Kristen A. Keefe - US grants

Alterations in the function of striatal neurons projecting directly (striatonigral) and indirectly (Striatopallidal) to basal ganglia output nuclei contribute to motor dysfunctions associated with neurological disorders such as Parkinson's disease. Recent finding suggest that antagonists of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor may be effective in treating Parkinsonism. NMDA receptors in striatum receive excitatory input from cortex, and are comprised of multiple subunits, the expression of which determines the pharmacology of the receptor. For example, inclusion of the 2a subunit confers an "antagonist-preferring" profile to the receptor, whereas inclusion of the 2b subunit produces one that is "agonist-preferring." These two NMDA receptor subunits how different regional distributions in striatum. The implications of these differences in pharmacology and regional distribution for the functioning of the basal ganglia are unknown. Some data suggests that NMDA receptor antagonists that distinguish between subtypes of NMDA receptors have differential effects on striatal neuron function. This proposal will test the hypothesis that blockade of specific subtypes of the NMDA receptor, either alone or in combination with dopamine receptor manipulation, will have differential effects on striatal efferent function. First, the expression of the 2a and 2b subunits by defined striatal neuron populations will be determined using in situ hybridization histochemistry with double labeling. Second, different classes of NMDA receptor antagonists will be given over a range of doses to both intact and dopamine-depleted rats to determine how blockade of different subtypes of NMDA receptors affects the function of striatal efferent neurons. The antagonists will be given alone and in combination with D1- and D2-dopamine receptor agents, which selectively affect striatonigral and striatopallidal neurons, respectively. The techniques of in vivo microdialysis and in situ hybridization histochemistry will be combined to measure GABA and neuropeptide release from, and immediate early gene expression in, the two striatal efferent pathways as measures of striatal neuron function. Clarifying the relation between NMDA receptors and striatal efferents will improve our understanding of cortical influences on basal ganglia function and may contribute to the development of more effective therapies for disorders of basal ganglia, such as Parkinson's disease.

[unreadable] DESCRIPTION (provided by applicant): Drug-seeking in response to stimuli previously associated with drug use contributes significantly to recidivism in drug-addicted individuals, as presentation of drug-associated stimuli can elicit intense craving for drug, which contributes to relapse. Drug-associated cues also act as conditioned reinforcers to maintain or establish complex drug-seeking behaviors. In animal models of addiction, drug-associated stimuli can both elicit and maintain drug-seeking behavior. Considerable research has examined neural circuits underlying the role of such drug-associated conditioned reinforcers in the maintenance of drug-seeking behavior. Significantly less research has examined neural substrates underlying the ability of drug-associated discriminative stimuli to elicit drug-seeking behavior. Furthermore, whether the neural circuits in which plasticity takes place and subsequent neural activity produces these differing effects of drug-associated cues on drug-seeking behavior overlap or are distinct is not well defined. Evidence suggests, however, that there are likely to be differences in the circuits involved. Finally, activation of molecular substrates, such as activity regulated cytoskeletal- associated protein (arc), zif268, and c-fos, thought to underlie synaptic plasticity fundamental for learning and memory, has only been examined to a very limited extent in studies investigating the role of drug-associated cues in the initiation and maintenance of drug-seeking behavior. This proposal therefore will test the hypothesis that cocaine-associated discriminative stimuli that elicit drug-seeking behavior activate molecular cascades underlying synaptic plasticity in neuronal populations that are distinct from, yet somewhat overlapping with, those in which cocaine-associated conditioned reinforcers that maintain drug-seeking behavior induce these molecules. This hypothesis will be examined using cellular compartment analysis of temporal activity with fluorescent in situ hybridization (catFISH) to assess the brain regions activated by these two types of stimuli in animals exposed to both drug-associated stimuli and whether these genes are activated in the same neuronal populations in a given brain region. Such information will allow greater understanding of systems-level and molecular substrates underlying stimulus-induced drug-seeking behavior, which should translate into improved therapeutic management of stimulus-induced relapse in drug addiction. Drug seeking in response to stimuli previously associated with drug use contributes significantly to recidivism in drug-addicted individuals, as presentation of drug-associated stimuli can elicit intense craving for drug, which contributes to relapse. This project will examine the neural circuits engaged by drug-associated stimuli that elicit and maintain drug seeking behavior and the activation of molecular substrates thought to underlie synaptic plasticity fundamental for learning and memory. The data to be obtained will provide critical, novel insight into the systems-level and molecular substrates underlying stimulus-induced drug-seeking behavior, which should translate into improved therapeutic management of stimulus-induced relapse in drug addiction. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The long-term consequences of methamphetamine (METH) abuse include a persistent, partial loss of monoamine systems in the brain, particularly the dopamine innervation of the striatum. Despite the apparent relative sparing of function at smaller sizes of dopamine depletion, several lines of evidence suggest that there is a significant impact of such partial dopamine loss on central nervous system function. Both abstinent METH abusers with documented decreases in dopamine uptake sites in striatum and patients early in the course of Parkinson's disease show deficits on cognitive tasks. Studies in animals with partial monoamine depletions have revealed deficits in learning and memory functions dependent on striatal, hippocampal, and cortical function, and recent data from our laboratory suggest that such deficits may be associated with impaired activation of the effector immediate early gene arc. Partial dopamine depletions also are associated with decreased dopamine concentrations evoked by electrical stimulation mimicking phasic dopamine signaling. Finally, partial monoamine depletions are associated with changes in measures of the function of striatonigral (direct pathway) efferent neurons of striatum. Taken together, these data suggest that partial dopamine depletions of striatum selectively alter striatonigral neuron function and, consequently, basal ganglia- dependent learning and memory function by impairing phasic dopamine neurotransmission. This hypothesis will be examined by 1) further characterizing the impact of METH-induced neurotoxicity on basal ganglia-mediated learning and memory processes; 2) determining whether METH-induced neurotoxicity is associated with degradation of dopamine transients and whether activating phasic dopamine transmission will selectively enhance striatonigral neuron function; and 3) examining the impact of METH-induced neurotoxicity on arc induction and cytoplasmic distribution as well as on the involvement of striatal Arc in learning and memory. Completion of these experiments will provide improved understanding of the molecular, cellular, and behavioral impact of METH-induced neurotoxicity to central dopamine systems on basal ganglia function. Such understanding will be critical to allow for the development of targeted strategies to therapeutically manage the long-term effects of such stimulant abuse. PUBLIC HEALTH RELEVANCE It is now established that methamphetamine (METH) abuse leads to long-lasting decreases in the dopamine innervation of the caudate-putamen in humans, as well as other species. The goal of this project is to further determine the impact of METH- induced neurotoxicity on basal ganglia function and basal ganglia-mediated learning and memory processes and to assess whether changes in phasic dopamine neurotransmission underlie the long-term effects of METH on basal ganglia function and behavior.

DESCRIPTION (provided by applicant): Research in the neurosciences has become increasingly interdisciplinary: it is not possible to understand the nervous system by focusing on a narrow area of expertise. The principle underlying the graduate education offered by the University of Utah Interdepartmental Neuroscience Program is that students approach a problem from a very broad perspective. The approach includes the application of a diversity of techniques that range across molecular biology, electrophysiology, classical embryology and behavioral protocols. The diverse techniques are unified by two intellectual approaches that have made the University of Utah famous: the study of human disease and a tradition of excellence in the field of genetics. Fifty one NIH- funded training faculty members, chosen from among 76 neuroscientists in 13 departments at the University of Utah, are aligned in five specific areas of research in neuroscience: Molecular Neurobiology, Cellular Neuroscience, Development, Brain and Behavior, and Neurobiology. These five areas reflect the breadth of the program and provide exceptional opportunities to the 45 current students. This renewal application requests funding for three first year and three second year Ph.D. students in the Interdepartmental Program in Neuroscience. All of the training positions in the previous award period were filled and funding for the same number of trainees is requested in the present application. Due to the outstanding neuroscience training program, recent graduates have been placed in excellent postdoctoral fellowships at Yale, Stanford, Harvard, U Mass, UCSD, and UCSF. There is no doubt that trainees from the Interdepartmental Neuroscience Program will continue to become future leaders in neuroscience research.

? DESCRIPTION (provided by applicant): As of 2012, approximately 22 million Americans required treatment for drug dependence. Drug addiction arises as a consequence of changes in brain circuitry induced by drug exposure and resultant dopamine release. Synaptic plasticity processes underlie numerous aspects of drug abuse and addiction, including the formation of drug-clue, action-outcome, and stimulus-response associations affecting instrumental behavior, as well as extinction of drug-seeking behavior. Synaptic plasticity is heavily dependent on the expression and function of immediate early genes (IEGs), in particular activity-regulated, cytoskeletal-associated (Arc) and early growth response-1 (egr1/zif268) genes. These genes critically mediate consolidation and reconsolidation of long-term memories, including memories implicated in aspects of drug abuse and addiction. Understanding the regulation of these IEGs is therefore significant in its potential to identify novel therapeutic targets to disrupt aberrant plastic changes contributing to drug abuse and addiction. Many studies have focused on understanding processes mediating transcriptional regulation of IEGs, as well as on dendritic trafficking and local translation of Arc mRNA. However, to date there are no studies in the extant literature examining the basic cellular process of nuclear export of mRNAs in general, much less IEG RNAs in particular, in the adult mammalian brain. Our experimental evidence suggests that nucleocytoplasmic export of these IEG mRNAs differs in striatonigral (direct pathway) vs. striatopallidal (indirect pathway) neurons in dorsal striatum. Further, our recent data suggest that the nuclear export of IEG mRNAs in striatonigral efferent neurons may be regulated by dopamine. The overall goal of the proposed studies is thus to test the novel hypothesis that there are differences in nucleocytoplasmic export of IEG mRNAs in striatal efferent neuron subpopulations. In Specific Aim 1, we will determine the phenotypic distribution of known mediators of nucleocytoplasmic mRNA export and their co-localization with IEG mRNAs in striatal efferent neuron subpopulations. In Specific Aim 2, we will differential centrifugation and FACS to separate nuclei from striatonigral and striatopallidal neurons, followed by ribonucleoprotein immunoprecipitation and RT-PCR to biochemically assess the interaction of Arc and zif268 with specific ribonucleoprotein complexes involved in nuclear export. In Specific Aim 3, we will determine the role of dopamine D1 receptor activation in the regulation of nuclear export of IEGs in striatonigral efferent neurons. Given the lack of data regarding the regulation of nuclear export of mRNAs in neurons of the adult mammalian brain and potential regulation of this process by neurotransmitter systems, including DA, successful completion of the proposed studies will provide seminal insight into the regulation of these critical plasticity-related IEGs n striatal circuitry intricately involved in drug abuse and addiction. These findings thus have the potential to reveal novel therapeutic targets to modulate striatal plasticity so as to improve treatment outcomes in individuals with histories of drug abuse and addiction.

The mission of the NIH is to ??seek fundamental knowledge about the nature and behavior of living systems and the application of that knowledge to enhance health, lengthen life, and reduce illness and disability.? Achieving this end requires scientists who are not only capable of generating that fundamental knowledge, but also driving the successful translation of that knowledge into therapeutic interventions to reduce illness and disability. The objective of the proposed two-year training program is to provide one pre-doctoral and one post- doctoral/clinical fellow per year with formal education in the translational processes needed for moving basic science discoveries to clinical practice. The program will capitalize on strong translational training opportunities already available at the University of Utah and will complement those opportunities with new programming. Specifically, trainees will benefit from: 1) a series of monthly workshops delivering formal education in the process of moving pharmaceutics, devices, and apps from discovery to clinical use; 2) substantive experiences in clinics, in which patients with disorders of relevance to the trainee?s research are seen and with whom the trainee will engage in the clinic and in patient support groups to gain greater insight into the needs of patients; and 3) an internship to gain first-hand knowledge of the regulatory requirements and practices required to move products through to clinical use. Trainees will conduct their primary research under the direction of a translational supervisory team consisting of a basic scientist, a clinician/clinical scientist, and a researcher with experience in translating basic research discoveries into the clinic. In consultation with this mentoring team, the trainee will design and conduct preclinical research using best practices for enhancing the translational potential of their work. At the end of this training, participants will be able to: 1) Describe the issues to be considered in designing basic, preclinical studies and making decisions about whether to move a preclinical study forward with respect to development of a therapeutic agent, device, or app; 2) Apply knowledge of these considerations to the development of their research proposal; 3) Evaluate the research proposals of others with respect to translational considerations; and 4) Create new knowledge through research and publications that incorporate appropriate translational principles. Together, these experiences will develop burgeoning ?professional translators? poised to promote the translation of basic research findings to improved therapeutic outcomes in patients.