John R. Mantsch, Ph.D - US grants

The characterization of factors that contrbute to the onset and persistence of compulsive drug-seeking behavior is crucual for the constrution of effective treatment strategies for drug abuse. Likewise, the determination of the mechanisms through which these factors interact with this behavior is petinent to the development of potential pharmacotherapeutic agenits. The experiments described in this proposal are designes to investigate the interaction between stressful environmental stimuli, adrenocorticosteroids stimulus effects of cocaine and therefore may provide a substrate through which the environment may influence this behavior. Four specific amis designes to test this hypothesis are outlines in this proposal. The purpose of the experiments proposed in Specific Aim 1 is to determine the role of Type I and Type II adrenocorticosteroid receptors in the favilitation on intravenous cocaine self-administration by cortocosterone. The role of corticosterone in the determinative stimulus of cocaine and in the stressor-induced generalization to cocaine will be investigated in Specific Aim 2 using the corticosterone synthesis inhibitor, keteconzole. Specific Aim3 consists of experiments desgined to assess the reinstatement of cocaine self-administration by a conditioned stressor following chronic extinction. The role of corticosterone in this reinstatement will be investigated using ketoconozole. Changes in Type I and Type II receptors following chronic cocaine self-administration will be investigated in experiments proposed in Specific Aim 4.

DESCRIPTION (provided by applicant): This proposal investigates how behavioral and neurobiological responses to stressors are altered in individuals addicted to cocaine. It is hypothesized that cocaine addiction is associated with an increased behavioral reactivity to stressors that is a consequence of maladaptive alterations in the responsiveness of the hypothalamic-pituitary-adrenal (HPA) axis due to disrupted adrenocorticosteroid receptor-mediated feedback inhibition. Considering the established role of stress as a determinant of drug craving and relapse, persistent behavioral hyper-reactivity to stressors could contribute to the high incidence of relapse in abstinent addicts, a major obstacle for the effective management of cocaine addiction. The core feature of the present proposal is the use of a rat self-administration (SA) model of cocaine addiction in which addiction-related changes in stress responses will be implied from differences between rats self-administering cocaine under long-access (LgA) conditions that give rise to escalating patterns of SA and increased susceptibility to cocaine-induced relapse (i.e., 10-h access to 2.0 mg/kg/inf), rats tested under short-access (ShA rats) conditions that do not produce these effects (i.e., 3-h access to a lower cocaine dose) and non-self-administering, yoked-saline controls. Stress responses in these groups will be investigated using a series of integrated behavioral, pharmacological, and molecular studies. Differences in the behavioral reponses to stressors will be assessed by examining the reinstatement of extinguished cocaine-seeking behavior by a stressor, electric footshock, "anxiety"-Iike behaviors measured in the elevated plus-maze and defensive burying paradigms, and locomotor responses to novelty. Parallel studies will examine the activation of the HPA axis by the same stressors measured as increases in hormonal secretion, mRNA expression and glucocorticoid (GR) and/or mineralocortioid (MR) receptor activation. In order to test the hypothesis that altered responsiveness of the HPA axis is the consequence of disturbances in MR and/or GR-mediated negative feedback, MR and GR mRNA and protein expression in the pituitary gland and various brain feedback sites will be measured using in situ hybridization histochemistry and Western blot analysis. Negative feedback will be further analyzed at a functional level in experiments investigating dexamethasone-induced suppression and metyrapone-induced disinhibition of the HPA axis.

Addiction to cocaine and other illicit drugs is estimated to cost our society $181 billion which equates to $603 per U.S. citizen. The cost of addiction can be dramatically lowered through the use of treatments;unfortunately, many drugs of abuse, including cocaine, lack a single approved pharmacotherapy. Addiction to psychomotor stimulants, such as cocaine, is marked by a transition in drug consumption from a casual, recreational style of use to a more compulsive, excessive pattern that arises as a result of drug-induced changes in brain functioning. In order to develop effective treatments, it will likely be necessary to identify and target altered brain functioning underlying addiction. Towards this end, drug-induced changes in glutamate release from cystine-glutamate antiporters have been linked to pathological alterations in neural transmission and normalizing cystine-glutamate exchange blocks compulsive drug-seeking in preclinical models. Further, small-scale clinical studies using acetylcysteine to target cystine-glutamate exchange have shown modest efficacy including reduced drug craving and cocaine use. The efficacy of N-acetyl cysteine is limited due to extensive metabolism in the liver and poor passive transport into the brain. As a result, the present proposal seeks to develop novel chemical entities that are more potent and effective in targeting cystine-glutamate exchange in the brain. Aim 1 will involve the design of 32-40 compounds. Aim 2 will utilize in vitro and in vivo screening techniques to determine which compounds are most effective and potent in targeting cystine-glutamate exchange. Specifically, we will use pure glial cortical cultures to determine the capacity of brain cells to utilize the novel ligands to target cystine-glutamate exchange. Next, we will screen the most promising compounds in vivo by assessing the capacity of these ligands to bypass hepatic metabolism, enter into the brain, and target cystine-glutamate antiporters. Aim 3 will determine the potency and efficacy of these novel compounds in blocking cocaineprimed, stress-primed, and cocaine-paired cue primed reinstatement of cocaine-seeking in preclinical models of compulsive drug seeking. Collectively, these experiments have the potential to identify cystine-glutamate antiporters as a novel target in the treatment of addiction and to generate a series of compounds that may ultimately be effective in treating cocaine addiction.

DESCRIPTION (provided by applicant): Despite intense research efforts, drug addiction persists as a serious public health problem for which no effective treatment exists. Although they are likely beneficial, traditional herbal preparations are not widely considered to be viable options for the management of drug addiction, due in part to the paucity of clinical and preclinical studies examining their efficacy and safety. Recent studies by our research team and others have demonstrated that the tetrahydroprotoberberine (THPB) alkaloid, levo-tetrahydropalmatine (l-THP), isolated from crushed roots of the herb Stephania delavayi Diels (di bu long) may be useful for treating drug addiction. THPBs are naturally occurring alkaloids found in plants from the botanical genera Corydalis and Stephania and are contained in many traditional Chinese herbal preparations. Two of these species, Corydalis ambigua (yan hu suo) and Stephania tetranda (fang ji) are among the 50 fundamental herbs in Chinese herbology and have been used for their sedative, neuroleptic and analgesic properties. Several THPBs, most notably l-THP and the levo isomer of stepholidine (l-SPD) have pharmacological profiles that make them ideally suited for treating drug addiction, including antagonist and/or partial agonist effects at a variety of dopamine (DA) receptors. Further characterization of the effectiveness of THPBs in preclinical models of drug abuse should facilitate our understanding of which THPB-containing preparations will have the greatest utility for treating drug-dependent populations. This proposal builds on preliminary data demonstrating that l-THP reduces cocaine-seeking behavior in preclinical rat models and that l-THP reduces drug craving and relapse in recovering heroin addicts. The two isolated THPB constituents selected for testing (l-THP and l-SPD) have distinct properties that may make them safer and more effective compared to existing DA receptor antagonist drugs. The proposed studies will use preclinical rat self-administration based procedures to compare the effects of l-THP and l-SPD with those of the non-selective DA receptor antagonist, fluphenazine, and an "atypical" anti- psychotic in paradigms designed to examine the ability of these agents, when administered acutely or chronically, to curb cocaine use and to prevent the relapse of cocaine seeking induced by drug re-exposure, the onset of stress, and exposure to drug-associated conditioned cues. The occurrence of potential undesirable effects as a consequence of chronic administration of these compounds, including sedation, extrapyramidal symptoms, weight gain abuse liability, and hepatotoxicity will also be examined. It is anticipated that, through preclinical investigation of the effectiveness and safety of isolated THPBs, we will be able to advance the use of herbal preparations containing these THPBs as viable treatments for drug addiction.

DESCRIPTION (provided by applicant): For many cocaine addicts, drug use is a stress-driven behavior. Previous research has shown that stressor- induced cocaine seeking is mediated in part by corticotropin-releasing factor (CRF) stimulation of a neurobiological pathway involving dopamine (DA) neurons in the ventral tegmental area (VTA) that likely project to nucleus accumbens (NA). The regulation of this pathway by stressors and CRF appears to emerge as a consequence of prior cocaine use and therefore may be relevant to the onset of addiction. We have demonstrated that rats provided long access to cocaine for self-administration (SA) each day (LgA rats) display greater reinstatement in response to a stressor (electric footshock; EFS) or administration of CRF directly into the VTA, compared to rats provided shorter daily drug access (ShA rats). Furthermore, we have found that that the establishment of heightened stressor-induced reinstatement appears to require elevation of glucocorticoids (GCs) at the time of earlier LgA SA, suggesting that the induction of addiction-related neuroplasticity leading to heightened stressor-induced drug seeking following excessive patterns of cocaine use is a GC-dependent process. The goal of this proposal is to test the hypothesis that cocaine addiction is associated with an emergent or augmented CRF regulation of dopaminergic neurons projecting from the VTA to a subregion of the NA, the shell, that is attributable to increased VTA CRF receptor (CRF-R) expression or function and leads to a heightened susceptibility to stressor-induced craving and relapse. Furthermore, we hypothesize that the establishment of heightened CRF regulation is dependent upon elevated GCs and activation of GC receptors (GR) at the time of earlier drug exposure. These hypotheses will be tested in this proposal in three specific aims. In the first aim we will further investigate the relationship between augmented VTA CRF sensitivity and stressor-induced reinstatement through a series of experiments that examine the relative time-courses of altered CRF- and EFS-induced reinstatement as they relate to changes in CRF-R expression and trafficking and determine the involvement of CRF-R subtypes in the VTA in stressor-induced cocaine seeking through antagonist administration and receptor knockdown by RNA-interference. In the second aim we will examine the role of altered CRF actions in the VTA in the augmented stressor-induced regulation of NA DA and its involvement in stressor-induced cocaine seeking using in vivo microdialysis in and administration of DA receptor antagonists into the NA core and shell. In the final aim, we will examine the GC- dependence of the effects of LgA SA on stressor-induced cocaine seeking and NA DA neurotransmission and CRF-R expression/trafficking through a surgical adrenalectomy and diurnal GC replacement approach that eliminates evoked GC secretion while maintaining normal diurnal patterns of plasma GCs and through central infusion of the GR antagonist, RU-486 via osmotic minipump. Understanding the neurobiological processes through which stressor-induced regulation of cocaine use is established in cocaine addicts should facilitate the development of new and more effective treatment approaches, particularly for subpopulations of cocaine addicts whose drug use is stress-driven.

DESCRIPTION (provided by applicant): An insidious aspect of addiction is that afflicted individuals are at risk of relapse even after extended periods of abstinence. Stressful life events are important contributors to relapse in recovering cocaine addicts, but the mechanisms by which they influence motivational systems are poorly understood. Studies suggest that stress may set the stage for relapse by increasing the sensitivity of brain reward circuits to drug-associated stimuli. This proposal seeks to elucidate the mechanisms by which stress, through increases in glucocorticoid hormones, influences relapse vulnerability. We have previously shown that treatment of rodents with stress levels of glucocorticoids does not lead to reinstatement of drug-seeking behavior, but potentiates reinstatement in response to a dose of cocaine that, by itself, is not sufficient to trigger relapse. In parallel to its behavioral effect, corticosterone pretreatment also potentiates the effects of low-dose cocaine on extracellular dopamine concentration in the nucleus accumbens, suggesting that glucocorticoids may potentiate drug seeking by enhancing dopaminergic neurotransmission in this critical reward-processing brain region. We are examining the role of organic cation transporter 3, a high-capacity dopamine transporter that is acutely and directly inhibited by glucocorticoids, in mediating the effects of glucocorticoids on dopaminergic neurotransmission, cocaine relapse, and motivated behavior in rodents. Because of a lack of pharmacologically specific inhibitors for OCT3, we are using two different genetic approaches to test the hypothesis that corticosterone potentiates cocaine-induced dopaminergic neurotransmission and drug-seeking behavior by inhibiting OCT3-mediated clearance of dopamine in the nucleus accumbens. In the first aim, we will determine the impact of corticosterone-induced inhibition of dopamine clearance in the nucleus accumbens on dopamine signaling and drug relapse by using in vivo microdialysis and fast-scan cyclic voltammetry to measure dopamine concentration and clearance in cocaine-seeking animals. In the second aim, we will determine the role of OCT3 in the behavioral and neurochemical effects of corticosterone by examining corticosterone effects on drug-seeking behavior and nucleus accumbens dopamine signaling in animals genetically modified to lack OCT3 expression either globally or specifically in the nucleus accumbens. In the third aim, we will test the hypothesis that corticosterone-induced decreases in dopamine clearance modulate reward sensitivity and natural reward processing. These findings will thoroughly characterize a novel mechanism by which stress hormones can rapidly regulate dopamine signaling and contribute to the impact of stress on drug intake and motivated behavior in general.

DESCRIPTION (provided by applicant): Despite its prevalence, decades of research have failed to yield an FDA-approved medication for the treatment of cocaine addiction. The lack of viable pharmacotherapeutic approaches is attributable, in part, to fundamental gaps in our understanding of the situations and underlying neurobiological processes that promote relapse to drug use in abstinent cocaine addicts. It is well established that stress is an important contributor to drug relapse. Considering its pervasive and unavoidable nature, this relationship between stress and drug use is highly problematic. Recent findings indicate that the role of stress in relapse is more complex than once believed and that, rather than simply triggering cocaine use, stress can indirectly promote drug relapse by heightening sensitivity to drug-associated stimuli. Our team has established a self-administration/reinstatement rat model for examining this stage-setting role for stress in cocaine seeking. Using this model, we have demonstrated that the ability of a stressor (electric foot shock) to promote reinstatement by an otherwise subthreshold priming dose of cocaine requires increases in corticosterone and activation of CB1R cannabinoid receptors. Moreover, our preliminary findings have localized this mechanism to the prelimbic cortex (PLC), a source of glutamatergic projections to the nucleus accumbens core that have been shown to be critical for cocaine use. This collaborative multi-PI proposal brings together a multi-disciplinary team of scientists to test the hypothesis that, durin stress, corticosterone enhance endocannabinoid signaling in the PLC, thereby suppressing GABAergic neurotransmission and disinhibiting pyramidal neurons that project to the nucleus accumbens core. This stage- setting mechanism allows for subthreshold doses of cocaine to induce reinstatement. Aim 1 of the proposal will examine the role of stress-induced increases in endocannabinoids in the PLC, with a focus on 2-AG, and the resulting activation of CB1 receptors in the stress-induced potentiation of cocaine seeking. Aim 2 will investigate the role of corticosterone regulation of endocannabinoid signaling in the PLC in the effects of stress on cocaine seeking and the mechanisms through which this regulation occurs. Aim 3 of the proposal will examine how these stress-induced alterations in the PLC disrupt GABAergic regulation of pyramidal neurons that project to the nucleus accumbens core to promote cocaine use. The findings from these proposed experiments have the potential to lead to the development of new and more effective treatment approaches for the management of cocaine addiction. However, the importance of defining the mechanisms through which stress alters cortical regulation of this pathway extends beyond addiction and should guide our understanding of how stress regulates motivated behavior in general and therefore how it contributes to a range of neuropsychiatric conditions.

DESCRIPTION (provided by applicant): Despite its prevalence, decades of research have failed to yield an FDA-approved medication for the treatment of cocaine addiction. The lack of viable pharmacotherapeutic approaches is attributable, in part, to fundamental gaps in our understanding of the situations and underlying neurobiological processes that promote relapse to drug use in abstinent cocaine addicts. It is well established that stress is an important contributor to drug relapse. Considering its pervasive and unavoidable nature, this relationship between stress and drug use is highly problematic. Recent findings indicate that the role of stress in relapse is more complex than once believed and that, rather than simply triggering cocaine use, stress can indirectly promote drug relapse by heightening sensitivity to drug-associated stimuli. Our team has established a self-administration/reinstatement rat model for examining this stage-setting role for stress in cocaine seeking. Using this model, we have demonstrated that the ability of a stressor (electric foot shock) to promote reinstatement by an otherwise subthreshold priming dose of cocaine requires increases in corticosterone and activation of CB1R cannabinoid receptors. Moreover, our preliminary findings have localized this mechanism to the prelimbic cortex (PLC), a source of glutamatergic projections to the nucleus accumbens core that have been shown to be critical for cocaine use. This collaborative multi-PI proposal brings together a multi-disciplinary team of scientists to test the hypothesis that, durin stress, corticosterone enhance endocannabinoid signaling in the PLC, thereby suppressing GABAergic neurotransmission and disinhibiting pyramidal neurons that project to the nucleus accumbens core. This stage- setting mechanism allows for subthreshold doses of cocaine to induce reinstatement. Aim 1 of the proposal will examine the role of stress-induced increases in endocannabinoids in the PLC, with a focus on 2-AG, and the resulting activation of CB1 receptors in the stress-induced potentiation of cocaine seeking. Aim 2 will investigate the role of corticosterone regulation of endocannabinoid signaling in the PLC in the effects of stress on cocaine seeking and the mechanisms through which this regulation occurs. Aim 3 of the proposal will examine how these stress-induced alterations in the PLC disrupt GABAergic regulation of pyramidal neurons that project to the nucleus accumbens core to promote cocaine use. The findings from these proposed experiments have the potential to lead to the development of new and more effective treatment approaches for the management of cocaine addiction. However, the importance of defining the mechanisms through which stress alters cortical regulation of this pathway extends beyond addiction and should guide our understanding of how stress regulates motivated behavior in general and therefore how it contributes to a range of neuropsychiatric conditions.

PROJECT SUMMARY Aversive environmental events influence the daily lives of all people by altering their emotional states, decision making, and motivated behavior. For individuals with substance abuse disorders who are attempting to remain abstinent, these unfortunate events are clinically relevent, as they are frequently cited as a principle cause of relapse. In order to develop strategies to protect against this important determinant of relapse, it is essential to characterize the mechanisms through which aversive stimuli influence motivational neural circuitry. To this end, several decades of research have identified the nucleus accumbens (NAc) as a critical limbic/motor interface, heavily regulated by dopamine, where affective and associative reward information directly influence behavioral output. Unfortunately, the manner by which aversive stimuli regulate dopamine signaling remains poorly understood, with several studies producing conflicting results. We have identified an aversion signal, initiated by aversion-induced reductions in dopamine concentration, that is associated with increased striatal activity and drug seeking. The objective of this proposal is to determine how aversive stimuli regulate NAc dopamine signaling and the mechanisms through which reductions in dopamine alter neuronal activity in the NAc to shape behavior. To accomplish this objective, the proposal brings together a multi-disciplinary team that will use in vivo fast scan cyclic voltammetry, in vivo electrophysiology, and ex vivo slice electrophysiology to examine the independent contributions of reduced dopamine signaling and increased striatal activity to a panel of aversion- related behaviors, including drug seeking, and both reward and punishment sensitivity. In Aim 1 we will test the hypothesis that aversive stimuli, via reductions in NAc dopamine activate a subpopulation of aversion-responsive D2-like receptor-expressing NAc neurons to produce aversion-related behavioral responses. In Aim 2 we test the hypothesis that aversive stimuli increase corticotropin releasing factor (CRF) in the ventral tegmental area (VTA) and reduce dopamine in the NAc through a CRFR1- and GABAB receptor-dependent regulation of Girk channels on VTA DA neurons that project to the NAc shell. In Aim 3 we will examine upstream pathways that mediate the effects of aversive stimuli on NAc dopamine and behavior and will test the hypothesis that a beta adrenergic receptor-regulated pathway from the ventral bed nucleus of the stria terminalis to the VTA represents one such pathway. Understanding how aversive stimuli alter NAc dopamine signaling and how such alterations encode behavior has implications for understanding and treating a range of stress-related neuropsychiatric conditions including addiction and depression.