Thomas Louis Kash - US grants

Abstract Alcohol abuse is an enormous public health problem. While there have been significant gains in our knowledge of this disorder, we lack fundamental knowledge pertaining to the biological mechanisms that contribute to the chronic relapsing pathology of alcohol abuse. It has been hypothesized that alcohol exposure modifies function in brain regions critical for regulation of emotion, and that these changes underlie persistent alterations in behavior. The serotonin (5HT) system has been implicated in the pathophysiology of a range of psychiatric conditions, most notably anxiety disorders and depression (two conditions co-morbid with alcohol use disorders). Altered 5HT signaling has been suggested to contribute to cravings and relapses, as well as the increased negative affective state, manifested as the anxiety-like behavior and dysphoria, associated with alcohol abuse. In the previous submission, we found that chronic intermittent ethanol exposure drives changes in 5HT systems in the BNST, one brain region linked with anxiety-like behavior. Additionally, we identified a 5HT sensitive micro-circuit in the BNST that regulates anxiety-like behavior. We propose here to use multiple converging approaches to test the impact of alcohol exposure on discrete aspects of this 5HT sensitive circuit. We further propose to explore the impact of disruption of these processes on alcohol-induced anxiety-like behaviors and alcohol drinking. We will test the central hypothesis that chronic intermittent alcohol exposure leads to persistent adaptations in this 5HT sensitive circuit in the BNST to drive increased anxiety-like behavior and alcohol consumption.

DESCRIPTION (provided by applicant): Alcoholism and alcohol abuse are major health problems and represent a tremendous financial burden on our society. A growing literature indicates that chronic alcohol exposure leads to an imbalance between central stress and anti-stress systems in key brain circuits that regulate emotional behavior. These imbalances can lead to pathological behavior, including increased anxiety, stress-responsivity and enhanced risk of relapse. Despite these advances identifying the role these systems play in alcohol related behaviors, there remains a gap in our knowledge of the fundamental biological, cellular and circuit mechanisms that contribute to this dysregulated behavior. In order to more effectively treat alcohol abuse, it is necessary to define the impact of chronic alcohol exposure on the in the circuitry that is critical for regulation of this behavior. Here, we propose to characterize the impact of chronic alcohol exposure on anti-stress systems, specifically neuropeptide Y (NPY) and GABAergic neuroactive steroids, in the amygdala and extended amygdala, brain regions critical for regulation of stress and anxiety-like behavior. Additionally, we will utilize inducible channel rhodopsin viruses in combination with neurochemically specific Cre-recombinase driver lines to determine the impact of chronic alcohol exposure on GABAergic circuits in these brain regions. In total, the proposed work will begin to define specific alcohol-induced cellular and circuit adaptations that are likely to play key roles in pathological behaviors associated with alcoholism.

Repeated binge alcohol drinking is a major public health problem and is thought to lead to pathophysiological alterations in brain circuitry that contribute to alcohol dependence and addiction. While complex behaviors such as ethanol consumption are likely controlled by a distributed, interconnected network of brain nuclei, corticotropin releasing factor (CRF) producing neurons within the central nucleus of the amygdala (CeA) are thought to play a crucial role in progressively driving pathological ethanol consumption. The CeA is composed of numerous neurochemically distinct neurons, and therefore determining how endogenous CRF signaling modulates neural circuits via their functional connectivity with postsynaptic targets has proven difficult due to technical limitations in evaluating specific long-range synaptic projections. To circumvent this, we propose to use optogenetic techniques coupled with brain slice electrophysiology and behavioral assays to examine the properties of CRF neuronal circuits in the extended amygdala and to determine whether activation or inhibition of CRF containing neural circuit elements can alter binge ethanol intake. We hypothesize that CRF producing neurons within the CeA project to the bed nucleus of the stria terminalis (BNST), and that activation of this pathway will be enhanced and required for repeated binge ethanol intake. We will test this hypothesis using a multi-disiclplinary approach combining both in vivo and ex vivo analysis of function. In total, the proposed research will provide essential information concerning the role that the CRF projection from the CeA to the BNST plays in binge ethanol drinking.

? DESCRIPTION (provided by applicant): The Brain Research through Advancing Innovative Neurotechnologies (BRAIN) Initiative has the ambitious goal of elucidating how neuronal ensembles interactively encode higher brain processes. To accomplish this goal, new and improved methods for both recording and manipulating neuronal activity will be needed. In this application, we focus on technologies for manipulating neuronal activity. The major significance of this application is that we will provide an enhanced chemogenetic toolbox that allows non-invasive, multiplexed spatiotemporal control of neuronal activity in domains ranging from single synapses to ensembles of neurons. To achieve this, we will provide: Chemical actuators with improved pharmacokinetics and pharmacodyamics suited for use with current DREADDs in eukaryotes ranging from Drosophila to primates (Specific Aim #1) Photo-caged CNO and other chemical actuators to provide millisecond-scale control (Specific Aim #1) Novel DREADDs and 'split-DREADDs' targeted to distinct neuronal pathways to enable multiplexed interrogation of neuronal circuits (Specific Aims #2 and 3) Chemogenetic platforms with minimized desensitization and down-regulation (Specific Aim #3)

It has been well established that alcohol can modulate a variety of molecular targets resulting in changes in synaptic function and circuit activity. Much of this body of work has focused on relatively high alcohol concentrations, leaving a gap in knowledge regarding the molecular and circuit targets of low dose alcohol. This is a critical topic, as a mechanistic understanding of these highly sensitive targets can provide an opportunity to probe how alcohol initially impacts the brain. Because of the limited data on the neuronal circuits sensitive to low dose alcohol, we are electing to approach this problem using primarily unbiased anatomical approaches. Specifically, leveraging expertise of multiple laboratories, we will capitalize on cutting edge molecular approaches and advances in whole-brain in vivo imaging to identify and interrogate highly conserved neuronal circuits and molecular targets that are sensitive to low dose alcohol. We will then use converging approaches to test the causal role of these circuits and molecules in regulating sensitivity to low dose alcohol. Therefore, taken together, these aims will provide an unprecedented opportunity to identify (Aims 1 and 2) and test the causal role (Aim 3) of brain circuits and molecules that are activated by low dose alcohol.

A growing literature indicates that chronic alcohol exposure leads to recruitment of central stress systems, such as corticotrophin releasing factor (CRF) and Dynorphin (DYN), in key brain circuits including the bed nucleus of the stria terminalis (BNST) and Central nucleus of the amygdala (CeA), leading to altered functional connectivity and pathological behavior. However, there remains a gap in our knowledge of the broader circuit mechanisms that contribute to this dysregulated behavior. Our goal is to identify alcohol induced adaptations in stress circuitry to provide a circuit based template for treatment of alcohol use disorders and co-morbid conditions. In the previous funding period, we found that activation of CRF neurons in the BNST are required for excessive alcohol consumption. Here, we propose to determine the impact of intermittent alcohol exposure and stress on plasticity in subpopulations of neurons in both the BNST and the CeA. The central hypothesis to be tested is that repeated alcohol exposure leads to increased stress induced recruitment of extended amygdala CRF and DYN, driving pathological behavior.

Chronic ethanol exposure and stress alter synaptic transmission and neuronal excitability in a number of brain circuits that control acquisition, maintenance, relapse and escalation of ethanol seeking and drinking. Work from previous INIA cycles/projects has generated hypotheses about how specific physiological changes in defined neuronal populations and circuits affect these ethanol-related behaviors. In the INIA-Stress renewal, multiple projects will test these hypotheses by manipulating and measuring neuronal and circuit function using multiple viral based genetic approaches in several distinct mouse Cre- recombinase driver lines. The central goal of this core is to provide initial validation of all mouse lines, and ongoing validation of all viral tools used in the consortium. While multiple viral tools will be validated, the Designer Receptor Activated by Designer Drug (DREADD) based chemogenetic approach has emerged as a common tool across multiple projects and will represent the bulk of the effort. The DREADD technique, based on neuromodulation by G protein-coupled receptors, has many attractive features for neuron/circuit control and has been shown to be highly effective for probing circuit function in animal models. The services of this core will be provided to fill significant gaps in the literature and provide assistance to many of the INIA-Stress projects.

It has been well established that alcohol can modulate a variety of molecular targets resulting in changes in synaptic function and circuit activity. Much of this body of work has focused on relatively high alcohol concentrations, leaving a gap in knowledge regarding the molecular and circuit targets of low dose alcohol. This is a critical topic, as a mechanistic understanding of these highly sensitive targets can provide an opportunity to probe how alcohol initially impacts the brain. Because of the limited data on the neuronal circuits sensitive to low dose alcohol, we are electing to approach this problem using primarily unbiased anatomical approaches. Specifically, leveraging expertise of multiple laboratories, we will capitalize on cutting edge molecular approaches and advances in whole-brain in vivo imaging to identify and interrogate highly conserved neuronal circuits and molecular targets that are sensitive to low dose alcohol. We will then use converging approaches to test the causal role of these circuits and molecules in regulating sensitivity to low dose alcohol. Therefore, taken together, these aims will provide an unprecedented opportunity to identify (Aims 1 and 2) and test the causal role (Aim 3) of brain circuits and molecules that are activated by low dose alcohol.

Abstract Excessive drinking cost the United States $249 billion in 2010 with 77% attributed to lost productivity, health care costs, and accidents as a result of binge drinking. Despite this high cost, the precise neural mechanisms that underlie escalated drinking remain elusive. The intermittent access (IA) paradigm is a rodent schedule of alcohol drinking that reliably leads to voluntary escalated alcohol intake and preference as well as dysregulated emotional behavior. While numerous neurochemical systems have been identified as playing a role in alcohol- related behavioral pathology, one of the most promising leads for treatment is the Kappa Opioid Receptor (KOR) and its endogenous ligand Dynorphin (Dyn). Consistent with the important role of this system in excessive alcohol drinking, we found that pharmacological blockade of KOR leads to a suppression of escalated IA drinking. Following IA, we found that dynorphin expressing neurons in the Insular Cortex show evidence of increased recruitment. Further, we found that Dyn containing neurons in the Insular Cortex (ICDyn) project to the Substantia Nigra (SN). Taken together, these preliminary data suggest that KOR signaling in the the IC to SN pathway plays a key role in alcohol abuse, potentially via regulation of dysphoric behavioral states. In this proposal we will use a strong multidisciplinary approach to test central hypothesis: Intermittent access to alcohol causes dysregulation of Dyn / KOR systems in the insular cortex to substantia nigra circuit that drive increased ethanol consumption.

The high incidence of alcohol abuse that occurs in patients with chronic pain underscores the importance of continued research towards effective treatments for both pain and alcohol use disorders. It has been found in animal models that chronic alcohol exposure can drive adaptations in the brain, which drive increased pain- related behaviors. Our long-term goal is to understand how chronic alcohol exposure can alter the neuronal circuits that regulate pain-related behavior in order to develop more effective approaches to treat alcohol induced pain. One region of particular interest for these studies is the periaqueductal gray (PAG). We have previously shown that a subpopulation of dopamine neurons in the ventrolateral PAG (PAGDA) are activated by acute alcohol and activation of these same neurons induces an anti-nociceptive effect in the hot-plate test. Furthermore, in our preliminary data, we found that activation of the outputs to the bed nucleus of the stria terminalis (BNST) could replicate this anti-nociceptive effect. These findings are noteworthy, as they identify a novel ascending anti-nociceptive pathway (PAGDA to BNST) distinct from the well-characterized descending anti-nociceptive pathway (PAG to Medulla). In keeping with the critical role of the PAGDA to BNST pathway, we found that viral deletion of CRF from the BNST can alter pain related behaviors. In addition, we have found that intermittent alcohol drinking can drive changes in pain-related behavior in mice. Taken together, these studies support the testable hypothesis that intermittent alcohol drinking drives alterations in pain related behavior, in part through disruptions in the PAGDA to BNST pathway, and that in vivo activation of this pathway can ameliorate alcohol- induced hyperalgesia.

Project Summary Alzheimer?s disease (AD) has a current economic impact of over $277 billion per year, and its prevalence is on the rise with more individuals dying from AD each year (up over 120% from 2000-2015). Recent studies have highlighted that stressful life experiences, including alcohol use disorder (AUD) may accelerate the progression of AD in the brain. Tangles of the microtubule binding protein tau forms the defining hallmark of all AD brains, and its pathological accumulation mediates AD progression. The earliest evidence of tau accumulation, decades prior to cognitive decline, occurs in a brain region that is highly engaged by stress: the pontine noradrenergic nucleus called the locus coeruleus (LC). Work in cell-based and animal models have demonstrated that 1) alcohol exposure may be linked to early tau pathogenesis and 2) pathological tau may originate in the LC and spread to synaptically connected brain regions including the bed nucleus of the stria terminalis (BNST) and the amygdala. Since withdrawal from alcohol is highly stressful, we hypothesize that intermittent exposure to high level alcohol will drive increased tau pathology in a mouse model of AD. This administrative supplement is in support of our U01 focused on examining how withdrawal from chronic intermittent ethanol (CIE) alters noradrenergic function within regions like the BNST and amygdala. Our preliminary data suggests that withdrawal from CIE bi-directionally regulates LC activity, with enhanced activation 6-8 hours in acute withdrawal, and suppression 24 hours in withdrawal. Repeated cycles of withdrawal from CIE may result in transiently high LC activation, spreading prion-like tauopathy, and further impinging LC physiology and behavior. Here we propose to test the impact of CIE in an AD setting. We will evaluate tau pathology, LC function, and AD-related learning and memory in three related but independent aims.

Abstract: This is an administrative supplement to P60-AA011605 ?Molecular and Circuit Pathogenesis of Alcohol Addiction? in response to PA-20-227 ?Administrative Supplements for Research on Dietary Supplements (Admin Supp Clinical Trial Not Allowed).? The supplement will add an aim to Project 3, ?Frontolimbic circuitry, behavioral flexibility, and adolescent alcohol history.? The parent project investigates how adolescent binge drinking (humans) or ethanol exposure (rats) impairs behavioral flexibility, with effects persisting into adulthood. We use a unique translational approach to probe the neurobiological bases of the ability to form and to flexibly overcome automatic actions and to evaluate theoretically based interventions to bidirectionally modulate behavioral flexibility. Our core hypothesis is that adolescent binge alcohol exposure promotes both an overreliance on stimulus-response action selection strategy (habit) and hypersensitivity to reward conditioning in adulthood via common alterations in shared underlying neural circuits. Moreover, the relationship between reliance on habit and sensitivity to reward conditioning is mediated by neural circuit changes impairing top-down control of responses to salient exogenous cues. The parent project uses resting-state fMRI and electrophysiology to identify differences in brain circuit function associated with impairment in overriding automatic S- R associations and sensitivity to reward conditioning. It also tests whether bidirectional manipulation of frontal cortex can promote or reduce top-down control over behavior, thereby ameliorating or mimicking the impairment associated with adolescent alcohol exposure. This supplement adds an aim to determine whether dietary choline supplementation can prevent or reverse the impairments in behavioral flexibility and associated neurochemistry induced by adolescent ethanol exposure. Overall, this work will identify objective targets for use in developing novel treatments to promote flexible, goal-directed actions over deleterious automatic actions. This approach may substantially improve our ability to cope with the public health challenges of AUDs, a leading cause worldwide of preventable death.

This NIAAA Alcohol Research Center (ARC) Grant is the catalytic element that integrates a large group of investigators across the University of North Carolina at Chapel Hill (UNC). The UNC School of Medicine Bowles Center for Alcohol Studies (BCAS), provides a foundation of administrative support and dedicated Bowles building space for alcohol research. The UNC ARC fosters interdisciplinary collaborative research on alcohol use disorders, alcohol abuse and the impact of alcohol on health and disease - exactly the goal of an NIAAA ARC. The ARC has facilitated the growth and development of UNC into an outstanding alcohol research University, among the best in the world. Research and education have always centered on a theme of molecular and cellular pathology in alcohol use disorders. This renewal focuses on the molecular mechanisms that underlie alcohol-induced circuit pathology across the stages of addiction. Ultimately, our guiding hypothesis is that alcohol-induced dysregulation of neural circuitry drives pathological behaviors and is thus the key cause of all alcohol-related pathology. This 4th renewal of the UNC ARC continues an emphasis on alcohol use disorder pathology, integrating existing and new faculty to investigate changes in neural circuits and molecular signaling in models of drinking across the proposed stages of addiction. The scope of these studies addresses the critical neurobiological changes leading to all alcoholic pathologies, i.e. the mechanisms leading to heavy chronic drinking. The ARC integrates multiple signaling systems and neurocircuits that each focus on specific mechanisms within and across brain regions. The research components also include the translational endpoint, functional magnetic resonance imaging (fMRI) connectivity of each component?s pathological circuit model in the Scientific Resource Core. This approach is expected to increase discovery, improve models and gain strength from common assessments across preclinical models to the ARC human studies. The ARC through the Information Translation Core informs practicing health professionals, health professional and college students as well as youth through specific alcohol curricula for each group to have the greatest impact on health. This proposal connects principle investigators of involving 15 independent funded faculty. By design, each research component of this ARC will focus on specific models that capture distinct endophenotypes associated with alcohol abuse. A range of molecular mechanisms that drive these circuit alterations will be explored, including kinases, cytokines and neuropeptides. This ARC renewal continues to be the catalytic element that integrates a broad group of investigators, pairing senior and junior faculty within ARC components that promote discovery across the BCAS and UNC as well as educating many within NC. This ARC proposal continues a research focus on pathogenesis of alcohol addiction with emphasis on molecular and circuit mechanisms that lead to dysfunctional brain networks, a theme at the cutting edge of neuroscience. The ARC will conduct, promote, support, and mentor research on the pathology of alcohol use disorders and educate broad groups of the public, including health professionals, families, college students and youth in North Carolina on the causes and prevention of these disorders.

Pain is currently the most common cause of long-term disability in the United States, affecting over 70 million Americans and 1.5 billion people worldwide. The first line of treatment for pain has been opioids, however their use is associated with addiction, dependence and overdose. A strategy for reducing the reliance on opioids for treatment of chronic pain is to better understand the circuits that mediate different aspects of chronic pain and identify non-opioidergic mechanisms to restore normal circuit function and behavior. Corticotropin releasing factor (CRF) signaling in the bed nucleus of the stria terminalis (BNST) has been shown to regulate both nociceptive and affective/motivational behaviors associated with pain, however the clinical utility of pharmacologically targeting the CRF system has yet to be explored. Given the critical need to develop treatments to target affective and motivational aspects of chronic pain, this could be an important path forward. We hypothesize that persistent inflammatory pain leads to increased activation of CRF signaling in the BNST leading to disrupted affective behaviors and reduced motivation. We posit that modulators of BNST function that oppose CRF signaling could provide a novel approach to investigate and treat both nociceptive and motivational/affective aspects of pain. Relevant to this, we have found in preliminary studies, a population of periaqueductal gray dopamine (PAGDA) neurons that project to the BNST that exhibit anti-nociceptive properties when activated, highlighting the possibility that dopamine in the BNST is a critical suppressor of pain-related behaviors. Previous studies have found that these PAGDA neurons play a key role in opioid induced anti-nociception. The objective of this proposal is to rigorously and mechanistically determine the role of the PAGDA to BNSTCRF circuit in inflammatory pain-driven changes in behavior, with the long term goal of identifying new treatments for inflammatory pain. We have collected strong data showing that acute pain engages this circuit, and hypothesize that this initial engagement leads to plasticity contributing to the persistence of inflammatory pain, and the development of emotional behaviors associated with inflammatory pain. This will be accomplished via three convergent aims