Kathleen A Grant - US grants

The aim of the research project is to assess the contribution of specific ligand-gated ion channels in mediating the perceptible effects of ethanol in the CNS. Three of these receptor/channel complexes that are particularly sensitive to the effects of ethanol in in vitro assays are the GABAA receptor complex, the NMDA subtype of glutamate receptor complex, and the 5-HT3 subtype of serotonin receptor. These receptor systems encompass components of the major inhibitory and excitatory neurotransmitter systems, and a component of the serotonin system, implicated in many behavioral effects of ethanol. The interaction of ethanol with these receptor systems in electrophysiological and biochemical assays occurs at concentrations of 5-50 mM, corresponding to a range of blood ethanol concentrations associated with mild intoxication to loss of righting reflex. The sensitivity of these receptor-linked ion channels to ethanol indicates a specificity to the pharmacological actions of ethanol. The interaction of ethanol at several different receptor systems can be expected to give rise to a mixture of internal stimulus effects, composed of distinguishable component parts. Understanding how ethanol acts at each component receptor system to produce its discriminable effects will advance our understanding of ethanol's pharmacological actions and may be helpful in the design of potential pharmacotherapies to alter and possibly limit excessive ethanol intake. The proposed studies utilize the drug discrimination procedure to 1) determine the modulatory sites on the GABAA receptor complex that are sufficient to produce ethanol-like discriminative effects 2) determine if antagonism of NMDA transmission by compounds acting at the NMDA, channel, glycine or polyamine modulatory sites on the NMDA receptor complex results in similar discriminative stimulus effects as ethanol. 3) Determine the structural requirements of the 5-HT3 antagonists to block the discriminative effects of ethanol, and if this blockade can be due to action of the 5-HT3 antagonists at the nicotinic cholinergic or GABAA channels. 4) Determine if the dose of ethanol used in training the discrimination determines the relative contribution of each receptor system in mediating the discriminative effects of ethanol. 5) Whether a component of the compound discriminative stimulus effects of ethanol can be isolated from the action of ethanol at other receptor systems.

The overall goal of the proposed research is to characterize the pharmacological basis of the subjective effects of alcohol in a non-human primate model. This model uses a drug discrimination procedures to define receptor mechanisms that mediate the interoceptive (or subjective) stimulus effects of ethanol. Studies in the preceding period of funding have suggested that positive modulation of GABAA receptors appears to produce the most robust ethanol-like stimulus across training dose and gender. Of the GABAA ligands, the endogenous neurosteroids offer a unique opportunity to study mechanisms of physiological states that alter sensitivity to the subjective effects of ethanol. The proposed studies will use neurosteroids to define GABAA, subtypes of receptors that mediate the discriminative stimulus effects of ethanol; test novel steroid analogs to produce discriminative stimulus effects though NMDA inhibition; and use menstrual cycle phase to test hypotheses concerning alterations in GABAA receptor subtypes leading to changes in sensitivity to ethanol. Perhaps functionally linked to the GABAA receptor system are specific serotonin receptor subtypes that produce opposite effects on sensitivity to ethanol's discriminative stimulus effects. The proposed studies will further characterize these interactions. Overall, the results from the proposed studies should advance our understanding of endogenous mechanisms that alter sensitivity to the subjective effects of ethanol in a non-human primate species. The specific aims are the research are: 1) To characterize the neurosteroid modulation of GABAA receptors and the discriminative stimulus effects of ethanol. 2) To characterize the neurosteroid modulation of NMDA receptors and the discriminative stimulus effects of ethanol. 3) To characterize the influence of menstrual cycle phase on neurosteroid modulation of the discriminative stimulus effects of ethanol. 4) To characterize the 5-HT1D and 5-HT2C modulation of the discriminative stimulus effects of ethanol.

Epidemiological data of alcohol drinking patterns in the U.S. suggest men are more likely to drink alcohol, drink heavily, and have more legal and psychosocial problems related to their consumption of alcohol compared to women. The biological basis for these gender differences in problem drinking is unknown. It is possible that there are gender differences in either vulnerability to the reinforcing effects of ethanol or sensitivity to the behavioral effects of ethanol; these differences could, in turn, result in differential alcohol consumption. Although some studies suggest that the consumption of alcohol is influenced by menstrual cycle phase, the relationship between reproductive function and alcohol is difficult to study in human being for ethical, logistic, and financial reasons. It is also difficult to study changes in behavior over the menstrual cycle in women because both experimenter and subject bias may affect the outcome of the study. Likewise, behavioral responses to alcohol are difficult to study because expectations about the effects of alcohol on behavior strongly influence social and affective behavior. An alternative is to circumvent examining these difficulties by using an appropriate animal model. Macaques are a useful model because they develop blood alcohol levels similar to human beings in response to a given dose, they metabolize alcohol at approximately the same rate as human beings, they can be trained to self-administer alcohol in significant amounts, they rely on complex social relationships, they are capable of complex cognitive function, and females have menstrual cycles that are similar in length and pattern of gonadotropin and sex steroid secretion to those of women. In addition, while reports are variable, the majority of the available data suggests that several broad classes of behavior (e.g., aggression, affiliation, food intake, sexual interaction, and social withdrawal) vary with the menstrual cycle in macaques. Behavioral variability coincident with menstrual cyclicity provides the potential plasticity necessary to observe female reproductive function effects on alcohol consumption and on associated behavioral responses. We propose to investigate the effects of gender, menstrual cycle phase, ethanol dose, and social setting on oral ethanol self-administration and behavior in this nonhuman primate model.

DESCRIPTION: Alcohol Abuse and Alcoholism pose serious health problems world-wide. Animal models have aided in our understanding of the addiction process and the development of therapeutic strategies to treat alcoholism. Two of these animal models include selective breeding for high ethanol intakes and drug discrimination procedures. We propose to use a combination of these two models to characterize potential receptor mechanisms that may predispose individuals to seek or avoid high intakes of ethanol. Sardinian alcohol-preferring (sP) and Sardinian alcohol-non preferring (sNP) rats were selectively bred for divergent ethanol drinking behavior at the University of Cagliari, Italy. The sP rats display more behaviors indicative of anxiety compared to the sNP rats, and the voluntary ethanol intake in sP rats significantly reverses their anxiogenic state, suggesting that an anxiogenic phenotype may be linked to high ethanol preference and consumption. Because it is well accepted as an assay of receptor mediated behavioral action of a drug, we propose to use drug discrimination procedures to investigate the receptor systems that are prominent in mediating ethanol's behavioral effects associated with an "anxious state" in these selected lines. Two specific aims are proposed. One is to characterize the receptor systems that mediate the discriminative stimulus effects of ethanol associated with doses that are known to be anxiolytic. The second is to characterize the "anxious state" in sP and sNP rats using a PTZ discrimination. The data gathered from the present study are expected to demonstrate that the receptors involved in mediating the effects of ethanol in sP rats are related to ethanol anxiolytic effects. Furthermore, the data will provide a receptor profiling of the behavioral effects of a genetically selected line that appears to have a strong association between ethanol consumption and the relief of anxiety.

Ethanol produces internal stimulus effects that are reinforcing and that maintain either social or pathological drinking in a large number of individuals. The drug discrimination procedure is a particularly useful technique to characterize the receptor mechanisms that mediate the internal stimulus effects of drugs. The overall goal of the proposed research is to assess the contribution of specific ligand-gated ion channels (GABAA and NMDA) in mediating the mixed discriminative stimulus effects of ethanol. Thus, the aims of the present proposal are to continue the development of a model to characterize ethanol's direct and concurrent activity at a number of receptor systems and explore behavioral correlates of some newly identified cellular mechanisms of ethanol's activity. These five new Specific Aims are: l. To investigate the discriminative stimulus effects of ethanol mediated by NMDA and GABAA receptor systems as a function of ethanol training dose. 2. To investigate the optimal ligand to represent the modulatory effects of ethanol at the GABAA (or NMDA) receptor in characterizing receptor interactions. 3. To determine if discriminative stimulus effects that are unique to ethanol are due, in part, to an interaction between ethanol-sensitive GABAA and NMDA receptor systems. 4. To determine whether the ethanol-like effects of the 5-HTIB agonists are secondary to ethanol's effects of GABAA receptors. 5. To assess the potentiation of ethanol's activity at a ligand-gated ion channel by activating metabotropic receptors with drug discrimination. The immediate significance of the proposed studies will be to extend information on ethanol as a mixed stimulus in the CNS, composed of, but not limited to, actions at specific sites on ligand-gated ion channels. These findings will suggest variables that determine the effectiveness of pharmacological agents to block ethanol's stimulus effects, and may suggest the appropriate receptor system(s) to target.

Ethanol produces internal stimulus effects that are reinforcing and that maintain either social or pathological drinking in a large number of individuals. The drug discrimination procedure is a particularly useful technique to characterize the receptor mechanisms that mediate the internal stimulus effects of drugs. The overall goal of the proposed research is to assess the contribution of specific ligand-gated ion channels (GABAA and NMDA) in mediating the mixed discriminative stimulus effects of ethanol. Thus, the aims of the present proposal are to continue the development of a model to characterize ethanol's direct and concurrent activity at a number of receptor systems and explore behavioral correlates of some newly identified cellular mechanisms of ethanol's activity. These five new Specific Aims are: l. To investigate the discriminative stimulus effects of ethanol mediated by NMDA and GABAA receptor systems as a function of ethanol training dose. 2. To investigate the optimal ligand to represent the modulatory effects of ethanol at the GABAA (or NMDA) receptor in characterizing receptor interactions. 3. To determine if discriminative stimulus effects that are unique to ethanol are due, in part, to an interaction between ethanol-sensitive GABAA and NMDA receptor systems. 4. To determine whether the ethanol-like effects of the 5-HTIB agonists are secondary to ethanol's effects of GABAA receptors. 5. To assess the potentiation of ethanol's activity at a ligand-gated ion channel by activating metabotropic receptors with drug discrimination. The immediate significance of the proposed studies will be to extend information on ethanol as a mixed stimulus in the CNS, composed of, but not limited to, actions at specific sites on ligand-gated ion channels. These findings will suggest variables that determine the effectiveness of pharmacological agents to block ethanol's stimulus effects, and may suggest the appropriate receptor system(s) to target.

While a relationship between dopamine and ethanol is widely accepted, this interaction remains relatively undocumented in primates, including humans. The proposed research is critical to the understanding of factors influencing the association between ethanol reinforcement and midbrain dopamine response. Background studies suggest that one mechanism by which ethanol can affect dopaminergic function if through modulation of GABAergic tone in the central nervous system. It is known that the endogenous neurosteroid allopregnanolone acts as a positive modulator of GABAA/benzodiazepine receptors and shares significant overlap with ethanol in producing similar discriminative stimulus effects. There is also evidence that there may be a sex difference in sensitivity to the ethanol-like stimulus effects of allopreganolone. Based on our current knowledge of the interactions between ethanol, dopamine and neurosteroids we hypothesize that we can use positron emission tomography (PET) to document ethanol's increase synaptic dopamine under acute exposure and that these effects of ethanol will be mimicked by allopregnanolone. Further, we hypothesize that chronic ethanol self-administration of ethanol will result in a neuroadaptation of dopaminergic response, reflected in a functional down-regulation of D2 receptor availability. Finally, we hypothesize that the neuroadaptation to ethanol will be slowly reversed by abstinence from ethanol self-administration. With this model and the proposed PET methodology, we can explore neuroadaptation of midbrain dopamine neurons to chronic ethanol self-administration in a longitudinal study. Thus, this research provides a critical step in the understanding the mechanisms underlying the association between dopamine and ethanol reinforcement.

The rapid development of pre-clinical studies on alcohol abuse and alcoholism across many diverse disciplines presents an ever increasing training problem. Because of this multi-disciplinary data base, knowledge from molecular biology, biochemistry, neuroscience and behavior are required for appropriate synthesis and future study design. The proposed training program provides both the pre- and postdoctoral students with exposure to all facets of basic science alcohol research by a group of well-funded, committed alcohol researchers. The training faculty consists of 11 full-time members of either the Department of Biochemistry or the Department of Physiology and Pharmacology at Wake Forest University School of Medicine. Most of these members are also faculty of the interdisciplinary Neuroscience Program. The graduate trainees will have a choice between these three graduate programs. Trainee recruitment will be extensive and include an active attempt to involve minority students in the program. Given the faculty, resources and the multi-disciplinary nature of the training opportunity, be believe that each trainee will be able to continue a career in alcohol specific research following their tenure in the program. It is the primary goal of the program to assure that each trainee becomes able to understand the complex nature of alcohol's actions, with an appreciation for analysis from the molecular to the behavioral level.

DESCRIPTION (provided by applicant): Stress is believed to be an etiological factor in the abuse of ethanol. Chronic and acute stress are known to alter the behavioral effects of ethanol, including the reinforcing effects. Available evidence also suggests that chronic stress alters neurotransmission in specific brain regions that are important for mediating the reinforcing effects of many drugs of abuse, including ethanol. However, there is a need to more completely characterize the limbic, hypothalamic, pituitary and adrenal response to stress an how this response predicts heavy ethanol self-administration. In addition, the source of stress can determine the endocrine and nervous systems response and adaptation. Macaque monkeys are social animals and form stable, liner, social dominance hierarchies. The relative rank of an individual within social hierarchies has trait-like qualities and remains constant, even if the monkeys are separated for some time. Many studies have shown that social subordination in macaque troops results in elevated signs of stress. Socially-derived stress in monkeys can be categorized as psychogenic and, in subordinate monkeys, uncontrollable. Stress of this nature is most associated with stress-induced psychiatric pathology, including alcoholism. We propose to investigate the effects of socially-derived stress, specifically social subordination and limited social contact on ethanol self-administration. We have developed a model of alcohol self-administration in macaque monkeys that produces excessive ethanol? consumption in a proportion of the population. The heavy drinkers are largely male and consume an average of 3.0-4. 0 g/kg/day (I 2-16 drinks/day) with average blood ethanol concentrations of 160 mg% 8 hours into the daily drinking episode. With this model we propose to characterize the limbic, hypothalamic, pituitary and adrenal response to stress in naive monkeys and then explore how this response predicts heavy ethanol self-administration.

DESCRIPTION (provided by applicant): The Administrative Core Component will make sure that all aspects of the INIA consortium work at an optimal level. The core will serve as an identifiable center for entire INIA consortium. This core will be responsible for decisions about research directions, including review of component progress and inclusion and exclusion of investigators. The core will also ensure the proper flow of information between the different consortium components, and flow of information to the larger research community in collaboration with the Bioinformatics Core. The duties of this core will also include organizing and financing all INIA committee meetings, retreats and research presentations. The administrative core will also track the INIA consortium finances and assist in budget planning for the different research components and cores. Stress contributes to excessive drinking and alcoholism, but the molecular and cellular mechanisms that underlie alcohol-stress interactions are not well understood. The brain circuitry involved in coordinating and producing responses to stress is known, and includes the extended amygdala (amygdala, bed nucleus of the stria terminalis and nucleus accumbens (NAc)], the hippocampus (HPC), the prefrontal cortex (PFC) and the hypothalamus. There is also considerable evidence implicating the neurotransmitters glutamate, GABA and serotonin (5-HT) in stress-induced neurophysiological responses in these brain regions. Alcohol effects on synaptic transmission in stress-related brain regions have not been characterized in much detail. Furthermore the role of the aforementioned neurotransmitters in these responses has not been examined. INIA component I will focus on examining synaptic transmission in amygdala, HPC, NAc and PFC in wild-type mice and mice with gene-targeted knockouts of the GABAAbeta3. Delta and gamma2 subunits, the NMDAR2A subunit, theGluRA subunit, the5-HTIA receptor and SERT. Similar experiments will be performed in selected randomly mutagenized mice identified in INIA component 3, and mice from selected BxD recombinant strains identified in INIA component 4. Brain slice preparations and isolated neurons will be examined from all of these groups of mice following chronic alcohol exposure and withdrawal. chronic alcohol self-administration and stress-induced reinstatement of alcohol drinking. Excitatory and inhibitory synaptic transmission will be measured using field potential and intracellular recording in brain slices containing the regions of interest. Synaptic plasticity will also be examined in brain slices. Acutely isolated neurons will be used to examine changes in neurotransmitter receptor function, and single-cell MRNA profiling will be used to identify neuronal subtypes. Serotonin release and reuptake will be examined in brain slices and synaptosomal preparations from the brain regions of interest. These studies will provide crucial information about neuroadaptive changes in synaptic transmission and plasticity induced by ETOH and stress in brain regions implicated in stress responses. We will also gather information about the role of particular neurotransmitter receptors and transporters in these neuroadaptive changes. Findings from this project " Ill be compared to data gathered in INIA components 2, 3 and 4 to generate more integrated information about the neuroadaptive changes related to alcohol-stress interactions. In future studies we will use mice developed by the Knockout Mouse Core and in component 7 in studies such as those proposed at present. Information gained in this component will be compared with that gained in the primate studies proposed in component 5, and will be shared with the larger research community on the INIA web page with the help of the Bioinformatics Core. It is predicted that information from this project will contribute to a better under- standing of the mechanisms underlying stress-alcohol interactions that may lead to better therapies for treating excessive drinking and alcoholism.

DESCRIPTION (provided by applicant): The fundamental basis for the public health concern surrounding alcohol abuse and alcoholism is the excessive self-administration of alcohol and the adverse consequences of this abusive drinking. However, our understanding of the biological basis of risk factors for and neurobiological consequences of excessive self-administration of ethanol remain limited. Advances in our understanding the primary factors that establish, maintain, and are consequences of excessive alcohol self-administration are greatly enhanced by the use of appropriate animal models. A primary strength of this Center for the Neurobehavioral Study of Alcohol (CNSA) is in developing and utilizing animal models of alcohol abuse and alcoholism to identify neurobiological factors as targets for prevention, harm reduction or treatment strategies related to the course of alcoholism. The Specific Aims are: AIM 1: To provide an integrated, multi-disciplinary approach to study the risk for and neurobiological consequences of excessive ethanol self-administration. AIM 2: To provide a national resource for the dissemination of research findings and training in the area of the neurobehavioral analysis of excessive alcohol consumption resulting in alcohol abuse and alcoholism. These aims will be addressed through the functions of an Administrative, Pilot and an Animal Core as well as six research projects. In the proposed studies of the CNSA we will study excessive ethanol self-administration in two animal models: (1) monkeys that drink ethanol excessively in the presence of a concurrent choice of water and (2) rats whose ethanol self-administration is induced and maintained under a schedule-induced polydipsia procedure. The projects are highly integrated and provide a multidisciplinary approach ranging from functional genomics (Project 1), to synaptic transmission (Projects 2 & 3), to functional and anatomical changes in brain circuitry (Projects 4 and 6), to assessing behavioral and physiological correlates (Project 5). We will disseminate this information through our educational efforts within the Administrative Core and in the larger alcohol research community that exists at Wake Forest University School of Medicine.

Human alcohol research and clinical practice demonstrate that, without question, individual variation in risk for excessive drinking, in sensitivity to alcohol effects, and in response to treatment strategies are critical. Our model of ethanol self-administration in cynomolgus monkeys provides a unique and important model of alcohol abuse. Because macaque monkeys are so similar to human beings in their complex physiology, endocrinology, neurobiology and genetic code they can play a pivotal role in testing hypotheses generated from behavioral models that employ other species, particularly rodents. Further, the non-human primate can then be a key step in translating candidate mechanisms of ethanors effects into the human realm through the use of imaging techniques, functional genomics and behavioral outcomes. Finally, macaques can be effectively used to target mechanistic hypotheses generated by studies in humans. Thus, our primary focus of the next funding cycle for the Center for the Neurobehavioral Study of Alcohol (CNSA) is to concentrate efforts on characterizing antecedent and consequent factors related to excessive ethanol self-administration in this monkey model. To complement the non-human model of self-administration, we will also study a second animal model. Rats will be maintained under a scheduled-induced polydipsia procedure that produces excessive alcohol self-administration as evidenced by physical dependence upon withdrawal. We will use this procedure to investigate additional and complimentary mechanisms of neurobiological adaptations in response to chronic ethanol self-administration. Taken together, the CNSA will focus efforts on understanding antecedent behavioral and physiological variables as well as consequent molecular, cellular, neuroanatomical, physiological and behavioral effects of excessive self-administration of ethanol in understanding two established models of alcohol self-administration. Specific Aim 1: To provide CNSA Investigators with adult cynomolgus monkeys (Macaca fascicularis) that have undergone the following standardized ethanol self-administration procedure. Specific Aim 2: To provide CNSA Investigators with adult rats (Long Evans) that have been subjected to a standardized schedule-induced polydipsia procedure resulting in physical dependence upon ethanol.

[unreadable] DESCRIPTION (provided by applicant): Required Functions from Notice of Limited Competition AA-06-101: The Administrative Coordinating Core will continue to provide the organizational framework for the management, direction, and overall coordination of the consortium. It will coordinate use of core resources to the Research Project Components and will acquire and direct data from the Research Project Components to the Informatics core. The consortium coordinator will manage the administrative core. The Administrative Coordinating Core will include an Administrative Management plan and this core will also be responsible for the collaborative responsiblities such as the functions of the Scientific Advisory panel and the overseeing of a Steering Committee to help develop interactive protocols, evaluate results, and suggest future directions. The core will also administer the Pilot Projects Component of INIA as well as the sharing of data and information between the INIA components. This core is responsible for scientific enrichment activities such as workshops for the researchers and investigators of INIA. In addition, the core will be responsible for organizing an annual meeting of the INIA investigators. The specific aims of the Administrative Core are to: 1. Provide scientific leadership on the INIA consortium's theme of stress, anxiety and alcoholism. 2. Provide the administrative center for the INIA Consortium in order to provide a framework for the overall management, direction and coordination of the consortium. This includes insuring scientific oversight by the Scientific Advisory Panel and the INIA Steering Committee, as well as coordinating the interactions of the different research and core components of the INIA. The project management plan will optimize synergism and sharing of resources and services. The Administrative Management plan will also be responsible for scientific enrichment activities such as organizing an annual retreat, workshops, and conferences. 3. Oversee the Pilot Projects Component of INIA. 4. Promote the inclusion of neuroscientists in the area of stress and anxiety who are new to the alcohol field as well as established alcohol investigators that are interested in studying the interaction of stress and anxiety on mechanisms of alcohol. [unreadable] [unreadable] [unreadable]

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Stress is believed to be an etiological factor in the abuse of ethanol. However, the role of stress in the risk for excessive ethanol consumption is difficult to untangle from the stress derived from excessively drinking alcohol. A starting point is to operationally define stress as activation of the hypothalamic-pituitary-adrenal (HPA) axis through measurable changes in circulating levels of the hormones adrenocorticotropin (ACTH) from the pituitary and cortisol from the adrenals. Monkeys show clear individual differences in endocrine response of the HPA axis to stressful events and also clear individual differences in the amount of ethanol they choose to self-administer. To address the causal interaction of stress and excessive ethanol interaction, we propose to characterize individual differences in HPA response to stress prior to, during and following chronic ethanol self administration. Further, the very nature of endocrine response to stress brings into focus the concept of neurocircuitries underlying information flow, integration and functional output. Viewing the HPA response as an intermediate determinant of behavior guides a translational endeavor into the realm of intermediate phenotypes or "endophenotypes". To address the predictive validity of an HPA response as an endophenotype underlying the risk of excessive ethanol self-administration, we will screen a large population of monkeys for specific HPA responses. Individuals that are on the extreme ends of the population distribution of the potential endophenotype will be characterized in the ethanol self-administration procedure. Finally, we will screen gene polymorphisms to identify those associated with an HPA response endophenotype.Finally, we will screen gene polymorphisms to identify those associated with an HPA response endophenotype.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This project is aimed at determining the molecular mechanisms that mediate acute and long term actions of alcohol on the brain. Identifying the molecular targets of alcohol is fundamental to developing strategies for preventing the development of abusive use of alcohol, and for designing therapies for alcohol addiction. We will use patch-clamp recording from granule cells in acutely prepared slices of cerebellum to identify molecular and cellular responses to acute alcohol exposure. Specifically, we are testing the role of four molecular mechanisms, which are known to interact with the GABAergic system in the cerebellum, in mediating the alcohol-induced increase in cerebellar granule cell GABAergic transmission. We will also determine if chronic, voluntary alcohol consumption leads to long-term molecular adaptations in cerebellar GABAergic transmission. Molecular adaptation to chronic alcohol consumption may contribute to withdrawal symptoms, and hence contribute to addiction. Finally, we will determine if individual variability in cerebellar granule cell sensitivity to alcohol underlies individual variability in sensitivity to alcohol-induced motor impairment. Sensitivity to alcohol-induced motor impairment is known to be a heritable predictor of alcohol abuse in humans, and thus determining its molecular underpinnings should help screen humans for predilection for alcohol abuse. The successful completion of the proposed experiments will improve our understanding of the molecular targets of alcohol, and will identify molecular aspects of cerebellar contribution to alcohol abuse and addiction.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Perhaps the most significant barrier in the treatment of alcohol abuse and alcoholism is the paucity of tools available for diagnosing and monitoring excessive alcohol consumption. For many reasons, (e.g., social stigma associated with alcohol abuse, and the potential legal/social ramifications of frank alcoholism), self-reported use, though informative, is known to be inconsistent. Indeed, there is a motivation for patients to deny drinking. This has led to the search for biochemical markers of alcohol abuse. Several biochemical markers of alcoholism have been proposed, but there are limitations to the accuracy and sensitivity of these assays. These biomarkers include: mean corpuscular volume, carbohydrate-deficient transferrin, gamma-glutamyl transferase, aspartate aminotransferase, and ethylglucuronide. The present study proposes to combine the power of a non-human primate model of alcohol self-administration with new, high-throughput proteomic techniques to identify additional diagnostic plasma protein biomarker signatures of excessive alcohol consumption. Blood samples from monkeys prior to and during ethanol self-adminstration have been sent and are currently being analyzed. These results will be combined with the recently completed sampling taken after one year of ethanol self-adminstration to determine the accuracy of this blood biomarkers thoughtout alcohol consumption.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. This project aims at understanding individual variation in risk for excessive drinking, with a focus on impulsive behaviors. A history of heavy ethanol intake appears to be related to increases in measures of impulsivity in humans. However human subject studies have been unable to distinguish antecedent baseline measures of impulsivity from the consequential effects of a history of heavy ethanol consumption. Our model of ethanol self-administration in cynomolgus monkeys provides a unique and important model of alcohol abuse, and reflects the individual differences in propensity to drink alcohol noted in the human population. Because of genetic similarities between humans and non-human primates, these studies can then be a key step in translating candidate mechanisms of ethanol's effects into the human condition through functional genomics. Thus, our primary focus of this PARC project is to use the monkey model to characterize antecedent and consequent measures of two aspects of impulsivity (the ability to rapidly stop, or withhold, responding and the aversion to a delay in reinforcement) with genetic factors related to excessive ethanol self-administration. Our Specific Aims are: (1) To determine if measures of impulsivity prior to alcohol exposure will predict chronic alcohol self-administration over a 12 month period;(2) To determine if chronic ethanol self-administration increases measures of impulsivity;and (3) To determine the expression of key gene networks in a brain area related to impulsive behaviors (the orbital medial prefrontal cortex) both prior to and following chronic alcohol drinking.

DESCRIPTION (provided by applicant): Excessive alcohol ingestion, occasionally or chronically, is co-morbid with medical disorders affecting the brain and behavior as well as other organ damage. Much of what is known about risk for and the consequence of heavy alcohol consumption, including mechanisms of organ damage, is derived from rodent studies or retrospective human accounts. This application proposes establishing a unique resource for alcohol research, a Monkey Alcohol Tissue Research Resource (MATRR). From this resource both tissue and associated bioinformatics tools will be made readily available to the wider alcohol research community. The tissue is derived from a standard protocol of ethanol self-administration in 3 species of monkeys. This resource will provide novel data for hypothesis testing relating the risk for and consequences of alcohol consumption and serve to bi-directionally bridge the gap between rodent and human studies. The basis of the MATRR is that non human primates, specifically monkeys, show a range of drinking excessive amounts of alcohol (>3.0 g/kg or a 12 drink equivalent/day) over long periods of time (12-30 months) with concomitant pathological changes in endocrine, hepatic and central nervous system (CNS) processes. These longitudinal designs span stages of drinking from ethanol-nai've to early alcohol exposure to chronic abuse. The CNS and peripheral organs from these animals comprise a unique translational resource for mechanistic and genetic studies of ethanol-induced pathologies. The state-of-the-art necropsy protocol will provide fresh, fixed and frozen tissue that are appropriate for ex vivo electrophysiology and neurochemical recordings, histological studies and genomic, proteomic and metabolomic approaches, respectively. The demand for, and the quality of, the tissues are already high as reflected in the number of requests and publications. Nevertheless, this resource needs further development in order to fulfill its potential as a cutting edge translational tool in alcoholism research. Thus, the primary goal of this proposal is to build the resources of this tissue bank and distribute these tissues and/or associated bioinformatics, to the broader alcohol research community. RELEVANCE (See instructions): The research outlined in this proposal will establish a unique post-mortem tissue bank for alcohol research. Using sophisticated techniques tissues from populations of monkeys that have been chronically drinking alcohol and the related drinking and genetic information will be disseminated to the wider alcohol research community to better understand disease processes associated with alcoholism.

The goal of this project is to develop methods that will permit researchers to remotely and automatically monitor behavior of primates and other highly social animals. The PIs will collect behavioral data from cameras and microphones. They will then develop statistical models and computational algorithms to track the individuals in the group and to recognize facial expressions and vocalizations. Patterns in movements, expressions, and vocalizations will be used to develop behavior-identifying algorithms that will recognize different behaviors such as aggression, submission, grooming, eating and sleeping. The project is a collaboration between computer scientists and primatologists. A key element of this project is the observation that complex social interactions can often be regarded as being composed of sequences of elementary behaviors which occur frequently and consist of relatively simple and distinct gestures. Thus, the task of modeling complex social interactions can be broken down into two regimes - elementary behaviors spanning short duration, and their stochastic sequences spanning relatively longer time duration.

Apart from advancing computational science, the new methods for recording behavior unobtrusively and analyzing them at a high data rate are likely to be of interest to behavioral ecologists, socio-biologists and neuroscientists in studies of primates and other highly social animals. With these new tools, scientists can study and understand behavior, for example, in the context of planning conservation efforts for threatened species, building accurate animal models for health research, and supporting animal husbandry decisions in zoos. The project will provide an extensive, annotated data repository and associated algorithms and will also fund graduate students who will gain hands-on training in all aspects of the project.

Human alcohol research and clinical practice demonstrate that, without question, there is a wide range of individual variation in risk for excessive drinking, in sensitivity to alcohol effects, and in response to treatment strategies. Impulsive behaviors are recognized as one risk factor, particularly if the construct of impulsive behaviors encompasses measures of both response inhibition and temporal discounting. A history of heavy ethanol intake appears to be related to increases in these measures of impulsivity. However human studies have been unable to distinguish antecedent baseline measures of impulsivity from the consequential effects of a history of heavy ethanol consumption. Our model of ethanol self-administration in cynomolgus monkeys provides a unique and important model of alcohol abuse, and reflects the individual differences in propensity to drink alcohol noted in the human population. Because of genetic similarities between humans and nonhuman primates, these studies can then be a key step in translating candidate mechanisms of ethanol's effects into the human condition through functional genomics. Thus, our primary focus of this PARC project is to use the monkey model to characterize antecedent and consequent measures of two aspects of impulsivity (response inhibition and aversion to a delay in reinforcement) with genetic factors related to excessive ethanol self-administration.

DESCRIPTION (provided by applicant): This application is in response to the RFA-AA-11-006 to provide support for the continuation of our consortium Integrative Neuroscience Initiative on Alcoholism: Stress, Anxiety and Excessive Drinking (a.k.a. INIAstress). Since its inception in 2002, INIAstress has had the primary goal of coordinating and facilitating translational, multidisciplinary and integrative research aimed at elucidating genetic and environmental influences on brain mechanisms that mediate excessive alcohol (ethanol) consumption, the response to stress, and the reciprocal relationship between excessive drinking, the physiological state of stress, and the subjective state of anxiety. Through this characterization we have helped to define factors that contribute to an individual's risk for the development of alcoholism, revealed underlying mechanisms and conditions that promote excessive and harmful drinking, and forged progress towards discovering novel, more effective, and tailored treatment strategies. In this renewal, we continue our cross-species approach and have further refined our INIAstress projects and cores to inform us about unique adaptations in brain circuitry following chronic intermittent ethanol exposure (ethanol-allostasis) that impact subsequent interaction of stress and ethanol to promote further excessive drinking. Collectively, these collaborative studies directly integrate behavioral, endocrine, neural and genetic data from animal models to humans within a scope of expertise and thematic inquiry that would not be possible using more traditional grant mechanisms (ROs or P50).

DESCRIPTION (provided by applicant): The self-administration of ethanol assessed under open-access conditions allows the characterization of how chronic ethanol intoxication pushes the organism beyond the normal limits of homeostasis (a constant internal environment) and into chronic, variable, stress responses (allostasis) and disease states. A critical barrier to understanding the apparent reciprocal relationship between stress and excessive ethanol drinking is the availability of within-subject comprehensive, longitudinal data sets on the neurogenetic, neurochemical, neurophysiological, neuroendocrine, and behavioral adaptations to chronic ethanol. The macaque monkey that self-administers high doses of ethanol for >20 months can provide these data sets; optimally within a highly collaborative and integrative environment such as the Integrative Neuroscience Initiative on Alcoholism (INIA). Therefore, in this renewal, we will continue to provide a nonhuman primate link in the INIA consortium to address fundamental aspects of what is known (or suspected) in humans with respect to stress, anxiety and excessive alcohol drinking. We propose to extend our study on longitudinal adaptations to excessive ethanol self-administration from cynomolgus monkeys to the rhesus monkey pedigree population at the Oregon National Primate Research Center (ONPRC). The studies will address fundamental questions of stress and risk for excessive drinking including consumption patterns following stressful provocations and repeated periods of prolonged abstinence from alcohol. Translational variables will include changes in the patterns of drinking alcohol as monkeys transition from moderate to heavy drinking, specific prefrontal cortical epigenetic adaptations, circulating endocrine and peptide markers, and functional connectivity MRI (fcMRl) studies. In addition, the research designs allow for unprecedented opportunities to investigate underlying synaptic changes in key regions of an integrative neural circuitry associated with the consequences of chronic alcohol self-intoxication.

DESCRIPTION (provided by applicant): The primary goal of the conference is to customize an informatic platform to produce new knowledge in the long-term adaptation of multiple organ systems to chronic, excessive, voluntary alcohol self-administration in order to understand the dynamic contributions of these adaptations to adverse biomedical outcomes. We will accomplish this goal by bringing together scientists that have collaborated and used the resources within the Monkey Alcohol Tissue Research Resource (www.MATRR.com) and the Integrative Neuroscience Initiative on Alcoholism (INIA): Stress, anxiety and excessive alcohol (www.INIAstress.org). The data sets that have been generated on the tissue to date are cross-disciplinary and cross-institutional, but all have the common factor of the same alcohol self-administration procedure, same antecedent and subsequent longitudinal genomic, endocrine, imaging and behavioral protocols. Indeed, the monkey model of alcohol self-administration has produced novel data for hypothesis testing relating the risk for and consequences of alcohol consumption and serve to bi-directionally bridge the gap between rodent and human studies. The symposium will focus on datasets generated from two species of monkeys (cynomolgus and rhesus macaques), particularly the rapid expansion of the data from different disciplines in the past 3 years. An outcome of the meeting will be to further understand from the users how to develop an informatics system that can accept summarized experimental outcomes across many disciplines and experimental protocols in order to integrate the analysis of genomic, genetic and phenotypic information and produce a comprehensive picture of dynamic interactions of risk factors, alcohol exposure, and adverse biomedical outcome. The gathering of geneticists, molecular biologists, neurophysiologists, endocrinologists, immunologists, osteopathologists, in vivo imagers, and behaviorists for a two day meeting will efficiently accomplish this goal.

Project Summary / Abstract Alcoholism and other addictive disorders have been categorized as brain diseases, implying that the brain is dysfunctional in addicted individuals. However, the extent to which the brains of people with alcohol use disorders (AUDs) were dysfunctional prior to being introduced to alcohol or became dysfunctional as a result of acquiring an AUD is not clear. Non human primates (NHPs) are particularly useful in linking brain circuitry to risk factors for and consequences of AUD because 1) in vivo imaging techniques used in humans can be applied to NHPs and are informative of circuitry involving different cortical fields (Kroenke et al., 2014; Miranda- Dominguez et al., 2014 a, b) (2) macaque monkeys show stable individual differences in the amount of alcohol they will self-administer over extended access (Grant et al, 2008a; Baker et al. 2014) (3) their genetic composition is highly similar to humans (Ferguson et al. 2012). Translational risk factors between NHP and humans that lead to excessive alcohol drinking have been identified, including drinking topography (Grant et al., 2008b), endocrine factors (Helms, Park and Grant, 2014) and age at the onset of drinking (Helms et al. 2014), but these risk factors have not been linked to brain circuitry. We propose to structural and functional connectivity between subcortical and cortical fields as potential biomarkers for risk and consequence of chronic binge drinking in both male and female cynomolgus monkeys. To our knowledge, the proposed studies will be the first to assess both structural diffusion tensor imaging (DTI) as well as functional brain aspects of resting state connectivity MRI in monkeys; the first to examine individual differences in resting state connectivity with the risk for heavy drinking; the first to quantify chronic ethanol intake on measures of resting state connectivity; the first to examine potential sex differences in these correlates of risk for and consequences of chronic ethanol drinking and the first to accumulate an informative dataset of within-subject genomic changes in the pre-frontal cortex of primates as a result of chronic ethanol self-administration. Collectively for the PARC, the studies will provide a translational bridge to the human subject project (P001: Nagel) and mouse project (P003: Hitzemann) while providing tissue to explore epigenetic changes across the brain and link them to changes in brain circuitry (P004: Carbone). Finally, because the brain imaging techniques proposed here are translational, the studies have the potential to inform prevention as well treatment for alcoholism and AUDs.

PROJECT SUMMARY The consortium Integrative Neuroscience Initiative on Alcoholism: Stress and Chronic Alcohol Interactions (a.k.a. INIAstress) is a collection of highly integrative and innovative projects and cores. The overarching goal of INIAstress is elucidating the changes in brain circuitry and mechanisms that underlie how chronic alcohol alters response to stress, which in turn can facilitate the transition from moderate/social drinking to heavy/hazardous drinking and associated problems. Through this characterization we have helped to define factors that contribute to an individual's risk for the development of alcoholism, revealed underlying mechanisms and conditions that promote excessive and harmful drinking, and forged progress towards discovering novel, more effective, and tailored treatment strategies. We continue our cross-species translational investigation and we will integrate this information to provide novel and valuable insights into diagnostic, prevention, and individualized treatment options for those suffering with alcohol use disorder (AUD) as well as co-morbidity with stress-related disorders such as post-traumatic stress disorders. Collectively, these collaborative studies directly integrate behavioral, endocrine, neural, and genetic data from animal models to humans within a scope of expertise and thematic inquiry that would not be possible using more traditional grant mechanisms. As the lead application, the Administrative Resource Core will provide scientific leadership on the consortium's main goals and serve as a centralized administrative structure to facilitate and coordinate the overall management, direction and integration of research activities within the INIAstress Consortium.

PROJECT SUMMARY Our longitudinal design of alcohol self-administration in the non-human primate has yielded evidence of stress axis risk factors associated in the development of heavy alcohol drinking as well as an impairment in brain regions involved in the control of voluntary actions. In this proposal, we will examine the ability of manipulating stress circuitry by inhibiting glucocorticoid receptors to decrease heavy ethanol drinking and relapse. We will also test the hypothesis that heavy alcohol drinking leads to impairments of the neural control of voluntary actions that involves a relative shift in activation of cortico-basal ganglia circuitry between the associative and sensorimotor subcircuits in response to context, contingencies and the predicted outcome of the action. The associative circuitry, involving prefrontal cortical projections to the caudate nucleus, is implicated in flexible adjustments to behavior. The sensorimotor circuitry, on the other hand, involves sensorimotor and motor cortical projections to the putamen and controls habitual behaviors. We will use baseline resting-state functional connectivity with MRI to investigate if individual differences in cortico-basal ganglia connectivity are associated with performance on a self-paced set shifting task as well as heavy alcohol consumption. Designer receptors exclusively activated by designer drug (DREADDs) will be implemented to alter cortico-basal ganglia circuits and examine effects on cognitive flexibility and heavy ethanol drinking. Resting-state fMRI will then be used to verify the changes in connectivity strength by the activation of DREADDs, so that this less invasive method can be positioned to identify abnormal functioning of cortico-basal ganglia subcircuits. This highly innovative research in macaque monkeys will advance the application of neurotechnologies to understand and modulate addiction, a maladaptive behavior.

PROJECT SUMMARY SARS-CoV-2, the causative agent of COVID-19, is responsible for a continuing pandemic with over 2 million infected individuals and 115,000 deaths in the US as of June 11, 2020 with cases still rising. Similar to the respiratory diseases caused by the SARS and MERS coronaviruses, COVID-19 is characterized by fatigue, cough, fever, sputum production, dyspnea, and acute respiratory distress syndrome (ARDS). ARDS is the result of excessive lung inflammation causing the accumulation of fluid and inflammatory cell debris and leading to decreased gas exchange and oxygen levels, and eventual multi-organ failure. The leading hypothesis for the molecular basis of SARS-CoV-2 pathogenesis is that an aberrant induction of pro- inflammatory cytokines, chemokines and soluble mediators lead to a debilitating cytokine storm that in turn results in severe lung tissue damage (3-6). Current studies suggest a major role for myeloid cell subsets in the development of the cytokine storm, but the precise roles of pro-inflammatory alveolar monocytes and macrophages have not been thoroughly examined and animal or human subject studies. SARS-CoV-2 disruption to daily routines and social settings have led to increased sales and consumption of alcoholic beverages. As chronic heavy alcohol consumption compromises lung health and immunity leading to increased susceptibility to both bacterial and viral pulmonary infections, and is a risk factor for ARDS. Thus, chronic heavy alcohol drinking could increase the chances of COVID-19 infection and exacerbate the disease course. However, there are no longitudinal studies in controlled populations that provide both precise measures of lung function with exact measures of alcohol consumption in human subjects. In this proposal we will obtain a pulmonary functional test (PFT), a clinical diagnostic of lung infection (CT) and alveolar macrophages to the R24 AA01943 Monkey Alcohol Tissue Research Resource (MATRR). This added capacity will be a resource for investigators to understand 19 the potentially complex relationships between alcohol consumption and COVID- related-outcomes and to enhance the nation's response to the current pandemic.