Charles Chavkin - US grants

The proposed research will explore the nature of the dynorphins, a class of endogenous opioid peptides, interaction with opioid receptors in a particular brain region of the rat and guinea pig. The goal of these studies is to provide a detailed understanding of the actions of an endogenous neuropeptide at a site of its synthesis and release, and thus ultimately to provide an important model of endogenous opioid action. Our specific hypothesis is that the dynorphin peptides are synthesized by dentate granule cells and released at their mossy fiber terminals to control the excitability of hippocampal pyramidal neurons. Preliminary evidence obtained has supported this idea: We have demonstrated the presence of the dynorphin opioid peptides in the rat hippocampus, the in vitro release of these peptides from hippocampal slices, and the effects of exogenously applied opioids on hippocampal cell excitability. These data strongly suggest that the dynorphin opioids are likely to be neurotransmitters in the rat hippocampus. We plan to test our hypothesis by further delineating the actions of opioids and the properties of the opioid receptors present in this brain region. Electrophysiological recording methods will be used to measure responses to the dynorphins in hippocampal slices; the pharmacological effects of opioids will be quantified; responses to endogenously released opioids will be identified, and the opioid receptor types mediating the effects of both exogenously applied and endogenously released opioids will be defined. To accomplish these goals we will record the effects of the dynorphins applied to likely hippocampal cell targets in the pyramidal and dentate granule cell layers. We propose to 1) define the effects of dynorphin by intracellularly recording cell excitability while applying peptide by micropipette; 2) characterize the specific opioid receptors that mediate the pharmacological effects of the dynorphins at opioid sensitive sites in the hippocampus; 3) identify the effects of endogenously released dynorphins by stimulating the peptide-containing fiber tracts and measuring the opioid antagonist sensitive effects.

We plan to study the effects of chronic morphine exposure on the opioid receptors in the hippocampal region of the rat central nervous system (CNS). Our hypothesis is that cellular tolerance to chronic morphine exposure is the result of a reduction in opioid receptor coupling to its signal transduction system rather than a reduction in receptor affinity or receptor number. This proposal describes a series of experiments designed to test that hypothesis. Using extracellular electrophysiological recording methods, the opioid receptor types mediating the response of hippocampal pyramidal cells to morphine-like (opioid) compounds will be defined. Quantitative dose-effect relationships will be determined for selected opioid agonists to measure their potency changes in the morphine-tolerant hippocampal preparation. Using electrophysiological measures, the molecular basis for the reduction in agonist potency will be determined by comparing the opioid receptor affinities and agonist efficacies of selected opioids in hippocampal slices prepared from morphine-tolerant and drug-naive rats. This will require the adaptation of partial receptor inactivation methods originally developed for use in opioid sensitive smooth muscle bioassays. Using in vitro ligand binding assays selective for mu and delta sites, we will also compare the opioid receptor affinities and binding site densities in morphine-tolerant and naive rat hippocampus. If tolerance is a consequence of a change in the post receptor binding events as we predict, a likely site of change would be the coupling between the receptor and the signal transduction system. The proposed link between opioid receptors and GTP-coupling protein (Ni) will be determined by measuring the sensitivity of opioid effect to pertussis toxin treatment of the rat hippocampal slice. We will determine whether morphine-tolerance increases the pertussis toxin sensitivity as might be predicted by the expected change in agonist efficacy. The primary goal of these studies is an understanding of the cellular and molecular mechanisms used by the brain to adapt to chronic opiate exposure. Opiate addiction is known to have behavioral, cellular, and molecular components. Information will be obtained in this study to describe the latter mechanisms. By defining the specific molecular events underlying tolerance development in a model CNS system, we expect ultimately to have a clearer concept of the relevant clinical processes.

Endogenous Opioid Regulation of Synaptic Transmission in the Dentate Gyrus An important role of endogenous neuropeptide transmitters in brain function is suggested by their prevalence and diversity. Understanding how these molecules may normally function as potential neurotransmitters requires that we know what controls their release, where they act, and what effects they have. We have made substantial progress in answering these questions for endogenous opioid peptides in the hippocampus by characterizing the molecular forms of the peptides present, the distribution of the receptor sites, and the pharmacological effects of opioids in each of the principal regions of the hippocampus. Further work is required to define the effects of endogenously- released opioids on hippocampal neurons. Information obtained from this study will allow us to construct a detailed description of the role of endogenous opioid peptides in the hippocampal neural network and provide a model of neuropeptide action in normal hippocampal physiology associated with learning and memory processes.

Phencyclidine is a widely abused drug having dramatic hallucinogenic, intoxicant, and CNS stimulant effects. The cellular mechanisms of these effects are not yet known, but three possible sites of action have been proposed. Phencyclidine has been shown to have specific effects on a type of glutamate receptor, on a class of potassium channels, and on the sigma receptor system. The latter site of action is the least well understood but is particularly interesting because other drugs able to bind to the sigma site (e.g. N-allyl-normetazocine and cyclazocine) also have hallucinogenic effects and also because haloperidol (an important antipsychotic phenothiazine) has a very high affinity for the sigma receptor. These observations suggest that some of the psychotomimetic effects of phencyclidine may be mediated by the sigma receptor rather than the glutamate receptor or the potassium channel interactions. We are ultimately interested in studying the properties of the sigma receptor system: 1) by defining the electrophysiological effects of sigma drugs on neuronal physiology in the central nervous system, 2) by isolating the putative endogenous sigma transmitter, and 3) by identifying the role of the sigma system in the control of neurotransmission in the neocortex. This proposal describes a series of experiments designed to extend our initial observations of the effects of sigma drugs on rat hippocampal neurons in the in vitro brain slice preparation, and to extend our preliminary results which suggested that an endogenous sigma transmitter is released from nerve terminals to bind to sigma receptors in the rat hippocampus. Our specific goals during the proposed period of support are to accomplish two key tasks: we will provide a novel chemical characterization of the putative endogenous sigma factor by comparing the properties of the sigma activity in brain extracts with those released from brain slices in a calcium-dependent manner. [This information will ultimately lead to the isolation and identification of the sigma factor]. The second goal is to develop three bioassays of sigma receptor action that can be used to define the effects of sigma receptor activation, define which sigma drugs are agonists and which antagonists, and can be used to follow the purification of the endogenous sigma factor.

Understanding how the widely-abused opiate drugs influence brain function requires a better understanding of how the endogenous opioid peptides normally act as neurotransmitters and how their function is altered by chronic opiate abuse. Substantial progress has been made in defining the molecular forms, tissue distribution, specific receptors, and pharmacological actions of the endogenous opioid peptides, yet little is known about their normal physiological role in the mammalian nervous system. We need to know what controls opioid peptide release, where endogenous opioids act, and what physiological effects they normally have. During the past several years, we have been using the rodent hippocampal slice preparation as a model to study the actions of endogenous opioid peptides. In this study, we will define the effects of endogenously released opioid peptides (the dynorphins and enkephalins) on the electrophysiological properties of identified hippocampal neurons. The studies described will examine anatomical structure and physiological properties of the opioid peptide synapses present in the hippocampus. We will determine the distribution of the opioid peptides and opioid receptors using specific antisera, and we will identify the electrophysiological actions of endogenous opioid peptides at three different sites in the hippocampus. We propose to characterize the transmitter properties of 1) the endogenous dynorphins present in the granule cells which regulate excitatory transmission in the dentate gyrus, 2) the endogenous enkephalins present in perforant path axons which regulate inhibitory transmission in the dentate gyrus, 3) the endogenous dynorphins present in the mossy fiber axons which regulate excitatory transmission in the CA3 region of the hippocampus. The effects of chronic morphine and kappa agonist exposure on the functioning of the endogenous opioid system will also be determined. Information obtained from this study will allow us to construct a detailed description of the role of endogenous opioid peptides in the hippocampal neural network and provide a model of opioid neuropeptide action in normal and opiate tolerant hippocampal physiology.

This proposal requests support to continue a successful predoctoral and postdoctoral training program designed to provide training in molecular and cellular aspects of drug abuse research. The University of Washington School of Medicine has strong research programs studying molecular aspects of drug receptor signaling mechanisms in several departments, and this training program has dramatically facilitated the coordination of training and collaboration of research effort among the drug abuse researchers at this institution. Examples of ongoing studies include: signal transduction by opiate receptor tolerance, cannabinoid receptors signaling and tolerance, the development of novel opiate drugs, the regulation of ionic channels by psychotomimetic agents, the effects of drugs of abuse on G-protein coupled enzyme and channel activities, and the molecular consequences of chronic drug exposure. We expect that the continued application of increasingly sophisticated biochemical and physiological methods will provide important advances in our understanding of the mechanisms by which specific drugs of abuse act. It is the intent of this program to identify and support three predoctoral students and three postdoctoral fellows interested in studying molecular and cellular mechanisms of drug action of specific abused drugs. Graduate students in their second or third years of study who identify a thesis project of direct relevance to the research mission of NIDA (characterization of the actions of pharmacological agents subject to the non-medical use) will be supported and encouraged through this training program. Postdoctoral fellows similarly working on a research project of direct relevance will be supported. The training program will be strongly research oriented but will also include seminars, journal clubs and didactic presentations on broader issues relevant to the study of drug abuse. Beyond the directly beneficial effects on the careers of the trainees, one of the most significant successes of the previously funded program has been its catalytic effect on the research environment at this institution: it has provide a means to support new investigators (Mackie and Terman) who are establishing new programs in cannabinoid and opiate research; it has encouraged established investigators to expand their research on drug abuse (Storm, Nathanson, Tempel); it has provided a platform to bring the molecular scientists in better contact with the behavioral and clinical drug abuse researchers at this institution. Although this has been a relatively small program, it has had a big impact here, and its renewal would encourage the further development of the drug abuse research community here. A funded Drug Abuse Research Training Program will continue to serve as an important catalyst to focus research at this institution on the basic neurobiology of a significant health issue.

DESCRIPTION (Adapted from the applicant's abstract): This research plan is designed to examine the role of the endogenous dynorphin peptides under a number of normal and pathological conditions. The dynorphins are a family of inhibitory neuropeptides expressed at high concentration within the hippocampal formation. The investigators are guided by two key observations: first, that the dynorphin opioids can be released from granule cells to inhibit excitatory amino acid release both from presynaptic afferents of the perforant path (retrograde inhibition) and from granule cells (autoinhibition). Second, that in both human and animal models of temporal lobe epilepsy, there is often an expansion of the dynorphin-containing fibers in the dentate gyrus as a consequence of synaptic reorganization. These observations lead to the following two hypotheses: that the dynorphin opioids may be neuroprotective by reducing glutamate release during periods of hyperactivity, and that dynorphin-mediated inhibition may serve an expanded role under conditions in which the GABAergic tone is reduced. Pilocarpine-treatment provides an animal model of temporal lobe epilepsy that duplicates many features of human pathology including a loss of a group of hilar GABAergic neurons and an expansion of the dynorphin-containing mossy fibers in the dentate gyrus. The investigators plan to use this model to address the following specific aims: 1) To use anti-opioid receptor antibodies, which can act as specific markers for a subpopulation of GABAergic neurons and excitatory afferents in this region, to identify anatomical changes within the rat dentate gyrus after pilocarpine-induced neurotoxicity. 2) To determine if the anatomical changes in opioid receptor expression correlate with changes in the pharmacological response to opioid agonists on granule cell excitability. 3) To determine whether infusion of kappa opioid receptor selective agonists or antagonists during the pilocarpine-induction of temporal lobe epilepsy will protect or exacerbate the neurodegeneration assessed by anti-receptor antibody labeling. 4) To measure the amplitude and kinetics of the actions of endogenously released dynorphins which regulate excitatory neurotransmission in the dentate gyrus. These approaches will provide a new characterization of the anatomical and physiological changes in neuronal circuitry underlying this animal model of temporal lobe epilepsy. The ultimate goal of these studies is to develop novel strategies for the control or amelioration of this seriously debilitating seizure disorder.

DESCRIPTION (from applicant's abstract): Prolonged agonist exposure causes desensitization of the mu, delta, and kappa opioid receptors by mechanisms that are still incompletely understood. Opioid receptor desensitization is likely to involve a reduction in the efficiency by which agonist-bound receptor catalyzes the activation of G proteins. Evidence suggests that prolonged agonist exposure results in opioid receptor phosphorylation by a G protein coupled receptor kinase followed by the binding of beta-arrestin, although desensitization mediated by other kinases has also been suggested. We p ropose to extend our studies of opioid receptor desensitization mechanisms by defining the effects of specific kinases on the coupling between opioid receptor activation and inwardly rectifying potassium channels heterologously expressed in Xenopus oocytes, by comparing the effects of specific receptor mutations on desensitization in AtT20, a mammalian cell line, and by defining the regulation of coupling between opioid receptors and potassium channels naturally expressed in hippocampal neurons. Using these three cellular systems as models for components in vivo tolerance processes, we will address the questions: which kinases are effective in mediating homologous desensitization, and which domains of the receptors are the critical targets of phosphorylation. Because desensitization is likely to be an important component of opioid tolerance, the identification of the key mediators of desensitization would be significant and would suggest potential targets of drug development that might be useful in controlling opioid tolerance. The resulting knowledge is expected to provide a more rational basis for treating the consequences of prolonged opioid exposure.

DESCRIPTION: (provided by applicant) This NIDA P50 Center would foster research interactions, promote exchange of ideas and technology and enhance the training environment in ways that will strongly advance drug abuse research at the University of Washington and will generate resources that will be made widely available. The Center would bring together a group of established investigators to focus on developing mouse models to resolve the molecular components underlying, drug abuse. Three projects within the Center address related questions: molecular mechanisms underlying opiate tolerance (Chavkin), molecular mechanisms underlying cannabinoid tolerance (Mackie), the specific role of protein kinase A in drug preference and sensitivity to amphetamine and cocaine (McKnight). These projects focus on the molecular basis of animal behavior controlled by drugs of abuse, and the design of the Center includes extensive collaborative interactions between these related projects. The Center would support core facilities required for efficient resource utilization and technique transfer within the projects: 1) a Mouse Genetics Core (Binion/McKnight) would facilitate the generation of new strains having tissue-specific, inducible mutations in key components of the animal?s response to opioid, cocaine and cannabinoid drugs; 2) the Behavioral Core (Bernstein & External Advisors) would standardize the analysis of drug effects and drug tolerance; and 3) the Anatomy Core (Westenbroek) would perform a generalized neuroanatomical characterization and maintain access to a confocal microscope facility. The third element of the Center is a Pilot Project component that initially would bring a distinguished scientist (Palmiter) into the drug abuse field, potentially bring other outstanding scientists (Catterall, Storm) into drug abuse research, and would help junior faculty (Stella, Brot) develop research programs in this area. All of the individuals listed would actively participate in regular progress meetings, training sessions, journal club and seminar programs, and Center retreats. The Center would expand the educational and training activities of an existing, NIDA Institutional training grant on the molecular pharmacology of abused drugs. Our prime motivation for establishing this Center is to enhance the intellectual, educational, and developmental interactions that would stimulate and expand drug abuse research. Synergy within the proposed Center would be derived from the transfer of ideas, techniques, expertise, perspective and mice between members of the group.

DESCRIPTION (provided by applicant): In this application, we outline a set of experiments focusing on the effects of stress-induced release of endogenous opioids on the behavioral responses to cocaine. The application is based on the hypothesis that kappa opioid receptor activation by repeated stress induces release of endogenous dynorphin peptides that underlie a significant component of the stress-induced potentiation of the rewarding properties of cocaine. Results from our preliminary studies have demonstrated that repeated swim stress potentiates cocaine conditioned place preference (CPP) of wild type male C57BI6 mice. Stress-induced potentiation was blocked by the kappa opioid receptor antagonist nor-binaltorphimine (nor-BNI) and was not evident in prodynorphin knockout mice. Results will be extended by determining whether two other stressors (repeated restraint stress and chronic social stress) also produce similar kappa-mediated potentiation. Second, stress-induced reinstatement of cocaine CPP as a model of relapse will be assessed, and the effects of kappa receptor antagonism on stress-induced reinstatement will be determined. We propose to use a phosphospecific antibody to immunolabel brain sections from stressed and unstressed mice with the objective of developing an anatomical assay for activated kappa opioid receptors. The neural pathways containing activated KORs following swim, restraint and social stresses will be compared to define those common circuits likely to mediate endogenous dynorphin regulation of the cocaine response. Lastly, we propose to study the cellular effects of kappa receptor activation on neuronal excitability in the brain regions identified in the previous aim to be potential sites of endogenous opioid action. Brain slices of the identified regions taken from mice previously subjected to the behavioral paradigms defined will be compared by measuring effects of kappa receptor activation on electrophysiological responses from slices from unstressed, control mice. WCVC recordings from cell types will be made. The results should provide essential insight to the possible cellular and molecular mechanisms underlying the regulation of cocaine addiction by kappa opioid systems.

This application seeks support for the continuation of a successful grant designed to study the effects of phosphorylation on opioid receptor activation of G-protein coupled potassium channels (Kit3) In the previously funded period, the effects of both serine/threonine phosphorylation and tyrosine phosphorylation on opioid receptor desensitization and Kir3 channel activation were described. We propose to extend these studies by focusing our effort on the specific effects of tyrosine phosphorylation (Y-PO4) on mu opioid receptor (MOR) activation of Kir3. Our previous site directed mutagenesis studies identified potential Y-PO4 Isites within the receptor and channel likely to control the response to opioid agonists. Initially, the mechanisms of this regulation will be studied using cDNA expression of MOR and Kir3.1 in AtT20 cells and primary hippocampal cultures. Studies would be extended by in vitro electrophysiological recording oi DAMGO activated Kir3 responses and confocal imaging of receptor and channel trafficking. We would use pharmacological inhibitors to identify the kinases and phosphatases responsible the Y-PO4 mediated effects in these malleable in vitro systems. Results of this aim would test the hypothesis that Y-PO4 of specific sites within MOR and Kir3.1 regulates the efficiency of opioid signaling. The phosphorylation state of specific sites within the MOR and Kir3.1 sequences would be assessed by 32p-incorporation and by probing with novel phosphospecific antibodies. Regulation of opioid receptor signaling by tyrosine phosphorylation has important implications for understanding opioid responses during physiological stress; thus, moving from simple in vitro analyses to more complex, in vivo systems would be a priority. After characterizing the specificity and utility of the phosphospecific antibodies (MOR-YP and Kir3.1-YP) in the in vitro systems, we will test the hypothesis that nerve trauma results in growth factor-induced changes in tyrosine phosphorylation of MOR and Kir3.1 detectable by changes in MOR-YP and Kir3.1-YP immunostaining within nociceptive circuits in spinal cord and brain. Results of the proposed studies are likely to provide additional understanding of the mechanisms mediating the plasticity of opioid signaling.

DESCRIPTION (provided by applicant): This proposal requests support to continue a successful predoctoral and postdoctoral training program designed to provide training in molecular and cellular aspects of drug abuse research. The University of Washington School of Medicine has strong research programs studying molecular aspects of drug receptor signaling mechanisms in several departments, and this training program has dramatically facilitated the coordination of training and collaboration of research effort among the drug abuse researchers at this institution. We expect that the continued application of increasingly sophisticated biochemical and physiological methods will provide important advances in our understanding of the mechanisms by which specific drugs of abuse act. It is the intent of this program to identify and support four predoctoral students and three postdoctoral fellows interested in studying molecular and cellular mechanisms of drug action of specific abused drugs. Beyond the directly beneficial effects on the careers of the trainees, one of the most significant successes of the previously funded program has been its catalytic effect on the research environment at this institution. A funded Drug Abuse Research Training Program will continue to serve as an important catalyst to focus research effort at this institution on the basic neurobiology of a significant health issue.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] This is an application for a Senior Scientist K05 award for the research career development of Dr. Charles Chavkin who is the Allan and Phyllis Treuer Chair of Pain Research in the Department of Pharmacology at the University of Washington. Dr. Chavkin received his PhD training in pharmacology at Stanford University, completed postdoctoral training at the Salk Institute and Scripps Clinic before joining the faculty at the University of Washington. He is currently the principal investigator of RO1-DA11672-06, R01-DA16898-02, P01-DA15916-03, and director of T32-DA07278-11. The research aims of these grants are respectively designed to 1) study the phosphorylation mechanisms regulating of opioid receptor activation of Kir3 type potassium channels, 2) study the cellular mechanisms responsible for behavioral stress-induced potentiation of the cocaine reward, 3) study the mechanisms of opioid analgesic tolerance using transgenic mouse models, and 4) supervise training of predoctoral and postdoctoral fellows in molecular approaches of drug abuse research. The purpose of this K05 application is to consolidate salary support for Dr. Chavkin and to reduce his non-research related administrative responsibilities. This plan has the full support of the department of pharmacology Chair and the Vice-Dean for Research in the School of Medicine. The long term goals of the proposed research plan are to further develop a multidisciplinary approach to the study of the actions of endogenous dynorphin opioid neuropeptides in drug addiction. The dynorphin/kappa opioid system seems to function as key mediators of the response to chronic stress in ways that potentiate the rewarding properties of cocaine. Understanding how chronic stress increases the risk of drug addiction and increases the risk of relapse has important therapeutic implications. The multidisciplinary approach necessary to address these questions requires molecular biological tools to define the targets of drug action, molecular pharmacological methods to resolve the cellular events underlying the responses, electrophysiological methods to define the actions of drugs on the neurons affected, anatomical methods to define the neural circuits and sites of drug action, and behavioral methods to provide relevant and valid stimuli to the nervous system. The award would greatly strengthen the candidate's ability to provide leadership in this integrative effort. [unreadable] [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Our prior studies have shown that the endogenous dynorphin opioid peptides, released during exposure to repeated behavioral stressors (repeated forced swim, repeated social defeat, or foot shock), strongly potentiate the rewarding effects of cocaine in mice (as measured by an enhanced conditioned place preference to cocaine) and reinstate extinguished drug seeking behavior in mice (as measured by reinstatement of extinguished cocaine conditioned place preference). We previously found that stress-induced dynorphin release activates kappa opioid receptors (KOR) that stimulate phosphorylation of p38 MAPK expressed by both astroglial cells and GABAergic neurons in brain. KOR-dependent activation of p38 MAPK was required for astrocyte activation (increased GFAP-ir and proliferation) and for the KOR-dependent potentiation of the behavioral responses to cocaine. However, a specific role of astrocyte activation in mediating the enhanced behavioral response to cocaine is not clear. In this exploratory study, we propose to use transgenic GFAP-CreERT2 mice in which Crerecombinase expression is regulated both by a tamoxifen-inducible DNA recombinase variant and by the Glial acidic fibrillary protein (GFAP) promoter. These mice will be crossed with a floxed p38???conditional knockout mouse to generate adult mice in which KOR-dependent activation of p38???may be selectively blocked in astrocytes. Using the GFAP-CreERT2/floxed p38?? CKO mice, we propose to determine: 1) whether induction of Cre expression by tamoxifen in adult mice can selectively excise the p38???gene in astrocytes, but not in neurons; 2) whether the reduction in p38?? expression effectively blocks stress-induced, KOR-dependent increases in GFAP-ir expression in brain; and 3) whether astrocyte-selective reduction in p38?? expression blocks reinstatement of extinguished cocaine conditioned place preference. The proposed studies are high-risk (cell-type selective regulation of gene expression is still an art form, and a specific role for the alpha isoform of p38 MAPK is not established), but high pay-off (selective manipulation of astrocyte activation by dynorphin during stress-responses would strongly advance our understanding of the role of astrocyte activation in relapse of drug abuse in humans). The feasibility of the proposed studies is established (we have all of the mice and experimental tools in hand). Pilot funding is required because genotyping, breeding, housing, and testing of these mice in the immunohistochemical and behavioral assays proposed require an intensive effort. PUBLIC HEALTH RELEVANCE: This application is designed to understand the ways in which astrocytes in brain, that have been activated by stress-induced dynorphin release, contribute to addiction. Astrocytes express kappa opioid receptors and are strongly activated by stress-induced dynorphin release. Kappa receptor activation potentiates the reinforcing effects of cocaine and reinstates drug seeking behaviors in cocaine conditioned rodents. This study would identify the connection between astrocyte activation and reinstatement of extinguished cocaine place preference by assessing the effects of selectively disrupting kappa opioid signaling in astroctyes on behavior. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Understanding the basis of addiction requires insights at every level of neuroscience from defining the initial drug responses, the adaptive changes in cellular function resulting from drug exposure, the learning and memory plasticity changes at the neural circuit level encoding changes in motivation, the neuroeconomic calculations underlying aberrant choice, and the neuropathological changes resulting in co-morbid psychiatric disease. The ultimate goal of these studies is to identify new targets of intervention that can protect individuals from addiction, prevent the relapse of existing addiction behaviors, predict an individual's addiction risk, and thus reduce the huge personal, familial and societal costs of addiction. The next generation of scientists approaching the problem of addiction will use increasingly integrative approaches that actively combine molecular, genetic, and behavioral techniques to develop an understanding of how an addicted person's brain functions differently from non-addicted persons. Genetic tools to define drug addiction risk and proteomic tools to define functional changes in signaling complexes will play important roles. The University of Washington has research strength in many aspects of neuroscience, genome sciences, and psychiatry that comprise an outstanding and collaborative research environment. Some of the existing faculty have developed strong programs in drug-abuse research that build on this rich environment. However, we have important programmatic gaps that this proposed recruitment would help us address. Specifically, the university has made major investments during the last 15 years in genome sciences and proteomic research that have not yet been integrated into our equally strong investments in addiction research. The time is right to bridge this gap by recruiting someone interested in using the emerging tools of functional proteomics to better define the structural and functional changes distinguishing an addicted from normal brain. Identification of these key signaling structures and the processes responsible for their changes in the addicted state would hopefully provide new targets for rational therapeutic intervention. PUBLIC HEALTH RELEVANCE: Consistent with the aims of this ARRA Stimulus initiative, the University of Washington, School of Medicine proposes to recruit a tenure-track assistant professor in the area of drug abuse research. The newly hired individual would complement an existing group of NlDA-funded investigators having strengths in molecular pharmacology, targeted genetics, signal transduction and behavioral pharmacological approaches. We are seeking an individual using emerging proteomic or molecular genomic approaches to address questions of addictive drug actions on the structure and function of signaling complexes in brain.

? DESCRIPTION (provided by applicant): Recent advances in molecular and behavioral pharmacology suggest that novel opioid analgesics selective for the kappa opioid receptor system may provide effective pain relief without having the addiction risk common to mu-selective opioid agonists. New insights from molecular signaling studies suggest strategies to develop kappa opioid agonists lacking the dysphoric effects of the currently available compounds. In addition, the role of the endogenous dynorphin/kappa opioid systems in mediating the dysphoric effects of stress that increase addiction risk and precipitate relapse of drug taking has also strongly stimulated interest in the therapeutic potential of selective kappa opioid antagonists. Progress in the development of novel therapeutics based on these insights has been greatly stimulated by bringing all of the scientists from academic and corporate research units together for intensive and open discussions in two prior conferences on the Therapeutic Potential of Kappa Opioids for the Treatment of Pain and Addiction held in Seattle WA (2011) and Cambridge, MA (2013). No other venue invites all the 1) medicinal chemists developing novel ligands based on recent kappa receptor crystal structure insights, 2) molecular, cellular & behavioral pharmacologists doing preclinical characterizations of kappa opioid functional selectivity, 3) clinical scientists identifying the roles of dynorphin/kappa in human addictions and mood disorders, and 4) corporate scientists conducting clinical trials and drug development studies. Support is now requested to continue this series of conferences in 2015, 2017 and 2019. Plans for the next conference on the Therapeutic Potential of Kappa Opioids for the Treatment of Pain and Addiction to be held in Chapel Hill, NC on April 21-24, 2015 are developing well. The draft-program and plans to use the conference facilities at the Carolina Inn in historic Chapel Hill are coalescing nicely. Plans are also being developed by the program committee for future meetings in Philadelphia (2017) and San Diego (2019). The conference organizers have successfully involved both the established leaders in the kappa opioid field as well as junior scientists. The meeting has achieved reasonable gender balance of its speakers by actively welcoming all interested participants. Active support of minority scientists' participation has also helped ensure the diversity of the meeting. The goal of the conference is to help build the momentum in the field necessary to translate basic research findings into new therapeutic options for the nonaddictive treatment of pain, new treatments for stress-induced mood disorders, and effective treatments that can reduce addiction risk by enhancing stress-resilience.

DESCRIPTION (provided by applicant): Pharmacological activation of kappa opioid receptors (KOR) in humans elicits reports of dysphoria, and KOR activation by agonists or by stress-evoked dynorphin release in rodents produces aversion. These dysphoric/aversive effects of KOR activation have been shown to increase the rewarding effects of cocaine, increase drug self-administration, and reinstate extinguished drug seeking behaviors. The cellular and molecular mechanisms responsible for KOR-dependent aversion are not fully understood, but a better understanding may suggest new therapeutic approaches to the treatment and prevention of stress-related diseases including some forms of drug addiction. Evidence strongly supports a role for KOR-dependent inhibition of dopamine release in the nucleus Accumbens (NAc), however recent evidence further suggests that dynorphin activation of p38 MAPK in the serotonergic dorsal raphe nucleus (DRN) is also required for KOR-dependent aversion. Because a complementary role of serotonin in the regulation of affective state seems plausible, we propose to understand how activation of p38 MAPK by KOR stimulation, either by stress-induced dynorphin release in the DRN or systemic administration of a selective KOR agonist, results in conditioned place aversion. To accomplish these aims we propose 1) to measure KOR-dependent aversion in conditional knockout (CKO) mice having floxed p38a MAPK excised by PET1-promoter driven Cre recombinase, 2) to assess the effects of p38a MAPK activation by KOR on serotonin transporter reuptake efficiency in synaptosomes prepared from swim stressed mice having either normal or genetically modified dynorphin/KOR system function, and 3) to measure the effects of KOR- dependent p38 MAPK activation on electrically evoked release of serotonin and dopamine in vivo and in brain slices using fast scan cyclic voltammetry. Our hypothesis is that the aversive effects of KOR activation is a consequence of a shift in the balance between serotonergic and dopaminergic tone in the NAc caused by p38a MAPK activation of the serotonin transporter.

DESCRIPTION (provided by applicant): Repeated stress exposure produces a dysphoria-like response in mice that manifests behaviorally as stress-induced immobility, aversion and social avoidance. In a series of recent studies, we found that exposure to different forms of sustained stress activates a cascade of neurochemical responses in brain involving CRF activation of dynorphin release, dynorphin activation of kappa opioid receptors, kappa stimulation of p38alpha MAPK, and subsequent translocation of the serotonin transporter (SERT) from an endosomal compartment to the plasma membrane. Conditional gene deletion approaches and neurochemical studies revealed that the stress-induced aversion response specifically required the dynorphin/Kappa/p38alpha MAPK/SERT in the dorsal raphe serotonergic nerve terminals innervating the ventral striatum; disruption of any one of these components genetically or pharmacologically blocked the stress-induced dysphoric response and conferred stress-resilience in these animal models. Because stress-vulnerability is a known risk factor for clinical depression in humans, the proposed studies are designed to further characterize the mechanisms responsible for SERT translocation in the serotonergic nerve terminals. Specifically, we propose to express the human-SERT gene by local injection of a lentiviral construct containing the normal hSERT coding sequence into dorsal raphe neurons of SERT(-/-) mice. We previously showed that this restores SERT functionality and stress-vulnerability in the mice. In aim 1, we would alter the hSERT coding region in the lenti-hSERT by a systematic site-directed mutagenesis approach to define the structural features of SERT necessary for stress-induced translocation. Natural variants of hSERT have been identified in the human population by genome sequencing, and some of these variants have been described as conferring disease risk to the affected individuals. In aim 2, we would engineer lenti-hSERT to express these natural variants, then determine the effects of the resulting structural changes on the stress-response in mice. SERT(-/-) mice expressing different forms of the lenti-hSERT would be assessed 1) behaviorally for stress-induced aversion responses, 2) neurochemically for stress-induced translocation of SERT protein to plasma membrane of synaptosomes isolated from ventral striata, and 3) biophysically using rotating disk electrovoltammetry to define the effects f stress on serotonin transport kinetics. The proposed studies would better define how stress-induced changes in serotonin transport in the ventral striatum might control the risk of depression-like behaviors in mice, and the proposed analysis of the natural human variants of SERT might help describe a possible genetic basis for individual differences in stress-resilience.

Project Summary The proposed Conte Center ?Stress Mechanisms Increasing Risk of Mood-Disorders? will involve the coordination by an Administrative Core of five research projects and one research core. This Administrative Core will have six principal responsibilities: 1) facilitate effective communication between the project teams, 2) coordinate communication between the research teams and the external advisors, 3) maintain a public website that presents the research progress in broadly accessible terms, 4) coordinate professional development for pre-doctoral graduate students and postdoctoral fellows working in the research teams, 5) coordinate local outreach efforts communicating advances in mental health research to local high school and middle school students, and 6) provide efficient resource sharing to interested members of the scientific community.

Project Summary Extensive research has established that kappa opioid receptor (KOR) antagonists can block the dysphoria, anxiety and cognitive disruptions caused by behavioral stress exposure and that KOR agonists produce strong anxiogenic, aversive and psychotomimetic effects in humans and animal models of human behaviors. Our current understanding is that the dynorphin-KOR system produces its depressive effects on mood and cognition through actions on the serotonergic (5HT) projections from Dorsal Raphe Nucleus (DRN) to the Nucleus Accumbens (NAc) and Ventral Tegmental Area (VTA). In addition, published results show that the dynorphin-KOR system regulates the dopaminergic (DA) inputs from the VTA to NAc. The studies proposed in Project 1 would use a combination of optogenetic and mouse genetic approaches to test the hypothesis that endogenous dynorphins, locally released within the DRN-VTA-NAc circuit during stress-exposure, produce aversion behaviors and disrupt cognition.

? DESCRIPTION (provided by applicant): The Silvio O. Conte Center at the University of Washington is designed with a specific goal of understanding how stress-exposure can exacerbate depressive symptoms and increase the risk of a depressive episode in vulnerable individuals. Clinical experience has established a strong interaction between a history of unregulated stress exposure and the risk of mood disorders, but how the stress- mediators produce enduring changes in brain function that result in depressive symptoms is not clear. Novel therapeutics based on new insights to these mechanisms have the potential to enhance stress-resilience in this vulnerable population. The research aims of this Center are designed to understand 1) how the dynorphins, which are released in response to repeated stress, cause aversive effects in mice that have been suggested to be responsible for a component of stress-induced dysphoria in humans; 2) how the serotonergic inputs from the dorsal raphe nucleus (DRN) that regulate the dopaminergic system in the ventral tegmental area (VTA) affect neuronal excitability and are changed by repeated social defeat stress exposure; 3) how stress exposure alters gene expression in the serotonergic neurons of the DRN; and 4) how repeated stress exposure alters the regulation by CRF of the dopamine release by VTA neurons projecting to the nucleus accumbens (NAc). These studies in mice will combined the use of transgenic Cre driver lines, conditional gene inactivation, optogenetics, and DREADD techniques to selectively regulate specific components of the DRN-> VTA -> NAc neuronal circuit known to be important in mood regulation. The studies will use behavioral, electrophysiological and fast-scan voltammetry techniques to measure the responses mediated by the different components of the circuit. Both male and female mice will be studied to identify sex-specific responses. The Center will include translational studies that attempt to link the mouse studies to stress responses in Women with treatment-resistant depression. A plausible hypothesis underlying this clinical study component is that the inflammatory mediators affecting gene expression in peripheral blood lymphocytes may also be acting as stress-mediators in brain. For example, both cytokines and dynorphins regulate the serotonin transporter function, and a link between their effects on neuronal circuits may suggest novel therapeutic approaches. The UW-Conte Center will support basic science and clinical research, as well as community Outreach and Training missions in a coordinated and synergistic mode.

Research Support Core Summary The Research Support Core will include: 1) a viral production core that will generate the viral constructs and particles required by the individual projects; 2) a consolidated mouse breeding colony and genotyping core that will maintain stocks of transgenic mice shared by different projects; 3) a shared mouse behavioral facility enables the project investigators to standardize their protocols, efficiently train new investigators, and avoid inefficient duplication of equipment; and 4) an electrode fabrication and implantation facility managed by a dedicated equipment manager will help ensure that carbon fiber electrodes, single unit electrophysiology recording tetrodes, head stages and swivels for in vivo recording, stereotaxic devices for electrode implantation and also for viral injection are appropriately maintained and new users are trained and supervised. The Research Support Core will enable the Center investigators to efficiently share equipment resources, manage shared behavioral testing space, produce shared viral constructs, maintain the mouse breeding colony. The Research Support Core would expand the existing research capacities to accommodate investigators working on other Projects in the Center. It would fund support staff for training and supervision of new investigators. It would facilitate new collaborations. Furthermore, centralizing these capabilities in the Research Support Core would enable a more efficient use of these resources.

Project Summary: Pharmacological activation of kappa opioid receptors (KOR) in humans elicits reports of dysphoria and cognitive disruption. KOR activation in rodents by agonists or by stress-evoked dynorphin release have been shown to produce aversion, increase anxiety-like behaviors, increase the rewarding effects of drugs of abuse (e.g. cocaine, ethanol & nicotine), increase addictive drug self-administration, and reinstate extinguished drug-seeking behaviors. The cellular and molecular mechanisms responsible for these dynorphin- dependent, pro-addictive behaviors are not fully understood, and a better understanding may suggest new therapeutic approaches to the treatment and prevention of stress-related diseases including some forms of drug addiction. Prior studies supported by this award demonstrated that KOR activation by stress-induced release of dynorphin or pharmacological KOR agonist administration produces aversion in mice and potentiates cocaine conditioned place preference by activating p38? MAPK in serotonergic and dopaminergic neurons to regulate excitability and serotonin transport. Conditional knockout of p38? MAPK in either serotonergic neurons of the dorsal raphe or dopaminergic neurons in the ventral tegmental area (VTA), blocked the aversive and pro-addictive effects of KOR activation. It is not yet clear how KOR regulation of the serotonergic, glutamatergic, and dopaminergic circuits individually contribute to the behaviors, and the studies proposed in this renewal are designed to better define how dynorphin regulation of each of these neural circuits results in the dysphoric and pro-addictive effects of stress. To accomplish these aims, we propose to: 1) use optogenetic activation and inhibition of dorsal raphe neurons that project to nucleus accumbens or VTA to define the circuits and mechanisms responsible for dynorphin-dependent, stress-induced potentiation of cocaine place preference; 2) identify the sources of dynorphin responsible for these behavioral effects by excising prodynorphin in pDynflox mice from candidate neurons then assessing the effects on stress-induced potentiation of cocaine place preference; 3) use whole cell voltage clamp recordings of VTA dopamine neurons to characterize the pre and postsynaptic effects of KOR activation of p38? MAPK, which has been previously shown to be required for these stress-induced behaviors. Kappa opioid receptor antagonists may have therapeutic potential in the treatment of addiction by promoting stress resilience, and the proposed studies would advance that concept by defining the actions of stress-induced dynorphin on the reward circuit. Functionally selective kappa opioid receptor agonists that activate G-protein signaling without stimulating p38? MAPK may be effective analgesics without producing the euphorigenic effects of mu opioid agonists or the dysphoric effects of conventional kappa agonists. The proposed studies would advance our understanding of the therapeutic potential of kappa selective ligands in the treatment of chronic pain and drug addiction.

Pilot Project Summary The University of Washington Center of Excellence in Opioid Addiction Research would establish a pilot project program designed to support 2 new projects per year. Proposed pilot projects will be solicited annually from the NIDA P30 Investigators. These proposals would be ranked by the External Advisory Committee based on a simple set of criteria: Priority will be given to proposals that 1) address an aspect of opioid use disorder, 2) support young investigators, 3) utilize the Imaging and Molecular Genetics resources, and 4) emphasize innovation/high risk. Low priority will be given to proposals that supplement existing funding. We expect that some of the pilot project proposals will come from trainees supported by our NIDA-T32 DA 007278. Providing research support to fellows already having stipend support from the T32 or an individual NRSA award will leverage the Pilot Project funds and strengthen our training program. Based on these criteria, the top 2 projects will be selected and the applicants will be asked to expand their outlines to a 5-page description that includes specific aims, research plan and deliverables. These proposals will again be reviewed by the members of the External Advisory Committee who will provide constructive input. Once these suggestions are incorporated, the 2 proposals will be forwarded to NIDA Program for additional consideration prior to funding. Based on these criteria, two proposals have been selected in this initial submission. Pilot Project 1 describes studies proposed by Mr. Tim O'Neal, a graduate student working in Dr. Susan Ferguson's lab (Mr. O'Neal has stipend support from a NIDA F31 NRSA). This project would use in vivo calcium imaging to analyze responses and excitability changes in PFC neurons during heroin self-administration. Pilot Project 2 describes studies proposed by Dr. Antony Abraham, a postdoctoral fellow working in Dr. Charles Chavkin's lab. (Dr. Abraham has been supported by the UW's NIDA T32). His project would evaluate novel activity sensors designed to characterize kappa opioid receptor activation in PFC neurons during morphine-withdrawal induced dynorphin release. Stress-induced dynorphin release has been shown to promote a dysphoric response that triggers craving and relapse, but the mechanisms and sites of action have not yet been characterized. Future Pilot Projects will be initiated annually after review.

Molecular Genetics Resource Core ? Summary The Molecular Genetics Resource Core (MGRC) will provide a variety of services for the University of Washington Center for Excellence in Opioid Research. We will provide state-of-the-art viral vectors for neural circuit dissection, generate numerous viral-based CRISPR/Cas9 targeting vectors for in vivo molecular circuit analysis, and provide access to platforms for the viral delivery of novel sensors and actuators developed by UW addiction researchers. The UW MGRC will also generate novel mouse lines for UW addiction researchers and provide access to existing mouse lines for researcher initiated investigations. A major provision of the UW MGRC will be hands on training in molecular genetics techniques for graduate students, postdoctoral fellows, and qualified undergraduates. We will also provide a Summer Workshop for the education and training of UW addiction research trainees. Collectively the resources, education, and training provided by the UW MGRC will facilitate the rapid dissemination of cutting-edge research on the molecular and genetic basis of opioid addiction and prepare the next generation of scientists for careers in substance use disorder research.

Summary: Opioid abuse is a major epidemic in the United States. In 2017 alone, abuse of prescription and illicit opioids resulted in over 60,000 deaths. The transition from therapeutic use of opioid pain relievers to destructive substance abuse occurs, in part, through maladaptive activation of mesocorticolimbic brain circuits. How these various sensory, reward, and stress neural circuits act at the systems, cellular and molecular levels remain poorly understood. As we work to advance our understanding of the development of opioid use disorder, we need to develop fundamental insights into how opioid circuits function in reward and aversion. In addition, we need to learn how these circuits are modulated, altered, and adapted over time in animal models of opioid addiction. We aim to leverage the diverse team of investigators at UW towards answering these questions. We propose to establish an advanced Imaging and Addiction Neural Circuits Core (INCC) to provide UW addiction researchers, neuroscientists, and the wider community with a suite of advanced neurotechnologies, for in vivo neural circuit perturbation and analysis. Beyond building and continually renewing a state-of-the-art imaging facility, a principal role of the Core will be to train the NIDA P30 Investigators in its uses in order to enhance the research of their NIDA funded projects. We will also disseminate these tools to the larger research community by providing on-site training. Collectivity, these resources will enable researchers at UW and beyond to identify and correct neurocircuit maladaptation observed in opioid addiction models.

Administrative Core Summary/Abstract The main goals of the Administrative Core would be to 1) organize monthly Research Progress meetings, 2) host external seminar speakers, 3) provide public outreach and website support, 4) ensure training in rigor & reproducibility of investigators working within the P30 Center, 5) facilitate training and professional development of fellows, 6) archive complete data sets underlying publications generated by the P30 Center and facilitate sharing of resources generated by the P30 Center. The Center would recruit External and Internal Advisors who would help ensure strategic and fair allocation of shared resources, guide training sessions, select and monitor progress of pilot projects. The Administrative Core would monitor budgets to ensure that expenditures match the projections, ensure compliance with Environmental Health & Safety, IACUC and OSHA rules for the Core Spaces, make the travel arrangements and meeting schedules of our External Advisors when they visit the UW Addiction Center, and provide up-to-date information about Seminars and meetings for the P30 Investigators and resource availability.

Overall Summary The ?University of Washington Center of Excellence in Opioid Addiction Research? is designed to provide shared resources that would enhance efficiency and facilitate collaborative research for the study of the effects of opioids on neural circuits with the goal of understanding opioid addiction mechanisms and developing novel treatments for drug addiction. Center participants from 16 UW laboratories are using optogenetic control of rodent behavior, in vivo neuroimaging of single cell calcium signals and other receptor signaling probes, viral gene expression and CRISPR/cas9 manipulations to deconstruct and study opioid self-administration. This group has a long-history of highly productive, collaborative, cutting-edge research and training that will be strengthened by the shared resources provided by this award. The proposed Center would be comprised of four components: The Administrative Core will coordinate resource utilization, organize monthly Research Progress Meetings, organize training and outreach efforts. The Imaging and Neural Circuits Core would develop shared resources for in vivo brain imaging in rats and mice using 1-Photon endoscopy, Inscopix imaging, 2-Photon confocal microscopy, Spatial Light Modulation, fiber photometry and operant behaviors. The Molecular Genetics Resource Core would provide DIO-AAV / CRISPR-cas9 / Canine Adeno Viral reagents, develop new activity actuators and sensors and provide advanced training in cell-specific genetic manipulation coupled with behavioral and computational analysis. A Pilot Project Core would enable participants and trainees to initiate new projects utilizing the Imaging and Genetic Cores and would foster training and collaborations within the Center laboratory groups and university community at large. The Center would become a national neuroscience resource by providing training to visiting scientists, reagents for genetic manipulation and novel actuators/sensors on request from investigators at other institutions. It will provide workshops and summer courses for training advanced undergraduate and graduate students in optogenetics, computational neuroscience, and viral design & construction techniques. All of these components would be focused on understanding the changes in neural circuitry responsible for opioid addiction and on the development of new therapeutic tools based on these insights.