Marisa Roberto - US grants

DESCRIPTION (provided by applicant): The central nucleus of the amygdala (CeA) functions as a hub that converts emotionally relevant sensory information into appropriate behavioral and physiological responses, and plays a crucial role in anxiety-like behavior associated with ethanol dependence and withdrawal. Notably, both the corticotropin-releasing factor (CRF) and norepinephrine (NE) systems are critical in behavioral aspects of addiction, including the anxiogenic effects of drug withdrawal. We hypothesize that ethanol dependence induces neuroadaptations in the CeA and that many of these neuroadaptations may persist after cessation of ethanol exposure. Therefore, we propose to investigate whether or not the changes in signal transduction and synaptic functions seen in the CeA persist after prolonged ethanol withdrawal (abstinence) for 1 and 2 weeks. The previous funding period identified critical findings in the understanding of acute alcohol effects and alcohol dependence in the medial subdivision of the CeA. We demonstrated that corticotropin-releasing factor 1 (CRF1) receptors are involved in the ethanol-induced increase in GABA release in CeA and the CRF system is up-regulated after ethanol dependence. Notably, chronic CRF1 antagonist treatment blocked alcohol dependence-induced increases in ethanol consumption. Both the protein kinase C epsilon (PKCepsilon) and adenylate cyclase/protein kinase A (AC/PKA) transduction pathways are involved in mediating the CRF and ethanol effects on GABA release. Ethanol dependence downregulated the GABAB system and gabapentin (a structural analogue of GABA) reversed several behavioral aspects of ethanol dependence. We also identified long-lasting neuroadaptations induced by ethanol dependence in the glutamatergic systems. Based on these combined findings, the research plan of the present competitive renewal will be to continue studying (in vitro and in vivo) the presynaptic mechanisms of ethanol and the neuroadaptations within the CeA during the development of alcohol dependence, with a focus on newly identified intracellular pathways involved in ethanol, CRF and NE effects in the CeA. Since there is no data on the persistence of such neuroadaptive changes after cessation of ethanol exposure, we propose to study neural function during ethanol abstinence and characterize how long those neuroadaptative changes last. The general hypothesis is that chronic ethanol and dependence alter the CRF and NE systems and their effects on CeA GABA and glutamate signaling through common cellular systems to induce a maladaptation in neural function that sensitizes ethanol withdrawal-induced anxiety-like behavior. Given the extreme importance of this observation, it is imperative to provide data that can elucidate the cellular basis of the susceptibility of alcoholics to stress and relapse. A better understanding of the neuroadaptations shaping the synaptic networks involved in ethanol dependence represents a challenge to alcohol researchers and will be useful toward uncovering new therapeutic agents to alleviate alcohol dependence and prevent relapse.

[unreadable] DESCRIPTION (provided by applicant): This is a proposal to support young investigators from the United States and registration fees for all the speakers to attend the International Conference entitled Alcoholism and Stress: A Framework for Future Treatment Strategies. This Congress will be one of the first international meetings directed at bridging ongoing independent programs on stress mechanisms and alcoholism in Europe and the USA. We expect that it will be of broad interest to basic and clinical researchers around the world. The meeting will be held on May 6-8, 2008 in Volterra, Pisa, Italy. The attractive location of the meeting will provide an excellent opportunity for scientists from the USA to present and discuss their research with international colleagues and also provide opportunities for new collaborative research. The target audience consists of clinicians and researchers interested in the advancement of biomedical research on alcoholism and alcohol-related biomedical phenomena with the goal of developing new and innovative treatment strategies. This meeting will give participants the chance to present their latest findings related to alcohol and stress research through abstract, symposia, and roundtable submissions, as well as plenary lectures. Most importantly, it will provide a unique opportunity for alcohol researchers from all over the world to meet on a personal level and interact with pharmaceutical researchers to develop drug strategies for alcoholism treatment. Approximately 150 attendees are expected from all over the world, including, but not limited to the following countries: Australia, Austria, Belgium, Canada, China, Finland, France, Germany, Greece, Italy, Japan, Russia, Spain, Sweden, Switzerland, United Kingdom, and the United States. This application requests funding for travel and registration fee for 20 US scientists to attend this important meeting on Stress and Alcohol. This application also requests coverage of registration fee for the invited speakers and for publication costs. [unreadable] [unreadable] Relevance to Public Health: Alcoholism is an etiologically and clinically heterogeneous disorder in which compulsive alcohol seeking and use represent core symptoms. Environmental stress has been recognized as one of the major factors for alcohol abuse, including binge drinking and alcohol dependence. However, the interaction between environmental stress and heritable factors in the development of alcoholism is still largely unexplored. Understanding the nature of this interaction in regulating individual risk of becoming an alcohol abuser represents a major challenge in this research area and may provide invaluable help for the development of preventive strategies or pharmacotherapeutic remedies. This application seeks funding from NIAAA to enable American and foreign scientists to attend the World Conference entitled Alcoholism and Stress: A Framework for Future Treatment Strategies. The aim of this meeting is to bridge ongoing independent programs on alcoholism and stress mechanisms in Europe and the United States of America (USA). [unreadable] [unreadable] [unreadable]

project proposes to continue cellular studies of the role of neurotransmitters and their receptors in alcohol drinking and dependence, and is based on behavioral findings that the central amygdala nucleus (CeA) is a key brain area involved in stress reactions and alcohol dependence. These behaviors involve several transmitters, including GABA, glutamate, CRF, opioids, endocannabinoids (eCBs) and neuropeptide Y (NPY). With the rationale that the synapse is the most sensitive site of ethanol action, and particularly those synapses in CeA mediated or regulated by these transmitters, we hypothesize that these same neuropharmacological systems within the CeA are involved in excessive alcohol drinking and dependence. Therefore, we propose 3 Specific Aims to clarify the cellular underpinnings of these behaviors, using in vitro electrophysiological recording and in vivo microdialysis in CeA neurons of 2 TSRI-ARC rat models of selfadministered binge drinking and chronic ethanol-induced dependence (CEID), by studying: 1) the synaptic and cellular effects and interactions of opioids with CRF and acute and chronic ethanol exposure via these binge and dependence models. 2) the effects and interactions of cannabinoids and eCBs with CRF and acute and chronic ethanol exposure via these two models;and 3) the effects and interactions of NPY with CRF and acute ethanol in dependence, withdrawal and protracted abstinence. The electrophysiological studies will use CeA brain slices and involve standard intracellular and whole-cell clamp methods and a battery of measures to assess the pre- versus postsynaptic sites of action of ethanol and ligand effects. Microdialysis will be used to verify transmitter release. This project should provide important new information on the possible sequelae of ethanol binge drinking to dependence, at the cellular, synaptic and ion channel levels.

DESCRIPTION (provided by applicant): Behavioral evidence indicates that the neuropeptide nociceptin/orphaninFQ (NOC) attenuates ethanol intake and relapse associated with ethanol exposure and stressful events. Because the central nucleus of the amygdala (CeA) plays a crucial role in ethanol dependence, we speculate that the anti-alcohol and anti-stress properties of NOC are mediated via its interactions with the GABA and corticotropin-releasing factor (CRF) systems within the CeA. Our research is centered on the effects of acute and chronic ethanol on CeA neurons. We demonstrated that acute ethanol increases GABA transmission in CeA of both na[unreadable]ve and ethanol-dependent rats by increasing GABA release, and that this ethanol effect involves intermediary substrates such as presynaptic CRF1 receptors. We recently reported that NOC decreases GABAergic transmission and release and prevents the ethanol-induced enhancement of GABAergic transmission in CeA. Our preliminary studies indicate that NOC opposes the actions of CRF, and that neuroadaptations such as increased sensitivity of the NOC system occur during the development of ethanol dependence in CeA. So far the mechanism of NOC action is unknown, and the interactions of the NOC and CRF systems likely to take place to regulate the synaptic effects of ethanol have not been investigated. Based on these combined findings, in this research proposal we will use electrophysiological, molecular biological and in vivo microdialysis approaches to study the antagonistic role of NOC in the ethanol effects on GABAergic transmission in CeA and the molecular mechanisms that mediate such NOC effects (Aims 1 and 2). We also intend to uncover the neuroadaptive response(s) of the NOC system associated with ethanol dependence (Aims 1 and 2). In Aim 3, we will test our hypothesis that NOC opposes CRF effects at a presynaptic site, and we will identify the interactions of NOC and CRF on GABAergic synapses in CeA. Emphasis will be given to determine the neuroadaptations that take place between the NOC and CRF systems in association with ethanol-dependence. A better understanding of the neuroadaptations shaping the synaptic networks involved in ethanol-dependence represents a challenge to alcohol researchers and will be useful toward the uncovering of new therapeutic agents to alleviate alcohol dependence. PUBLIC HEATLH RELEVANCE: Recent behavioral studies have revealed a modulatory effect of the neuropeptide nociceptin on ethanol- and stress-related phenomena, suggesting an important role of nociceptin in ethanol dependence. However, until now there have been no physiological data on the possible interactions of nociceptin and ethanol at the synaptic level. Therefore, the experiments proposed in this application will use electrophysiological, molecular biological and in vivo microdialysis approaches to study for the first time the cellular interactions of nociceptin, ethanol and corticotropin-releasing factor (CRF) systems at GABAergic synapses in the central nucleus of the amygdala, a brain region highly implicated in alcohol dependence. Emphasis will be given to determining the neuroadaptations that take place between the nociceptin and CRF systems in association with ethanol dependence. A better understanding of the neuroadaptations shaping the synaptic networks involved in ethanol dependence will be useful toward revealing new therapeutic agents to alleviate alcohol dependence.

ABSTRACT: Alcoholism is a chronically relapsing disorder that develops over time and is characterized by the transition from recreational alcohol use to abuse and dependence. Negative emotional states, such as post- traumatic stress disorder (PTSD) or anxiety, influenced by genetic factors or determined by environmental conditions contribute to shaping this transition. On the other hand, chronic exposure to alcohol is a major determinant for the occurrence of mood disorders (e.g., anxiety, depression, PTSD) and negative emotional states (i.e., dysphoria, irritability). The amygdalar nuclei [both the central nucleus of the amygdala (CeA) and basolateral amygdala (BLA)] are considered a hub for negative emotional circuitry, and the role of the stress peptide corticotropin-releasing factor (CRF) in this brain structure is critical for both development of alcohol dependence and mood disorders/negative affect. During the previous funding period we provided essential new insight into the relationship between innate overexpression of the CRF1 receptor system, stress hypersensitivity and excessive ethanol consumption in genetically selected Marchigian Sardinian (msP) rats. Our most recent results show that enhanced CRF signaling in msP rats is responsible for increased hydrolytic activity of fatty acid amide hydrolase (FAAH) and blunted endocannabinoid (eCB) signaling in the CeA/BLA, leading to enhanced GABA and glutamate transmission in the amygdala. Our hypothesis is that such alterations contribute to enhance stress sensitivity and to exacerbate anxiety-like symptoms in the msP rats, which may increase drinking to alleviate these negative conditions. Understanding the mechanisms through with innate and environmental factors act/interact to dysregulate CRF/eCB transmission in the BLA and CeA will provide new insight into the etiopathology of alcoholism, aiding the development of new therapeutics for this still largely untreated medical condition. The research plan for this competitive renewal is to investigate how alteration of eCB signaling in the amygdala triggered by innate (in msP rats) or EtOH-induced (post-dependent Wistars) upregulation of the CRF1 system contributes to excessive alcohol drinking and exacerbates maladaptive conditioned fear responses, similar to symptoms of PTSD in humans. The ability to restore normal eCB function by FAAH inhibition, and therefore to counteract excessive drinking and normalize fear responses, will be also studied. A better understanding of the molecular mechanism underlying genotypic differences of the msP compared to outbred Wistar rats and of neuroadaptations following exposure to alcohol, will provide novel insight into the innate susceptibility to develop Alcohol Use Disorder and will be useful toward the development of new therapeutic agents to alleviate alcohol dependence. The key personnel involved in the present study possess all the necessary expertise needed to accomplish this multidisciplinary program. In particular, Dr. R. Ciccocioppo will supervise behavioral experiments, breeding programs and animal selections. Dr. M. Roberto will be dedicated to the electrophysiology experiments and the research program coordination. Dr. L. Parsons will perform the neurochemistry experiments, and will also perform behavioral tests at TSRI to ensure cross-lab reproducibility and provide the animals for electrophysiology. These three key personnel each have extensive publication histories in their respective areas of expertise, and have ongoing collaborative interactions, documented by common publications, that will be invaluable for the successful completion of the proposed experiments.

DESCRIPTION (provided by applicant): This is a proposal to support investigators from the United States and enable them to attend the International Conference entitled "Alcoholism and Stress: A Framework for Future Treatment Strategies." This Congress is one of the first international meetings directed at bridging ongoing independent programs on stress mechanisms and alcoholism in Europe and the USA. We expect that it will be of broad interest to basic and clinical researchers around the world. This Conference is a sequel to our highly successful inaugural meeting on Alcohol and Stress that was held in Volterra in May 2008. The meeting will be held on May 3-6, 2011 in Volterra, Pisa, Italy. The attractive location of the meeting will provide an excellent opportunity for scientists from the USA to present and discuss their research with international colleagues and also provide opportunities for new collaborative research. The target audience consists of clinicians and researchers interested in the advancement of biomedical research on alcoholism and alcohol-related biomedical phenomena with the goal of developing new and innovative treatment strategies. This meeting will give participants the chance to present their latest findings related to alcohol and stress research through abstract, symposia, and roundtable discussions, as well as plenary lectures. Most importantly, it will provide a unique opportunity for alcohol researchers from all over the world to meet on a personal level and interact with clinical researchers to develop drug strategies for alcoholism treatment and to foster alcohol-related research collaboration within a community that otherwise would have had limited access to such a venue and body of knowledge (this includes the European countries). Approximately 150 attendees are expected from all over the world, including, but not limited to the following countries: Australia, Austria, Belgium, Canada, China, Finland, France, Germany, Greece, Italy, Japan, Russia, Spain, Sweden, Switzerland, United Kingdom, and the United States. This application requests funding for travel and registration fees for 20 US scientists to attend this important meeting on Stress and Alcohol. This application also requests coverage of registration fees for the invited speakers and for publication costs (abstract book). PUBLIC HEALTH RELEVANCE: Alcohol abuse and anxiety disorders are global health problems. This proposal is for the organization of an international conference that aims to bridge ongoing independent research programs on alcoholism and stress, with the overall goal of developing treatment strategies for alcoholism. This meeting will allow participants the opportunity to present current work in the alcohol and stress fields, and will also allow researchers to identify and forge collaborative efforts between European and American research programs.

DESCRIPTION (provided by applicant): This competitive U01 renewal application, under the INIA-West consortium, is based on behavioral findings that the central amygdala (CeA) is a key brain area underlying stress reactions and alcohol dependence, and that these behaviors involve several CeA transmitters (GABA, glutamate) and neuropeptides (CRF, opioids and galanin). Our published electrophysiological studies of these systems in CeA provide an entry point for proposed new studies: a stated need to pursue physiological evaluation of the function of gene products suggested by the molecular components (e.g., of Y. Blednov and others) of the INIA-West to be involved in excessive alcohol drinking. Therefore, we now propose to use electrophysiological and cytochemical methods to investigate the hypothesis of a role for neuroinflammatory factors (lipopolysaccharide {LPS}, toll-like receptor 4 {TLR4}, CD14, cytokines) in alcohol preference and excessive drinking. To test this hypothesis at the cellular level, we propose 4 Specific Aims: 1) To assess the role of TLR4 activation in effects of ethanol and CRF on GABAergic and glutamatergic transmission in CeA slices by LPS superfusion or i.p. injection in CeA of wild type (WT) and CD14 knockout (KO) mice; 2) To assess effects of the TLR4-g en e rated inflammatory cytokines IL-ip, TNFa, and IL-6 on membrane and synaptic measures in CeA of WT mice; 3) To determine if the LPS, CRF or chronic ethanol increase cytochemical signs of inflammation in CeA; 4) To determine if the electrophysiological or cytochemical effects of LPS, cytokines, ethanol or CRF on CeA seen in Specific Aims 1-3 can be reversed by pre-treatment with certain anti-inflammatory drugs. These proposed studies thus represent new steps toward evaluating the cellular sites and mechanisms of action of the emerging gene targets suggested by other INIA West components to underlie alcohol preference or excessive drinking, and may further validate drug targets for reversal or prevention of alcohol effects and excessive drinking.

DESCRIPTION (provided by applicant): This is a competing renewal application for a Consortium for the Integrative Neuroscience Initiative on Alcoholism (INIA)-West (Notice# RFA-AA-11-006) to identify the molecular, cellular, and behavioral neuroadaptations that occur in specific brain neurocircuitries that result in excessive alcohol consumption. This multidisciplinary initiative focuses on the molecular and cellular neuroadaptations in brain addiction circuits associated with the basal forebrain, including reward circuitry in the ventral striatum and dependence circuitry in the extended amygdala. The overall hypothesis for INIA-West is that genetic differences and neuroadaptations in reward circuitry are responsible for individual differences in the vulnerability to the excessive alcohol consumption. Sixteen research components and six scientific cores will use excessive drinking models in animals to mimic the binge- and dependence-induced excessive drinking of alcohol abuse disorders. The overall goals of INIA-West are (1) to confirm gene targets nominated by expression assays or other methods by use of transgenic, knockout, inducible knockout, site-specific knockout, RNAi, in situ hybridization, in vivo electrophysiology, in vivo imaging, and next-generation sequencing, (2) to identify druggable targets that are most promising for medication development for the treatment of alcoholism by use of novel molecules in concert with molecules with existing FDA approval in animal models with the most predictive ability, and (3) to attract new and innovative investigators to the field of alcohol research by recruiting individuals for U01 grants and Pilot projects and by making the informatics integrated datasets accessible, searchable, and interactive with other databases for all scientists interested in alcoholism research. Core facilities are proposed that provide molecular genetic support and target assessment translation for medications target development. A Pilot Project program is proposed to identify exciting new areas of research and the continual recruitment of new investigators to the alcohol field. The INIA program will be directed by the Administrative Core in close cooperation with the Executive Committee and Steering Committee and with the continual advice of a distinguished Scientific Advisory Board.

DESCRIPTION (provided by applicant): Recent research has highlighted the role of the central nucleus of the amygdala (CeA) and the recruitment of the corticotropin-releasing factor (CRF) system in the development of ethanol dependence. Particularly, CRF type 1 receptor (CRF1) antagonists block the anxiogenic effects and the increase in ethanol self-administration produced by stressors and ethanol withdrawal. Notably, chronic CRF1 antagonism abolishes dependence-induced escalation of ethanol drinking in rats exposed to chronic intermittent ethanol. We demonstrated that acute ethanol and CRF increase GABAergic transmission in mouse and rat CeA via CRF1 activation, and the effects of CRF and CRF1 antagonists on GABAergic transmission are enhanced in CeA neurons from ethanol-dependent rats. However, the CeA is comprised of heterogeneous cell types and technical limitations have precluded the identification and further characterization of neurons from which recordings were obtained. Thus, despite the vast knowledge accumulated on the CRF1-mediated effects of acute and chronic ethanol in this brain region, the underlying local neuronal microcircuitry needs to be delineated. Thus, in this research proposal we will use an innovative transgenic mouse model expressing green fluorescent protein (GFP) in neurons expressing CRF1 (CRF1:GFP mice) to compare the electrophysiological, neurochemical, morphological and hodological properties of CeA CRF1-expressing neurons to those of unlabeled neurons. Using electrophysiology in combination with histochemical and neurotracing methods, we will obtain novel information on functionality and connectivity of these neurons, thereby providing mechanistic insights into the role of the CRF system in the development of alcohol dependence. Drs. Contet and Mandyam, key personnel involved in the present study, possess all the necessary expertise needed to accomplish such a multidisciplinary project. A better understanding of the molecular mechanisms underlying ethanol effects and the neuroadaptations shaping the CRF neurocircuitry involved in ethanol-dependence represent a challenge to alcohol researchers and will be useful toward uncovering new therapeutic agents to alleviate alcoholism.

The Cellular Electrophysiology Project (Director: Marisa Roberto, Co-Director: George Siggins) proposes to continue cellular studies of the role of neurotransmission in alcohol drinking and dependence and is based on behavioral findings that the basolateral (BLA) and central (CeA) amygdala nuclei are key brain areas involved in stress reactions and alcohol dependence. This renewal application will focus on the overall hypotheses that the transition from low levels of drinking to chronic binge drinking is driven by decreased activity in endocannabinoid systems in ¿reward circuits¿ in the amygdala and that the transition from binge drinking to dependence is also driven by recruitment of a dysregulated stress system driven by corticotropinreleasing factor (CRF) and the neuronal pentraxin Narp. We will use amygdala slice preparations to functionally and morphologically characterize CeA and BLA neurons (Specific Aims 1 and 2) and neurocircuitry (Specific Aims 1-3) involved in responses to ethanol, cannabinoids, and CRF and in the progression from binge drinking to dependence. Specific Aim 1 is designed to test the hypothesis that binge drinking and/or dependence will differentially alter responses to cannabinoids (CB1 receptor agonists and antagonists) in specific neurons and synapses in the BLA and CeA. Specific Aim 2 will test the hypothesis that withdrawal from alcohol bingeing or dependence will alter synaptic glutamatergic transmission both within and between the BLA and CeA and/or their responses to CRF and acute ethanol via changes in Narp levels. Specific Aim 3 is designed to use neuronal filling with biocytin and tracing via retrograde labeling from brain-region targets of BLA and CeA neurons, such as the bed nucleus of stria terminalis, to test the hypothesis that alterations in synaptic properties within specific neurons and neurocircuitries are involved in binge drinking, dependence, and withdrawal and may be associated with changes in the endocannabinoid and CRF or Narp systems. This project will use BLA and CeA brain slices and standard intracellular and whole-cell clamp methods and a battery of measures to assess the pre- vs. postsynaptic sites of action of ethanol and ligand effects and will involve collaborations with the Parsons, Zorrilla, and Mandyam components and Viral Vector and Animal Models/Biological Measurement Cores. The project should provide important new information on the possible sequelae of ethanol binge drinking to dependence at the cellular, microcircuitry, synaptic, and ion channel levels.

Abstract     Our TSRI-ARC has focused on preclinical work on the cellular, neurochemical, and molecular mechanisms of alcohol dependence, with a major interest in the role of the central nucleus of the amygdala (CeA) in excessive alcohol drinking. However, there  is  a  great  need  to  understand  the  mechanisms  that  mediate  dependence-­ induced  drinking  motivated  directly  by  withdrawal/abstinence  symptoms.  The  Neurophysiology  Project  will  continue  to  study  the  neuroadaptations  induced  by  alcohol  dependence  in  the  CeA,  and  will  now  investigate  upstream neuronal circuits that are responsible for dysregulation of the CeA during alcohol abstinence. Corticotropin-­releasing factor (CRF) and CRF1 receptors are involved in the ethanol-­induced increase in GABA  release in the CeA and the CRF system is upregulated after ethanol dependence. Chronic CRF1 antagonism  blocked  alcohol  dependence-­induced  increases  in  ethanol  consumption.  Notably,  the  transition  to  alcohol  dependence also dysregulates executive function, and the infralimbic (IL) subdivision of the mPFC exerts ?top-­ down? control over the amygdala to regulate emotional aspects of goal-­directed behaviors. Thus, this renewal  application  focuses  on  the  overall  hypothesis  that  ethanol  dependence  and  withdrawal  are  driven  by  the  recruitment of CRF and serotonin (5-­HT) signaling in cortical-­amygdala circuits. We will study neural function  during  ethanol  abstinence  and  characterize  how  long  those  neuroadaptive  changes  persist.  In  particular,  our  goal  is  to  characterize  neuroadaptations  in  the  CRF  and  5-­HT  systems  and  their  effects  in  the  CeA  and  IL  through  common  cellular  systems  to  induce  a  maladaptation  in  neural  function  that  promotes  ethanol  withdrawal-­induced anxiety-­like behavior. Our project is designed to test the hypothesis that dependence and  withdrawal (early withdrawal = 2-­8 h;? late withdrawal = 2 weeks) differentially alter responses to 5-­HT and CRF  in CeA neurons (Specific Aim 1) and will affect the excitability of IL pyramidal neurons in layer V that project to  the  CeA  (Specific  Aim  2).  Finally,  Specific  Aim  3  will  electrophysiologically  assess  the  ethanol-­induced  synaptic  and  molecular  mechanisms  of  candidate  drugs  that  are  identified  and  tested  in  preclinical  animal  models  (Contet  and  Animal  Models  Core)  and  subsequently  tested  in  the  clinical  component  (Mason).  The  project  will  use  IL  and  CeA  brain  slices  and  standard  whole-­cell  patch-­clamp  and  cell-­attached  electrophysiological methods, as well as measures of protein levels and chemogenetic and behavioral testing  that  will  involve  continued  collaboration  with  the  George/Zorrilla,  Contet,  Martin-­Fardon,  and  Mason  projects  and Animal Models Core. It is imperative to provide data that elucidate the cellular basis of the susceptibility of  alcoholics  to  stress  and  relapse.  A  better  understanding  of  the  neuroadaptations  that  shape  the  synaptic  networks that are involved in ethanol dependence represents a challenge to alcohol researchers and will lead  to the development of new therapeutic agents to alleviate alcohol dependence and prevent relapse.         PHS 398/2590 (Rev. 06/09)  Page        Continuation Format Page 

Stress is a risk factor for alcohol use disorders (AUDs). Generally, individuals with anxiety disorders such as posttraumatic stress disorder (PTSD) also have elevated rates of AUD, more severe alcohol withdrawal symptoms, and greater relapse risk. Accumulating evidence indicates that immune-related pathways are critical biological components of CNS function and dysfunction. Our published and preliminary results show that canonical cytokines, such as Interleukin (IL)-18, have a profound capacity to affect neuronal function and regulate GABA and glutamate transmission within the amygdala. Notably, IL-18 and its receptors are highly expressed in the amygdala, a brain region that strongly contributes to anxiety- and addictive-related behaviors, and stress history and alcohol exposure increase IL-18 expression. Recent studies in humans found that IL-18 receptor gene expression is associated with distinct PTSD subtypes and that a single nucleotide polymorphism (SNP) in the IL-18 gene (rs1946518) is associated with AUD in a highly traumatized civilian cohort largely comorbid for PTSD. Notably, the same SNP in the IL-18 gene is also associated with amygdala reactivity in anxiety. The goal of this proposal is to 1) identify the cellular mechanisms underlying the essential role of IL-18 in homeostatic regulation of normal neuronal activities and amygdala circuits, and 2) test the hypothesis that IL-18 signaling contributes to the development of maladaptive stress-induced anxiety and AUDs. To accomplish our goal, we will employ innovative and complementary techniques: behavior, electrophysiology, ribosome profiling combined with next generation sequencing, in situ hybridization/RNAScope and immunohistochemistry, as well as pharmacological and viral vector-mediated knock down of the IL-18 system in amygdala. We will determine the interactions of peripheral and central immune elements in healthy and pathological function, comparing Vulnerable vs. Resilient subjects, at the molecular, cellular, circuit and behavioral levels. We will identify the role of IL-18 signaling on synaptic functions in amygdala circuits in both male and female rats using an adapted ?2-hit? rat model of stress to generate escalated drinking and high anxiety-like phenotypes for behavioral and physiological studies. Collectively, these studies will elucidate the mechanisms that drive IL-18 dysregulation to contribute to stress-induced anxiety and AUDs, and may identify promising targets for treatment strategies for anxiety disorders and alcoholism.

The purpose of this program is to provide promising young scientists with mentor-based training in a dynamic, highly collaborative and interactive environment to promote their development as independent investigators engaged in alcohol research. Highly qualified candidates are competitively selected from a large applicant pool recruited through advertisements in addiction- and neuroscience-related societies and publications, referrals from established colleagues in the field of alcohol research, and from letters of inquiry resulting from the Training Faculty?s publications and public presentations. Mechanisms are in place to encourage applications from underrepresented groups, to foster diversity in the training program and the field of addiction research. A rigorous training program has been established to develop trainees? skills in (i) identifying important, relevant and testable hypotheses, (ii) experimental design and conduct, (iii) data analysis and interpretation, and (iv) scientific communication through written and oral presentation. Emphasis is placed on career development, and trainees complete an Individual Development Plan (IDP) upon admission to the program to generate trainee-mentor discussions of career plans and goals, strategies and timelines for achieving these goals, and ways in which these goals may have changed during the training period. Trainees are encouraged to engage in numerous workshops and resources available at The Scripps Research Institute (TSRI) on effective CV development, networking, job applications and interviewing techniques. In addition, mentors and Program Faculty actively engage trainees in discussions of each of these skills on an ongoing basis. Throughout their tenure in the program, trainees are educated in the responsible conduct of research, and this occurs via courses offered through the TSRI Career and Postdoctoral Services Office, the annual Neuropsychopharmacology of Addiction course taught by the Program Faculty, and through day-to-day input from mentors and Program Faculty regarding ethical practices in laboratory research. The highly collaborative group of 16 Program Faculty members is characterized by strong, well-funded programs in alcohol and addiction research incorporating multidisciplinary experimental approaches combining biochemical, morphological, physiological, and behavioral techniques. This is a translational training program associated with the longstanding TSRI P60 Alcohol Research Center, and trainees can engage in studies spanning the molecular and cellular domains, through investigations of neurobiological mechanism in whole animals, and clinical studies in humans on the vulnerability to alcoholism, and the development of therapeutics for this prevalent disorder. The training program has functioned continuously since 1979 under three successive Program Directors, and has a consistent record of preparing outstanding young investigators for a rapid transition to independent and impactful careers in alcohol research and addiction neuroscience.