Olivier George, Ph.D. - US grants

The Animal Models/Biochemical Measurement Core of The Scripps Research Institute Alcohol Research Center (TSRI-ARC) will provide a variety of behavioral and bioanalytical services to meet the specific needs of the Center at large. The first goal of the Core is to provide animals engaged in excessive drinking or have a history of excessive drinking using the intermittent access to ethanol drinking (IAE) model or chronic ethanol-induced dependence (CEID) model to TSRI-ARC and Center at Large investigators (Specific Aim 1). In addition, the Core will also supervise all changes in equipment and procedures and any refinement of the current animal models to ensure that standardized procedures are used across all laboratories. The second goal of the Core is to perform biochemical measurements, such as blood alcohol, cortisol/corticosterone, and ACTH levels and brain amino acid and endocannabinoid content, in support of all Center-related projects by establishing state-of-the-art techniques and an efficient, user-friendly website to schedule services, monitor progress, and receive data (Specific Aim 2). Finally, the last goal of the Core is to further characterize the animal models of excessive drinking to be utilized by the TSRI-ARC, including exploring the effect of excessive drinking on the transition to dependence and the effect of excessive drinking on the brain stress system by characterizing the consequence of a history of binge drinking (IAE model) on the brain stress system and the transition to alcohol dependence. Translational dependent measures also used in the Clinical Neurobehavioral Research Component will be employed (Specific Aim 3). The Animal Models/Biochemical Measurement Core will enable Center investigators to enrich the interpretational power of their experiments through investigations of behavioral, neurochemical, neuroendocrine, and pharmacokinetic processes contributing to alcohol dependence.

DESCRIPTION (provided by applicant): Drug addiction, and cocaine addiction in particular, is considered a chronic relapsing disorder in which subjects episodically administer the drug and ultimately transition from nondependent drug use to the compulsive drug use of addiction. Knowledge of the neurochemical/neurocircuitry changes that provide the motivational basis for vulnerability for increased drug intake with extended access are beginning to provide insights into the neurobiological changes that may lead to vulnerability to escalation in drug intake and relapse. During the previous funding period an animal model of extended access to cocaine self-administration that resembles the compulsivity of addiction has been established and validated. Animals with extended access increase their drug intake (escalation) over time and show increased motivation to obtain the drug (increased progressive- ratio [PR] responding) and show reward deficits during abstinence from the drug (elevation in brain reward thresholds). Work during the previous funding period also has shown that extended access to cocaine decreases basal release in the mesolimbic dopamine system, and inhibitory G-protein function, and increased sensitivity to dopamine antagonists and partial agonists. Chronic cocaine, cocaine withdrawal, and stress- induced reinstatement of cocaine self-administration also were associated with increases in CRF, norepinephrine, and dynorphin function in the extended amygdala. The overall hypothesis under test is that the neuroplastic changes in the extended amygdala lead to a cascade of neurobiological changes that initially involve excessive dopamine release, subsequent loss of dopamine function, and subsequent activation of CRF and dynorphin brain stress systems. A subhypothesis is that the binge-induced loss of dopamine function observed with psychostimulant drugs interacts with the brain stress systems in the basal forebrain to further promote and sustain the increased cocaine intake with extended access. To test this hypothesis, the present proposal will validate a stress-induced increase in cocaine self-administration paradigm and explore the role of CRF and dynorphin in stress-induced escalation (Specific Aims 1 and 2), explore the role of decreases in dopamine function measured by Go in the dopamine system in drug and stress-induced escalation (Specific Aim 3) and explore the interaction of Go in the ventral striatum and CRF and dynorphin function in the extended amygdala in stress-induced escalation (Specific Aim 4). These studies will not only provide new data in the role of stress in the compulsivity that is associated with cocaine dependence, but may also provide key markers for the development of dependence and ultimately key targets for understanding vulnerability and developing novel treatments.

DESCRIPTION (provided by applicant): Alcoholism is a chronically relapsing disorder characterized by a compulsion to seek and take alcohol and has been linked to dysregulation of the brain arousal and emotional systems critically involved in both the positive and negative reinforcement important for the development of alcoholism. Dependence and the vulnerability to relapse has been argued to include counteradaptive neurochemical events within the brain emotional systems normally used to maintain emotional homeostasis (Koob and Le Moal, 2005, 2008, Appendix) and produce compulsive drinking via negative reinforcement mechanisms. In the previous funding period, we characterized key roles of increased activity of the brain stress systems corticotropin-releasing factor (CRF) and norepinephrine and decreased activity in the neuropeptide Y anti-stress system in dependence-induced drinking. The research plan of the present competitive renewal will be to continue the studies on the mechanisms of neuroadaptation within brain arousal-stress systems during the development of excessive drinking induced by dependence with a focus on newly identified brain arousal-stress systems within the neurocircuitry of the extended amygdala: vasopressin, hypocretin (orexin) and nociceptin. The overall hypothesis under test in the present proposal is that increased vasopressin and hypocretin activity and decreased nociceptin activity in the central nucleus of the amygdala and/or basolateral amygdala, bed nucleus of the stria terminalis, and nucleus accumbens are responsible for the enhanced drinking associated with a dependence, and that these systems interact via an activation of CRF in the extended amydala. The Specific Aims are: To explore the role of vasopressin (SpA 1), orexin (SpA 2), and nociceptin (SpA 3) in the extended amygdala on increased ethanol self-administration during withdrawal in rats using administration of selective antagonists/agonists, and to explore the role of corticotropin releasing factor (CRF) in the actions of vasopressin, orexin and nociceptin in rats during dependence (SpA 4). To accomplish these aims, a series of studies with administration of selective receptor subtype antagonists and/or agonists in rats and neuroanatomical studies with measures of neuronal activation (cFos) using a reliable animal paradigm of excessive ethanol self-administration in dependent rats will be employed. Results will provide novel and innovative insights into the neural substrates of emotional dysregulation in key brain motivational areas that form the basis of excessive drinking associated with dependence, and as such, will provide new targets for diagnosi of vulnerability, prevention and treatment of alcohol dependence.

The Pilot Component The Scripps Research Institute Alcohol Research Center- (TSRI-ARC) will provide a program for conducting pilot studies that advance the research agenda of the TSRI-ARC on neuroadaptive mechanisms associated with the transition from binge drinking to dependence and potentially generate independent grant applications relevant to the focus of the Center at Large. The principal goals of the Pilot Project Program are to enable the TSRI-ARC to explore new directions for innovative research related to the center's goals (Specific Aim 1) and to recruit scientists new to alcohol research into the field, thereby exposing center investigators to fresh perspectives and methods (Specific Aim 2). In general, it is expected that the Pilot projects will provide seed funding to qualified investigators to enable them to gather sufficient preliminary data to attract support for testing a new hypothesis through R01, R21, KO1 or similar mechanisms (or to eliminate a new hypothesis as not worth pursuing). Ultimately the aim of the Pilot Studies component is to provide the TSRI-ARC with a flexible means to develop and explore new research activities or directions and unique scientific opportunities that have the potential to evolve into independently-funded research projects. The proposed pilot studies are integrated into the overall TSRI-ARC program and involve innovative approaches, which will have translational impact across the research components. Emphasis has been placed, in the first two years, on studies that will develop innovative new exciting neurobiological approaches to neurocircuitry targets and on human translational studies for the neuroadapatations associated with excessive drinking and dependence. As demonstrated with the success of our previous Pilot programs, we anticipate that the results of the pilots will help launch innovative lines of research and in parallel new careers in the study of the neurobiology of alcoholism.

DESCRIPTION (provided by applicant): Alcoholism is a chronic relapsing disorder characterized by compulsive use and loss of control over intake. Alcoholism produces significant cost to society in the United States and worldwide. The excessive use of alcohol has long been shown to have detrimental effects on prefrontal cortex function including impairment in decision making, executive function, and memory and learning. In addition, many studies have established that brain stress systems are activated by excessive drinking. However, few studies have explored how chronic alcohol and activation of the brain stress system interacts with the prefrontal cortex to produce cognitive dysfunction and contribute to compulsive alcohol intake. The overall hypothesis of this project is that activation of the brain stress systems [corticotropin releasing factor (CRF) and norepinephrine (NE)] in the prefrontal cortex disrupts cognitive function that exacerbates the powerful motivation for alcohol seeking associated with compulsive use. To address this hypothesis, the present proposal has been designed to (1) To further characterize the time course of development of cognitive dysfunction and compulsive drinking in animal models of excessive drinking. (2) To determine the pattern of changes in the stress systems in the prefrontal cortex in the development of compulsive drinking and (3) To test if chronic inactivation of the stress systems in the prefrontal cortex prevents cognitive deficits and the development of compulsive alcohol drinking. The approach combines neuroanatomical, neuropharmacological, and molecular techniques and the use of innovative animal models of alcohol dependence, such as the escalation-binge and dependence-induced drinking models, combined with very specific measures of compulsive alcohol drinking, working memory and perseverative responding. Understanding the neurobiological mechanisms within the prefrontal cortex that produce cognitive deficits and contribute to the compulsivity of ethanol dependence will provide key information for understanding the individual differences in vulnerability to develop alcoholism and new targets for the treatment and prevention of alcoholism.

Project Summary / Abstract A major issue in the alcohol field is the lack of animal models of the voluntary induction and maintenance of alcohol dependence. Rats will readily self-administer alcohol, but the amount of alcohol consumed is very low and thus does not produce blood alcohol levels that are clinically relevant for alcoholism (100-200 mg% for several hours per day). In the previous funding period, we successfully developed a novel model of the voluntary induction and maintenance of alcohol dependence in rats using chronic intermittent ethanol vapor self- administration (EVSA). In this model, animals exhibit severe addiction-like behaviors, including somatic signs of withdrawal, anxiety-like behavior, hyperalgesia, and responding despite adverse consequences (on a progressive-ratio schedule of reinforcement) after 6 weeks of EVSA. The current proposal seeks to further develop this paradigm, identify the neuronal networks of the voluntary induction of alcohol dependence, and characterize a novel model of voluntary ?extreme binging.? Extreme alcohol binging is a critical societal issue and one of the priorities of the NIAAA Strategic Plan 2017-2021. Binge and extreme binge drinking are particularly troubling because they increase the risks for blackouts, alcohol poisoning, sexual assault, sexually transmitted diseases, poor academic performance, and developing AUD. By combining alcohol vapor self- administration with state-of-the-art brain mapping techniques, we will identify neuronal networks that drive alcohol drinking and relapse after the voluntary induction of alcohol dependence. Our data show that both the passive and active administration of alcohol vapor produces the escalation of alcohol drinking, increases the motivation to obtain alcohol, and increases relapse, but the voluntary induction of dependence is characterized by the specific recruitment of dorsomedial striatum (DMS) and dorsolateral striatum (DLS) neurons during withdrawal. We also propose to further characterize alcohol drinking and relapse in animals that are previously made dependent by EVSA vs. animals that are made dependent by passive exposure to alcohol vapor. Finally, we propose to validate and fully characterize a novel model of extreme alcohol binging, in which animals self- administer alcohol vapor to the point of reaching blood alcohol levels of ~400 mg%, losing consciousness (?blacking out?), and exhibiting short-term memory loss. Results from these studies will provide a full characterization of alcohol drinking and relapse in animals that voluntarily develop dependence and will unveil neuronal circuits that underlie the voluntary induction and maintenance of alcohol dependence. Results from this proposal will also provide a novel animal model to study and characterize extreme alcohol binging in rodents. The proposed studies have the potential to have a sustained and powerful impact on the field of addiction because they could unveil neuronal targets that are specifically recruited during the voluntary induction of alcohol dependence and extreme binging that could be used to develop novel therapeutic approaches.

DESCRIPTION (provided by applicant): Tobacco use continues to be a major public health problem in the United States, with one-third of users becoming dependent, and tobacco smoking is the leading avoidable cause of death in the United States. In the United States alone, exposure to tobacco smoke by nonsmokers (second-hand smoke exposure) is believed to cause an additional 50,000 deaths from heart disease and cancer, 1 million lung and ear infections, and increased asthma episodes in an additional 1 million children. The rapid growth of electronic cigarette use may have dramatic consequences on the population. The electronic cigarette industry generated over $1 billion in revenue in the United States last year and is predicted to pass traditional cigarette sales by 2047. Moreover, the danger of electronic cigarette is that they are marketed as safe to use, but virtually no research has been conducted on the consequences of nicotine vapor exposure on the brain and development of nicotine dependence. Second-hand smoke and electronic cigarette use lead to clinically significant blood nicotine levels. We hypothesize that they will produce important neuroadaptations that contribute to increased risk of developing dependence and facilitating relapse. The present proposal focuses on animal studies to investigate the specific effects of inhalation of nicotine at levels similar to electronic cigarettes on the development of nicotine dependence and relapse. The first hypothesis is that chronic exposure to nicotine vapor at concentrations similar to electronic cigarettes, or even lower, will affect key behaviors related to nicotine dependence (anxiety-like behavior, hyperalgesia). The second hypothesis is that chronic exposure to nicotine vapor at concentrations similar to electronic cigarettes, or even lower, will increase the escalation of nicotine intake and provoke relapse after a period of abstinence. To determine the exact effect of nicotine inhalation, we developed an innovative inhalation system to expose rats to known concentrations of nicotine in the air. The use of this new animal model of nicotine inhalation will provide a unique opportunity to determine the lowest level of nicotine exposure that is required to produce behavioral changes that may facilitate the acquisition of and relapse to nicotine dependence. The results of these studies will provide (i) the lowest dose of nicotine vapor required to increase the risk for nicotine dependence, (ii) evidence of whether electronic cigarette use is associated with increased risk of the acquisition of and relapse to nicotine dependence, and (iii) important information for nicotine dependence prevention efforts and policymakers.

Abstract  Twin studies suggest that approximately 50% of the vulnerability to cocaine use disorder is determined by  genetic factors, but genome-­wide association studies (GWAS) in humans have only begun to identify specific  genes that confer this risk. One major impediment to studies of cocaine use disorder is the complexity of the  phenotype and the lack of control of environmental variables. We propose a complementary approach that  leverages a multidisciplinary, highly collaborative consortium that combines next-­generation sequencing with  state-­of-­the-­art behavioral screening in a unique, genetically diverse, nonhuman animal model. The primary  goal of this proposal is to identify gene variants that are associated with increased vulnerability to compulsive  cocaine use by performing a GWAS in N/NIH heterogeneous stock rats. We will use the most relevant animal  model of cocaine use disorder (i.e., escalation of intravenous cocaine self-­administration) and highly  standardized measures of controlled and compulsive cocaine self-­administration. To increase the impact of  these findings and facilitate translational and basic research studies on the mechanisms underlying compulsive  cocaine use, we will also establish a data/tissue repository (CocaineBioBank.org) from behaviorally and  genetically characterized animals that will allow researchers to further investigate the cellular and molecular  mechanisms underlying compulsive cocaine use and identify the biological changes associated with the  expression of specific gene variants. This project is likely to have a sustained and powerful impact on the field  because it will (1) characterize the transition from controlled to compulsive cocaine use in male and female  outbred rats, (2) identify genes associated with compulsive cocaine use, (3) create the CocainBioBank which  will provide free access to brain, kidney, liver, spleen, ovary, testis, adrenal, and blood samples with a variety  of tissue preservation protocols that will allow the generation of induced pluripotent stem cells as well as  neuroanatomical, molecular, biochemical, and pharmacological studies on behaviorally/genetically  characterized animals.   

Project Summary/Abstract  A  key  issue  in  the  alcohol  field  is  the  lack  of  knowledge  on  the  discreet  neuronal  ensembles  that  are  responsible  for  excessive  alcohol  drinking  in  alcohol-­dependent  subjects.  This  is  a  major  obstacle  for  the  alcohol field because investigations of the neuronal ensembles that mediate excessive alcohol drinking would  provide  a  comprehensive  understanding  of  the  neuronal  circuits  that  causally  contribute  to  alcohol  dependence. The recent development of pharmacogenetic and optogenetic tools that allow selective targeting  of  specific  neuronal  ensembles  is  a  tremendous  opportunity  to  bridge  this  gap  in  the  literature.  The  central  hypothesis  of  this  proposal  is  that  activation  of  the  parabrachial  nucleus  (PBN)  and  central  nucleus  of  the  amygdala  (CeA)  during  withdrawal  is  responsible  for  the  recruitment  of  a  set  of  discreet  neuronal  ensembles  that are scattered throughout the brain and ultimately responsible for excessive drinking and the emergence of  negative emotional states in dependent rats. We obtained robust preliminary results that show that selectively  targeting these neuronal ensembles produces long-­lasting reversal of excessive alcohol drinking in dependent  rats,  identifies  a  causal  relationship  between  these  ensembles,  and  reveals  novel  neuronal  pathways  that  contribute  to  alcohol  dependence.  Specific  Aim  1  characterizes  the  role  of  the  CeA  and  PBN  withdrawal  neuronal  ensembles  in  excessive  alcohol  drinking  in  dependent  rats.  Specific  Aim  2  dissects  the  role  of  the  different  CeA  CRF  pathways  in  the  recruitment  of  the  neuronal  ensembles  and  excessive  alcohol  drinking  in  alcohol dependence. Results from these studies have the potential to have a strong and lasting impact in the  field  because  our  approach  will  improve  our  understanding  of  the  neurobiological  mechanisms  of  alcohol  dependence and identify novel neuronal populations and circuits that specifically control behaviors associated  with alcohol dependence.                                     

  Abstract A key issue in the alcohol field is identification of the neuronal circuits responsible for the loss of control over alcohol drinking in dependence. Loss of control has long been hypothesized to result from dysfunction of the frontal lobes and subsequent disinhibition (or activation) of subcortical systems that underlie stress, anxiety, reward, pain, and habits. Recent work has also causally implicated an ensemble in the central nucleus of the amygdala as required for excessive drinking and anxiety-like behavior during abstinence. The goal of the Neurocircuitry Component is to integrate these views by identifying the cortical pathways upstream (cortico- amygdalar) and downstream (amygdalo-cortical) of the CeA that contribute to the compulsivity of alcohol- drinking and negative emotional behavior. The first hypothesis in this proposal is that reduced infralimbic and greater anterior insula glutamatergic outflow to the amygdala promotes more compulsive drinking, with greater anxiety- and irritability-like behavior in abstinence. Reciprocally, the second hypothesis is that dysregulation of the prefrontal cortex partially results from activation of a neuronal ensemble in the CeA that promotes compulsive alcohol drinking and abstinence symptoms via recruitment of direct and indirect amygdalo-cortical projections to the infralimbic cortex and anterior insula. The third hypothesis is that dysregulated activation in cortico-amygdalo-cortical loops can be normalized by a selective glucocorticoid receptor antagonist or microtubule-associated protein-type 2 (MAP-2) ligand. This project may have a sustained and powerful impact on the field by (1) causally implicating specific cortico-amygdalo-cortico loops in the compulsivity of alcohol drinking and emergence of negative emotional states during abstinence and 2) identifying a circuit mechanism for the efficacy of glucocorticoid receptor antagonists and MAP-2 ligands to reduce drinking and negative emotional symptoms.  

Project SummaryAbstract Epidemiological studies suggest that genetic variability significantly contributes (20-60%) to the analgesic response to opioids and vulnerability to opioid use disorder. However, genome-wide association studies (GWASs) in humans have only begun to identify specific genes that confer this risk. One major impediment to studies of opioid use disorder is the complexity of the phenotype and lack of control of environmental variables. We propose a complementary approach that leverages a multidisciplinary, highly collaborative consortium that combines next-generation sequencing with state-of-the-art behavioral screening in a unique, genetically diverse, nonhuman animal model. The primary goal of this proposal is to identify gene variants that are associated with greater vulnerability to compulsive oxycodone use, tolerance to the analgesic effects of oxycodone, development of withdrawal-induced hyperalgesia, and sensitivity to FDA-approved medications by performing a GWAS in N/NIH heterogeneous stock rats. We will use the most relevant animal model of oxycodone use disorder (i.e., escalation of intravenous oxycodone self-administration) and highly standardized measures of compulsive oxycodone self-administration combined with longitudinal assessments of pain thresholds. This project is likely to have a sustained and powerful impact on the field because it will (1) characterize the transition from controlled to compulsive oxycodone use and its comorbidity with hyperalgesia in male and female outbred rats, (2) identify genes associated with compulsive oxycodone use, the preclinical efficacy of current medication (e.g., buprenorphine), and the analgesic/hyperalgesic effects of chronic oxycodone use, and (3) facilitate follow-up studies by creating a repository that contains brain and blood with a variety of tissue preservation protocols that will facilitate follow-up and replicative studies by allowing the generation of induced pluripotent stem cells and neuroanatomical, molecular, biochemical, and pharmacological studies on behaviorally/genetically characterized animals.

Abstract  Twin studies suggest that approximately 50% of the vulnerability to cocaine use disorder is determined by  genetic factors, but genome-­wide association studies (GWAS) in humans have only begun to identify specific  genes that confer this risk. One major impediment to studies of cocaine use disorder is the complexity of the  phenotype and the lack of control of environmental variables. We propose a complementary approach that  leverages a multidisciplinary, highly collaborative consortium that combines next-­generation sequencing with  state-­of-­the-­art behavioral screening in a unique, genetically diverse, nonhuman animal model. The primary  goal of this proposal is to identify gene variants that are associated with increased vulnerability to compulsive  cocaine use by performing a GWAS in N/NIH heterogeneous stock rats. We will use the most relevant animal  model of cocaine use disorder (i.e., escalation of intravenous cocaine self-­administration) and highly  standardized measures of controlled and compulsive cocaine self-­administration. To increase the impact of  these findings and facilitate translational and basic research studies on the mechanisms underlying compulsive  cocaine use, we will also establish a data/tissue repository (CocaineBioBank.org) from behaviorally and  genetically characterized animals that will allow researchers to further investigate the cellular and molecular  mechanisms underlying compulsive cocaine use and identify the biological changes associated with the  expression of specific gene variants. This project is likely to have a sustained and powerful impact on the field  because it will (1) characterize the transition from controlled to compulsive cocaine use in male and female  outbred rats, (2) identify genes associated with compulsive cocaine use, (3) create the CocainBioBank which  will provide free access to brain, kidney, liver, spleen, ovary, testis, adrenal, and blood samples with a variety  of tissue preservation protocols that will allow the generation of induced pluripotent stem cells as well as  neuroanatomical, molecular, biochemical, and pharmacological studies on behaviorally/genetically  characterized animals.   

Project SummaryAbstract Epidemiological studies suggest that genetic variability significantly contributes (20-60%) to the analgesic response to opioids and vulnerability to opioid use disorder. However, genome-wide association studies (GWASs) in humans have only begun to identify specific genes that confer this risk. One major impediment to studies of opioid use disorder is the complexity of the phenotype and lack of control of environmental variables. We propose a complementary approach that leverages a multidisciplinary, highly collaborative consortium that combines next-generation sequencing with state-of-the-art behavioral screening in a unique, genetically diverse, nonhuman animal model. The primary goal of this proposal is to identify gene variants that are associated with greater vulnerability to compulsive oxycodone use, tolerance to the analgesic effects of oxycodone, development of withdrawal-induced hyperalgesia, and sensitivity to FDA-approved medications by performing a GWAS in N/NIH heterogeneous stock rats. We will use the most relevant animal model of oxycodone use disorder (i.e., escalation of intravenous oxycodone self-administration) and highly standardized measures of compulsive oxycodone self-administration combined with longitudinal assessments of pain thresholds. This project is likely to have a sustained and powerful impact on the field because it will (1) characterize the transition from controlled to compulsive oxycodone use and its comorbidity with hyperalgesia in male and female outbred rats, (2) identify genes associated with compulsive oxycodone use, the preclinical efficacy of current medication (e.g., buprenorphine), and the analgesic/hyperalgesic effects of chronic oxycodone use, and (3) facilitate follow-up studies by creating a repository that contains brain and blood with a variety of tissue preservation protocols that will facilitate follow-up and replicative studies by allowing the generation of induced pluripotent stem cells and neuroanatomical, molecular, biochemical, and pharmacological studies on behaviorally/genetically characterized animals.