Howard C. Becker - US grants

The past decade has evidenced a great deal of research related to the effect of prenatal alcohol exposure on fetal growth and development in animals. As a result, it is clear that ethanol is a teratogen in the classical sense of producing birth defects. Also, some types of behaviors are abnormal and an elevated stress response follows some types of stressors. Data regarding whether prenatal alcohol exposure alters the offspring's response to subsequent alcohol challenge are inconsistent. The present proposal attempts to develop a more complete picture of the teratogenic effects of ethanol by utilizing a mouse model to address the above three issues, namely behavioral impairment, alcohol sensitivity, and stress responsiveness. Moreover, a single appetitively-motivated conflict task that does not employ shock will be used. The behavioral paradigm is referred to as negative contrast. This task permits quantification of how animals respond to an abrupt reduction in expected reward. Specifically, the consummatory performance of animals that are downshifted from a preferred to a less preferred reward is greatly depressed in comparison to animals that are not shifted, but receive experience with only the less preferred or smaller reward. That is, while the smaller reward is relatively not as rewarding as that previously experienced by shifted animals, it does, itself, have some intrinsic rewarding property since unshifted animals will readily consume it. Thus, the task engenders a consummatory conflict situation (as evidenced by elevated plasma corticosterone levels), since both rewards may be considered to be "rewarding." In fact, ethanol has been shown to act as an anti-conflict agent in this paradigm, alleviating the depressed performance in downshifted animals. As such, the negative contrast paradigm can be employed as a useful tool to probe for long-term effects of in utero alcohol exposure in mice. It will permit a biochemical correlate of a behavioral task which is "frustrating" and permit an evaluation of the anxiolytic properties of ethanol as a function of prenatal alcohol exposure. Thus, these studies will examine the behavioral, pharmacological, and neuroendocrinological ramification of prenatal alcohol exposure in mice using a single test paradigm. These issues are of clinical importance and, therefore, of interest to examine in an animal model with the ultimate goal of generating data of significance to children with Fetal Alcohol Syndrome.

While a growing body of literature has characterized the physiological and morphological consequences of prenatal ethanol (EtOH) exposure, few studies have examined the influence of such prenatal treatment on subsequent responsiveness to EtOH challenge. Moreover, there is a clear void in the literature pertaining to the influence of prenatal EtOH exposure on subsequent responsiveness to chronic EtOH treatment, particularly as such treatment relates to the development of tolerance and dependence. Given the anomalies in neurochemistry, neuroanatomy, endocrinology, and behavior reported in animals exposed to EtOH prenatally, combined with preliminary data from our laboratory, there is good reason to hypothesize that group differences in EtOH sensitivity should be observed. The general hypothesis being tested is that animals exposed to EtOH in utero will differ from controls in subsequent response to acute and chronic EtOH challenge. To test this hypothesis, a total of ten studies have been designed to investigate the influence of prenatal EtOH exposure on subsequent postnatal sensitivity to (1) acute EtOH challenge, (2) the development and expression of EtOH tolerance, and (3) the development and expression of physical dependence. More specifically, a mouse model system will be used to determine whether prenatal EtOH exposure effects acute sensitivity to EtOH-induced stimulation prenatal EtOH exposure effects acute sensitivity to EtOH- induced stimulation of locomotor activity and EtOH-induced motor incoordination, the development and/or expression of tolerance to the motor incoordinating action of EtOH, and development and/or expression of EtOH dependence, as assessed by intensity of the withdrawal response. Further, additional studies are aimed at identifying potential neurochemical mechanisms underlying altered CNS sensitivity to acute and chronic EtOH challenge by employing a psychopharmacologic approach. Taken together, these studies will fill a critical void in the fetal EtOH literature and begin to address possible CNS mechanisms underlying alteration in neurosensitivity to EtOH in offspring exposed to EtOH in utero.

APPLICANT'S ABSTRACT: This is a request for an ADAMHA Research Scientist Development Award (RSDA). The candidate has demonstrated a career commitment to the alcohol research area, and this RSDA award will provide him with the time and support needed to expand and enhance his skills as a scientist. In particular, the RSDA award will afford the candidate the opportunity to develop and establish neurochemical skills which will complement his demonstrated expertise in psychopharmacology, and make him a more well- rounded, competitive, and productive scientist in the alcohol field. The acquisition of such research tools will provide the candidate with a powerful repertoire of research skills that will better enable him to realize his scientific goals, as well as his full potential as a scientist. Hence, this award will provide the candidate with both the resources and time commitment necessary to see professional growth come to fruition. With support from this RSDA proposal, the candidate intends to develop research expertise in neuropharmacology techniques so that they may be incorporated in his overall alcohol research program. Currently, the candidate has two active funded research themes. One research project is focused on the behavioral teratogenic effects of ethanol and the other theme is directed toward examining the role of the GABA-A receptor complex in mediating acute and chronic effects of ethanol. As detailed in the research plan of this application and a corresponding R01 proposal, the candidate intends to apply newly acquired neurochemical skills to more directly examine the role of GABA-A (and NMDA) receptors in a model of potentiated ethanol withdrawal that he has established in his laboratory. This includes, but is not limited to, radioligand binding procedures and measurement of GABA-A receptor-operated 36Cl-flux in brain tissue homogenate samples. To accomplish these goals, the candidate will collaborate with Dr. Marlene Wilson of the Department of Pharmacology at the University of South Carolina School of Medicine, who has extensive experience in conducting these neurochemical procedures. In addition, although not a primary focus of this application, these same neurochemical skills may also be incorporated in other aspects of the candidate's research program. For example, receptor binding procedures may be employed to more directly address neurochemical alterations in prenatal ethanol-exposed offspring that have been indirectly identified by the candidate through his psychopharmacology work in this area. Thus, funding this RSDA application will not only foster continued professional growth and maturity as a researcher in the alcohol field, but will also greatly contribute to the overall enhancement of the quality and breadth of his research program. This, along with his improved ability to train graduate and postdoctoral students in modern- day multidisciplinary alcohol research, will hopefully contribute to advancing our knowledge of the CNS mechanism underlying acute and chronic actions of ethanol.

THIS IS A SHANNON AWARD PROVIDING PARTIAL SUPPORT FOR THE RESEARCH PROJECTS THAT FALL SHORT OF THE ASSIGNED INSTITUTE'S FUNDING RANGE BUT ARE IN THE MARGIN OF EXCELLENCE. THE SHANNON AWARD IS INTENDED TO PROVIDE SUPPORT TO TEST THE FEASIBILITY OF THE APPROACH; DEVELOP FURTHER TESTS AND REFINE RESEARCH TECHNIQUES; PERFORM SECONDARY ANALYSIS OF AVAILABLE DATA SETS; OR CONDUCT DISCRETE PROJECTS THAT CAN DEMONSTRATE THE PI'S RESEARCH CAPABILITIES OR LEAD ADDITIONAL WEIGHT TO AN ALREADY MERITORIOUS APPLICATION. THE APPLICATION BELOW IS TAKEN FROM THE ORIGINAL DOCUMENT SUBMITTED BY THE PRINCIPAL INVESTIGATOR. Alcoholism represents a major medical as well as an economic and social burden in our society. One of the serious consequences of chronic alcohol use is the development of physical dependence and the possibility of experiencing a life-threatening withdrawal syndrome. The natural course of the disease is episodic in that, invariably, periodic interruptions in chronic alcohol consumption occur. Thus, not only is experience with a characteristic withdrawal syndrome common in the alcoholic, but multiple such occurrences are likely. Among other variables, prior episodes of ethanol (EtOH) withdrawal may increase the severity of future withdrawal reactions, an effect that is viewed as being somewhat akin to a "kindling-like" phenomenon. Both clinical and experimental evidence have demonstrated a progressive intensification of the withdrawal syndrome following repeated episodes of EtOH intoxication and withdrawal. The focus of this proposal is directed toward using an established animal model of potentiated EtOH withdrawal to begin to probe for neurochemical mechanisms underlying the phenomenon. As an initial step in the search for underlying mechanisms, the role of the GABAa and NMDA receptor systems will be examined in potentiated EtOH withdrawal. The general hypothesis to be tested is that decreased GABAa receptor activity and increased NMDA receptor activity progressively intensify with each successive EtOH withdrawal episode and the magnification of these changes is ultimately manifested as an observed potentiation of withdrawal seizures following repeated EtOH withdrawal experience. Studies proposed in this application are designed to test this hypothesis by comparing both behavioral sensitivity and neurochemical changes in animals with multiple or single EtOH withdrawal experience. Altered behavioral sensitivity (measured as chemoconvulsant seizure threshold dosage) to drugs active at the GABAa and/or NMDA receptor complexes will provide indirect support for the general hypothesis while more direct evidence for the involvement of these receptor systems will be obtained by measuring sensitivity to drug- induced modification of GABAa receptor-operated chloride (36C1-) influx, as well as radioligand binding parameters at each of the receptor complexes. Taken together, the proposed work will provide important information about the potential role of the GABAa and NMDA receptor systems in mediating potentiated EtOH withdrawal associated with repeated withdrawal experience. Moreover, results from these studies may provide clinically- relevant insight regarding more effective pharmacotherapy strategies for treating EtOH withdrawal.

Alcohol abuse and alcoholism are significant problems in society, and ethanol (EtOH) withdrawal represents one of many serious consequences of chronic drinking. Given the fact that many individuals experience multiple EtOH withdrawal, and that repeated detoxifications have been shown to progressively intensity in severity, it is clear that treatment strategies need to be evaluated for their ability to effectively and (and safely) address this sensitization of "kindling" of EtOH withdrawal syndromes. Our models of EtOH withdrawals is really suited to address this clinically relevant research issues since evaluations of pharmacologic treatments in preventing or blocking behavioral indices of sensitized withdrawal hyperexcitability may also be extended to evaluation of their effects against electrophysiological and molecular correlates of this withdrawal sensitization phenomenon. A series of anticonvulsant drugs that influence activity at GABAa and NMDA receptor systems (important neural substrates of EtOH withdrawal) will be evaluated for their ability to block or attenuate the development and/or expression of 1) behavioral indices of sensitized withdrawal-related CNS or attenuate the development and/or expression of 1) behavioral indices of sensitized withdrawal-related CNS hyperexcitability, 2) sensitization of electrographic measures of withdrawal-related CNS hyperexcitability and 3) molecular alterations (mRNA and protein expression) of GABAA and NMDA receptor subunits associated with repeated ethanol intoxication and withdrawal. Taken together, the proposed work will employ an established model of multiple EtOH withdrawals and a multi-disciplinary approach in evaluating the efficacy of pharmacologic agents in treating behavioral, electrophysiological and molecular indices of withdrawal sensitization. Results from these studies will provide clinically-relevant information about pharmacotherapy strategies for treating EtOH withdrawal and importantly, the potentially long-term deleterious consequences related to multiple EtOH withdrawal.

DESCRIPTION (provided by applicant): Alcohol abuse continues to be a substantial medical and social problem in the United States. A complex interplay among numerous biological and environmental factors influences ETOH drinking behavior. Chronic excessive consumption of ETOH can lead to the development of dependence, and repeated experience with associated withdrawal episodes may constitute a powerful motivational force that perpetuates continued ETOH use/abuse, as well as contributing to relapse (i.e., the re-initiation of drinking after periods of abstinence). Given the high rate of recidivism in alcoholism, it is clear that relapse represents a major problem for most alcoholics, and relapse continues to represent a major challenge to treatment efforts. Currently, there is no therapeutic intervention (including pharmacotherapy) that is fully satisfactory in preventing relapse and sustaining abstinence. Thus, the development and use of preclinical models of ETOH relapse that closely approximate the clinical situation is essential for advancing investigations aimed at elucidating neural substrates and environmental circumstances that precipitate and/or mediate relapse, as well as facilitating the evaluation of more effective treatment strategies. The focus and overall objective of this proposal is to utilize a mouse model of repeated ETOH withdrawals and relapse to evaluate the efficacy of pharmacotherapies for relapse prevention, as well as identify molecular neurobiological events related to increased relapse susceptibility. Conceivably, changes in gene and protein expression that endure well beyond the presence of ETOH in the brain may represent key molecular adaptive events that are critical in mediating fundamental changes in brain function that define relapse susceptibility. Thus, it follows that identification of long-term changes in gene expression following repeated cycles of chronic ETOH exposure and withdrawal would provide valuable insight about potentially novel targets for new treatment approaches that are especially relevant to battling relapse. To date, there is very little information available about changes in gene expression that may relate to ETOH relapse. Therefore, a unique and novel feature of this proposal is to combine a robust behavioral model of ETOH relapse with sophisticated molecular techniques that involve applying gene arrays for identification of a 'relapse gene profile', followed by immunoblotting to validate the relevance of candidate changes. Further, identifying common molecular targets of pharmacotherapies for relapse prevention has the potential to lead to the development of new treatment strategies that are more effective than current ones in preventing relapse and sustaining abstinence. Thus, the aims and objectives of this proposal reflect an overarching experimental strategy that embodies a behavior-gene-behavior approach. The overall goal of this proposal is to utilize functional genomics/proteomics in a new model of ETOH relapse to evaluate, on a molecular level, the efficacy of various pharmacotherapies, as well as to identify new and novel targets for therapeutic intervention.

DESCRIPTION (provided by applicant): A complex interplay among numerous biological and environmental factors govern both ethanol (EtOH) seeking and drinking behavior. Among these, genetics and stress certainly play prominent roles in shaping vulnerability and susceptibility for future problems with EtOH, as well as risk of developing alcoholism. While stress has been viewed as an important contributing factor to EtOH abuse and alcoholism, the interaction between stress and EtOH drinking behavior is not well understood. This is especially true when one considers the role/impact of stress on EtOH self-administration in the context of EtOH dependence. Chronic excessive consumption of EtOH can lead to the development of dependence, and repeated experience with associated withdrawal episodes may constitute a powerful motivational force that contributes to the perpetuation of EtOH use/abuse. Further, functional changes in brain/neuroendocrine stress and reward systems as a result of chronic EtOH exposure may render subjects not only more vulnerable to engage in excessive uncontrollable EtOH drinking, but also more susceptible to events (stress) that may trigger re-initiation of EtOH use after periods of abstinence (relapse). Unfortunately, little is known about the dynamics associated with the relationship between stress and EtOH consumption after dependence has been established. While a substantial emphasis of the existing NIAAA-supported "Stress, Anxiety and Alcohol Abuse" (WFUSM-INIA) Consortium is focused on elucidating the interaction of stress on EtOH consumption in various genetic mouse models, none of this work involves looking at the stress-EtOH relationship in dependent animals. The overall goal of this project is to utilize our established mouse model of EtOH dependence to investigate whether stress associated with repeated cycles of chronic EtOH exposure/withdrawal experience influences EtOH self-administration behavior, and whether such experience subsequently influences the ability of acute stress to modify EtOH seeking and drinking behavior. As such, this project not only fills a void in the literature related to EtOH dependence and stress, but importantly, it targets the major overarching theme of the WFUSM-INIA Consortium. Furthermore, the proposed work not only complements ongoing work in the Consortium, but it enhances the Consortium by expanding its focus to include the full spectrum of the addiction process, from use, to abuse, and to dependence. The overall goal of this proposal is to provide new information about the interaction between EtOH dependence, stress, and EtOH drinking behavior that is relevant to the WFUSM-IN1A Consortium, as well as the alcohol field in general.

Alcoholism is a substantial medical and social problem in the U.S. and relapse represents a major challenge to treatment efforts. Currently, there is no therapeutic intervention that is fully satisfactory in preventing relapse and sustaining abstinence. While dependence is known to contribute to the problem of relapse, this issue has not been thoroughly studied. The focus and overall objective of this proposal is to utilize a mouse model of EtOH dependence and relapse to evaluate the ability of pharmacotherapies to reduce voluntary EtOH drinking, as well as neurochemical alterations that may underlie motivation to drink in dependent compared to non-dependent animals. A contemporary view of alcohol and drug addiction indicates that activation of different neurochemical systems within the brain reward ("motive") circuitry play a critical role in establishing the initial reinforcing effects of EtOH, as well as shaping motivational forces that perpetuate EtOH use/abuse during later stages in addiction. The mesolimbic dopamine pathway, with the nucleus accumbens being a key target structure, is a prominent component of this circuitry. Among several neurotransmitter systems that impinge on this dopamine pathway, glutamate transmission has emerged as a key player in contributing to the motivational effects of EtOH. A guiding principle of this proposal is that activation of the mesolimbic dopamine pathway plays an important role in establishing the acute reinforcing properties of EtOH, while adaptive changes in dopamine and glutamate transmission contribute to conditions that promote relapse and engender excessive EtOH drinking behavior characteristic of dependence. The research plan entails use of in vivo microdialysis procedures to characterize changes in extracellular levels of dopamine and glutamate in nucleus accumbens associated with voluntary EtOH drinking, as well as evaluation of various pharmacological agents for their ability to influence concomitant measures of behavior (drinking) and associated neurochemical changes in the model of dependence and relapse. The overall goal of the proposal is to provide new information about neural substrates underlying EtOH drinking in dependent compared to nondependent subjects, as well as evaluate the ability of potential pharmacotherapeutics to impact both neurochemical and behavioral (drinking) changes related to dependence and relapse.

DESCRIPTION (provided by applicant): A complex interplay among numerous biological and environmental factors govern both ethanol (EtOH) seeking and drinking behavior. While stress has been viewed as an important contributing factor to EtOH abuse and alcoholism, the interaction between stress and EtOH drinking behavior is not well understood. This is especially true when one considers the role/impact of stress on EtOH self-administration in the context of EtOH dependence. Chronic excessive consumption of EtOH can lead to the development of dependence, and repeated experience with associated withdrawal episodes may constitute a powerful motivational force that contributes to the perpetuation of EtOH use/abuse. Further, functional changes in brain/neuroendocrine stress and reward systems as a result of chronic EtOH exposure may render subjects not only more vulnerable to engage in excessive EtOH drinking, but also more susceptible to events (stress) that may trigger re-initiation of EtOH use after periods of abstinence (relapse). Unfortunately, little is known about the dynamics associated with the relationship between stress and EtOH consumption, as well as mechanisms underlying this interaction in the context of dependence. During the current funding period, we linked a model of EtOH dependence and drinking to demonstrate that repeated cycles of chronic EtOH exposure and withdrawal experience enhances subsequent voluntary EtOH consumption. This proposal builds and expands on this work, with an emphasis on elucidating mechanisms underlying the phenomenon. Specifically, proposed studies will focus on the neuropeptide CRF and the neuroactive steroid allopregnanolone because they are intimately related to stress responsiveness (involving both neuroendocrine-related and independent brain stress pathways), as well as EtOH dependence and EtOH self-administration behavior. The overall focus of this proposal is aimed at utilizing an established mouse model of EtOH dependence to examine mechanisms by which stress associated with repeated cycles of chronic EtOH exposure/withdrawal (changes in brain CRF and allopreqnanolone activity) influence subsequent EtOH self-administration behavior, as well as stress-induced relapse behavior. As such, this project not only fills a void in the literature related to EtOH dependence and stress, but importantly, it targets the major overarching theme of the INIA-Stress Consortium as well as complements other projects with a similar research focus in the Consortium. Results from this research proposal will provide new and novel information about possible mechanisms underlying/mediating the interaction between EtOH dependence, stress, and EtOH drinking/relapse behavior that is relevant to the INIA-Stress Consortium, as well as the alcohol field in general. The overall goal is to provide a more complete and comprehensive analysis of the biological underpinnings and environmental circumstances in which stress contributes to excessive EtOH drinking and the development of alcoholism.

DESCRIPTION (provided by applicant): Ethanol tolerance is a complex phenomenon that encompasses a wide range of ethanol-induced processes. The role of ethanol tolerance in sustaining excessive drinking behavior that consequently can lead to the development of ethanol dependence remains to be determined. Studies proposed in this application are responsive to the RFA (AA-08-009) in that they test a novel hypothesis related to the role of tolerance to the aversive properties of ethanol in the context of ethanol dependence. More specifically, we propose that tolerance develops to the aversive properties of ethanol and that this tolerance, in turn, maintains excessive drinking in dependent animals. Moreover, we hypothesize that an underlying mechanism for tolerance to the aversive properties of ethanol relates to neuroadaptation in glutamatergic neurotransmission in the basolateral amygdala (BLA). Our overall research strategy and approach involves employing a well-characterized mouse model of ethanol dependence that reliably produces excessive voluntary ethanol consumption. Using this model, studies will be conducted to: (a) examine tolerance to the aversive properties of ethanol, as defined by reduced sensitivity to ethanol-induced condition taste aversion (Specific Aim I);(b) measure basal levels and the capacity for ethanol to stimulate extracellular levels of glutamate in the BLA (Specific Aim II);and (c) examine the effects of direct manipulation of BLA glutamatergic neurotransmission on ethanol-induced conditioned taste aversion and drinking behavior (Specific Aim III) in ethanol dependent and non-dependent mice. The findings will fill a general void in the literature regarding the role of glutamate in the BLA for tolerance to the aversive consequences of ethanol, and should delineate a potential link to increased risk for excessive ethanol consumption and increased relapse associated with dependence. PUBLIC HEALTH RELEVANCE: Tolerance to the aversive properties of alcohol may facilitate increased consumption that, in turn, can lead to the development of dependence along with sustained excessive drinking. Using an animal model of alcohol dependence and drinking, we aim to advance knowledge regarding factors and mechanisms associated with tolerance and dependence that promote excessive drinking behavior. Further discovery about mechanisms underlying tolerance to the aversive properties of alcohol in animals may lead to a better understanding of alcohol tolerance and dependence in humans and, ultimately, better treatment strategies and outcomes for those suffering with alcohol dependence.

DESCRIPTION (provided by applicant): This research proposal is submitted in response to the NIH announcement (NOT-OD-09-058) on availability of Recovery Act Funds for Competitive Revision Applications. The currently funded parent grant (UOI AA014095) to this competitive revision application is a research component of the NIAAA supported INIA- Stress Consortium, which is focused on stress-ethanol interactions in relation to excessive drinking behavior. Specifically, the project aims to examine mechanisms by which stress associated with repeated cycles of chronic ethanol exposure and withdrawal promotes excessive ethanol drinking and increased vulnerability to relapse associated with ethanol dependence. Utilizing a mouse model of ethanol dependence and relapse/excessive drinking, major emphasis is focused on studying the role of CRF. This competitive revision proposal is designed to extend and enhance our efforts to elucidate adaptive changes in CRF activity within brain reward and stress pathways that are associated with ethanol dependence and escalation of drinking behavior. While studies in the parent grant focus on neuroadaptive changes in CRF in the mouse model of ethanol dependence and relapse drinking, these studies are limited to measuring brain regional changes in mRNA (by quantitative real-time PCR (qRT-PCR) analysis) and peptide content (by ELISA) of CRF itself. Studies proposed in this competitive revision application will extend and complement work conducted in connection with the parent grant by focusing on transcriptional and protein changes in CRFI and CRF2 receptors using the same ethanol dependence and relapse drinking model. Further, in addition to analysis of brain CRF1 and CRF2 receptor mRNA and protein by qRT-PCR and immunoblotting, in situ hybridization procedures will be used to gain greater anatomical resolution of neuroadaptive changes in CRF function associated with the model of ethanol dependence and excessive drinking. Overall, the goal is to provide a more comprehensive analysis of adaptive changes in functioning of the CRF stress neuropeptide system within brain reward and stress pathways that result from stress associated with ethanol dependence and contribute to excessive drinking behavior and the development of alcoholism. PUBLIC HEALTH RELEVANCE: While stress has been viewed as an important contributing factor to alcohol abuse and alcoholism, the interaction between stress and ethanol drinking behavior is not well understood. This is especially true when one considers the role/impact of stress on ethanol self-administration in the context of dependence. The overall goal of this research project is to gain a better understanding about the role of stress in promoting excessive ethanol drinking and increased vulnerability to relapse associated with ethanol dependence.

DESCRIPTION (provided by applicant): This application is submitted in response to the NIH Announcement (RFA-OD-09-003) and the proposal addresses the broad Challenge Area (08): Genomics, and the specific Challenge Topic, 08-AA-104: Regional Central Nervous System (CNS) Gene Expression. Alcohol (ethanol) abuse and dependence are substantial medical and social problems in the U.S. and constitute a significant public health concern. Alcoholism is a chronic relapsing disease, and relapse represents a major challenge to treatment efforts. Despite significant advancements in our understanding about neural substrates and environmental factors that drive this insidious cycle of addiction, few treatments are available and none have proven to be fully satisfactory in tackling this problem. Thus, a major challenge to the field is to employ preclinical models that not only facilitate advancing our knowledge about etiological factors involved in perpetuating heavy ethanol use/abuse, but also identifying new potential therapeutic targets that will be key in the development of the next generation of treatments. We have developed a mouse model of ethanol dependence and relapse drinking that is ideally suited for studies aimed at identifying molecular neurobiological events that contribute to enhanced relapse vulnerability as well as driving escalation of ethanol drinking. This research proposal is aimed at utilizing this established mouse model of ethanol dependence and relapse drinking in combination with microarray technology and informatics to profile changes in gene expression related to excessive drinking associated with ethanol dependence. A novel and innovative feature of the proposal is that it entails applying sophisticated analytical tools to facilitate identification of unique brain regional and time-dependent molecular events (mRNA abundance) related to chronic ethanol exposure, acute and protracted phases of withdrawal, and the potential for ethanol consumption to impact these changes. Thus, this comprehensive experimental approach will generate a very rich data set indicating changes in gene expression that are unique to each of these aspects of the model as well as transcriptional alterations in pathways/functions that are common across time points and brain regions. This highly unique data set will, in turn, not only provide new and valuable insights about mechanisms relevant to the problem of dependence and harmful drinking, but also provide a valuable resource to the field in revealing potential new and novel therapeutic targets that may be especially informative for efforts to develop more effective treatments for this major medical and social problem, namely alcohol abuse and alcoholism. PUBLIC HEALTH RELEVANCE: This project aims to identify genetic and molecular neuroadaptive events related to excessive drinking associated, with alcohol dependence with the hope of revealing potential new and novel therapeutic targets for development of new treatments for alcoholism.

The Shared Core provides centralized services needed by investigators in the Charleston Alcohol Research Center (ARC). Since its inception, the Shared Core has been a valuable part of the ARC, achieving its goals and refining its services to best meet the needs of a dynamic research environment. The proposed Shared Core contains two new services identified as important by Center researchers, the Animal Model Core and the Genotyping Core, while retaining the highly valued and widely utilized Biostatistics Core. - The Animal Model Core will provide basic science researchers in the Center with mice that have been treated in a standardized, well-validated ethanol dependence model. This will facilitate integration of data collected from the various Center projects including electrophysiological, neurochemical, and behavioral measures that will be related to consequences of chronic intermittent ethanol exposure using a single model. - The Genotyping Core will support the Center's increasing needs for advanced genetic analysis. Although this service will predominantly support clinical research projects in the ARC, extending this core function to other clinical as well as preclinical projects will enhance translational efforts in the Center. - The Biostatistics Core will continue to provide data management and expert statistical analysis and consulting service to all Center research projects through the Office of Statistics and Database Management. By centralizing these services, several important benefits are realized. First, the Shared Core provides needed expertise in specialized areas. Second, it affords a more efficient and standardized research environment, as it prevents the duplication of effort by individual research groups. Third, the Shared Core provides time- and cost-savings for all research studies that utilize the services. Finally, the Shared Core system fosters greater integration of Center research activities and it provides a central authority for quality control over provision and utilization of services as well as ensuring that its services meet the needs of Center investigators.

Alcoholism is a chronic relapsing disorder that continues to be a serious medical and social problem in the U.S. and enhanced efforts are needed to treat it. Advances in our understanding about mechanisms underlying motivation to drink and, in particular, mechanisms that drive transition from moderate drinking to more excessive and uncontrolled drinking are key to developing new and more effective treatment strategies. A contemporary view of alcohol addiction is that adaptations in glutamate (GLU) and dopamine (DA) transmission within regions of the striatum (key components of neurocircuitry that mediate motivated behavior) play a significant role in regulation of ethanol drinking behavior. While our current research efforts have focused on neurochemical adaptations in the ventral striatum (i.e., nucleus accumbens;NAc), recent evidence suggests that adaptations in GLU and DA transmission in the dorsolateral striatum (DLS) may play a prominent role in mediating the transition from controlled drinking to uncontrolled compulsive drinking that is characteristic of ethanol dependence. Accordingly, the overall objective of this proposal is to examine adaptations in GLU and DA transmission in dorsal striatum that may qualitatively or quantitatively differ from those in ventral striatum, as well as evaluate the influence of pharmacotherapeutics on voluntary ethanol drinking and neurochemical alterations that may underlie motivation to drink in dependent compared to nondependent animals. A guiding principle of the proposal is that ethanol dependence is associated with adaptations in GLU and DA transmission within dorsolateral striatum and that play an integral role in driving transition from moderate controlled drinking to uncontrolled excessive drinking. The research plan entails use of an established mouse model of ethanol dependence and relapse drinking along with in vivo microdialysis procedures to characterize changes in extracellular levels of GLU and DA in dorsal compared to ventral regions of the striatum that are associated with escalation of voluntary ethanol drinking in dependent mice compared to stable intake exhibited by nondependent mice. Additionally, these studies will provide new information on potential neuroanatomical sites and neurochemical mechanisms underlying the ability of pharmacological treatments to reduce excessive ethanol drinking associated with dependence, which could lead to new insights and approaches in the development of more effective pharmacotherapies for the treatment of alcoholism.

DESCRIPTION (provided by applicant): While stress is known to be an important contributing factor to alcohol abuse and alcoholism, the interaction between stress and ethanol drinking behavior, as well as mechanisms underlying this interaction in the context of dependence are not well understood. Prolonged ethanol exposure constitutes a potent stressor, producing a state of allostasis whereby chronic intoxication continually taxes the organism beyond normal homeostatic limits, rendering it ill-equipped to exert appropriate behavioral control over ethanol consumption, as well as appropriately respond to other (additional) stressful events that may provoke return to excessive drinking. During the current funding period, we have used a mouse model of chronic intermittent ethanol (CIE) exposure to demonstrate that resulting significant escalation of drinking is associated with profound alterations in neuroendocrine-related (HPA axis activity) and independent (extrahypothalamic corticotropin releasing factor, CRF) stress systems. However, mechanisms underlying dependence-related alterations in stress responsiveness and, in particular, the ability of stress to modulate drinking in the context of dependence have not been extensively studied. Accordingly, this proposal will build and expand on our current work by utilizing our CIE model of dependence to examine the role of CRF and CRF1 receptor activity in brain regions within cortical-limbic-HPA circuitry (brain structures and pathways integral to stress and reward/motivational processes) that mediate/contribute to: (a) escalation of ethanol drinking in dependent mice; (b) altered (compromised) behavioral responsiveness to stress challenge; and (c) the ability of stress to modulate ethanol consumption in dependent compared to nondependent mice. A unique feature of this proposal is use of various tools and approaches in examining behavioral, physiological, neurochemical, and molecular responses to stress overlaid on the CIE model. As such, this project not only fills a void in the literature related to ethanol dependence and stress, but importantly, it targets the major overarching theme of the INIA-Stress Consortium as well as complements other projects with a similar research focus in the Consortium. The overall goal of the project is to provide a more comprehensive understanding of neuroadapfive changes in CRF funcfion that underlie stress-ethanol interactions in the context of dependence, and gain a better understanding of how such changes contribute to excessive and harmful drinking behavior as well as the development of alcoholism. PUBLIC HEALTH RELEVANCE: Alcoholism is a major health concern as well as a significant social and economic burden on society in the U.S., and stress is known to contribute to the problem. This research project aims to enhance our understanding about mechanisms by which stress associated with alcohol dependence promotes excessive drinking. The goal is to facilitate development of new and more effective treatment strategies for alcohol abuse and alcoholism.

DESCRIPTION (provided by the applicant) Overall Center Abstract: The Charleston Alcohol Research Center has had, and continues to have, alcohol treatment as the Center's overarching theme. Our Center continues to embrace a multidisciplinary approach to accomplish its objectives, with basic scientists working side-by-side with psychiatrists or clinical psychologists on athematic program of research. Junior investigators recruited into the Center are teamed with more experienced investigators to provide them with a unique mentoring opportunity aimed at arming them with the necessary experience to become independent researchers. For this application, the research teams have taken advantage of developing or refining cutting-edge technologies (e.g., brain imaging, genetics, in vivo microdialysis, multi-array recording, and laboratory paradigms to study stress-alcohol interactions and/or voluntary drinking) to address their specific research questions. In the next five years, the Research Components are tied together by either a focus on neuroanatomical and/or neurochemical adaptations that accompany the transition from controlled to uncontrolled drinking, the neurocircuitry underlying reward processes, or trait personality factors that may mediate the risk for development of alcohol dependence or the response to medication. Pharmacotherapy, or implications for pharmacotherapy, remains the major focus of the Charleston Alcohol Research Center, and is the area where we have developed a national/international reputation. The Research Components are supported by two Cores and two other components. The Administrative Core provides the leadership and infrastructure to facilitate the mission of the Center as a Whole; the Shared Core provides and manages common services needed by the researchers to maximize resources and increase productivity; the Pilot Project Component attracts new talent or new ideas to the Center; and the Research Translation/Information Dissemination Component accomplishes the aim and responsibility of a Center for information dissemination on advances in alcohol research to the general public, as well as to health care providers.

Treating alcohol dependence is a great unmet need worldwide and developing medications to treat alcohol dependence remains a high priority of the National Institute on Alcohol Abuse and Alcoholism (NIAAA). To help fulfill this goal, NIAAA has implemented a preclinical medication screening program to foster discovery of lead compounds, targets and mechanisms, and to entice the pharmaceutical industry to develop their best marketable compounds for alcoholism treatment. Previously, ¿proof-of-principle¿ studies were conducted to demonstrate the feasibility for successful testing using the clinically effective compounds naltrexone and acamprosate in rodent models of dispositional heavy drinking. The current contract seeks to test potential medications in heavy drinking paradigms which have demonstrated utility for medication screening.

Alcoholism is a chronic relapsing disorder that continues to be a serious medical and social problem in the U.S. and enhanced efforts are needed to treat it. Advances in our understanding about mechanisms underlying motivation to drink and, in particular, mechanisms that drive transition from moderate drinking to more excessive and uncontrolled drinking are key to developing new and more effective treatment strategies. A contemporary view of alcohol addiction is that adaptations in glutamate (GLU) and dopamine (DA) transmission within regions of the striatum (key components of neurocircuitry that mediate motivated behavior) play a significant role in regulation of ethanol drinking behavior. While our current research efforts have focused on neurochemical adaptations in the ventral striatum (i.e., nucleus accumbens; NAc), recent evidence suggests that adaptations in GLU and DA transmission in the dorsolateral striatum (DLS) may play a prominent role in mediating the transition from controlled drinking to uncontrolled compulsive drinking that is characteristic of ethanol dependence. Accordingly, the overall objective of this proposal is to examine adaptations in GLU and DA transmission in dorsal striatum that may qualitatively or quantitatively differ from those in ventral striatum, as well as evaluate the influence of pharmacotherapeutics on voluntary ethanol drinking and neurochemical alterations that may underlie motivation to drink in dependent compared to nondependent animals. A guiding principle of the proposal is that ethanol dependence is associated with adaptations in GLU and DA transmission within dorsolateral striatum and that play an integral role in driving transition from moderate controlled drinking to uncontrolled excessive drinking. The research plan entails use of an established mouse model of ethanol dependence and relapse drinking along with in vivo microdialysis procedures to characterize changes in extracellular levels of GLU and DA in dorsal compared to ventral regions of the striatum that are associated with escalation of voluntary ethanol drinking in dependent mice compared to stable intake exhibited by nondependent mice. Additionally, these studies will provide new information on potential neuroanatomical sites and neurochemical mechanisms underlying the ability of pharmacological treatments to reduce excessive ethanol drinking associated with dependence, which could lead to new insights and approaches in the development of more effective pharmacotherapies for the treatment of alcoholism.

The National Institute on Alcohol Abuse and Alcoholism (NIAAA) seeks a contractor to address the present and anticipated future needs of NIAAA's Medication Development Program. The Division of Neuroscience and Behavior's (DNB) Preclinical Medication Efficacy Testing Program seeks to re-compete a contract for the blind testing of reference compounds and proprietary compounds on alcohol drinking in mice using models of various aspects of alcohol dependence. The contract will use mechanisms having intellectual property conditions favorable to compound providers. This will eliminate a major barrier to testing optimized compounds having preclinical, as well as clinical toxicology data, and will stimulate interest by the pharmaceutical industry in developing high quality, marketable compounds for alcoholism. Overall, the contractor will assess effects of proprietary compounds in standardized models of alcohol dependence-induced alcohol intake using mice for subjects. Specifically, the contractor will be required to test effects of compounds submitted to them by NIAAA staff members in voluntary limited-access 2 bottle choice drinking of unsweetened ethanol solutions in mice made dependent through repeated cycles of ethanol vapor exposure and withdrawal. The contractor will also develop and implement testing of proprietary compounds in additional mouse models, which may include drinking in the dark (DID), intermittent access drinking model, alcohol deprivation model (ADE), and conditioned place preference (CPP) models. The contractor will also collect, analyze and display data from these tests and report them to designated NIAAA personnel. The data generated by this contract will be used in the identification of compounds for further development. Capability statements must conform to the Response Guidelines stated herein and should demonstrate a respondent's qualifications and experience, specifically providing evidence of their capability to perform the following requirements: https://www.fbo.gov/index?s=opportunity&mode=form&id=9a3afcb0812bbb2f105da81b112b728d&tab=core&_cview=0

PROJECT SUMMARY The Administrative Core serves as the backbone of the Charleston Alcohol Research Center (ARC), providing the organizational framework necessary for effective management of all administrative, research, and educational activities connected to the ARC. As the centralized hub of the ARC, the Administrative Core functions to coordinate activities of all components within the ARC, including two basic research projects, two clinical research projects, the Shared Resource Core, and the Pilot Project Core. The Administrative Core is responsible for the day-to-day management of all facets of the ARC to ensure it operates in a smooth, efficient, and productive manner. To accomplish its goals, the Administrative Core provides: (a) scientific and administrative leadership along with infrastructure that fosters cohesion and integration of multidisciplinary translational research efforts; (b) management and support for handling all administrative and fiscal matters, and decisions about resource allocation; (c) mechanisms for internal and external oversight and monitoring of progress to ensure high-level quality control of all Center activities; and (d) central coordination of training opportunities, professional development, and scientific enrichment activities, as well as educational outreach efforts that project the ARC as a regional and national resource. The Center Director, Scientific Director, and the Administrative Core staff have a long-standing and well- established working relationship, having provided effective stewardship of the ARC for many years. The scientific leadership team represents both basic science and clinical research backgrounds. This blend of scientific expertise reflects the ARC's philosophy of embracing translational and interdisciplinary research. The Administrative Core facilitates communication among ARC investigators through internal web-based links on the ARC website, as well as regularly scheduled Steering Committee meetings where scientific or operational issues can be raised and discussed. The Administrative Core also coordinates meetings with the Program Advisory Committee, which provides critical feedback on scientific progress and direction. Collectively, these critical functions enable the Administrative Core to play a central role in fostering multidisciplinary and translational research efforts that are thematically-focused on the topic of treatment and treatment implications, enhancing research collaborations and facilitating use of state-of-the-art experimental approaches, attracting new (especially early-stage) investigators into the Center that serves to invigorate its research efforts, and providing a stimulating environment that enriches training opportunities and professional development in alcohol research.

? DESCRIPTION (provided by applicant): The Charleston Alcohol Research Center (ARC) continues to focus on treatment and treatment implications as an overarching theme. The ARC continues to embrace multidisciplinary and translational research approaches, integrating both basic research and clinical investigations all centered on this common theme. The ARC also continues its tradition of teaming junior faculty with more experienced investigators, capitalizing on new talent and bringing sophisticated cutting-edge technologies and research approaches that enhance research efforts in addressing the Center's overall scientific goals. The ARC is comprised of four research projects and three cores. The Administrative Core provides the leadership and infrastructure to facilitate the overall scientific and educational mission of the Center as a whole. The Shared Resource Core provides vital scientific services needed by Center researchers to facilitate integration, maximize resources, and increase productivity. The Pilot Project Core attracts new investigators and new ideas to the Center, thereby broadening and augmenting its research and training activities. In this renewal application, proposed preclinical and clinical research projects all center on a common research focus - neuroadaptations in cortical processes that underlie transition from controlled to uncontrolled drinking. Two basic research projects will use sophisticated circuitry mapping, and cellular and molecular biology techniques to examine how chronic alcohol exposure alters the function of cortical areas, projections and networks relevant to behavioral control and motivational effects of alcohol. This work will lay the critical foundation for future efforts to target these cortical-subcortical circuitry changes in developing new treatments. Two clinical research projects will employ sophisticated neuroimaging techniques to focus on similar cortical areas and projections in evaluating the ability of different treatment modalities (pharmacological and non- pharmacological) to alter the circuitry and reduce alcohol cue-induced brain activation, craving, and drinking. The Charleston ARC is poised to continue its national leadership role and demonstrated success in: (a) fostering multidisciplinary and translational state-of-the-art research efforts that are thematically-focused on the topic of treatment and treatment implications; (b) attracting new (especially early-stage) investigators into the Center, thereby invigorating its research efforts; and (c) providing a stimulating environment that enriches training opportunities and professional development for the next generation of researchers in the alcohol field.

PROJECT SUMMARY The Shared Resource Core provides centralized resources and services that are tailored to the research needs of investigators in the Charleston Alcohol Research Center (ARC). In doing so, the Shared Resource Core continues to serve the critical role of promoting scientific integration, increasing efficient use of resources, and facilitating use of high quality contemporary technological and investigatory approaches that, collectively, are key to meeting the dynamic research environment fostered by the ARC. The Shared Resource Core is comprised of the Animal Core, the Clinical Intake and Assessment Core, and the Biostatistics Core. The Animal Core provides basic research projects and pilot projects with animals treated in a well-validated and established mouse model of alcohol dependence that involves repeated cycles of chronic intermittent ethanol (CIE) exposure. The Animal Core also will execute stereotaxic surgery and histological evaluation functions to support research incorporating direct brain site-specific injections of pharmacological agents, retrograde tracers, and viral vectors for optogenetic and pharmacosynthetic manipulations. The Clinical Intake and Assessment Core will expand current genotyping work to centralize advertisement and recruitment efforts, perform initial screening of potential study participants, and utilize assessment procedures (including genotyping) to best match and optimize allocation of subjects for enrollment in clinical research projects and pilot projects in the Center. The Biostatistics Core will continue to provide data management and statistical service to both basic and clinical research projects and pilots in the Center. This includes assisting in study design development, performing power analyses at the front end of the projects, construction of appropriate databases, conducting analyses utilizing various contemporary statistical approaches, and assisting in preparation of manuscripts and presentations for dissemination of study results. By centralizing these vital scientific functions, the Core avoids duplication of efforts, increases efficient use of resources, facilitates use of state-of-the-art research approaches and techniques, and improves quality control through oversight of centralized functions - all contributing to maximizing productivity in the ARC. Overall, the Shared Resource Core plays a pivotal role in enhancing integration and cohesion of ARC research activities, as well as stimulating and supporting the vibrant alcohol research and training environment at MUSC.

PROJECT SUMMARY While there is a high prevalence for the co-occurrence of alcohol use disorders (AUD) and post-traumatic stress disorder (PTSD), the mechanisms underlying these disorders are not fully understood. Further, few treatments are effective for those suffering with PTSD-AUD comorbidity. Animal models that closely mimic the key clinical features of these disorders are critical for elucidating mechanisms and factors that underlie the co-occurrence of these illnesses and facilitate development of new and more effective therapeutics. We have recently conducted a series of pilot studies with the goal of developing such a model. Specifically, we have demonstrated that chronic predator odor (TMT) exposure sensitizes male and female mice to later acute stress (TMT)-induced reinstatement of alcohol relapse-like behavior. This effect is long lasting (>60 days) and the sensitized effect generalizes to exposure to context cues associated with prior chronic TMT exposure. We have also demonstrated that a novel model of chronic early-life stress (CES) has long-lasting effects on anxiety behavior and stress responsiveness, as well as increased stress-related alcohol consumption. Finally, our pilot work shows that chronic TMT exposure produces long-last alterations in oxytocin (OT) expression in hypothalamus (PVN) as well as oxytocin receptor mRNA expression in stress-relevant projection brain regions (central amygdala; CeA, bed nucleus of stria terminalis; BNST). Further, systemic administration of OT blocked stress (TMT)-induced alcohol relapse in mice with and without a history of chronic stress exposure. Proposed studies in this application are designed to build on and expand these compelling and supportive pilot findings. The overall objective of this proposal is to optimize and further characterize our model of PTSD that captures many key clinical features of the disorder, link consequences of the model to alcohol self- administration and relapse behavior, and examine the role of the neurohormone oxytocin in this mouse model of PTSD-AUD comorbidity. Specifically, after more fully characterizing the chronic predator odor (TMT) model, we will examine whether CES similarly sensitizes mice to stress-induced alcohol relapse and then use these procedures in combination to examine whether CES experience further augments the ability of chronic TMT exposure to subsequently sensitize mice to acute stress challenge provoking alcohol relapse-like behavior. This unique mouse model of PTSD-AUD will then be used to probe involvement of the oxytocin system, with studies also examining the capacity of exogenous OT treatment to block and/or prevent sensitized stress-induced alcohol relapse in the CES-TMT model. The overall goal is to establish and utilize a clinically relevant mouse model of PTSD-AUD that will advance our understanding of these co- occurring disorders and facilitate development of more effective treatments for PTSD-AUD comorbidity.

PROJECT SUMMARY While stress is known to be a significant factor in triggering relapse and promoting heavy alcohol (ethanol) consumption, mechanisms underlying the interaction between stress and ethanol drinking, particularly within the context of dependence, are not well understood. During the current funding period, we examined stress- ethanol interactions using our well-established dependence model that involves repeated weekly cycles of chronic intermittent ethanol (CIE) exposure alternating with weekly voluntary limited access drinking test sessions. Specifically, we found that repeated brief exposure to forced swim stress (FSS) prior to ethanol drinking test sessions significantly increased drinking in dependent (CIE-exposed) mice, but did not alter intake in nondependent mice. Additional preliminary data we collected implicate a role for the dynorphin/kappa opioid receptor (DYN/KOR) system in mediating this reliable and robust stress-induced selective enhancement of drinking in CIE-exposed mice. Stress and chronic ethanol exposure are known to produce adaptations in stress-related neuropeptides (DYN/KOR and CRF systems) in the central amygdala (CeA) and the bed nucleus of the stria terminalis (BNST), key structures within extended amygdala circuitry that are integral to stress reactivity and motivational/reward processes. The overall objective of this proposal is to utilize our CIE- FSS Drinking model to examine the role of adaptations in DYN/KOR activity and potential CRF-DYN/KOR interactions within extended amygdala circuitry (CeA and BNST) in mediating the ability of stress to promote excessive ethanol consumption in male and female mice. Specifically, studies will utilize molecular biology (qPCR) procedures to examine changes in Pdyn and Crf mRNA expression in CeA and BNST at different time points in the model. Studies also will employ chemogenetic (DREADDs) and pharmacological approaches to selectively modulate DYN/KOR activity in the CeA and the CeA-BNST pathway, as well as examine interactions of CRF and DYN in mediating stress-enhanced drinking associated with dependence. Site-specific administration of KOR and CRF1R/CRF2R ligands in combination with the chemogenetic approach will probe for specificity of effects on drinking in the model. Thus, proposed studies will employ molecular biology, chemogenetic, and pharmacological approaches to interrogate the contribution of DYN/KOR activity and potential CRF-DYN/KOR interactions in the CeA and the CeA-BNST pathway in mediating stress- enhanced drinking associated with dependence. Collectively, results from this project will provide new insights on mechanisms underlying adaptations in stress-related neuropeptides within extended amygdala circuitry that underlie the ability of stress to further escalate drinking associated with ethanol dependence. The overall goal is to identify new targets that will aid in development of more effective treatment strategies for controlling stress-related excessive drinking, and alcohol use disorders in general.