Clyde W. Hodge, Ph.D. - US grants

The central nervous system mechanisms that control the subjective effects of alcohol are not well characterized. Research has implicated ligand- gated ion channels, particularly GABA/A and NMDA receptor systems, in producing the discriminative stimulus properties of ethanol. Pilot data indicate that centrally administered ethanol partially generalizes to, and augments, the systemically administered ethanol discriminative stimulus. However, the brain regions and neurochemical systems involved in this process have not been studied. The limbic system has been implicated in reinforcement by many drugs, and in discrimination of cocaine, amphetamine, and benzodiazepines. This suggests that the reinforcing and subjective effects of these drugs may be mediated by the same neural systems. Our research has shown that a portion of this system is involved in ethanol reinforcement. The overall aim of this proposal is to examine the involvement of 1) the direct actions of ethanol, and 2) GABA/A and NMDA neurotransmission in specific regions of the limbic system in the discriminative stimulus effects of ethanol. Two specific aims, using microinjection techniques and procedures common to the behavioral analysis of drug discrimination, are proposed to address this question. Specific Aim 1 will determine the role of limbic brain regions in ethanol discrimination by testing whether ethanol administered into the 1) medial prefrontal cortex, 2) amygdala, 3) hippocampus, or 4) nucleus accumbens will substitute for and/or augment the discriminative stimulus produced by systemic ethanol administration. These studies will provide a test of whether the direct actions of ethanol in limbic brain regions contribute to its discriminative stimulus properties. Because GABA/A and NMDA neurotransmitter systems are known to be involved in ethanol discrimination. Specific Aim 2 will determine the role of these transmitters in limbic brain regions in determining the discriminative stimulus effects of ethanol. These studies will test (1) whether GABA/A agonist or NMDA antagonist injections in limbic regions will substitute for systemically administered ethanol, (2) if GABA/A antagonist or an NMDA agonist injections will block the discriminative stimulus properties of systemically administered ethanol. Finally, because ethanol has simultaneous effects on both GABA and NMDA systems, studies will also be conducted to test the ability of combinations of GABA/A and NMDA drugs, administered into limbic regions, to substitute for or block the ethanol discriminative stimulus.

DESCRIPTION: Alcohol abuse and alcoholism are major health problems that cost our society numerous lives and billions of dollars each year. Understanding how alcohol can come to control an individual's behavior is key to the development of effective treatment. Our research program has begun to identify specific brain regions, and chemicals in these regions, that are involved in regulating how much alcohol an individual will consume. The region of the brain known as the hypothalamus controls most of the body's regulatory processes, such as feeding, drinking, and mating. For instance, it has been shown that direct injections of the neurotransmitter noradrenaline into this area will increase carbohydrate intake and result in obesity (serotonin has the opposite effect) in rats. Obesity, like alcoholism, involves the excessive consumption of a substance. Thus, the hypothesis of these studies is that alcohol may result in excessive intake by usurping the brain is natural feeding and drinking mechanisms which can result in a loss of control. The operant self-administration technique is considered to be one of the most valid measures of alcohol-seeking behavior. When combined with CNS microinjection techniques, the role of specific brain regions and receptor systems can be characterized. Three specific aims are proposed to characterize the involvement of hypothalamic regulatory systems in alcohol-seeking behavior. Specific Aim I will characterize the role of noradrenergic receptors located in the paraventricular nucleus (PVN) of the hypothalamus in regulating alcohol and sucrose reinforced responding. These studies will be conducted in ethanol-experienced and ethanol-naive rats and will test whether alcohol experience shifts the function of the PVN from regulation of carbohydrate intake to the control of alcohol seeking behavior. Specific Aim 2 will test the role of serotonin receptors, and Specific Aim 3 will test the influence of opiate receptors, in the same behavioral procedures. These studies will examine the degree to which the primary function of the PVN may be altered as a function of alcohol experience. The results from these studies will help clarify how homeostatic brain systems, such as the hypothalamus, may become involved in alcohol-seeking behavior.

DESCRIPTION (provided by applicant): The primary goal of this project is to understand molecular mechanisms that influence adaptive changes in alcohol self-administration and dependence that result from repeated alcohol exposure and withdrawal. Substantial evidence indicates that neuronal gamma-amino-butyric acid type A (GABAA) receptors modulate alcohol self- administration and alcohol withdrawal severity. Our research has shown that mutant mice lacking the epsilon isoform of protein kinase C (PKCepsilon) are supersensitive to acute alcohol and GABAA allosteric positive modulators. This supersensitivity is associated with reduced alcohol self-administration, absence of the alcohol deprivation effect (ADE), and lack of progression of ethanol withdrawal seizure severity. By contrast, PKCgamma null mutant mice are less sensitive to ethanol modulation of GABAA receptor function and drink more ethanol suggesting that specific isoforms of PKC may differentially modulate GABAA mediated effects of ethanol. The present proposal extends these findings by focusing on alcohol self-administration during relapse and dependence. Using null mutant mice, we will test the hypothesis that PKC epsilon and gamma differentially modulate alcohol self-administration, and withdrawal-related changes in alcohol self-administration, via GABAA receptor activity. Specific Aim 1 will test the hypothesis that specific PKC isoforms differentially modulate alcohol self-administration during repeated relapse via GABAergic mechanisms. We will first examine operant alcohol self-administration by PKC epsilon and gamma null mutant mice and controls using a model of repeated alcohol deprivation, which we have established in mice. Specific Aim 2 will examine repeated ethanol withdrawal seizure severity in PKC epsilon and gamma null mice. Studies of Aim 3 will extend the withdrawal findings of Aims 1 and 2 by examining relapse to alcohol self-administration during episodes of repeated dependence and withdrawal. Collaborative studies in each aim will examine multiple brain regions for changes in GABAA receptor subunit expression in the membrane fraction by Western Blot analysis, and MAPK/ERK signaling and neuropathology (Crews component). To confirm functional relevance of molecular changes, we will administer pharmacological agents (i.e., the nonselective GABAA alpha-antagonist flumazenil or the alpha1 selective agonist zolpidem) during repeated relapse and withdrawal. These studies are expected to elucidate molecular mechanisms of adaptive processes that influence alcohol self-administration after deprivation or withdrawal. This information might lead to the identification of novel pharmacological therapeutics for treatment of problems associated with alcoholism, such as loss of control and relapse.

DESCRIPTION (provided by applicant): The primary goal of this application is to characterize the involvement of metabotropic glutamate receptor subtype-5 receptors (mGluR5) in alcohol's reinforcing effects. Preliminary evidence indicates that chronic alcohol drinking selectively increases mGluR5 mRNA expression in the nucleus accumbens (NAcb) of rats, which is consistent with chronic receptor inhibition by alcohol in a brain region that is fundamental to development of addiction. Experiments in Aim 1 of this project will more fully characterize the involvement of mGluR5 in alcohol reinforcement. These studies will determine if operant alcohol self-administration alters mGluR5 protein expression and if mGluR5 receptor activity functionally regulates ethanol reinforcement in rats. Other preliminary data indicate that microinjection of an mGluR5 antagonist in the NAcb decreases the reinforcing effects of ethanol. Studies in Aim 2 will extend this observation by determining the functional significance of mGluR5 receptor activity in limbic brain regions that express mGluR5. Experiments will determine the effects of site-specific infusion of an mGluR5 antagonist in the ventral tegmental area (VTA), NAcb (core and shell), or medial prefrontal cortex (mPFC) on ethanol reinforced responding. These studies will determine if mGluR5 regulation of alcohol reinforcement is brain-region specific. Evidence indicates that ethanol inhibits mGluR5 function in vitro through a PKC-dependent mechanism. Similarly, our preliminary data indicate that mGluR5 blockade decreases ethanol self-administration in wildtype mice but has no effect in mice carrying a null mutation for PKC-epsilon. Thus, specific Aim 3 will characterize a potential cell- signaling based mechanism in mGluR5 regulation of alcohol reinforcement. Using PKC-epsilon knockout and wildtype mice, experiments will determine if mGluR5 blockade decreases ethanol reinforcement in a PKC-epsilon dependent manner. These studies will examine the effects of mGluR antagonists on alcohol and sucrose reinforced responding, and on ethanol-induced changes in locomotor activity to determine behavioral specificity. Finally, chronic ethanol exposure alters numerous molecular events in the brain including the gene transcription factor cyclic AMP- responsive element binding protein (CREB), which has been implicated in addictive behavior. Activation of mGluR5 increases p-CREB levels via a PKC dependent mechanism, whereas chronic ethanol drinking decreases p-CREB levels in the NAcb. Since ethanol is known to inhibit mGluR5 activity through a PKC-dependent mechanism, these findings suggest that self-administered ethanol may decrease p-CREB through an mGluR5/PKC (possibly PKCepsilon) dependent mechanism. Experiments in Aim 4 will determine if ethanol-induced changes in mGluR5 and p-CREB expression are dependent on PKCe, and the extent to which this is modulated by a history of chronic operant ethanol self-administration. Another study will determine if ethanol inhibits mGluR5-mediated changes in p-CREB in a PKCepsilon dependent manner. These studies, examine the functional linkage between mGluR5 and PKCepsilon in the mediation of chronic ethanol effects on transcriptional regulation, which has implications for adaptive changes in addiction. These findings will aid development of pharmacotherapeutics to treat problems associated with alcoholism.

DESCRIPTION (provided by applicant): Alcohol abuse and dependence have been linked clinically with negative affect and depression. Variations in the clinical presentation of alcoholism and depression have complicated diagnoses of "co-morbid depression," "dual diagnosis," "abstinence depression" and other negative affect symptoms, although it is generally agreed that alcohol dependent patients experience depression when they stop drinking. Further, the co-occurrence of negative affect or depression symptoms with alcoholism predicts more severe disease and a poor response to treatment. Prevailing theories of depression include the classic monoamine hypothesis as well as emerging molecular theories involving CREB gene transcription, neurotrophic pathways, and hippocampal neurogenesis. To date, however, no preclinical studies have addressed the neurobiological mechanisms of alcohol-induced depression. We have discovered that abstinence from chronic voluntary alcohol drinking is associated with increased immobility in the Porsolt swim test (PST), a validated model of depression-like behavior, in mice. This behavioral pathology is completely blocked by chronic administration of the antidepressant desipramine. Studies in Specific Aim 1 of this application will utilize this animal model to further characterize the relationship between chronic alcohol drinking and the emergence of depression-like behavior during abstinence. Specific Aim 2 will determine the efficacy and dose-response of several antidepressant medications to gain insight into potential therapeutic mechanisms. Specific Aim 3 of this project is to conduct an integrative analysis of molecular adaptations that are associated with 1) alcohol-induced depression and 2) antidepressant treatment. In support of this approach, we have discovered that alcohol-induced depression-like behavior is associated with significant decreases in p-CREB and BDNF expression and neurogenesis in the dentate gyrus of the hippocampus, each of which has been hypothesized to underlie depression. We have also found that desipramine reverses the alcohol- induced reduction in p-CREB and BDNF specifically in the dentate gyrus. Finally, Specific Aim 4 of this application is to manipulate CREB phosphorylation and BDNF levels in the hippocampus to determine if molecular adaptations in this important brain region functionally regulate alcohol-induced depression. This project has the potential to elucidate factors that lead to co-morbid depression in alcoholism, identify effective medications, and characterize underlying neurobiological adaptations that are associated (or dissociated) with alcohol-induced depression and its treatment. This information may be of major significance for the development of therapies that may benefit depression and alcoholism, as well as establishing the molecular basis of the interaction of these two mental diseases.

Absolute ethanol; Abstinence; Accounting; Addiction, Drug; Address; Alcohol Drinking; Alcohol abuse; Alcohol consumption; Alcohol, Ethyl; Alcoholic; Alcoholism; Alcohols; Amygdala; Amygdaloid Body; Amygdaloid Nucleus; Amygdaloid structure; Animal Model; Animal Models and Related Studies; Behavior; Behavior Control; Behavioral; Behavioral Manipulation; Behavioral Mechanisms; Body Tissues; Boozer; Brain; Brain region; CSBP1; CSBP2; CSPB1; Cell Communication and Signaling; Cell Signaling; Chemical Class, Alcohol; Chemical Dependence; Chronic; Clinic; Clinical; Complex; Condition; Coupled; Dependence, Drug; Dependent drinker; Development; Disease; Disorder; Drug Addiction; Drug Dependency; Drugs; EC 2.7.2-; ETOH; EXIP; Encephalon; Encephalons; EtOH drinking; Ethanol; Exposure to; Extracellular Signal-Regulated Kinases; Gene Transcription; Genetic Transcription; Goals; Grain Alcohol; Grant; Intake; Intracellular Communication and Signaling; JNK; JNK1; JNK1A2; JNK21B1/2; Lead; Link; MAP Kinase 8 Gene; MAP kinase; MAP-ERK Kinase; MAPK; MAPK ERK Kinases; MAPK Inhibitors; MAPK14; MAPK14 gene; MAPK8; MAPK8 gene; MEKs; Maintenance; Maintenances; Mammals, Mice; Mechanisms of Behavior and Behavior Change; Mediating; Medication; Methylcarbinol; Mice; Microinjections; Mitogen-Activated Protein Kinase Inhibitor; Mitogen-Activated Protein Kinases; Molecular; Motivation; Murine; Mus; Mxi2; Nervous; Nervous System, Brain; Nucleus Accumbens; PRKM14; PRKM15; PRKM8; Pathogenesis; Pathology; Pathway interactions; Pb element; Pharmaceutic Preparations; Pharmaceutical Preparations; Phosphorylation; Positive Reinforcements; Positive Reinforcer; Predisposition; Process; Protein Phosphorylation; Psychological reinforcement; RNA Expression; Range; Regulation; Reinforcement; Reinforcement (Psychology); Reinforcement Schedule; Reinforcements, Positive; Relapse; Research; SAPK1; SAPK2A; Saccharose; Self Administration; Self-Administered; Signal Pathway; Signal Transduction; Signal Transduction Systems; Signaling; Site; Specificity; Staging; Sucrose; Susceptibility; System; System, LOINC Axis 4; Testing; Thinking; Thinking, function; Time; Tissues; Transcription; Transcription, Genetic; abused drugs; alcohol exposed; alcohol exposure; alcohol ingestion; alcohol intake; alcohol problem; alcohol product use; alcohol reinforcement; alcohol research; alcohol use; alcoholic beverage consumption; alcoholic drink intake; alpha-D-Glucopyranoside, beta-D-fructofuranosyl; amygdaloid nuclear complex; behavioral control; behavioral pharmacology; biological signal transduction; chronic EtOH drinking; chronic alcohol consumption; chronic alcohol drinking; chronic alcohol ingestion; chronic alcohol use; chronic ethanol consumption; chronic ethanol drinking; chronic ethanol ingestion; day; disease/disorder; drinking; drug of abuse; drug seeking behavior; drug/agent; drugs abused; drugs of abuse; ethanol abuse; ethanol consumption; ethanol drinking; ethanol exposed; ethanol exposure; ethanol ingestion; ethanol intake; ethanol product use; ethanol reinforcement; ethanol research; ethanol use; etoh use; experiment; experimental research; experimental study; exposed to alcohol; exposure to alcohol; extracellular; hazardous alcohol use; heavy metal Pb; heavy metal lead; immunoreactivity; inhibitor; inhibitor/antagonist; model organism; mouse model; neural; neural circuit; neural circuitry; neuroadaptation; neuropsychiatric; neuropsychiatry; novel; p38; p38 MAPK Gene; p38Alpha; pathway; preclinical study; problem drinker; problem drinking; relating to nervous system; research study

The development of alcohol addiction is characterized by repeated binge/ intoxication episodes. Prominent theories of addiction suggest that drugs gain control over the individual, in part, by usurping glutamate-linked mechanisms of neuroplasticity within brain reward circuits. The preclinical studies in this application are focused on identifying alcohol-induced pathologies in brain reward circuits that underlie addiction. Preliminary results show that binge-like alcohol self-administration activates primary glutamatergic mechanisms of neuroplasticity (i.e., AMPA GluRI; CaMKIIa) in amygdala and accumbens nuclei that, in turn, are required for alcohol reinforcement. These novel findings suggest the main hypothesis of this application that the binge/intoxication stage of alcohol addiction is associated with adaptations in glutamatergic cell signaling in amygdala-accumbens circuits that regulate alcohol reinforcement. Aim 1 will investigate the effects of binge-like alcohol self-administration on adaptive changes in AMPA GluRI and CaMKIIa protein expression in the amygdala and nucleus accumbens and will evaluate functional effects of alcohol self-administration on CaMKIIo-positive glutamatergic projections that are intrinsic (BLA to CeA) and extrinsic (BLA to AcbSh) to the amygdala using optogenetic and electrophysiological strategies. Aim 2 will determine the functional role of AMPAR/CaMKII in the CeA and AcbSh, using pharmacological techniques and identify the functional involvement of CaMKIla-positive glutamatergic projections that are intrinsic/extrinsic to the amygdala in relation to binge-like alcohol self-administration, using optogenetic techniques. These studies will identify novel plasticity-linked molecular mechanisms and functional neural circuits that regulate binge-like alcohol self-administration. Finally, Aim 3 will characterize the role of AMPAR positive modulation in the CeA and AcbSh on escalated alcohol self-administration using pharmacological and optogenetic techniques. These studies will identify specific nuclei and circuits in which increased glutamate signaling contributes to escalated binge drinking. This Component will identify and validate novel mechanisms of a critical behavioral pathology that pervades the development and progression of alcohol addiction.

The primary goal of the UNC Alcohol Research Center is to increase understanding of the molecular and cellular pathogenesis in alcoholism. To facilitate this integrated research effort, the Scientific Core will provide centralized facilities and technical assistance for the application of microscopy, molecular biology techniques, and methods for evaluation of neural circuit function. The Core fosters interaction among Center Investigators with the explicit purpose of increasing coordination and cohesiveness among individual research components. To accomplish this goal, the Core provides immunohistochemical and microscopy resources to facilitate evaluation of ethanol-induced changes in protein levels in specific loci from brain as proposed by the Research Components. Core faculty and staff provide training in histology and immunohlstochemistry, access and training for modern light, wide-field, and laser scanning confocal microscopes the conduct of immunohlstochemistry and use of light and/or confocal microscopes, and equipment maintenance. The Core also provides full access to state-of-the-art image analysis software and equipment for quantitative analysis and presentation of digital images. The Scientific Core will provide resources for quantification of protein and mRNA. Core staff will provide technical assistance, training, and/or collaborate with investigators on all aspects of the methods ranging from tissue extraction and preparation to data collection, analysis, and interpretation. In addition, the Core now provides facilities, resources, training, and services in the conduct of optogenetics and electrophysiological techniques for evaluation of neural circuits by Research Components. A final goal of the Core is to facilitate collaborative and Integrative research efforts of the Center. To accomplish this goal, the Core Director holds a monthly scientific meeting where Center investigators present research findings, review Core functions and progress, and keep Core staff up-to-date regarding needs. The Scientific Core is an evolving resource that serves an integrative role by providing a formal venue in which investigators can present and discuss findings of the research components, methodologies and training of new laboratory investigators as well as planning new directions. Overall, the centralized services and equipment provided by the Core play a critical role in the successful completion of the research projects in an efficient and effective manner.

Scientific Resource Core The primary goal of the UNC Alcohol Research Center (ARC) is to increase understanding of the molecular and cellular pathogenesis in alcoholism. The purpose of the Scientific Resource Core (SRC) is to facilitate and extend this integrated research effort by providing access to cutting-edge technology, shared facilities, resources and technical expertise. In addition, the SRC fosters interaction among ARC investigators with the explicit purpose of increasing coordination and cohesiveness among individual research components. In this renewal application, the SRC has two specific goals. First, the SRC will support functional connectivity magnetic resonance imaging (fcMRI) studies designed to discover novel alcohol-induced changes in neural circuit connectivity, and activity, spanning multiple phases of addiction and mammalian species. This work will be conducted in an expert-driven environment within the SRC using shared facilities provided by the UNC Biomedical Research Imaging Center (BRIC). Access to cutting-edge imaging technologies and high end instrumentation in the BRIC significantly enhances the capabilities of Research Components by providing rigorous methods, sophisticated facilities, user support, and quality control of imaging. Second, the SRC provides facilities, resources, and expertise to allow measurement of alcohol-induced changes in gene and protein expression, and neural circuit function. These centralized facilities allow rigorous, standardized, and quality-controlled data collection across a variety of models, and extends the capability of Research Components to include novel methods. To assure effective functioning of the Core and to facilitate the integrative efforts of the ARC, the SRC holds a monthly meeting (chaired by Dr. Hodge) of ARC investigators that evaluates progress and provides a venue for data and idea sharing. Access to these resources, and integrative environment, is a highly significant feature of the ARC that contributes to the accomplishment of our shared goals. Together, these services and resources provide the Research Components with the unique capability of delineating alcohol-induced molecular changes in specific brain regions that are associated with altered neural circuit connectivity. This synergistic effort will provide novel insight into mechanisms of maladaptive changes in neural circuitry that underlie alcohol addiction. Moreover, this innovative and integrative strategy represents a clear advantage to conducting the proposed research as Center rather than separate research efforts.

Project Summary/Abstract Research Component 1. Alcohol addiction is a dynamic multiphasic disorder that is characterized, in part, by repeated binge/intoxication episodes where the powerful positive reinforcing effects of the drug predominate. Although multiple neural systems regulate alcohol reinforcement, we have shown that repetitive binge episodes engage glutamatergic ?-amino-3-hydroxy-5-methyl-4-isooxazole receptor (AMPAR) mechanisms of synaptic plasticity in brain reward pathways. Specifically, binge-like operant alcohol self-administration (SA) is associated with a shift toward GluA2-lacking Ca2+-permeable (CP-AMPAR) activity (increased GluA1-S831 phosphorylation and reduced GluA2 protein expression) in the basolateral amygdala (BLA). Preliminary optogenetic data show that these alcohol-induced adaptations are associated with increased AMPAR synaptic activity in nucleus accumbens core (AcbC) neurons receiving projections from BLA; thus, strongly implicating the BLA?AcbC neural circuit. We have also shown that amygdala AMPAR activity is required for the positive reinforcing effects of alcohol, and activation of amygdala AMPARs promotes escalated operant alcohol SA. This convergence of molecular, physiological, and bi-directional behavioral data support the overall hypothesis that: CP-AMPAR activity in the BLA?AcbC pathway functionally regulates the positive reinforcing effects of alcohol. We propose to test this hypothesis in three separate but integrated aims using male and female C57Bl/6J mice. First, molecular studies are proposed to evaluate AMPAR subunit protein expression (GluA1 and GluA2) and phosphorylation (pGluA1-S831 and S845) in amygdala and accumbens subregions to determine if alcohol SA produces a shift toward GluA2-lacking CP-AMPAR activity in this reward pathway. Magnetic resonance imaging (MRI) will also be coupled with an optogenetic strategy to identify specific BLA projections that are altered by alcohol SA. Second, to assess alcohol-induced adaptations in CP-AMPAR synaptic activity, we will evaluate synaptic properties of BLA neurons projecting to AcbC, and use optogenetics to test BLA?AcbC circuit function in M/F C57BL/6J mice. We predict increased CP-AMPAR signaling in BLA and BLA?AcbC circuit of alcohol SA mice. Third, site-specific pharmacological studies will determine if CP- AMPAR activity in the BLA or AcbC is necessary for alcohol reinforcement and/or sufficient for escalated binge-like SA. A site-specific AAV approach will express a dominant negative form of the AMPAR GluA1 subunit (GluA1ct) to determine if activity dependent GluA1 membrane trafficking in the BLA or AcbC regulates the reinforcing effects of alcohol. These studies will elucidate novel molecular mechanism(s) of alcohol?s reinforcing effects, which has potential to lead to new therapeutic strategies for treating behavioral pathologies associated with alcohol addiction. The overall goal of the UNC ARC is to increase understanding of molecular and cellular pathogenesis in alcoholism. To address this goal, Research Component 1 seeks to elucidate novel molecular targets of initial binge-like alcohol SA that, in turn, gain control over reinforcement processes to drive repetitive use and escalated intake, two of the most significant behavioral pathologies in alcohol addiction.

Project Summary Pathological alcohol-seeking behavior is regulated in part by glutamate AMPA receptor (AMPAR) activity in the amygdala. Transmembrane AMPA receptor regulatory proteins (TARPs) profoundly affect the trafficking and function of AMPARs in synaptic and behavioral plasticity. Although the TARP family of proteins is expressed throughout the brain, the TARP ?-8 subtype is restricted to forebrain regions including the basolateral amygdala (BLA); a brain region that is critical to addiction. However, the role of TARP ?-8 in alcohol use disorders (AUD) or other addictions is unknown. To fill this gap in knowledge, we propose an innovative set of behavioral, genetic, bidirectional systemic and site-specific pharmacological, molecular, and physiological studies in mice to evaluate the mechanistic role of TARP ?-8 in alcohol reinforcement, escalated self- administration, and cue-induced reinstatement of alcohol-seeking behavior as a model of relapse. Elucidating the neural mechanisms of these three critical behavioral domains has high translational value for understanding the development, progression, and maintenance of AUD. Successful completion of the studies in this application will provide fundamental mechanistic insights into TARP ?-8 regulation of pathological alcohol-seeking behavior. Moreover, this work moves the field forward in understanding the molecular mechanisms by which alcohol hijacks reward processes and has potential to inform development of new pharmacotherapeutic strategies that target AMPAR function in a highly selective brain region-specific manner.