Rita Fuchs Lokensgard - US grants

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): High impulsivity is associated with vulnerability to drug dependence, drug relapse, and treatment failure in cocaine addicts. Using a novel animal model, our laboratory has demonstrated that, remarkably, exposure to a cocaine-paired environmental context alone produces a state of increased impulsivity, indicated by greater delay discounting behavior (i.e., choice of a small immediately available reward over a larger reward available with a time delay) in a cocaine-paired context than in an unpaired control context [11]. Based on additional exciting preliminary data and the existing impulsivity literature the proposed exploratory R21 project will test the overarching hypothesis that distinct neuronal ensembles within the lateral orbitofrontal cortex (lOFC) promote, whereas in the prelimbic cortex (PrL) oppose, drug context-induced impulsive decision making (DCIDM). Furthermore, bidirectional regulation of DCIDM arises from the differential activation of excitatory projection neurons and GABAergic interneurons within these neuronal ensembles upon exposure to the cocaine-paired context. Specific Aim 1 will be to test the hypothesis that drug context-activated neuronal ensembles in the lOFC and PrL are critical for promoting and suppressing DCIDM, respectively. Experiments will use the Daun02 pharmacogenetic method to site-selectively lesion putative cocaine context-activated neuronal ensembles within the lOFC or PrL in c-fos-lacZ transgenic rats. We will evaluate the effects of these manipulations on delay discounting behavior in the cocaine-paired or an unpaired control context. We predict that lesion of cocaine context-reactive lOFC neuronal ensembles will inhibit, whereas lesion of cocaine context-reactive PrL neuronal ensembles will potentiate DCIDM. Specific Aim 2 will be to characterize the phenotype of drug context-activated neuronal subpopulations within the neuronal ensembles that regulate DCIDM in c-fos-lacZ transgenic rats. We will use double-label immunohistochemistry with confocal microscopy to quantify Fos/CaMKII- and Fos/GAD67-immunoreactive (IR) cell bodies spared by Daun02 in the brains of rats from Aim 1. We predict that exposure to a cocaine-paired context elicits preferential excitatory neuronal activation in th lOFC, and preferential GABA-ergic neuronal activation in the PrL, neuronal ensembles of interest. This will be reflected by Daun02-induced preferential Fos/CaMKII-IR cell loss in the OFC and preferential Fos/GAD67-IR cell loss in the PrL, in tissue collected after cocaine-paired context exposure during the DCIDM test. Overall, this R21 project will explore the neurobiological underpinnings of the newly discovered DCIDM phenomenon using a novel animal model, state-of-the-art pharmacogenetic techniques, and double-label immunohistochemistry with confocal microscopy. This high risk/high gain proposal has the potential to initiate a new line of investigation, provide original insight into the consequences o cocaine abuse on impulsive decision making, and inform the development of treatments for cocaine addiction.

PROJECT SUMMARY/ABSTRACT Intractable cocaine craving precipitated by exposure to a cocaine-associated environmental context is a major factor contributing to drug relapse. This phenomenon depends on available long-term memories of context- response-drug associations. Recent findings indicate that associative memories become labile upon retrieval and need to undergo protein synthesis-dependent reconsolidation into long-term memory stores in order to be retained over time. Cocaine-induced pathology in memory reconsolidation may result in unusually salient or intrusive cocaine memories that manifest as increased cue reactivity and propensity for drug relapse in a drug- associated environment. Thus, the long-term goal of this research program is to enhance our understanding of the functional neuroanatomy and cellular mechanisms of cocaine memory reconsolidation. During the previous funding period, we have shown that protein synthesis-dependent memory reconsolidation occurs in the basolateral amygdala. Remarkably, this process is functionally dependent on neural activity in the dorsal hippocampus, even though the two brain regions do not share monosynaptic connections. Logically extending this line of research in this competitive renewal application, Specific Aim 1 will identify novel memory reconsolidation circuits. Based on our new preliminary findings, we will test the hypothesis that the locus coeruleus serves as a relay between the dorsal hippocampus and basolateral amygdala to permit cocaine memory reconsolidation. In addition, we will evaluate how the inhibition of specific pathways within this putative circuitry alters electrophysiological activity at the targeted terminal region of each pathway. During the previous funding period, we also identified cellular mechanisms that are necessary for cocaine memory reconsolidation. Systematically extending this line of research, Specific Aim 2 will identify novel cellular mechanisms of cocaine memory reconsolidation in the basolateral amygdala. Based on our preliminary findings, Aim 2 will focus on the endocannabinoids (eCB), anandamide (AEA) and 2-arachidonoylglycerol (2-AG), in the basolateral amygdala. We will evaluate the extent to which memory reconsolidation is sufficient to produce changes in eCB levels, eCB degradation, and pyramidal cell excitability within the basolateral amygdala. In addition, we will test the hypothesis that AEA inhibits - whereas 2-AG facilitates ? the reconsolidation of labile cocaine memories and the activation of a requisite cellular mediator of memory reconsolidation within the basolateral amygdala. To accomplish these Aims, we will utilize sophisticated behavioral, novel optogenetic functional disconnection, and electrophysiological recording protocols, as well as immunohistochemistry, quantitative Western blotting, and eCB biochemical assays. Overall, renewal of this productive research program has the potential to significantly advance our understanding of the neural basis of cocaine memory reconsolidation and to provide an essential conceptual framework for future research and addiction treatment development efforts.