Ryan K. Bachtell - US grants

The overall goal of this proposal is to characterize the response of the Edinger-Westphal nucleus (EW) to alcohol. It has been shown in our lab and others that EW is robustly responsive to alcohol by various routes of administration. Because EW is a prominent target for alcohol administration, the proposed studies are intended to elucidate its mechanism and role in alcohol intoxication. The specific aims of this proposal are: 1) to characterize the neurochemistry of EW response to alcohol administration, 2) to identify candidate neurotransmitter receptors mediating activation of EW cells following alcohol administration, and 3) to identify behavioral effects of EW microinjected receptor antagonists blocking activation of EW. Neurochemistry will be analyzed using immunohistochemical and in situ techniques. Identification of receptors will involve the systemic administration of various receptor antagonists and subsequent analysis of EW activation by immunohistochemical analysis of c-Fos. Several common behavioral assays (i.e. alcohol sensitivity measures, locomotor activity, and anxiety measures) will be used to assess the role of EW neural activity following alcohol administration. It is hypothesized that activation of EW is targeted to a specific subset of neurons that are activated by specific receptor subtypes. It is further hypothesized that activation of these neurons is involved in the generation of alcohol-induced behavioral effects. The findings of this proposal will be important in understanding the mechanistic role of EW activation and its involvement in behaviors associated with intoxication. Furthermore, this investigation will aid in the pursuit of pharmacological treatments for alcoholism.

DESCRIPTION (provided by applicant): The proposed project focuses on the role of AMPA-mediated excitatory input to the nucleus accumbens (NAc) in addictive behavior associated with cocaine sensitization and self-administration. Chronic cocaine use reduces excitatory glutamatergic input to medium spiny NAc neurons containing dopamine D1 and D2 receptors. The studies in this proposal will investigate distinct interactions between excitatory input mediated by AMPA glutamate receptors and the regulation of addictive behavior by cocaine, and D1 and D2 dopamine receptors. Studies will utilize viral-mediated gene transfer in vivo to up- and down-regulate AMPA glutamate receptor function via the GluR1 subunit in the NAc. Since D1 and D2 receptors may interact with AMPA receptors via phosphorylation-dependent mechanisms, PKA- and CaMKII/PKC-resistant mutants of the GluR1 subunit will also be tested. The proposed projects will investigate the role of the GluR1 subunit in regulating cocaine sensitization, reinforcement, drug intake, and relapse behavior measured using a self-administration procedure. Studies will also investigate the pharmacological interactions that underlie this regulation with tests of D1/D2 responsiveness in locomotion and reinstatement.

DESCRIPTION (provided by applicant): Drug addiction is a brain disorder having enormous costs on society, yet effective treatments have not been elucidated. The rewarding properties of drugs of abuse contribute to initial drug taking behaviors that over time form an addiction that is characterized by increasing drug consumption and increasing susceptibility to relapse during periods of abstinence. The primary goal of the studies in this application is to enhance our understanding of the pharmacological mechanisms involved in cocaine reward and relapse that may aid in the development of more effective treatments. Chronic or repeated drug use results in several enduring perturbations in the brain circuitry that regulate motivated behavior prompting relapse in addicts. The nucleus accumbens (NAc) is a brain structure known to regulate behaviors associated with addiction (i.e. drug self-administration, relapse and reward) in both humans and rodents. Within the nucleus accumbens, subtypes of dopamine (D1 and D2) and adenosine receptors (A1 and A2A) modulate neuronal activity in a complementary, yet opposing manner. The interplay between these receptors and their subtypes is intriguing because they are: 1) localized to distinct populations of NAc neurons and 2) play reciprocal roles on the activity of adenylyl cyclase, an intracellular enzyme mediating cellular activity. It remains unclear how this reciprocal activity at the cellular level translates to the behavioral level, especially in the context of addiction. Preliminary findings demonstrate that stimulation of adenosine A2A receptors with systemic administration of A2A receptor agonists reduces relapse to cocaine seeking. Therefore, the overriding hypothesis for this application is that dopamine actions in the NAc that induce relapse will be tempered by increasing the reciprocal adenosine system in the NAc. Aim 1 will evaluate effects of 1) elevating endogenous adenosine levels and 2) directly stimulating NAc adenosine A2A receptors on the cocaine reward using a place-conditioning paradigm and cocaine reinforcement using a progressive-ratio self- administration paradigm. Aim 2 will identify the effects of 1) elevating endogenous adenosine levels and 2) directly stimulating NAc adenosine A2A receptors on cocaine relapse following chronic cocaine self- administration. Together these studies will provide a foundation for future work to identify the molecular mechanisms associated with the reciprocity of dopamine and adenosine receptors within NAc that contribute to cessation of cocaine use. PUBLIC HEALTH RELEVANCE: Drug addiction is a serious mental illness that involves significant motivational disturbances resulting in a loss of behavioral control leading to destruction of the afflicted individual as well as their surrounding social networks. This disease affects millions of people and generates enormous social and economic costs to society. The goal of this research is to better understand the disease as a whole, identify specific strategies to reduce cocaine use and evaluate major biological targets for potential therapeutic intervention to promote abstinence.

DESCRIPTION (provided by applicant): Drug addiction is a brain disorder characterized by a progression toward compulsive drug use and relapse to drug seeking during abstinence. The primary goal of this new application is to enhance our understanding of the neurobiological and neurochemical mechanisms involved in methamphetamine abuse. The nucleus accumbens (NAc) is a brain region in a complex circuit that mediates initial drug reward and relapse during periods of abstinence. In the NAc, subtypes of dopamine (DA) and adenosine (ADO) receptors are co-localized in distinct subpopulations of neurons where they play antagonistic roles on cellular functioning. Thus, neurons having co-localization of either D1/A1 or D2/A2A receptor subtypes are known to form distinct output pathways, which influence specific aspects of behavior. The opposing actions of DA and ADO receptor subtypes can be mediated by direct physical interactions (i.e. heteromeric receptors), and/or through differential activation of G- protein mediated signaling cascades. How these opposing receptor subtypes localized to distinct neuronal populations regulate addictive behavior is unknown. We have evidence to suggest that stimulation of ADO A1, but not A2A, receptor subtype inhibits methamphetamine reinforcement and relapse. These effects differ from our findings that cocaine relapse is inhibited by non-selective stimulation of A1 and A2A receptors. Our overarching hypothesis is that chronic methamphetamine use specifically disrupts ADO A1 receptor signaling in the mesolimbic DA pathway, leaving DA D1 receptors unregulated contributing to methamphetamine reinforcement and relapse. In Aim 1, experiments will assess methamphetamine-induced changes on ADO receptor subtypes within the mesolimbic system. Additional studies will identify how methamphetamine intake alters the heteromeric interactions between ADO and DA receptor subtypes. Experiments in Aim 2 are designed to dissect the differential influence of specific ADO receptor subtypes on methamphetamine reward and reinforcement using place conditioning and progressive ratio responding, respectively. Aim 3 is designed to explore how ADO receptor subtypes may differentially influence reinstatement to methamphetamine seeking. Additional studies will explore how the differential influence of ADO receptor subtypes interact with methamphetamine seeking induced by specific DA receptor subtypes in the NAc. Together these studies offer the potential to better our understanding of the brain mechanisms involved in methamphetamine abuse that appear to be substantially different than mechanisms associated with another abused psychostimulant, cocaine. These studies offer the potential to create novel treatment strategies such as A1 receptor agonists or bivalent receptor ligands (e.g. D1 antagonist-A1 agonist) that could specifically target heteromeric receptors localized to specific subpopulations of neurons within specific neural circuits that undergo methamphetamine- induced alterations.

Project Summary Drug addiction is characterized by cycles of compulsive drug use followed by periods of abstinence and relapse. Cues associated with prior drug use elicits drug craving that is highly associated with drug relapse. Addicted individuals are also more likely to exhibit changes in behavior in nondrug situations, such as increased risk taking and impulsiveness, and decreased motivation toward effortful activities. These findings suggest that chronic drug use induces persistent changes in the neural circuits that normally process associative learning and motivated behavior more generally. The ability of stimuli to subsequently elicit approach behavior has largely been attributed to dopamine signaling in the nucleus accumbens. This proposal will open a new avenue of exploration by providing unprecedented analysis of rapid, transient adenosine signaling in the nucleus accumbens by studying adenosine release kinetics in cocaine-naïve and cocaine- experienced animals. Adenosine is known to produce a multitude of functions in the brain that have largely been attributed to the basal tone of intra- and extracellular adenosine. Our current knowledge of adenosine signaling in the mesolimbic circuit is derived from studies largely examining adenosine receptor functions using genetic models and pharmacological agents. Phasic adenosine release, however, is a poorly understood phenomenon largely due to the challenges associated with measuring adenosine release with sufficient sensitivity and spatiotemporal resolution. Our preliminary findings demonstrate that fast-scan cyclic voltammetry can be used successfully in awake, behaving animals to measure phasic adenosine release under naturalistic learning conditions. The studies in Aim 1 will characterize adenosine signaling kinetics in the nucleus accumbens in cocaine-naïve and cocaine-experienced rats. These studies are expected to provide novel data illustrating that prior cocaine experience disrupts phasic adenosine signaling kinetics in the nucleus accumbens and produces differential sensitivity on adenosine signaling in response to the adenosine antagonist caffeine. The studies in Aim 2 will evaluate the role of adenosine neurotransmission in the nucleus accumbens during a Pavlovian discrimination task. These studies are expected to provide novel data illustrating that 1) adenosine signaling is rapid (sub-second) and tracks behaviorally-relevant events in real time, 2) adenosine and dopamine co-transmission is temporally coordinated to encode distinct aspects of Pavlovian associations, and 3) that cocaine experience alters both the phasic release of adenosine and disrupts the functional balance in adenosine and DA signaling in the nucleus accumbens that is necessary for learning. Together, the proposed studies will significantly advance our understanding of adenosine signaling in the nucleus accumbens for both drug addiction and naturalistic learning.

PROJECT SUMMARY Over the past 5-10 years, the opioid epidemic has become a national crisis in the United States. Currently, few good treatment options exist, and little is known about the underlying mechanisms contributing to risk for addiction and to drug effects on the brain. This project addresses both of these issues using a rat genetic model to identify genetic contributions to phenotypes associated with the development of opioid use disorders. We will identify oxycodone-related phenotypic, genotypic, and RNA expression differences within the HXB/BXH RI strains and 15 additional inbred rat strains for which genetic data are available, drawn from the Hybrid Rat Diversity Panel (HRDP). Our preliminary phenotypic data suggest that the founder strains SHR/OlaIpcv and BN-Lx/Cub, along with the ACI strain, differ on many of the phenotypic traits assessed including the self-administration of oxycodone. In Aim 1, 48 inbred rat strains will be assessed for multiple oxycodone-related behavioral phenotypes, including measures of analgesia. Quantitative trait loci (QTL) associated with these behaviors will be identified using existing genetic data. In Aim 2, we will perform RNA sequencing using tissue from the nucleus accumbens and amygdala in naïve animals and in rats following oxycodone self-administration. This will identify genes that differ by strain, which will be informative about baseline risk by genotype, and also identify genes that differ in response to oxycodone (shared and unshared across strains). Because genes do not operate independently, but work in networks and pathways, Aim 3 will employ a systems genetics approach to identify genetic networks involved in baseline differences across strains and in the response to oxycodone self-administration. Across all aims, we will compare the QTL regions, RNA expression differences, and gene network pathways to those found by others in the field using complementary rodent models and/or human studies (including our collaborator Dr. Olivier George) in order to narrow focus on priority genes and pathways.