David A. Baker, Ph.D. - US grants

The development of effective treatments for cocaine addiction will likely await the identification of neuroadaptations produced by repeated administration of cocaine. Recently, it has been revealed that glutamate neurotransmission in the nucleus accumbens is critical for the expression of behavioral sensitization and reinstatement of cocaine-seeking behavior, two animal models of drug-induced craving and paranoid psychosis. The aim of this proposal is to characterize cocaine-induced neuroadaptations in the release and reuptake of extracellular glutamate. Toward this end, rats will receive repeated administration of saline or cocaine, and in vivo and in vitro techniques will be used to examine cocaine-induced alterations in a) vesicular release of glutamate, b) Na- dependent reuptake of glutamate, and c) cystine/glutamate exchange. The experiments outlined in this proposal will use microdialysis for in vivo measures of glutamate transmission, which is somewhat controversial due to the fact that basal levels of glutamate do not appear to be of vesicular origin. Thus, the proposed experiments will also endeavor to address this controversy by determining the origin of basal levels of extracellular glutamate as estimated by microdialysis.

DESCRIPTION (provided by applicant): Attempts to identify the neural basis of addiction have demonstrated a critical role for glutamate neurotransmission, particularly in the nucleus accumbens, in cocaine-seeking behavior. The experiments in the present proposal will examine the contribution of a novel source of glutamate, specifically nonvesicular glutamate release from cystineglutamate antiporters, to the behavioral and neurochemical effects of cocaine. These studies will test the primary hypothesis that cocaine-induced pathogenic neuroplasticity includes adaptations in cystine-glutamate antiporters, and targeting these adaptations represents a novel approach in treating addiction. Experiments in the first aim will determine whether glutamate released from cystine-glutamate antiporters blocks cocaine reinstatement by stimulating group 2/3 metabotropic glutamate receptors. This could potentially block cocaine reinstatement by preventing cocaine-induced elevations in extracellular glutamate and dopamine, which have been shown by others to be critical for cocaine reinstatement. Toward this end, the capacity of the group 2/3 mGluR antagonist to block N-acetylcysteine regulation of cocaine-induced elevations in extracellular glutamate and reinstatement will be examined. Experiments in the second aim will examine whether cocaine-induced plasticity involving cystine-glutamate antiporters emerges during the course of self-administration or withdrawal and whether these adaptations are sensitive to differential cocaine intake. In addition, these experiments will examine whether cocaine intake and length of withdrawal produce parallel changes in cocaine reinstatement and cocaine-induced plasticity involving cystine-glutamate antiporters. Finally, the last set of experiments will utilize a more clinically relevant procedure to examine the putative anti-craving efficacy of the cysteine prodrug N-acetylcysteine. Specifically, these experiments will examine the capacity of chronic administration of N-acetylcysteine to reverse the neurochemical and behavioral effects of cocaine. It is the goal of this proposal to reveal cystine-glutamate antiporters as a novel target for potential pharmacotherapies for cocaine addiction. Moreover, these experiments also have the potential to illustrate that nonvesicular release of glutamate by cystine-glutamate antiporters is a fundamental component of glutamate neurotransmission in both the normal and diseased states, which would have far reaching implications given the number of disorders that involve glutamate.

[unreadable] DESCRIPTION (provided by applicant): Schizophrenia is a debilitating disorder that affects almost 1% of the world's population. Unfortunately, current antipsychotics induce severe side effects and are ineffective in treating a number of the symptoms of schizophrenia. The development of more effective pharmacotherapies will likely await a better understanding of the neurobiology of schizophrenia. The present experiments will examine the contribution of a novel source of glutamate, specifically nonvesicular glutamate release from cystine-glutamate antiporters, to the behavioral and neurochemical effects of phencyclidine (PCP), which represents one of the most commonly used animal models of schizophrenia. The experiments in the first aim will test the hypothesis that targeting the cystine-glutamate antiporter using the cysteine prodrug N-acetylcysteine represents a novel treatment strategy for schizophrenia. Specifically, these experiments will examine the impact of N-acetylcysteine treatment on locomotor activity, deficits in working memory and social withdrawal) reduced by acute and subchronic administration of PCP. These behaviors have been used previously to gain insight into the neurobiology of schizophrenia. Additional experiments in this aim will utilize in vivo microdialysis to assess the capacity of N-acetylcysteine administration to reverse the neurochemical effects of PCP in the medial prefrontal cortex. The experiments in the second aim will test the hypothesis that a dysregulation in the activity of cystine-glutamate antiporters contributes to the pathophysiology of PCP, and potentially schizophrenia. Specifically these experiments will examine whether acute or repeated administration of PCP alters extracellular or tissue levels of cystine and glutathione in the medial prefrontal cortex. Collectively, these experiments have the potential to identify the cystine-glutamate antiporter as a novel cellular mechanism in the pathophysiology of schizophrenia, as well as to demonstrate the potential antipsychotic properties of N-acetylcysteine. [unreadable] [unreadable]

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): Abnormal glutamate signaling within corticostriatal pathways has been linked to craving in humans and cocaine seeking in rats. Unfortunately, our limited understanding of glutamate has contributed to the lack of effective, well-tolerated treatments for many CNS diseases, including drug addiction. While glutamate is described as the primary excitatory neurotransmitter in the brain, it is unclear how the many components of this complex network of transporters and release mechanisms function in an integrated manner to regulate excitatory signaling. Due to a lack of available tools that selectively target these novel mechanisms, it has been difficult to convincingly demonstrate the importance of these novel mechanisms. One such component is system xc-, a source of nonvesicular glutamate release that is primarily expressed on astrocytes. It functions by exchanging extracellular cysteine for intracellular glutamate. System xc influences synaptic activity and plasticity through the release of glutamate and dopamine in multiple brain regions. Repeated cocaine produces a persistent reduction in system xc- activity, which appears to be necessary for glutamate-induced compulsive drug seeking. In contrast, manipulations that prevent or reverse cocaine-induced changes in system xc- activity normalize glutamate levels and blunt cocaine-induced reinstatement. In humans, N-acetylcysteine has shown promise in the treatment of drug addiction and related compulsive disorders. Studies such as these indicate that system xc- function may have profound implications in revealing the cellular basis of addiction, as well as the role of astrocytes in central nervous system activity - especially if it is determined that system xc- is the primary mechanism of action for N-acetylcysteine. Efforts to manipulate system xc- in rats typically involve the use of pharmacological tools that are associated with predictable pharmacological concerns. Increasing system xc activity by direct infusion of cystine into the brain or systemic administration of a cysteine prodrug (e.g., N acetylcysteine) are both effective since the rate of cysteine-glutamate exchange is a function of the relative extracellular/intracellular concentration gradients of its substrates. Mutations in the gene giving rise to xCT, the active subunit for system xc, is present in multiple mouse strains. However, essentially every study linking system xc to glutamate homeostasis or addiction has been conducted in rats or primates. The goal of this proposal is to use the novel Zinc Finger Nucleases (ZFN) approach to mutate the Slc7a11 gene encoding xCT in rat. After creating an xCT deficient rat model (aim 1), we will verify and characterize the general phenotype (aim 2) as well as addiction-specific phenotypes (aim 3). The development and application of these technologies to generate transgenic rat strains may result in a major paradigm shift in studying the neural basis of addiction by enabling more sophisticated and highly specific manipulations in a species that better models critical aspects of human addiction.

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.

Project Summary/Abstract Dysregulation of glutamate signaling is a core component of the pathological basis of drug addiction involving cocaine and many other substances. Recent progress in neuroscience and other fields clearly establishes that the molecular and cellular basis of glutamate-encoded signaling is vastly more complex than previously recognized. In particular, it is becoming increasingly evident that astrocytes, which are among the most abundant cells in the human brain, release glutamate to produce complex regulation over neural circuit activity. This novel form of glutamate-encoded intercellular signaling may be critical to understanding the pathological glutamate produced by long-term drug use since astrocytic glutamate release mechanisms, such as system xc- (Sxc), are altered by cocaine. While these discoveries raise numerous questions that may be essential in understanding glutamate signaling in the human brain, this proposal will focus on the question, how do neurons regulate glutamate release from astrocytes to control neural network activity and behaviors relevant to cocaine addiction. We will test the hypothesis that this is achieved by the actions of the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP), which we believe to be an unrecognized component of glutamate signaling in the nucleus accumbens (NAc). In support, we have found that A) PACAP mRNA is expressed in NAc afferents, B) PACAP receptors are expressed in NAc astrocytes and neurons projecting to the substantia nigra (SN) but not the ventral pallidum (VP), C) PACAP stimulates glutamate release from astrocytes by increasing the activity of system xc- (Sxc), D) PACAP application depresses eEPSCs in NAc medium spiny neurons (MSNs) projecting to the substantia nigra (SN) but not the ventral pallidum, and E) intra-NAc micro-infusion of PACAP blocks cocaine reinstatement. In this proposal, we will test the hypothesis that PACAP is a neuropeptide that regulates glutamate release from astrocytes and glutamate receptors on neurons to provide a novel form of pathway-specific regulation of NAc efferent pathways. In Aim 1, we will examine the molecular basis of PACAP-induced increases in Sxc activity and determine whether Sxc regulation is necessary for PACAP to depress eEPSCs in NAc-SN MSNs and block cocaine reinstatement. In this aim, we will also examine if PACAP increases glutamate from astrocytic release mechanisms other than Sxc. In Aim 2, we will examine the possibility that the form of astrocyte-neuron signaling triggered by PACAP require the regulation of neuronal glutamate receptors to produce the relevant changes in physiology that gate the output of NAc efferents and behavior. We will also explore whether PACAP alters presynaptic glutamate release. In Aim 3, we will investigate the role of endogenous PACAP to learn whether this neuropeptide is an unrecognized component of glutamate signaling in the NAc, and whether it is a key determinant of drug- seeking behavior.