Nicholas W. Gilpin - US grants

[unreadable] DESCRIPTION (provided by applicant): The main objective of this proposal is to assess the role of neuropeptide Y (NPY) in the development and maintenance of alcoholism, and to assess whether brain NPY systems represent a viable pharmacotherapeutic target to combat alcoholism. These problems will be examined using a multidisciplinary approach in the context of an animal model of alcoholism that mimics the human condition. This animal model is defined by cycles of chronic exposure to high doses of alcohol, multiple withdrawals from alcohol, and relapse to alcohol drinking, presumably for its ability to alleviate aversive symptoms associated with the absence of alcohol. The proposed experiments will examine the role of NPY at the behavioral, cellular, and molecular levels in the development and maintenance of alcoholism. More specifically, the experiments proposed herein will examine [1] the ability of NPY in the central nucleus of the amygdala to suppress relapse ethanol drinking and withdrawal-related anxiety-like behavior, [2] possible interaction effects of NPY and ethanol on inhibitory neurotransmission in the central nucleus of the amygdala, and [3] changes in levels of NPY receptor subtypes in the extended amygdala during and following the development of alcohol dependence. The overall hypothesis is that NPY systems in the central nucleus of the amygdale are largely dysregulated following induction of alcohol dependence and that these pathological changes are a major factor in driving the dependent organism to relapse, specifically to alleviate the negative affective consequences associated with the absence of alcohol. In accordance with the NIAAA mission, these results will clarify the consequences of alcoholism on the role of brain NPY systems in mediating relapse behavior and may eventually contribute to the development of a novel and effective pharmacotherapeutic agent to combat alcoholism. Dysregulation of brain neuropeptide Y systems is increasingly thought to be a major factor in the maintenance of alcoholism, especially by contributing to the negative affective consequences experienced by alcoholics during the absence of alcohol. Neuropeptide Y has robust anxiety-reducing effects that may be compromised by constant exposure to high doses of alcohol, thus contributing to the motivational factors that lead to relapse alcohol drinking. For the same reasons, brain neuropeptide Y systems represent an excellent candidate for the development of pharmacotherapeutics capable of effectively combating alcoholism in humans. [unreadable] [unreadable] [unreadable]

Project Summary/Abstract The main objective of the current proposal is to examine the effects of post-traumatic stress disorder (PTSD) on alcohol dependence. The proposed career transition award will provide new training for the applicant in animal models of PTSD and in biochemical techniques that will complement the applicant's experience with behavioral animal models of alcoholism. The training phase of the award will leave the applicant prepared to lead an independent research program in the R00 phase of the award. The R00 phase of the award and the independent research career of the applicant beyond this award will continue to focus on the negative reinforcement aspects of alcoholism and their neural overlap with other psychiatric disorders. Following exposure to a traumatic stressor, PTSD is a long-term pathological state marked by increases in anxiety and arousal, decreases in the ability to perceive pleasure, and generalization and avoidance of trauma- related stimuli. Alcohol dependence is compulsive alcohol use despite adverse consequences, loss of control when taking alcohol, and development of tolerance that results in withdrawal symptoms in the absence of the drug. Excessive alcohol consumption can be driven by the ability of the drug to alleviate aversive symptoms, including those produced by a pre-existing emotional disorder (e.g., PTSD). It is hypothesized that animals that exhibit maladaptive stress responses will exhibit higher susceptibility to alcohol dependence. Because PTSD patients and alcoholics exhibit similar patterns of neural dysregulation in the limbic system, it is hypothesized that the amygdala and BNST serve as a neural interface for overlapping/additive effects of PTSD and alcohol dependence. It is hypothesized that neuromodulators involved in the brain stress response (i.e. corticotropin- releasing factor [CRF], neuropeptide Y [NPY], and norepinephrine [NE]) and ubiquitous in these limbic circuits are similarly and additively dysregulated in PTSD and alcohol dependence.

DESCRIPTION (provided by applicant): Alcohol Use Disorder (AUD) is responsible each year for more than 2.5 million deaths worldwide, more than 58 million life years lost worldwide, and $220 billion financial cost in the United States alone. Post-Traumatic Stress Disorder (PTSD) affects 7.7 million Americans, and PTSD costs the U.S. billions of dollars annually. These problems will increase in scope during a time of perpetual U.S. involvement in overseas military conflicts. There is a lack of research directly investigating the neurobiology of traumatic stress-induced escalation of alcohol use. This proposal seeks to identify the neurobiological basis for alcohol abuse in individuals with PTSD, with the ultimate goal of contributing to the tailoring of effective therapeutic strategies to reduce alcohol drinking in humans with PTSD. This project falls within the scope of the NIAAA mission to support biomedical and behavioral research on the causes and treatment of alcoholism. More specifically, this application proposes the use of rat models to investigate the biological basis for excessive alcohol drinking following exposure to traumatic stress by using a multi-disciplinary approach that integrates behavior, pharmacology, molecular biology, and optogenetics techniques. This question is particularly important for the mission of the NIAAA because of the high rate of alcohol abuse in individuals with traumatic stress disorders, and the fact that AUD and PTSD each promote mortality and shorten life spans in humans. The long-term goals of this application are to understand the neurobiological basis of co-morbid AUD and PTSD, and to identify pharmacotherapies with promise for reducing alcohol abuse in individuals with PTSD. The proposed aims will investigate the role of amygdala neuropeptides and specific amygdala output pathways in mediating traumatic stress-induced alcohol drinking and negative affect. The overarching hypothesis of this proposal is that neuropeptides in the amygdala mediate traumatic stress-induced escalation of alcohol drinking.

Abstract LSUHSC CARC Information Dissemination Core One of the missions of the Comprehensive Alcohol Research Center (CARC) of the Louisiana State University Health Sciences Center (LSUHSC) is to develop inter-disciplinary collaborative networks that will impact alcohol- and HIV-related knowledge, attitudes and behaviors by educating the scientific and lay communities on the neurobiological basis and biomedical consequences of alcohol use and abuse, and the risk factors and biological underpinnings of HIV. During current and prior CARC funding periods, we have established partnerships with local entities aimed at impacting knowledge, attitudes, and behavior of various populations toward HIV and Alcohol Use Disorder. The activities proposed here are organized into three major categories based on target populations: 1) those aimed at disseminating information to PLWHA and individuals at risk for HIV and/or AUD, 2) those aimed at disseminating information to practicing and in-training health care providers, and 3) those aimed at incorporating CARC research personnel (faculty and trainees) into dissemination activities, thereby facilitating the achievement of Aims 1 and 2, and simultaneously facilitating information dissemination between research personnel. The third aim also describes activities that will facilitate recruitment and training of promising young scientists into CARC labs that are focused on HIV and alcohol research, foster collaborative multi-disciplinary HIV-alcohol research at LSUHSC, and promote dissemination of HIV-alcohol findings to extramural scientists.

Project Summary Humans with post-traumatic stress disorder (PTSD) are more likely to develop alcohol use disorder (AUD) than the general population, and AUD is the most commonly co-occurring mental health disorder in humans with PTSD. These conditions, separately and combined, affect millions of American, cause millions of deaths worldwide, and cost society billions of dollars. This proposal will examine individual differences in traumatic stress effects on specific brain circuits in male and female alcohol drinkers, and will manipulate those circuits to test their role in post-stress escalation of alcohol drinking. Here, we focus specifically on amygdala disinhibition and endocannabinoid signaling, but we will collect blood and brain samples that allow for subsequent analysis of predictive biomarkers, as well as follow-up experiments in alternate brain circuits. This project falls within the scope of the NIAAA mission to support biological and behavioral research underlying addictive behaviors. More importantly, the current proposal addresses the research objectives outlined in RFA-AA-17-016 by assessing alcohol drinking in a well characterized animal model of PTSD, determining the association of pre-stress alcohol drikning with the development of post-stress escalation of alcohol drinking, systematically testing the effect of sex on post-stress escalation of alcohol drinking, identifying neurobiological processes that underlie the transition to post-stress escalation of alcohol drinking, and identifying neurobiological targets for potential treatment of alcohol abuse in PTSD patients. Specifically, this application proposes the use of rat models to examine the neurobiology underlying stress-induced escalation of alcohol drinking in male and female animals. This application proposes the use of behavioral, electrophysiology, biochemistry, and molecular biology techniques to identify brain circuits altered by traumatic stress in subsets of alcohol drinkers, and to directly test the role of those circuits in escalated alcohol drinking after stress. The overall goals of the proposed work are to test the role of 1) amygdala disnihibition and 2) brain endocannabinoid signaling in traumatic stress-induced escalation of alcohol drinking in a stress-susceptible subpopulation.

ABSTRACT Alzheimer's disease and Alzheimer's disease-related dementias are a growing challenge in the current aging population, with no known treatments capable of stopping or reversing the progression of neurodegeneration. Traumatic brain injury (TBI) results in enhanced neuronal loss, a hallmark of Alzheimer's disease (AD), and increases the risk of neurodegenerative disorders. The biochemical mechanism(s) underlying neuronal loss post TBI (in humans and rodents) remain to be determined. One possible mechanism underlying post-TBI neurodegeneration is altered neuronal protein degradation and mitochondrial function. Protein and mitochondrial turnover defects lead to accumulation of pathological proteins (e.g., tau, TDP-43, and amyloid-? [A?]), and are considered major contributing factors in the pathogenesis of neurodegenerative diseases, like AD. The ubiquitin pathway is essential for regulating neuronal protein and mitochondrial turnover, and defects in this pathway lead to abnormal protein deposition post TBI. Studies from the lab of our collaborator, Dr. Desai, have identified conjugation of ubiquitin-like protein ISG15 (interferon-stimulate gene 15) to cellular proteins, termed ISGylation, as a mechanism underlying neurodegeneration. Free ISG15 and ISGylation are elevated in fibroblasts and brains obtained postmortem from subjects diagnosed with ataxia telangiectasia, lymphocytes obtained from subjects diagnosed with AD, and spinal cords of veterans who suffered TBI and were later diagnosed with amyotrophic lateral sclerosis (ALS). ISG15 expression is regulated by type I interferons (IFN?), forming an IFN?/ISGylation axis. Our preliminary data collected from alcohol-naïve TBI rats (as described in the parent grant) show increased ISGylation in the dorsal hippocampus; an important brain region involved in memory and learning processes, relative to sham controls. Whether post-TBI upregulation of ISGylation inhibits neuronal protein and mitochondrial turnover remains to be investigated. Moreover, it is not known whether alcohol consumption post TBI exacerbates upregulation of the IFN?/ISGylation axis. However, neuroinflammation commonly precedes and is associated with neurodegeneration and our data show accentuated neuroinflammation at the site of injury in alcohol-exposed TBI animals. These findings support the prediction that alcohol-induced increases in neuroinflammation will enhance post-TBI neurodegeneration. We hypothesize that post-TBI activation of the IFN?/ISGylation axis inhibits ubiquitin-dependent neuronal protein and mitochondrial turnover resulting in accumulation of toxic proteins (i.e., tau, A?), defective mitochondria, and neurodegeneration, and that these effects will be exacerbated by alcohol consumption. Two specific aims will use the same TBI experimental model as the parent grant to measure neuropathological changes that increase risk for Alzheimer's disease in adult female and male rats. Results generated from these studies will form the basis of a grant application examining epigenetic mechanisms involved in activation of the IFN?-/ISGylation axis as an underlying mechanism increasing the risk for Alzheimer's neuropathology post TBI.

Project Summary Alcohol Use Disorder (AUD) is responsible each year for more than 2.5 million deaths worldwide, more than 58 million life years lost worldwide, and $220 billion in financial cost in the United States alone. Furthermore, AUD is highly co-morbid with various other conditions (e.g., traumatic brain injury, post-traumatic stress disorder, anxiety, depression) that are themselves increasing in prevalence, thanks in part to increased clinical awareness. Over the last 20 years, one neural system that has shown significant promise as a potential therapeutic target for AUD and co-morbid disorders is corticotropin-releasing factor (CRF), and especially CRF signaling through the CRF-1 receptor (CRFR1). Recent advances in neuroscience allow for the creation of sophisticated genetic animal models that allow experimenters to target specific neuronal populations and examine their role in network dynamics and behavior, based on afferent innervation, projection target, and/or molecular signature. Here we propose to create one such model by developing and validating a CRFR1:Cre rat for use in pre-clinical studies on the neurobiology of AUD and related psychiatric conditions. This project falls within the scope of the NIAAA mission to support alcohol-related research in neuroscience. This project also meets the criteria for the R21 funding mechanism by supporting investigation of new model systems with the potential for significant impact on biomedical research. The long-term goal of the work proposed in this application is to create a genetic animal model that can be distribited and used by multiple investigators in various pre-clinical neuroscience fields (including but not limited to pre-clinical AUD research) where CRFR1 signaling has been implicated as a promising therapeutic target. The proposed aims will use BAC recombineering to create a CRFR1:Cre rat in partnership with the University of Michigan Transgenics Core, will support creation of a breeding colony of CRFR1:Cre rats, and will support work that performs anatomical, electrophysiological, and behavioral validation of this new genetic animal model.

PROJECT SUMMARY This application seeks funding to enable American junior scientists to attend the next three meetings of the International Drug Abuse Research Society (IDARS). The primary goals of this unique meeting are to 1) disseminate cutting-edge addiction-related research findings to an audience that otherwise would have limited access to this information, and 2) create an international program that will foster new international pre-clinical and clinical addiction research collaborations among speakers and attendees. The first IDARS meeting occurred in 2007, and meetings have occurred every two years since that time. In 2019, the 7th IDARS meeting will convene in Casablanca, Morocco, and the site of the 2021 and 2023 meetings have not yet been determined. We are currently seeking funds to support each of the next three meetings (2019, 2021, and 2023). More specifically, we seek funds to support the travel of junior investigators from the U.S. to the next three IDARS meetings, as well as modest administrative support for the activities proposed in this application.

Adolescent alcohol use leads to persistent neural adaptations and behavioral dysregulation that increase the risk of developing alcohol use disorder (AUD). Acute alcohol reduces pain, and chronic pain (e.g., hyperalgesia) can promote alcohol drinking through negative reinforcing analgesic effects. Paradoxically, chronic alcohol produces hyperalgesia or worsen pre-existing pain states. Recently, we reported that medial central amygdala (CeA) projections to a midbrain region called the periaqueductal gray (vlPAG) are critical for mediating hyperalgesia in chronically alcohol exposed adult male rats. This chronic alcohol weakens synaptic connectivity between medial CeA and vlPAG in adult male rats, photostimulation of the CeA-vlPAG circuit rescues hyperalgesia in alcohol-dependent adult rats, and photoinhibition of this circuit produces hyperalgesia in naïve rats. Our lab and others find that antagonism of corticotropin-releasing factor type-1 receptors (CRFR1) in CeA reduces hyperalgesia associated with alcohol withdrawal, nicotine withdrawal and traumatic stress in adult rats. Our overarching hypotheses are that chronic intermittent alcohol exposure in adolescent rats (AIE) produces persist long-term effects on polymodal (i.e., mechanical and thermal) hyperalgesia that is mediated by weakened CeA-vlPAG connectivity and increased CRFR1 signaling in CeA. The CRFR1 signaling gating of CeA-vlPAG function is important for mediating AIE-induced hyperalgesia. We include preliminary data showing that 1) AIE produces rapid and long-lasting thermal and mechanical hyperalgesia during adolescence and that this effect persists into adulthood (Fig. 1), 2) AIE reduces synaptic drive and excitatory/inhibitory ratio on vlPAG-projecting medial CeA neurons in adulthood (Fig. 3), 3) CRFR1 is expressed on vlPAG projecting cells (Fig. 2), and 4) validation data for a CRFR1:cre rat for CRFR1+ cell type- specific modulation of CeA outputs (Fig. 4). Because we also propose to challenge rats with a short-lasting inflammatory pain challenge in adulthood, we have also piloted dose-response effects of carrageenan on nociception in adult Wistar rats. Here, we propose aims that will test the hypotheses that AIE produces hyperalgesia during adolescence that persists into adulthood (Specific Aim 1), that AIE reduces synaptic drive and excitatory/inhibitory balance of synaptic transmission onto vlPAG-projecting CeA neurons via a CRFR1- dependent (Specific Aim 2), and that pharmacological, circuit-based, and epigenetic modulation of CRFR1+ PAG- projecting CeA neurons will rescue AIE-induced hyperalgesia and CeA-vlPAG circuit plasticity (Specific Aim 3). Importantly, we propose specific collaborations with Research Component 6 (PI:Chandler) and 5 (PI: Crews) along with the Epigenetics Core (PI: Pandey) of the NADIA consortium. This proposal focuses on testing adolescent alcohol effects on pain-related outcomes and aligns with the overall goal of the NADIA consortium to examine the effects of adolescent alcohol exposure on the adult organism.

PROJECT SUMMARY While several medications have been approved for alcohol use disorder (AUD), many patients fail to respond or comply with the treatments. Relapse triggered by reminders of alcohol use is a particular challenge to prevent, as the underlying memories exert a powerful motivational influence over behavior and represent a lifelong relapse risk factor. Learning of these associations is supported by structural plasticity in dendritic spines, driven by training-induced actin polymerization. Memory stability is subsequently achieved by arresting actin dynamics, stabilizing the cytoskeleton. As a result, memory is impervious to actin depolymerization within minutes of learning. However, prior work in the lab discovered that the actin cytoskeleton supporting methamphetamine and amphetamine memories remains uniquely dynamic in the amygdala long after training. This enables selective, retrieval-independent disruption of these memories and associated drug seeking with a single administration of an actin depolymerizer. Because actin?s critical roles in the body limit its therapeutic potential, focus shifted to nonmuscle myosin II (NMII), a direct driver of learning-stimulated actin polymerization in spines. Genetic and pharmacologic targeting of NMII established it is a viable therapeutic target and an NIH- funded medication development project for a clinically safe NMII inhibitor is underway, currently at the stage of IND-enabling studies (UH3 NS096833). The central hypothesis in the current proposal is that a single administration of an NMII inhibitor will produce a long-lasting disruption of alcohol seeking. Aim 1 will determine the retrieval-independent ability of NMII inhibition to disrupt associations that trigger alcohol seeking, like methamphetamine and amphetamine. Interestingly, the retrieval-independent effect of NMII inhibition does not extend to memories for fear, food, spatial memory or several other drugs of abuse. However, it does disrupt the reconsolidation of memories associated with drugs of abuse, including cocaine. Aim 2 will test the effect of NMII inhibition on reconsolidation of alcohol-associated memories and seeking. However, the relatively unique approach of reactivating memory with the unconditioned stimulus (US; a small amount of alcohol), rather than the conditioned stimuli (CS; associated context and cues) will be utilized. This US-based approach circumvents the limitation inherent to CS-based reactivation strategies, which require that potentially hundreds of associations be recalled in a clinical setting to enable disruption. As proof-of-principle, preliminary data indicate that US-based reactivation renders cocaine (COC)-associated memory susceptible to NMII inhibition. The proposed work is expected to identify a new therapeutic approach to the prevention of relapse to alcohol seeking with the potential for rapid translation through the use of a first in class compound whose NIH- funded development is on track for FDA approval. Importantly, the proposed studies will also lay the groundwork for an in depth mechanistic investigation in a subsequent R01.