Loren H. Parsons - US grants

DESCRIPTION: (Applicant's Abstract) This application for a NIDA FIRST Award outlines a series of studies designed to examine the involvement of serotonin-lB receptors in the reinforcing and addictive properties of cocaine, and to determine the mechanisms by which these receptors mediate and/or modulate the behavioral effects of this psychostimulant. Cocaine is known to significantly alter serotonin neurotransmission, and chronic cocaine use results in clinical pathologies which are associated with generalized serotonergic dysfunction. However, very little is known about the role of serotonergic neurotransmission in the behavioral effects of cocaine. Recent work indicates that activation of serotonin-lB receptors enhances cocaine reinforcement, though the mechanisms mediating this effect are not known. The first series of experiments will utilize behavioral techniques to determine the neuroanatomical sites which mediate the enhancement in cocaine reward by serotonin-lB receptors (Specific Aim I). It is hypothesized that serotonin-lB receptor activation induces a cascade of neurochemical events involving GABA neurotransmission in the ventral segmental area, basolateral amygdala and sublenticular ventral pallidum, as well as glutamate neurotransmission in the nucleus accumbens, and these events result in an increased activity of the neuronal systems through which cocaine reward is mediated. Thus, a second series of experiments will employ in vivo neurochemical techniques to determine mechanisms by which serotonin-lB receptor stimulation activates brain reward pathways (Specific Aim II). Finally, preliminary evidence indicates that cocaine exposure results in alterations in serotonin-lB receptor function which may contribute to sensitization to the locomotor activating and reinforcing effects of further cocaine intake. A third series of experiments will determine the involvement of serotonin-lB receptors in these long-term effects of cocaine exposure (Specific Aim III). In total, this work will characterize a new link in the circuitry which mediates the reinforcing effects of cocaine. Because the rat serotonin-lB receptor is the homologue of the human serotonin-lD receptor, and these receptors display nearly identical pharmacological profiles, the information gathered from these experiments is likely to have direct relevance to the neurobiological mechanisms of reinforcement in humans. Of equal importance, these studies may identify cocaine-induced alterations in specific brain systems which have significant clinical relevance, thus providing important information for the development of medications for psychostimulant addiction.

This is a request for a Capillary Electrophoresis-Mass Spectrometry (CE- MS) instrument under the Shared Instrumentation Grant mechanism of The National Center for Research Resources. The CE-MS system will be utilized primarily by a major user group comprised of four investigators at The Scripps Research Institute (TSRI) (Drs. Weiss, Henriksen, Gruol and Parsons) and one investigator at the Veterans Administration Medical Center on the campus of UCSD (Dr. Purdy). Each of the investigators in the major user group will use the CE-MS instrument to advance research that is currently funded by NIH awards. There are several characteristics of CE-MS that will accelerate and advance the research of the major user group. First, CE has the ability to separate a wide variety of analytes such as steroids, large and small peptides, oligonucleotides, RNA transcripts and DNA fragments. Second, CE offers unparalleled resolution capabilities and will provide the separation of important analytes with a selectivity that is currently unavailable in our research. The coupling of mass spectrometry (MS) with CE presents a third advantage for using this analytical technique. The combination of the mass/charge (m/z) information and sensitivity provided by MS with the separation characteristics of CE provides a tremendous advance in the unambiguous identification and quantification of biological molecules. Moreover, MS scan spectra can be used for the structural elucidation of novel substances. Finally, the very small sample requirements for CE- MS analyses will enable a substantial reduction in the disturbance made to the physiological systems being monitored, and will increase sample analyte concentrations and improve the time resolution of our measures. These features of CE-MS will advance the research endeavors of the local scientific community in ways that are currently impossible. The CE-MS will be housed in 'core facility' laboratory space at TSRI, and an Internal Advisory Committee will assure the use and equitable sharing of the CE-MS among investigators both within and outside the major user group. This committee is comprised of the Principle Investigator (Dr. Parsons), the Director of the Mass Spectrometry Facility at TSRI (Dr. Siuzdak), a senior researcher with significant CE experience (Dr. Rivier) and the Director of both the Division of Psychopharmacology and the Alcohol Research Center at TSRI (Dr. Koob), organizations with which each of the major users are affiliated. The Senior Vice President of TSRI supports this application, and had agreed that TSRI will assume financial responsibility for the long-term operation and maintenance of the CE- MS.

The projects in this proposal are designed to elucidate the role of serotonin-1B (5-HT1B) receptors in the mediation of ethanol reinforcement. Ethanol is known to interact with the serotonin (5-HT), dopamine, GABA and glutamate neurotransmitter systems, and each of these are believed to be involved in the production of the reinforcing effects of this widely abused drug. It is hypothesized that 5-HT1B receptors play a pivotal role in regulating the interactions among these four neurotransmitters in brain reward circuitries which are believed to mediate ethanol reward. The proposed experiments will employ behavioral and neurochemical strategies in rats to study the role of 5HT1B receptors in the regulation of ethanol self-administration, and to identify the specific neural mechanisms through which 5HT1B receptors exert their effect on ethanol consumption. In Specific Aim I, the effects of 5HT1B receptor manipulations on oral ethanol self-administration under both fixed ratio and progressive ratio schedules of reinforcement will be examined. In these experiments, 5HT1B receptor-selective drugs will be administered both peripherally to assess the effects of global 5HT1B receptor manipulations on ethanol reinforcement, and locally into discrete brain regions to identify relevant substrates and circuitries through which 5-HT1B receptors regulate ethanol reward. Brain regions to be investigated include the ventral tegmental area nucleus accumbens, basolateral amygdala and prefrontal cortex. Specific Aim II will employ in vivo microdialysis to investigate the neurotransmitter mechanisms involved in the modulation of ethanol reinforcement by 5-HT1B receptors. As in the first Specific Aim, 5-1HT1B receptor-selective drugs will be administered both peripherally to assess the effects of global 5-HT1B receptor manipulations on the neurochemical response to ethanol, and locally into the brain regions listed above to investigate the specific circuitries through which 5-HT1B receptors regulate ethanol reinforcement. The results of these experiments will provide new insight into this novel receptor system as a mechanism for modulating the motivational effects of alcohol. Because rat 5-HT1B receptors are homologous to human 5-HT1Dbeta receptors, the information gained in these experiments will have direct relevance for the neurobiological mechanisms of ethanol reinforcement in humans, and may have implications for the development of pharmacotherapeutic treatments of alcoholism.

A considerable amount of evidence implicates the cannabinoid system in the mediation and/or modulation of the behavioral effects of ethanol. An opioid peptide link has been suggested in this process though the relative involvement of specific opioid receptors in the CB1 receptor-induced modulation of ethanol drinking is not known. Based on recent data from our work and from others it is hypothesized that CB1 receptors exert a positive control over ethanol self-administration, and that this influence is mediated in part by a CB1 receptor-induced regulation of opioid peptide release. The projects in this proposal are designed to further characterize the effects of cannabinoid manipulations on ethanol consumption, and to evaluate the relative involvement of mu, delta and kappa opioid receptors (and their associated endogenous peptide ligands) in the modulation of ethanol intake by CB1 receptors. Specific Aim 1 will evaluate the ability of selective opioid receptor antagonists to reverse CB1 agonist-induced increases in ethanol self-administration. Specific Aim 2 will characterize the effects of altered brain endocannabinoid neurotransmission on ethanol self-administration, and will investigate the influence of each class of opioid receptor in these effects. Inhibition of endocannabinoid reuptake and hydrolysis, respectively, will be used in these experiments to increase interstitial endocannabinoid levels, which has been found in our laboratory to increase ethanol self-administration. Specific Aim 3 will investigate alterations in ethanol self-administration produced by cannabinoid and opioid manipulations in three brain regions: the nucleus accumbens shell, the ventral tegmental area and the central nucleus of the amygdala. In vivo microdialysis will be used in these experiments to provide direct evidence for a CB1 receptor modulation of opioid peptide release. Finally, Specific Aim 4 will examine the effects produced by CB1 receptor manipulations in an animal model of ethanol relapse in an effort to evaluate the potential therapeutic utility of CB1 antagonists for the treatment of alcoholism.

[unreadable] DESCRIPTION (provided by applicant): Recent reports have demonstrated that the selective cannabinoid CB1 receptor antagonist SR141716A attenuates the ability of drug-associated conditioned cues and drug priming injections to reinstate extinguished drug-seeking behavior. Remarkably this effect of SR141716A has been observed across a wide range of drug classes including psychostimulants, opiates, nicotine and ethanol. This broad spectrum of influence suggests that the endocannabinoid system has great potential as a therapeutic target for the treatment of relapse. However, the neural substrates and neuropharmacological mechanisms through which endocannabinoids modulate relapse behavior have not been characterized, and direct evidence of altered endocannabinoid signaling during the reinstatement of extinguished drug-seeking behavior has not been shown. Thus, the goals of this project are to evaluate the influence of endocannabinoids on conditioned cue-induced heroin-seeking behavior in an operant response-reinstatement model of relapse. The neural substrates through which CB1 receptors modulate conditioned cue-induced heroin-seeking behavior will be investigated using site-directed microinfusions of a selective CB1 receptor antagonist, a selective inhibitor of endocannabinoid reuptake, and the endocannabinoid substances (R)-methanandamide and 2-arachidonoylglycerol. The brain regions to be investigated are the basolateral amygdala, the medial prefrontal cortex, and the nucleus accumbens core. In vivo microdialysis sampling will be employed to characterize alterations in interstitial endocannabinoid levels in these brain regions during conditioned cue-induced reinstatement of extinguished drug-seeking behavior, and additional neurochemcial studies will begin to characterize the neuropharmacological mechanisms through which endocannabinoids modulate drug-seeking behavior. [unreadable] [unreadable]

Cannabis is the most commonly used illicit drug in the United States. Chronic heavy cannabis use can result in impaired cognitive processing as characterized by deficits in attention, memory, decision-making and inhibitory control. Neuroimaging studies in humans have demonstrated that long-term heavy cannabis use produces alterations in the function of the prefrontal (orbitofrontal) cortex, hippocampus and components of the basal ganglia, and it has been proposed that neural dysfunction in these regions contributes to a loss of inhibitory control that propels continued cannabis use. However, the cellular and neurochemical mechanisms that underlie cannabis-induced cognitive dysfunction are not known. The overall goals of this project are to employ rodent behavioral tasks to evaluate the effect of chronic treatment with A9-tetrahydrocannabinol (A9- THC) on cognitive function in adolescent and adult rats and to characterize A9-THC-induced alterations in neurochemical signaling that underlie these behavioral abnormalities. A9-THC doses that produce plasma A9-THC concentrations which bracket those achieved by human cannabis smokers will be administered to periadolescent and adult rats in a repeating cycle of 14-day blocks in which A9-THC is administered 2x per day for 6 days, followed by an 8-day drug-free period in during which behavioral testing is performed. This cycle of A9-THC dosing and behavioral testing will be repeated for up to 6 months, and behavioral testing will continue for up to two months after cessation of A9-THC dosing. In the context of the rat lifespan this will provide a reasonable approximation of long-term cannabis use in humans. The experiments in Specific Aim 1 will evaluate the onset, progression and persistence of cognitive dysfunction during and after this A9-THC dosing regimen. Evaluations of attentional capacity (5-CSRTT), impulsive choice/decision making (delaydiscount task), impulsive action/inhibitory control (DRL testing) and executive function (set-shifting) will be made. The experiments in Specific Aim 2 will employ in vivo brain microdialysis to characterize altered neurochemical function in the frontal cortex and related areas that are associated with A9-THC-induced performance deficits in these behavioral tasks. Task-related alterations in interstitial levels of monoamines, excitatory and inhibitory amino acids, acetylcholine and endogenous cannabinoids will be compared between treatment groups. These rodent experiments will complement the neuropsychological testing and brain imaging to be performed in the periadolescent non-human primate, adolescent human and adult human projects of this Center. The establishment of these four, projects will provide a very strong scientific platform for investigations into the neuropsychological and neurobiological bases of cannabis abuse and dependence.

2-arachidonoyl-glycerol; 2-arachidonoylglycerol; 2-arachidonyl-glycerol; 2-arachidonylglycerol; 4-Aminobutanoic Acid; 4-Aminobutyric Acid; 5,8,11,14-Eicosatetraenamide, N-(2-hydroxyethyl)-, (all-Z)-; 5,8,11,14-Eicosatetraenoic acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester, (5Z,8Z,11Z,14Z)-; 5,8,11,14-eicosatetraenamide, N-(2-hydroxyethyl)-; 5,8,11,14-eicosatetraenoylethanolamide; Absolute ethanol; Acute; Affective; Alcohol Drinking; Alcohol consumption; Alcohol dependence; Alcohol withdrawal syndrome; Alcohol, Ethyl; Alcoholic; Aminalon; Aminalone; Amygdala; Amygdaloid Body; Amygdaloid Nucleus; Amygdaloid structure; Animals; Anti-Anxiety Agents; Anti-Anxiety Drugs; Anxiety; Anxiolytic Agents; Anxiolytics; Attenuated; Back; Behavior; Boozer; Brain; Brain region; Butanoic acid, 4-amino-; Cannabinoids; Cannabinoids, Endogenous; Cell Communication and Signaling; Cell Nucleus; Cell Signaling; Cellular Neurobiology; Chronic; Common Rat Strains; Corticosterone; Data; Dependence; Dependent drinker; Depression; Disruption; Dorsum; Drugs; ETOH; Emotional; Encephalon; Encephalons; Endocannabinoids; EtOH drinking; Ethanol; Ethanol dependence; Event; Funding; GABA; Genetic; Glutamates; Grain Alcohol; Heavy Drinking; Human; Human, General; Individual; Intracellular Communication and Signaling; L-Glutamate; Lateral; Light; Literature; Maintenance; Maintenances; Mammals, Mice; Mammals, Rats; Man (Taxonomy); Man, Modern; Mediating; Mediator; Mediator of Activation; Mediator of activation protein; Medication; Melia; Mental Depression; Methylcarbinol; Mice; Microdialysis; Monitor; Murine; Mus; N arachidonoyl 2 hydroxyethylamide; N-(2-hydroxyethyl)arachidonamide; Negative Reinforcements; Nerve Cells; Nerve Impulse Transmission; Nerve Transmission; Nerve Unit; Nervous System, Brain; Neural Cell; Neurobiology, Cellular; Neurochemistry; Neurocyte; Neuronal Transmission; Neurons; Nucleus; Nucleus Accumbens; Other Finding; Pharmaceutic Preparations; Pharmaceutical Preparations; Phenotype; Photoradiation; Physiologic; Physiological; Play; Prefrontal Cortex; Pregn-4-ene-3,20-dione, 11,21-dihydroxy-, (11beta)-; Rat; Rate; Rats, Wistar; Rattus; Receptor Protein; Regulation; Reinforcements, Negative; Relapse; Research; Role; Science of neurochemistry; Self Administration; Signal Transduction; Signal Transduction Systems; Signaling; Stress; Symptoms; System; System, LOINC Axis 4; Testing; Thinking; Thinking, function; Tranquilizing Agents, Minor; Transmission; Wistar Rats; Withdrawal; Work; alcohol addiction; alcohol dependency; alcohol exposed; alcohol exposure; alcohol ingestion; alcohol intake; alcohol product use; alcohol use; alcohol withdrawal; alcohol-dependent; alcoholic beverage consumption; alcoholic drink intake; amygdaloid nuclear complex; anandamide; anandamide (20.4,n-6); antianxiety agent; arachidonoyl ethanolamide; arachidonoylethanolamide; arachidonylethanolamide; base; behavior test; behavioral pharmacology; behavioral test; biological signal transduction; brain tissue; drink heavily; drinking; driving force; drug/agent; ethanol addiction; ethanol consumption; ethanol dependency; ethanol drinking; ethanol exposed; ethanol exposure; ethanol ingestion; ethanol intake; ethanol product use; ethanol use; ethanol withdrawal; ethanol-dependent; etoh use; excess alcohol consumption; excess alcohol ingestion; excess ethanol ingestion; excessive alcohol consumption; excessive alcohol ingestion; excessive alcohol intake; excessive behavior; excessive drinking; excessive ethanol ingestion; experiment; experimental research; experimental study; exposed to alcohol; exposure to alcohol; extracellular; extreme drinking; gamma-Aminobutyric Acid; heavy alcohol use; in vivo; insight; interstitial; neural mechanism; neurochemistry; neuromechanism; neuronal; neurotransmission; problem drinker; receptor; research study; response; restraint; restraint stress; social role; transmission process; withdrawal from alcohol

Agonist; Amino Acids; Animal Model; Animal Models and Related Studies; Area; Assay; Attention; Bioassay; Biochemical; Biologic Assays; Biological Assay; Blood Plasma; Brain; CRF receptor type 1; CRF-R1; CRF1 receptor; CRH-1; Cannabinoids, Endogenous; Chemicals; Common Rat Strains; Consultations; Crh1 receptor; Encephalon; Encephalons; Endocannabinoids; Ethanol dependence; Experimental Designs; Glutamates; Immunologic, Radioimmunoassay; Investigation; L-Glutamate; Ligands; Mammals, Primates; Mammals, Rats; Measurement; Methods; Methods and Techniques; Methods, Other; Microdialysis; Molecular; Nervous System, Brain; Neuropeptide Tyrosine; Pilot Projects; Plasma; Primates; RIA; Radioimmunoassay; Rat; Rattus; Reproducibility; Reticuloendothelial System, Serum, Plasma; Sampling; Serum, Plasma; Standardization; Standards; Standards of Weights and Measures; Steroid Compound; Steroids; Techniques; alcohol research; aminoacid; brain tissue; corticotropin-releasing factor receptor 1; design; designing; ethanol addiction; ethanol research; in vivo; interest; model organism; monoamine; neuropeptide Y; pilot study; repository; sample collection; specimen collection; tool

DESCRIPTION (provided by applicant): The brain is arguably the most complex organ in higher mammals in terms of diversity of resident cell types, anatomical organization, and intricate modes of intercellular communication and protein regulation. Each of these factors presents a tremendous technical challenge for researchers interested in applying large-scale biological approaches to the study of brain physiology and pathology. Proteomics, for instance, is most adept at mapping protein expression and post-translational events in biological samples enriched in a single cell type (e.g., cultured cells or relatively homogenous organs, such as liver). The brain, however, offers a much more complicated scenario. Indeed, brain disorders such as drug dependence and withdrawal may be due to changes in the molecular composition and function of only a small number of neurons or neuronal circuits dispersed among a large background of unaffected cells. Neuronal plasticity is furthermore known to rely heavily on post-translational events that regulate protein structure and activity, often within specific subcellular compartments (e.g., the synapse). Detecting and quantifying such 'rare'biochemical events is beyond the capacity of contemporary proteomics methods. As a result, the tremendous opportunity afforded by proteomics to discover new proteins and pathways that contribute to higher-order brain function has, to date, gone unrealized. This proposal addresses this Grand Opportunity (GO) through creation of the first proteomics platform for quantitative analysis of protein expression and post-translational modification in the mammalian brain. This platform, termed qPEMM (for quantitative Protein Expression and Modification in Mammals) will be applied to create a brain atlas of proteomic changes that occur in nicotine-dependent rodents. Tobacco usage remains one the most prevalent forms of substance abuse and exerts a tremendous cost on world health and economy. While it is known that nicotine is the major component in tobacco smoke responsible for addiction, we currently have only a limited understanding of the biochemical alterations caused by nicotine in the nervous system. This project will yield the first large-scale anatomical inventory of nicotine- induced changes in protein expression and post-translational modification in the mammalian brain. These changes will identify key protein pathways that are dysregulated by nicotine, which should in turn serve as fertile ground for future studies aimed at deciphering the neurochemical basis for nicotine dependence. In this way, these proteomic studies will serve as a powerful hypothesis-generating engine that can be leveraged many times over to spawn new basic and translational research programs aimed at understanding and eventually treating nicotine addiction. More generally, this project will deliver a fully integrated and portable quantitative proteomic platform that can be adopted by biological researchers around the world to accelerate the growth and pace of scientific discovery. PUBLIC HEALTH RELEVANCE: Tobacco usage remains one of the most prevalent forms of substance abuse and exerts a tremendous cost on world health and economy. Here, we propose to complete the first large-scale anatomical inventory of nicotine-induced changes in protein expression and post-translational modification in the mammalian brain. These studies should yield new hypotheses to explain the mechanistic basis for nicotine addiction and, through doing so, reveal novel diagnostic and therapeutic strategies to treat this debilitating disease.

DESCRIPTION (provided by applicant): The development of alcohol use disorders follows a transition from social use motivated by hedonic and anxiolytic effects to dependence motivated by increasing withdrawal symptoms and an evolving desire to drink during abstinence. In this latter stage of alcohol dependence, abstinence from drinking is often accompanied by negative emotional symptoms, such as increased anxiety and depression, and the alleviation of these negative emotional states is hypothesized to be a major driving force for continued alcohol consumption. This shift from positive to negative reinforcement mechanisms likely results from enduring changes in CNS function induced by excessive alcohol consumption. Although several signaling systems have been implicated in this process there is still an incomplete understanding of the neural mechanisms underlying alcohol dependence. We have gathered evidence that EtOH consumption increases levels of the endogenous cannabinoid (eCB) 2- arachidonoyl glycerol (2-AG) in rodent brain, while long-term intermittent EtOH exposure down-regulates eCB signaling in brain regions relevant to emotional processing. Dependence-associated anxiety-like behavior and excessive EtOH consumption are reduced by generalized enhancement of eCB tone, though similar manipulations do not produce these effects in non-dependent animals. Based on these findings, we hypothesize that eCB clearance inhibitors have therapeutic value for treating alcohol dependence and alcoholism. This hypothesis will be tested through three Specific Aims. Aim 1 will characterize the ability of highly selective eCB clearance inhibitors to alleviate anxiety-like behavior in EtOH dependent mice throughout a period of protracted withdrawal. Importantly, these experiments will characterize the relative influence of two primary eCB molecules, 2-AG and anandamide (AEA), by selectively inhibiting the distinct hydrolytic mechanisms that clear these lipids from the brain. The experiments in Aim 2 will employ biochemical and neurochemical approaches to characterize the mechanisms contributing to dependence-associated dysregulation of brain eCB signaling. Additional work in this Aim will evaluate the influence of eCB dysregulation on other neurotransmitter systems involved in withdrawal-associated anxiety-like behavior and excessive EtOH consumption (including glutamate, serotonin and norepinephrine). The experiments in Aim 3 will characterize the efficacy of selective eCB clearance inhibitors for reducing high levels of EtOH consumption associated with dependence and protracted withdrawal. These experiments will also characterize the influence of eCB signaling on binge-like ethanol intake in non-dependent mice. Completion of the proposed work is likely to highlight a previously unrecognized mechanism in the etiology of alcohol dependence and may identify novel therapeutic targets for alcoholism.

DESCRIPTION (provided by applicant): This is a request for a Liquid Chromatography - tandem Mass Spectrometry (LC- MS/MS) instrument that will be utilized by a major user group of five investigators at The Scripps Research Institute (TSRI) and also by a minor user group of investigators from TSRI and neighboring institutions. The requested LC-MS/MS will be used for the analysis of neuroactive peptides, endogenous cannabinoids and exogenous drugs and their metabolites in brain tissue, in vivo brain microdialysates and plasma. These analyses will directly advance research that is currently funded by NIH awards. Several characteristics of the LC-MS/MS will facilitate the research of the major and minor user groups. First, mass spectrometers provide a form of "universal detection" that offers much greater molecular specificity than is typically provided through electrochemical, spectrophotometric or immunoassay detection methods. Second, tandem mass spectrometers (MS/MS) provide substantially higher signal-to-noise ratios and considerably greater analyte specificity than do single quadrupole mass spectrometers, providing substantially lower limits of quantitation in complex biological matrices. Third, the requested capillary liquid chromatography (LC) system substantially enhances mass sensitivity and is very well suited for the separation of small volume (<10 ?l) samples containing extremely low analyte concentrations (pM - nM) while providing flexibility for analysis of larger sample volumes as needed. The LC-MS/MS will be housed in a shared facility at TSRI that is equipped for the immediate installation of the instrument without need of renovation. TSRI has made a financial commitment to the long-term maintenance and operation of the instrument inclusive of service contracts, supplies and support of the technical staff. A highly qualified technical staff will provide user training and instrument maintenance and an Internal Advisory Committee will assure equitable instrument use by investigators both within and outside the major user group. The LC- MS/MS instrument will have a major impact on the successful completion and renewal of 22 NIH-funded research projects within the major user group that include individual R01 research projects, two NIH-funded Research Centers and one NIH-funded Program Project. Importantly, the results gathered with the requested instrument are likely to provide novel insight into the neural mechanisms contributing to drug and alcohol abuse, eating disorders, chronic pain, and psychiatric disorders such as anxiety and depression. HEALTH RELEVANCE: The requested LC-MS/MS instrument will provide the ability to quantify very low levels of neuroactive substances in biological samples including brain microdialysates. This will directly impact the progress and success of more than 22 NIH-funded research projects. The results gathered with the requested instrument will provide novel insight into the neural mechanisms contributing to drug and alcohol abuse, eating disorders, chronic pain and psychiatric disorders such as anxiety and depression.

DESCRIPTION (provided by applicant): Impaired cognitive processing is a hallmark of addiction. Deficits in inhibitory control (impulsive action), poor evaluation of consequences (impulsive choice) and ineffective reversal of compulsive or habitual behaviors (cognitive flexibility) can propel continued drug use despite adverse consequences. A persistent question in alcohol research regards the relative influence of pre-existing cognitive disruptions that confe susceptibility to problem drinking versus the induction of cognitive impairment related to cortical damage induced by repeated alcohol intoxication and withdrawal. In this regard animal models can provide important insight into the etiology of alcohol-induced cognitive impairment and can provide a platform for mechanistic studies and rapid pharmacotherapy screening. Using behavioral paradigms analogous to clinically employed tasks we have gathered preliminary evidence of significant increases in impulsive action and impulsive choice behaviors that emerge in rats during protracted withdrawal from long-term intermittent alcohol consumption. These cognitive impairments persist for several weeks, and can be ameliorated by a pharmacological manipulation known to improve cognitive function in human alcoholics. Based on these findings and knowledge of the neural mechanisms governing these behaviors in rats we hypothesize that withdrawal-associated dysregulation of monoamine and amino acid signaling in frontal cortical regions contributes to increased impulsivity and deficient cognitive flexibility during protracted alcohol withdrawal. This hypothesis will be tested through three Specific Aims. Aim 1 will characterize the emergence, nature and persistence of cognitive disruption during protracted alcohol withdrawal. Different facets of impulsive action will be explored using a novel 5-Choice Continuous Performance Task (5C-CPT) and the Stop Signal Reaction Time task (SSRT). Impulsive choice behavior will be indexed using the Delay Discount Test, and cognitive flexibility will be assessed using an operant spatial reversal learning task. The experiments in Aim 2 will employ in vivo microdialysis and biochemical approaches to characterize monoamine, and amino acid function in the orbitofrontal and medial prefrontal cortices during protracted alcohol withdrawal. Aim 3 will evaluate the efficacy of pharmacologic agents for ameliorating withdrawal-associated increases in impulsive action and impulsive choice.

The overall objective of the Research Translation/Information Dissemination (Education) Component of The Scripps Research Institute Alcohol Research Center (TSRI ARC) is to advance the NIAAA¿s mission to translate and disseminate scientific research findings to researchers, healthcare professionals, policy makers, and the general public. The main goals of the Research Translation/Information Dissemination Component are to: 1) develop and evaluate an education program to the high risk Mexican American minority and EuroAmerican communities in San Diego greatly in need of tools to combat underage drinking and alcohol use disorders in their communities, and 2) expand the dissemination of knowledge about the scientific findings of the TSRI ARC and their translation to clinical alcohol diagnosis, treatment, and prevention to students, scientific and medical professionals and trainees, and the lay public. To accomplish these goals, we will perform research translation/Information Dissemination (Education and Outreach) to High Risk Mexican American Immigrant and Other Communities in San Diego (Specific Aim 1), perform Integration, Translation, Training, and Education in Alcohol Research to ARC scientists and trainees, undergraduates, psychology, pharmacy graduate students, and addiction professionals (Specific Aim 2), and effect the TSRI ARC High School/College/Graduate School Intern Program (Specific Aim 3). Finally, we will disseminate the scientific findings of the TSRI ARC, information on alcohol use disorders, and how recent neurobiological research is informing new approaches to clinical alcoholism to the general public (Specific Aim 4).

DESCRIPTION (provided by applicant): The Alcohol Research Center of The Scripps Research Institute (TSRI-ARC) proposes to continue its interdisciplinary program focused on the theme of the central nervous system effects of alcohol. For this renewal application, the TSRI-ARC will be a P60 consisting of 9 components plus an Educational Component. Four core components are proposed: Administrative, Animal Models/Biochemical Measures, Biochemical and Pilot. Five research components are proposed: Cellular Electrophysiology, Cellular Physiology, Neuroendocrinology, Neurochemistry, and Clinical Neurobehavioral. The overall hypothesis of the TSRI-ARC is that with chronic binge drinking, the brain reward systems become tolerant to alcohol while central stress systems become activated and that as a result, the neuroadaptive changes associated with chronic drinking produce an evolving set of neurobehavioral symptoms that include hypohedonia, anxiety, hyperarousal, sleep disturbances, and negative affect, conceptualized as the dark side of addiction. The subhypotheses for the present proposal are: (1) The transition from low levels of drinking (1-2 drinks within 2 hours in humans) to chronic binge drinking (4-5 drinks within 2 hours in humans) is driven by decreased activity of opioid peptide systems and endocannabinoid systems in reward circuits in the frontal cortex, and nucleus accumbens (Specific Aim 1). (2) The transition from binge drinking to dependence is driven by compromised function in the reward systems and recruitment of a dysregulated central stress system, most notably driven by changes in CRF, glutamate, and endocannabinoids in the extended amygdala (Specific Aim 2). (3) A particularly vulnerable local human clinical population has a phenotype of alcoholism that displays this transition from bingeing to dependence in young adulthood, allowing us to translate our findings in animals to humans and humans to animals (Specific Aim 3). We believe the proposed innovative approaches for testing these hypotheses will provide valuable insight into novel approaches for treating and preventing alcoholism in the human population. The TSRI-ARC also supports the Center at Large, which includes: 13 ROIs, 6 UOIs, one T32 NIAAA training grant, two R37s, one R13, one RC1 award. Members of the Center at Large have access to the Cores of the TSRI-ARC, the INIA Cores and the TSRI NIAAA Training Grant in Neuropsychopharmacology. Training and information dissemination to the San Diego community will be effected by the training opportunities of the Center including an NIAAA training grant and the Education Component.`

Endogenous cannabinoid (eCB) signaling in the brain plays a homeostatic role in the constraint and termination of stress responses. During the previous cycle of the TSRI-ARC we found that chronic intermittent EtOH exposure down-regulates eCB signaling in the central nucleus of the amygdala, a brain region critically involved in stress responses and emotional processing. We also found that dependenceassociated anxiety-like behavior and excessive EtOH consumption are alleviated by enhancement of eCB tone. Endocannabinoids are also present in other stress-responsive brain regions such as the basolateral amygdala (BLA) and bed nucleus of the stria terminalis (BNST) where they play a prominent role in the plasticity of excitatory and inhibitory signaling. Dysregulated synaptic function in these regions is believed to contribute to dependence-associated affective disorders and we have gathered preliminary evidence that eCB clearance mechanisms are disrupted in these regions by alcohol dependence. Based on these observations we hypothesize that dysregulated eCB signaling in the BLA and BNST contributes to affective dysregulation and excessive EtOH consumption associated with long-term EtOH exposure. This hypothesis will be tested through three Specific Aims. Aim 1 will employ biochemical and neurochemical approaches to characterize the nature and persistence of dysregulated eCB function resulting from excessive EtOH exposure. Aim 2 will characterize the influence of disrupted eCB function in the BLA and BNST on dependence-associated anxiety-like behavior over a period of protracted abstinence. The relative influence of two primary eCB molecules, 2-AG and anandamide (AEA) will be characterized using pharmacological and genetic manipulations of their respective clearance mechanisms. Aim 3 will characterize the efficacy of selective eCB clearance inhibitors for reducing high levels of EtOH consumption associated with dependence and protracted withdrawal. The experimental design incorporates two distinct animal models of excessive drinking to index the development of eCB disruptions along the trajectory from chronic binge drinking to excessive EtOH consumption motivated by dependence.

DESCRIPTION (provided by applicant): The purpose of this program is to train promising young scientists at the postdoctoral level in the multi- disciplinary strategies of molecular, cellular, behavioral, and clinical neuropsychopharmacology of ethanol. The Scripps Research Institute Alcohol Research Center (TSRI-ARC), combined with a very active independent but interactive San Diego Alcohol Research Community, has developed a conceptual framework for the study of the neurobiology of alcoholism and the neurobiological basis for individual differences in vulnerability to alcoholism. Methods have been developed by combining biochemical, morphological, physiological and behavioral research to arrive at broad based studies of the neuropsychopharmacology of alcoholism through such fields as molecular biology, immunocytochemistry, electrophysiologic analysis (in vitro and in vivo), neuroendocrinology, behavioral pharmacology, and cognitive and motivational testing applied to animal and human subjects, and clinical studies. In addition, we also attempt to develop other important skills for pathways to independence: creative research expression, critical selection of problems, experimental design, data recording, validation and security, data interpretation, manuscript and grant preparation, promotion of transition awards, and ethical conduct of research. Trainees participate in scientific project development, research seminars from visiting scientists and journal clubs is well as actively participating in research programs. Each trainee will also receive a course on ethanol neuropsychopharmacology and a course in ethical conduct of research. Both informal and formal recruitment mechanisms are well established and have provided a sufficient number of high quality applicants. Extensive programs are in place for recruitment of minority and for facilitating minority interest in alcohol research. Significant success in the past funding period has resulted in retaining and promoting two minority faculty to the training program, training of two minorities and recruitment of two young faculty members. An evaluation of the success of the program is in place charting the career development of pathways to independence in alcohol research of former fellows with use of a formal evaluation survey. Postdoctoral fellows and other advanced trainees are selected with preferential weighting towards those seeking interdisciplinary methodologies. They are then assigned to one of the Principal Investigators of the training grant who coordinates their initial research project selections. Depending upon a trainee's prior research skills, collaborations with more than one senior scientist are encouraged. All combined, the training program provides a dynamic environment for fellows to develop and pursue a foundation for career in the neuropsychopharmacology of alcoholism.

The Administrative Core will provide leadership in research, research methodology development, and information dissemination on topics relevant to our overall mission. It coordinates all TSRI-ARC activities, and will help support and coordinate the TSRI-ARC with the Center at Large. Functions of the Administrative Core will be to provide scientific and administrative leadership for the Alcohol Research Center (ARC) by monitoring and enhancing collaborative interactions among the support core components and research components, to facilitate the dissemination of expertise between support core components, pilots and research components by organizing regular TSRI-ARC meetings, seminars, conferences and workshops to stimulate exchange of scientific information among the Center faculty and staff, to promote the sharing of multidisciplinary resources and services among the support core components and investigators, to administer a pilot project program to stimulate new alcohol research programs within the TSRI-ARC at Large, and to disseminate new research knowledge to academic, treatment and lay communities by engaging in community outreach activities designed to improve awareness, prevention and treatment of alcoholism and alcoholic diseases, and supporting regional and national research initiatives aimed at improving prevention and treatment of alcohol use disorders and addiction. It will arrange regular meetings of the Steering Committee and Program Advisory Board and monthly meetings of the Participating Investigators. The Administrative Core is organized to ensure proper functioning of the Center to benefit investigators, their utilization of Cener resources to enhance and expand alcohol research throughout the TSRI-ARC and Center at Large, and to provide review and advisory functions

DESCRIPTION (provided by applicant): A major and growing problem in the field of drug abuse is prescription opioid abuse and dependence, which has reached epidemic proportions. A major question is whether there are any unique pharmacodynamics or neuroadaptations to explain this epidemic. One largely unexplored hypothesis to explain the high abuse potential of the prescription opioid medicines such as oxycodone is that there are neurobiological vulnerabilities in response to chronic administration of these drugs that predispose individuals to addiction on such opioids and that these neurobiological vulnerabilities are exaggerated by the pharmacodynamics of certain synthetic opioids. To test this hypothesis, in the present proposal, individual differences in compulsive drug seeking and dependence will be correlated with individual neuroadaptational changes in brain stress systems known to drive dependence. In Specific Aim 1, animal models of compulsive drug seeking and dependence will be developed for oxycodone and compared to drug seeking for heroin and buprenorphine. Using such animal models, individual differences in compulsive drug seeking, withdrawal and re-escalation of compulsive drug seeking will be characterized. In parallel, in Specific Aim 2 dysregulation of neural systems known to drive the development of compulsive opioid seeking will be characterized. These include alteration of the brain corticotropin releasing factor (CRF) systems and brain dynorphin kappa systems in brain reward and stress circuits. Finally, in Specific Aim 3 microinjection of antagonists of the CRF system and the kappa system in specific brain regions and gene silencing of specific CRF and dynorphin neurocircuits will be employed to reverse neuroadaptive changes and consequent re-escalation of drug seeking in subgroups of rats with high levels of compulsive drug seeking. The present proposal will go far towards elucidating the neurobiological systems within specific neurobiological circuits of the ventral striatum and extended amygdala, which are critical for the motivational aspects of prescription opioid abuse and dependence. The present proposal also will provide important information for identifying novel non- mu opioid treatments for opioid addiction.