide access to assistance in statistical evaluation of data generated by the COCA and genetics consultation. 3. Provide an Internal Advisory Board mechanism for constant internal evaluation of COCA progress, and to establish and arrange for visits from an External Advisory Board. 4. Provide for data sharing amongst COCA investigators by maintaining a private COCA server. 5. Provide public access to the National Resource Databases being generated by the COCA, both on the COCA website and appropriate national public databases. 6. Provide the financial infrastructure to assist in hiring personnel, and purchasing supplies and equipment. Mentoring and Outreach 1. Working with Pilot Core C to mentor junior faculty by expanding opportunities in addiction research. 2. Contribute to seminars and journal clubs for pre- and postdoctoral fellows, as well as to promote community outreach to disseminate the latest findings in addiction research. 3. Provide an outreach infrastructure, notably with the College of Charleston neuroscience undergraduates and to high school seniors, and facilitate ongoing international outreach programs. 4. Contribute monies for PIs to present data generated by the COCA at national meetings.

PROJECT SUMMARY ? Project 3 Addiction to drugs of abuse produces pathological changes in synaptic physiology that impair the capacity of the prefrontal cortex (PFC) to communicate with the nucleus accumbens, and impede the successful regulation of drug seeking. The Center for Opioid and Cocaine Addiction (COCA) studies the genetic, cellular and physiological mechanisms of relapse in the PFC and nucleus accumbens rodent models and bidirectionally translates this with COCA clinical studies using fMRI read-out of treatments with N-acetylcysteine (NAC) and PFC transmagnetic stimulation (TMS) for cue reactivity in cocaine users. Project 3 specifically explores the nucleus accumbens for synaptic adaptations after heroin and cocaine self-administration in mice and rats, and the adaptations that occur during cue-induced drug seeking. We find both enduring changes in withdrawal and transient changes during cue-induced heroin and cocaine seeking that are correlated with the intensity of the relapse event. These changes are present in all four compartments of the synapse (pre- and postsynapse, astroglia and extracellular matrix). By simultaneously examining the 4 synaptic compartments of the tetrapartite synapse, we are accessing processes heretofore poorly studied in relation to addiction, which may contain unique opportunities for therapeutic development. Animal & Validation Core B will provide rats and mice trained to self- administer heroin or cocaine. Our endpoints include cell morphology, cell signaling and physiological processes. Specifically, we will measure enduring and cue-induced transient changes in the morphology, synaptic glutamate-mediated currents and protein expression in specific cell types in the accumbens using transgenic rats and mice and cell-specific viral mediated expression of transgenes in the nucleus accumbens. Finally, pursuant to an overarching COCA goal to understand which cell type and neuronal subpopulation is mediating the effects of cue-induced heroin and cocaine seeking, we administer the treatments used in the clinical Project 4 (NAC and continuous theta burst stimulation) to provide bidirectional construct validity for the relapse-related discoveries in Project 3.

ABSTRACT The interactions between previous life stress and sex differences in substance use disorder (SUDs) are understudied, and essentially unstudied in terms of the neurobiological underpinnings of sex differences in heroin and cannabis use, as well as in the comorbidity between stress disorders such as PTSD and SUDs. Project 3 serves as the preclinical research arm of the Center. In Aim 1 we will establish the utility of a behavioral model combining an acute life-threatening stress (restraint) in rats with the use and seeking of heroin and cannabis. We will use a novel cannabis addiction model involving self-administration of the two primary cannabinoids found in cannabis, ?9-tetrahydrocannabinol (THC) and cannabidiol (CBD), and along with standard heroin self- administration, examine the acquisition and maintenance of self-administration, as well as context+drug cue induced drug seeking. When rats are pre-exposed to restraint stress or sham 3 weeks prior to beginning self- administration, it will be in combination with an odor that can serve as a stress conditioned stimulus during the drug seeking trial. This model parallels the protocols employed with the cannabis and opioid using human subjects in Center Projects 1 & 2. In Aim 2 of Project 3, the sex differences in drug seeking and interactions with stress will be evaluated for neurobiological mechanisms at the level of plasticity in glutamatergic synapses in the nucleus accumbens core (NAcore). These synapses are known to undergo enduring parallel changes in astroglia and glutamate uptake after drug self-administration that are consequential for cue-induced drug seeking. Moreover, acute stress produces parallel enduring changes that promote the acquisition of drug use. However, sex differences or interactions between stress and sex in this synaptic plasticity are unknown and will be evaluated in Aim 2 using the behavioral model characterized in Aim 1. Finally, in Aim 3 we link directly with the clinical projects by examining drugs to attenuate context+drug cue+stress cue induced heroin and cannabis seeking. Rats will be treated with a compound (N-acetylcysteine) known to ameliorate cue-induced heroin and cannabis seeking by restoring glial glutamate uptake. Separate rats will be administered progesterone or lofexidine in parallel with Center Projects 1 and 2, respectively. Through Aims 1-3 we hope to identify novel mechanisms in synaptic plasticity that will explain interactions between stress, sex and drug seeking. We will also provide mechanistic understanding for how medications used in Projects 1 & 2 successfully reduce stress responsivity in cannabis and opioid use disorders.

PROJECT SUMMARY - Overall The personal, social and criminal consequences of opioid and cocaine abuse are enormous problems in North America. This is most tragically seen in rising morbidity due to heroin, prescription opioids and fentanyl overdose in the USA. Addiction to drugs typically cycles between three phases, active drug use, withdrawal from drug use and relapse to drug use. A point in the cycle of addiction where pharmacological intervention can be particularly beneficial is to interfere with the overwhelming motivation by addicts to relapse to drug use, even after extended periods of abstinence when acute withdrawal symptoms have dissipated. However, the enduring state of relapse vulnerability arises from interdependent brain adaptations produced during all three phases of addiction. Thus, in order to develop biological rationales for treating relapse, it is necessary to understand not only the neurobiology of relapse itself, but to determine which changes produced by drug administration and drug withdrawal contribute to the final enduring state of relapse vulnerability. The overarching goal of the Center for Opioid and Cocaine Addiction (COCA) is to create and maintain mechanisms of scientific synergy that will facilitate discovering the neuropathologies that underpin the enduring and uncontrollable drive to seek opioids and cocaine, and thereby advance biological rationales needed to efficiently generate pharmacotherapies that inhibit drug relapse. This goal will be achieved through a bidirectional translational strategy that involves 3 Cores and 4 research Projects. In addition to the Administrative and Pilot Cores, the Animal & Validation Core makes available transgenic rodents that have been trained to self-administer heroin or cocaine, and have been instrumented with intracranial cannulae, fiber optics or GRIN lens. This Core will also validate all viral reagents and transgenic animals shared by the COCA Cores and Projects. The 4 Projects range from determining the epigenetic substrates of long- lasting drug-induced alterations to understanding the molecular and brain circuit mechanisms of cue-induced drug seeking in rodents and humans. The Projects are designed to be highly integrated and form a bidirectional translation strategy for providing biological rationales for new therapeutic approaches to relapse prevention.

PROJECT SUMMARY Opioid abuse is a serious global problem that affects the health, social and economic welfare of all societies. Opioid use disorder (OUD) is a medical condition characterized by the compulsive use of opioids despite adverse consequences from continued use and the development of a withdrawal syndrome when opioid use ends. OUD involves both addiction to and dependence upon opioids. Opioids include prescription pharmaceuticals such as morphine, codeine, oxycodone and hydrocodone as well as illicit drugs such as heroin. Although animal models provide a rigorous, convenient means to precisely control environmental context and drug exposure, and assess behavioral, genomic, biochemical and cognitive changes, effective utilization of such models as an efficient proxy for human addiction remains challenging. The identities of gene variants that mediate behavioral differences in laboratory animals remain largely unknown, greatly limiting interpretation of physiologic differences and understanding of the environmental effects on drug abuse, and hindering translation of genetic findings to humans. This limitation highlights the urgent need for an integrated genomic characterization of a robust animal model of opioid abuse. The goal of this project is to exploit an outbred animal model of opioid abuse research, coupled with comprehensive genomic characterization, to improve detection of the underlying phenotypic and genomic changes associated with transition to opioid abuse. The project will engage an international multidisciplinary team of experts in the areas of addiction science and behavioral studies, neurobiology, rodent genetics, computational genomics, bioinformatics, and high throughput computing and data modeling to accomplish three Specific Aims. In Aim 1, we will exploit a validated outbred animal model of heroin self- administration with extended access that produces behavioral phenotypes consistent with increased drug consumption, accelerated motivation for drug intake and elevated drug-seeking in periods of drug absence. Animals at two distinct geographical sites (one in the USA and one in Europe) will be stratified across a ?vulnerable? ? ?resistant? spectrum with the objective of performing genetic screening on all individuals. In Aim 2, we will carry out a genome-wide association study (GWAS) using genotyping by sequencing (GBS) on 1,000 animals sampled from the two locations. Validation of the most significant genetic variants will be performed in an independent validation cohort of 100 animals. In Aim 3, we will assess the genomic impact of the genetic variants uncovered in Aim 2 via high throughput RNA sequencing to assess gene expression of relevant functional genes and uncover expression quantitative trait loci (eQTLs). Chromosome conformation capture (3C) will be used to physically link an eQTL with its target gene assigning causality to a variant and its regulated gene.