Joyce Besheer, Ph.D. - US grants

Chronic tobacco use continues to be a worldwide public health problem. Presently, there is an emphasis on smoking cessation which is prompting the development of new pharmacotherapies and treatment programs. Unfortunately, relapse after a brief period of smoking cessation is common. Most people attribute the relapse to the occurrence of withdrawal symptoms. Examination of behavioral and neurochemical changes that occur during nicotine withdrawal will help provide insight into nicotine relapse. Better understanding of these processes will result in the development of more effective cessation and prevention programs. The experiments in the present proposal seek to examine the effects of nicotine withdrawal on two behavioral preparations widely used in our laboratory. The first preparation assesses place preference conditioned by access to novel objects (i.e., novelty reward). Rats receive repeated access to novel object in one environment and no objects in another equally experienced environment. In a post-conditioning test that occurs in the absence of any objects, rats show a preference for the environment previously paired with novel. The second preparation assesses preference for a novel object over a familiar object (i.e., novelty seeking). In this task rats are presented with an object for a specific amount of time. The rats are returned to the environment 1 h later, but now have free-access to a novel object and the previously experienced object. During this novel- object test rats spend more time interacting with the novel object than the familiar object. Based on related research and theory, we predict that nicotine withdrawal will affect rats' response to novelty as assessed by these tasks.

DESCRIPTION (provided by applicant): The overall goal of this proposal is to provide mentored career development for a young scientist. The training and research support detailed in this proposal will enable the Candidate to utilize a multidisciplinary approach to understand factors that modulate the stimulus properties of ethanol. Importantly, this training will contribute to her development as an independent investigator. The Candidate will receive broad-based training in alcoholism research as well as several methods that will be required to address the research plan including immunohistochemistry techniques, confocal microscopy, stereotaxic surgeries and microinjection of drugs. The research plan will focus on the role of metabotropic glutamate receptors (mGluRs) in ethanol's discriminative stimulus effects. mGluRs have been shown to influence GABAA and glutamate functioning which are major components of ethanol's stimulus properties. The Candidate will test the hypothesis that mGluRS and mGluR2/3 can modulate ethanol discrimination by interaction with GABAA and NMDA receptor systems. Specific Aim 1 will utilize behavioral pharmacology techniques to determine if an antagonist of mGluRS and an agonist of mGluR2/3 will modulate ethanol discrimination, and the ethanol-like stimulus properties of GABAA positive modulators and NMDA antagonists. Specific Aim 2 will use dual-label immunohistochemistry with confocal visualization to evaluate if mGluRS and mGluR2/3 are co-expressed with GABAA and NMDA receptor subunits in ethanol discrimination trained rats. Specific Aim 3 will use immunohistochemisty techniques (Fos-like immunoreactivity) to assess brain regional involvement in ethanol discrimination and in mGluRS and mGluR2/3 modulation of ethanol discrimination and ethanol substitutes (diazepam and MK-801). Specific Aim 4 will use microinjection techniques to assess ethanol discrimination and the substitution of diazepam and MK-801 after administration of the mGluRS antagonist and the mGluR2/3 agonist into specific brain regions that show high levels of mGluR co-expression with GABAA and NMDA receptors as determined from Aim 2 and show anatomical involvement as determined from Aim 3. We predict that these experiments will reveal a modulatory role of mGluRS and mGluR2/3 in ethanol discrimination. Further, incorporating these techniques is highly innovative, and these studies will generate novel findings regarding the involvement of mGluRS and mGluR2/3 in the stimulus properties of ethanol. Understanding these mechanisms is important to basic science and has numerous implications for development of therapeutic interventions in alcoholism, especially given that the discriminative stimulus properties of drugs can be important determinants of abuse liability.

DESCRIPTION (provided by applicant): Compromised HPA axis function, including dysregulation of glucocorticoid feedback and blunted response to stressors, is a characteristic feature of alcoholism. Impaired HPA axis function is also evident in populations that are at risk for increased alcohol drinking such as nonalcoholic individuals with a family history of alcoholism, and individuals suffering from some forms of depression and other mood and anxiety disorders. Given these key observations that link dysregulated HPA axis function and susceptibility to increased drinking, there is a necessity in the field to understand the mechanisms underlying the interaction. We propose that a possible behavioral mechanism is alteration of the interoceptive effects of alcohol. All drugs of abuse share the common attribute that they produce interoceptive/subjective effects in humans. These interoceptive cues can potently influence drug taking and seeking behaviors. Emerging evidence shows that metabotropic glutamate receptors, specifically subtype 5 (mGluR5) regulate the interoceptive effects of alcohol. Using a model of repeated HPA axis activation (corticosterone (CORT) in the drinking water) that results in HPA axis dysregulation, we show reduced sensitivity to the interoceptive effects of alcohol, and a parallel decrease in both mGluR5 and phosphorylated ERK1/2 immunoreactivity in the nucleus accumbens. These findings suggest the primary hypothesis of this application: repeated HPA axis activation/dysregulation leads to adaptations in mGluR5 in the n. accumbens that functionally regulate the interoceptive effects of alcohol. Given that interoceptive drug effects can influence drug taking behavior, and the associations between compromised HPA axis function and susceptibility to increased alcohol drinking, we also propose the corollary hypothesis that: the CORT-induced reduction in sensitivity to the interoceptive effects of alcohol is associated with increased drinking. The studies in this application have four separate by integrated Specific Aims. First, experiments will characterize the effects of CORT exposure on the interoceptive effects of alcohol. The information gained from these studies will establish a relation between HPA axis activation/dysregulation and the interoceptive effects of alcohol. Second, experiments will examine adaptations within the mGluR5 system that occur after CORT exposure, and will pharmacologically manipulate mGluR5, which will provide novel information as to the relationship between changes in mGluR5 expression, and the expression of ERK1/2 and CREB, which are downstream targets, and the interoceptive of effects of alcohol. Third, using brain site-specific microinjections, studies will assess the functional involvement of mGluR5 in the expression of alcohol's interoceptive effects after repeated HPA axis activation/dysregulation, by evaluating the efficacy of mGluR5 compounds to restore sensitivity to the interoceptive effects of alcohol. Lastly, experiments will address the relation between the interoceptive effects of self-administered alcohol and alcohol self-administration and will determine a functional role for mGluR5 in the nucleus accumbens and amygdala in these behaviors. Together, the studies in this proposal have the potential to move the field forward by providing insight into how HPA axis activation/dysregulation can influence the interoceptive effects of alcohol, a critical mechanism of drug seeking. PUBLIC HEALTH RELEVANCE: Compromised HPA axis function is a characteristic feature of alcoholism and is also evident in populations that are also at risk for increased alcohol drinking such as nonalcoholic individuals with a family history of alcoholism. HPA axis dysregulation can result from chronic HPA axis activation. The purpose of this application is to examine how HPA axis activation/dysregulation alters the interoceptive effects of alcohol (a major controlling process of drug seeking behavior), possible mechanisms underlying the changes, and how these changes can impact alcohol drinking.

DESCRIPTION (provided by applicant): Adolescence is a critical developmental period during which alcohol use is often initiated. In addition to the detrimental effects that alcohol exposure can have on the brain during this sensitive developmental period, adolescent alcohol use is also associated with increased risk of dependence in adulthood. Thus, understanding the mechanisms underlying adolescent drinking is critical. A possible behavioral mechanism for increased alcohol drinking during adolescence and later in adulthood is diminished sensitivity to the interoceptive effects of alcohol. All drugs of abuse share the common attribute that they produce interoceptive/subjective effects in humans and animals, and these interoceptive effects are a major controlling process of drug seeking behavior and abuse liability. The interoceptive effects of alcohol and other drugs are routinely measured in animals using operant drug discrimination methods, in which animals are trained to discriminate between drug and non-drug interoceptive cues. To date, the alcohol and drug abuse field has not had the appropriate tools by which to adequately evaluate drug interoceptive effects during the brief adolescent period in rodents. This is due, in part, to the lengthy training procedures required in operant drug discrimination techniques. As such, little is known about interoceptive drug effects in adolescence. To address this gap in knowledge, the studies in this proposal incorporate a Pavlovian discrimination training method. This procedure is ideal for assessment of the interoceptive effects of alcohol within a short developmental window because as we show in Preliminary Studies, the rats quickly acquire the discrimination in a time frame that spans the adolescent developmental period. The studies in this application have two separate but integrated Specific Aims. In Specific Aim 1, experiments assess sensitivity to the interoceptive effects of alcohol in adolescents using the Pavlovian discrimination procedure. Adolescence is a critical period during which GABAergic and NMDA receptor systems undergo neurochemical remodeling and maturation. Interestingly, these systems are known to be major components of the alcohol drug cue (in adults) using operant discrimination techniques. Therefore, experiments will determine mechanistic involvement of GABAA and NMDA receptor-related systems underlying differential sensitivity to alcohol. The information gained from these studies could suggest a behavioral mechanism for increased alcohol drinking by adolescents. That is, adolescents may drink more alcohol because they are less sensitive to the interoceptive effects of the consumed alcohol. Further, given that interoceptive drug effects are a primary determinant of abuse liability, it is possible that experiencing the effects of alcohol during adolescence alters the perception of the alcohol cue in adulthood. Accordingly, another advantage of the Pavlovian discrimination procedure is that direct comparison between sensitivity to alcohol in adolescence and later in adulthood can be made. Thus, in Specific Aim 2, experiments will examine whether sensitivity to the alcohol cue in adulthood is altered after experiencing alcohol in adolescence. Further, given the developmental changes in GABAergic and glutamatergic mechanisms during adolescence the underlying mechanism (GABA or NMDA-related systems) will also be examined. Together, the exploratory and highly novel studies in this proposal, that incorporate a training method that has not been utilized to examine the interoceptive effects of alcohol, aim to extend our understanding of the interoceptive effects of alcohol in adolescence, and potentially contribute a behavioral mechanism that can inform our understanding of alcohol drinking in adolescence and increased risk for alcohol use in adulthood.

DESCRIPTION (provided by applicant): Compromised HPA axis function, including dysregulation of glucocorticoid feedback and blunted response to stressors, is a characteristic feature of alcoholism. Impaired HPA axis function is also evident in populations that are at risk for increased alcohol drinking such as nonalcoholic individuals with a family history of alcoholism, and individuals suffering from some forms of depression and other mood and anxiety disorders. Given these key observations that link dysregulated HPA axis function and susceptibility to increased drinking, there is a necessity in the field to understand the mechanisms underlying the interaction. We propose that a possible behavioral mechanism is alteration of the interoceptive effects of alcohol. All drugs of abuse share the common attribute that they produce interoceptive/subjective effects in humans. These interoceptive cues can potently influence drug taking and seeking behaviors. Emerging evidence shows that metabotropic glutamate receptors, specifically subtype 5 (mGluR5) regulate the interoceptive effects of alcohol. Using a model of repeated HPA axis activation (corticosterone (CORT) in the drinking water) that results in HPA axis dysregulation, we show reduced sensitivity to the interoceptive effects of alcohol, and a parallel decrease in both mGluR5 and phosphorylated ERK1/2 immunoreactivity in the nucleus accumbens. These findings suggest the primary hypothesis of this application: repeated HPA axis activation/dysregulation leads to adaptations in mGluR5 in the n. accumbens that functionally regulate the interoceptive effects of alcohol. Given that interoceptive drug effects can influence drug taking behavior, and the associations between compromised HPA axis function and susceptibility to increased alcohol drinking, we also propose the corollary hypothesis that: the CORT-induced reduction in sensitivity to the interoceptive effects of alcohol is associated with increased drinking. The studies in this application have four separate by integrated Specific Aims. First, experiments will characterize the effects of CORT exposure on the interoceptive effects of alcohol. The information gained from these studies will establish a relation between HPA axis activation/dysregulation and the interoceptive effects of alcohol. Second, experiments will examine adaptations within the mGluR5 system that occur after CORT exposure, and will pharmacologically manipulate mGluR5, which will provide novel information as to the relationship between changes in mGluR5 expression, and the expression of ERK1/2 and CREB, which are downstream targets, and the interoceptive of effects of alcohol. Third, using brain site-specific microinjections, studies will assess the functional involvement of mGluR5 in the expression of alcohol's interoceptive effects after repeated HPA axis activation/dysregulation, by evaluating the efficacy of mGluR5 compounds to restore sensitivity to the interoceptive effects of alcohol. Lastly, experiments will address the relation between the interoceptive effects of self-administered alcohol and alcohol self-administration and will determine a functional role for mGluR5 in the nucleus accumbens and amygdala in these behaviors. Together, the studies in this proposal have the potential to move the field forward by providing insight into how HPA axis activation/dysregulation can influence the interoceptive effects of alcohol, a critical mechanism of drug seeking.

DESCRIPTION (provided by applicant): Compromised HPA axis function, including dysregulation of glucocorticoid feedback and blunted response to stressors, is a characteristic feature of alcoholism. Impaired HPA axis function is also evident in populations that are at risk for increased alcohol drinking such as nonalcoholic individuals with a family history of alcoholism, and individuals suffering from some forms of depression and other mood and anxiety disorders. Given these key observations that link dysregulated HPA axis function and susceptibility to increased drinking, there is a necessity in the field to understand the mechanisms underlying the interaction. We propose that a possible behavioral mechanism is alteration of the interoceptive effects of alcohol. All drugs of abuse share the common attribute that they produce interoceptive/subjective effects in humans. These interoceptive cues can potently influence drug taking and seeking behaviors. Emerging evidence shows that metabotropic glutamate receptors, specifically subtype 5 (mGluR5) regulate the interoceptive effects of alcohol. Using a model of repeated HPA axis activation (corticosterone (CORT) in the drinking water) that results in HPA axis dysregulation, we show reduced sensitivity to the interoceptive effects of alcohol, and a parallel decrease in both mGluR5 and phosphorylated ERK1/2 immunoreactivity in the nucleus accumbens. These findings suggest the primary hypothesis of this application: repeated HPA axis activation/dysregulation leads to adaptations in mGluR5 in the n. accumbens that functionally regulate the interoceptive effects of alcohol. Given that interoceptive drug effects can influence drug taking behavior, and the associations between compromised HPA axis function and susceptibility to increased alcohol drinking, we also propose the corollary hypothesis that: the CORT-induced reduction in sensitivity to the interoceptive effects of alcohol is associated with increased drinking. The studies in this application have four separate by integrated Specific Aims. First, experiments will characterize the effects of CORT exposure on the interoceptive effects of alcohol. The information gained from these studies will establish a relation between HPA axis activation/dysregulation and the interoceptive effects of alcohol. Second, experiments will examine adaptations within the mGluR5 system that occur after CORT exposure, and will pharmacologically manipulate mGluR5, which will provide novel information as to the relationship between changes in mGluR5 expression, and the expression of ERK1/2 and CREB, which are downstream targets, and the interoceptive of effects of alcohol. Third, using brain site-specific microinjections, studies will assess the functional involvement of mGluR5 in the expression of alcohol's interoceptive effects after repeated HPA axis activation/dysregulation, by evaluating the efficacy of mGluR5 compounds to restore sensitivity to the interoceptive effects of alcohol. Lastly, experiments will address the relation between the interoceptive effects of self-administered alcohol and alcohol self-administration and will determine a functional role for mGluR5 in the nucleus accumbens and amygdala in these behaviors. Together, the studies in this proposal have the potential to move the field forward by providing insight into how HPA axis activation/dysregulation can influence the interoceptive effects of alcohol, a critical mechanism of drug seeking.

? DESCRIPTION (provided by applicant): Smoking, ethamphetamine (meth), and alcohol use remain a significant health burden. Notably, clinical studies have identified sex differences in response and sensitivity to the drugs, as well as in abstinence and relapse rates. Interestingly, preclinical investigations on these topics have not been as prevalent. There is an extensive literature establishing that stimuli associated with drugs can be powerful modulators of drug-seeking behavior, and can promote relapse following a period of abstinence. One factor of importance to the development and tenacity of nicotine dependence is the ability of the pharmacological effects of nicotine to serve as an interoceptive stimulus for other reinforcing events (e.g., peer acceptance, alcohol, work breaks, stress relief, etc.). Consequently, over time a smoker has an opportunity to develop a rich appetitive conditioning history with the interoceptive stimulus effects of nicotine. An important prediction from theories of conditioned reinforcement and incentive motivation is that an excitatory conditioned stimulus, nicotine in our case, should be able to support other behavior. Using preclinical models, one goal of the present application is to directly test the hypothesis that following an appetitive interoceptive conditioning history with nicotine, the nicotine stimulus will acquire additional motivational valu that will exacerbate reinstatement of methamphetamine (Aim 1) and alcohol (Aim 2) seeking to a greater degree than not having this conditioning history. Another goal is to determine whether sex differences exist in these behaviors. The proposed studies will fill an important gap in the literature, as it is unknown whether interoceptive conditioning involving a drug stimulus can alter the extent of reinstatement that had been maintained by a different drug. This is an innovative conceptual framework by which to approach the subject of relapse in addiction. Accordingly, confirmation of this hypothesis will advance current theory as it reveals a potential new process that may serve as a risk factor in relapse. Opening a new area of scientific inquiry, advancement of theory, and improvement of animal models are consistent with the objectives of the R21 mechanism - Exploratory/Developmental Research Grant Award.

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.

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.

There is high comorbidity between the prevalence of post-traumatic stress disorder (PTSD) and alcohol use disorders (AUD) and this is a growing research area of interest. Current animal models have led to important insights into the neural circuits and molecular mechanisms involved in PTSD alone. However, despite the prevalence of AUD in patients with PTSD, there is a knowledge gap regarding the underlying neurobiology of comorbidity. This is due, in part, to the lack of animal models assessing the co-occurrence of PTSD and alcohol misuse. This is a critical topic, as validated animal models can lead to a better mechanistic understanding of the co-occurrence of these disorders, which may ultimately lead to more effective treatment strategies. In this application, we will combine a predator odor (PO) exposure model of PTSD with alcohol self- administration to model the emergence of maladaptive drinking patterns following development of PTSD from a traumatic experience. Studies in Aim 1 will focus on establishing and validating an animal model of PTSD and alcohol self-administration, along with cutoff behavioral criteria for use in the model. Studies in Aim 2 will probe adaptations in the Group II family of regulatory metabotropic glutamate receptors (mGluR2/3) as there is growing evidence for glutamatergic dysregulation in both PTSD and AUD. Studies in Aim 3 will focus on examining neural circuitry involving the nucleus reuniens, a midline thalamic nucleus, that has been emerging in the glutamatergic circuitry and symptom profile of PTSD, stress and depression. These studies represent an innovative strategy to advance understanding of mechanisms underlying susceptibility to increased alcohol drinking in PTSD.

Project Summary Animal and human studies suggest that elevation of neuroactive steroids may address many of the behavioral pathologies associated with alcohol use disorders. The goal of this project is to evaluate the hypotheses that elevated steroidogenesis in the ventral tegmental area will reduce operant ethanol self-administration and the escalation of voluntary drinking following deprivation in male and female alcohol preferring (P) rats. Endogenous neuroactive steroids will be elevated by viral vector-mediated gene delivery of the biosynthetic enzyme P450scc that converts cholesterol to pregnenolone. Our recent studies demonstrate that vector-mediated delivery of P450scc to the VTA reduces ethanol self-administration and increases local expression of (3?,5?)-3-hydroxypregnan-20-one (3?,5?-THP, allopregnanolone) (Cook et al., 2014). We now propose to extend these studies by examining effects in both male and female P rats, probing effects on deprivation-induced drinking and targeting the vector to tyrosine hydroxylase (TH) neurons in the VTA. We will examine vector and behavioral specificity as well as the persistence of effects. Aim 1 will investigate if elevation of steroidogenesis by gene delivery of P450scc to VTA alters A) operant ethanol self-administration in non?dependent male and female P rats or B) deprivation-induced drinking in ethanol dependent male and female P rats. Aim 2 will examine whether TH neuron-specific elevation of steroidogenesis in VTA alters A) operant ethanol self-administration in non?dependent male and female rats or B) deprivation-induced drinking in ethanol dependent male and female P rats. These studies will increase our understanding of the role of VTA neuroactive steroids in ethanol reinforcement, anxiety-like behavior and escalated drinking following ethanol deprivation.

Abstract The present application requests a diversity supplement to 1R01AA026537 (PI ? Joyce Besheer). The support is requested to facilitate the postdoctoral training of an outstanding and talented Hispanic American female postdoctoral student Dr. Laura Ornelas. The purpose of this career and research training plan is to facilitate the career development of Dr. Laura Ornelas, who has the explicit goal of becoming an independent academic researcher specializing in neurobehavioral aspects of alcohol abuse. With the quality research training and career development activities proposed and outlined in the application, she will acquire the skills necessary for advancing her training and contributing to the growth of an independent and critical thinker, and a scientist who will be a highly competitive faculty candidate. Dr. Ornelas? training will be under the supervision of Dr. Joyce Besheer, an Associate Professor in the Department of Psychiatry, and a member of the Bowles Center for Alcohol Studies (BCAS) at the University of North Carolina ? Chapel Hill. Dr. Besheer has shown a strong commitment to student mentoring at all training levels, undergraduate, graduate, postdoctoral. The proposed work is within the scope, but not redundant with the studies of the parent grant. The experiments which Dr. Laura Ornelas will conduct are designed to extend these studies by focusing on 1) the brain regional neuronal response to the predator odor (PO) and re-exposure to the PO-paired context, 2) the consequence of predator odor exposure and re-exposure to the context on the re-initiation of self-administration following an abstinence period (relapse-like drinking), 3) and the involvement of the nucleus reuniens related brain circuitry in this re-initiation of drinking. Consequently, the conduct of this work will provide a richer and more comprehensive understanding of neuroadaptive changes and the lasting effects of PO exposure on alcohol self-administration. This application includes a thorough and well-described mentoring plan for Dr. Ornelas? research and career development. This research/career development plan is designed to ensure that Dr. Ornelas will broaden her scientific skill set, and gain experience and develop skills necessary for a career in the biomedical sciences, specifically in alcohol research, which is Dr. Ornelas? career objective.

Alcohol has long been recognized as a teratogen. Cannabinoids (CBs), however, have been popularly believed to be safe during pregnancy. Behavioral observations in children prenatally exposed to marijuana, and animal studies of THC and the more recently introduced, highly potent synthetic cannabinoids (SCBs), contradict this belief and indicate that cannabinoid exposure is, indeed, teratogenic. Importantly, in rodents, early prenatal exposure to SCBs causes birth defects similar to those in fetal alcohol syndrome. During early gestation, both alcohol and cannabinoids independently induce brain abnormalities involving ventral midline-derived structures, including the hypothalamus, septal area and striatum. We hypothesize that these two drugs, when administered together during the early periods of gastrulation and neurulation, will have a significant persistent teratogenic effect as evidenced by altered response to novelty/habituation processes, corticosterone responses and alcohol self-administration in adolescent offspring. Using a rat model of prenatal teratogen exposure during the key developmental events of gastrulation and neurulation, the proposed research will explore this premise, addressing the following Specific Aims: Aim 1 will examine the effect of prenatal alcohol+SCB exposure on locomotor activity and habituation to a novel environment, and changes in corticosterone in response to a mild stressor. Aim 2 will test the hypothesis that early gestational alcohol+SCB exposure will potentiate alcohol self- administration, motivation to self-administer alcohol and alcohol-seeking behavior. These data will be important information on the functional effects of prenatal co-exposure to two widely used drugs and how this may increase adolescent drinking behaviors.

Project Summary/Abstract Neuroimmune activation and signaling contribute to increased ethanol drinking and possibly alcoholism. Preclinical studies have shown that ethanol preference and drinking are modulated by neuroimmune gene expression and that cycles of ethanol exposure lead to long-lasting changes in expression of neuroimmune genes, including neuronal Toll-like receptor 3 (TLR3). These are highly relevant findings given that we have shown that TLR3 expression in post-mortem human alcoholic brains correlates with lifetime ethanol consumption. Therefore it is important to understand the interaction between neuroimmune signaling and ethanol drinking, and the impact of neuroimmune activation on neurobiological changes that can drive escalated ethanol drinking. As such, the focus of this application is on the effects of the TLR3 agonist Polyinosinic:polycytidylic acid (Poly(I:C)) on escalations in operant ethanol self-administration (SA), the underlying neuroimmune and glutamatergic changes (specifically in relation to metabotropic glutamate receptor 2/3; mGluR2/3) in the nucleus accumbens (Acb) and insular cortex (IC), and the role of the IC?Acb circuit. As such, the primary hypothesis of this application is that neuroimmune signaling (i.e., TLR3 activation) induces glutamatergic adaptations in the Acb and IC?Acb circuitry that drive escalations in ethanol SA. Experiments in Aim 1 will examine changes in neuroimmune signaling and glutamatergic targets in Acb and IC in ethanol SA trained rats following Poly(I:C)-TLR3 activation, and parallel functional glutamatergic adaptations in the AcbC in collaboration with Component 5. Experiments in Aim 2 will examine the ability of an mGluR2/3 agonist and antagonist to block and potentiate, respectively, the escalation in ethanol SA following TLR3 activation, and the role of Acb mGluR2/3. Experiments in Aim 3 will utilize a chemogenetic approach by the incorporation of Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) to examine the effects of inhibition and activation of IC?AcbC projections on the escalation in ethanol SA following TLR3 activation. Next, fMRI will be used to assess cortico-accumbal functional connectively as a consequence of ethanol SA history, TLR3 activation, and mGluR2/3 activation in conjunction with the Scientific Core. This proposal tests novel neuroimmune signaling and IC-reward circuits that integrate into, benefit from and contribute to the Alcohol Research Center?s focus on neurocircuit plasticity induced by ethanol. The proposed studies investigate novel mechanisms and circuits associated with the development of alcohol use disorders that could lead to new therapies.