Ellen Unterwald - US grants

neuropeptide receptor; opioid receptor; drug addiction antagonist; receptor expression; narcotic antagonists; cocaine; neurotransmitter metabolism; species difference; endorphins; ethanol; buprenorphine; morphine; methadone; receptor binding; autoradiography; guinea pigs; laboratory rat; high performance liquid chromatography; radioimmunoassay;

DESCRIPTION (provided by applicant): Drug addiction is characterized by repeating cycles of drug intoxication, withdrawal, and relapse. The greatest challenge in the treatment of addiction is the prevention of relapse to further drug-seeking and drug-taking behaviors. The negative mood state produced by drug withdrawal, including heightened states of anxiety and anhedonia, is a major contributor to relapse. Relapse is also facilitated by exposure to external stressors. This is an application for a competitive renewal of our research project that investigates the impact of chronic exposure to cocaine on the delta opioid receptor system. During the prior award period, we have shown that acute withdrawal from repeated cocaine administration results in increases in anxiety- and depression-like behaviors in a rat model and these behavioral effects are accompanied by a desensitization of delta opioid receptor signaling. Delta opioid receptor agonists were shown to be effective anxiolytic agents under both baseline conditions and during cocaine withdrawal. The important role of delta opioid receptors in anxiety was further demonstrated by the ability of delta opioid receptor agonists to attenuate stress-induced anxiety when injected directly into the central nucleus of the amygdala. The research outlined in this application will investigate the interactions of stress and the anxiety-like state produced by withdrawal from cocaine. The studies will determine if heightened anxiety leads to increased susceptibility to stress-induced relapse to cocaine-seeking behaviors using the reinstatement to cocaine place preference model in the rat. The anatomical site of action of delta opioid receptor agonists in relieving withdrawal-induced anxiety will be determined with a focus on the extended amygdala. As anxiety and the negative affective state that occur during cocaine withdrawal can precipitate relapse, the proposed research will determine if delta opioid receptor agonists can prevent stress-induced reinstatement. Additional studies will begin to elucidate the cellular and molecular mechanisms that are involved in anxiety-like states produced by cocaine withdrawal and the mechanism through which delta opioid receptors are producing their beneficial actions. The focus of these studies will be on the interactions of delta opioid receptors with corticotrophin releasing factor and noradrenergic transmission. The overall objectives of the proposed research are to determine the role of anxiety states in stress-induced relapse to cocaine-seeking behaviors and to elucidate the neural substrates underlying anxiety produced by withdrawal from repeated administration of cocaine. The significance of the proposed research is the establishment of a novel target for the prevention of relapse and the elucidation of the functional role of delta opioid receptors in the extended amygdala in modulating the negative effects of cocaine withdrawal. Dysregulation of the delta opioid system following chronic cocaine use may play a critical role in abnormal responsiveness to stress and the long-lasting vulnerability to relapse.

DESCRIPTION (provided by applicant): We are requesting a 5-year competing renewal of our NIDA P30 Center with support for the 6 current Cores (Administrative, Animal, Biochemical Pharmacology, Cell and Immunology, Integrative Pharmacology, and Molecular Biology) and the addition of a seventh, the Database and Drug Interactions Core. Since its inception, the P30 Center has enhanced productivity, synergy, and multidisciplinary research, and enabled the recruitment of new faculty into the field of drugs of abuse. Major integrative themes of our Center include 1) Neuroimmunopharmacology including the study of the effects of drugs of abuse on HIV, 2) Study of drugs of abuse, particularly opioids and cannabinoids, in regulation of inflammation, pain, body temperature, reinforcement, and immune function, and 3) Drug interaction studies which are vital because individuals rarely abuse a single drug. New directions include increased emphasis on cannabinoids and additional studies on the relationship between drugs of abuse and HIV. The scope of the ongoing and future projects in the Center marries in vivo and in vitro approaches that examine effects of drugs at the cellular, biochemical, molecular and whole-organism levels. Integration of information ranging from measurements of behavior to gene activation has stimulated collaborations leading to novel hypotheses and results. The Cores of the P30 Center have fostered multidisciplinary interactions resulting in a true intellectual integration and synergy leading to hypothesis building and implementation of lines of experimentation not previously conceived. The results from the collaborative studies supported by the P30 Cores have been truly innovative. The Center also is a focus for training students, postdoctoral fellows, and junior faculty. Temple University has made major commitments to the Center that will sustain growth in the area of drug abuse research in the future. The Center is the glue that brings together investigators currently funded by NIH or other Federal or non-Federal sources and enhances and extends the effectiveness of research related to drug abuse and addiction in a cost-efficient manner. Cutting edge research being conducted by members of CSAR is supported and enhanced by utilizing the expertise, methodologies, and techniques provided by the Cores.

DESCRIPTION (provided by applicant): This application requests support for the continuation of the Training Program Drugs of Abuse and Related Neuropeptides at Temple University which is currently in its 20th year. The purpose of the Drug Abuse Training Program is to provide a comprehensive training experience for pre- and postdoctoral fellows in the area of drug abuse and addiction. The program is multidisciplinary in nature with 20 participating faculty residing in the Departments of Pharmacology, Microbiology and Immunology, Neuroscience, Molecular Biology, Anatomy and Cell Biology, Pathology, Physiology, Psychology, and Pharmaceutical Sciences. Historically the strengths of our faculty have been in the areas of opioid pharmacology and neuroimmunopharmacology, and these strengths continue with our researchers making substantial contributions to these fields. More recently, our expertise has expanded to include strong programs in the pharmacology of psychostimulants, cannabinoids, nicotine and HIV infectivity. The laboratory approaches are state-of-the-art and range from molecular and cellular biology to behavioral analyses. Our current program supports eight predoctoral and four postdoctoral trainees and we are requesting continuation at this level. Our predoctoral trainees are exposed to a rigorous program of didactic and laboratory experiences. Postdoctoral trainees concentrate on research during their training but participate in seminars and courses related to the field of drug abuse and additiction. The Drug Abuse Training Program also provides a variety of career development activities for all trainees, such as manuscript writing, oral communication skills, grant preparation, teaching experience, attendance at national scientific meetings, laboratory management skills, and training in the responsible conduct of science. The goal of our program is to facilitate our trainees to become productive and independent researchers highly knowledgeable in research areas that will help solve problems related to drug abuse and addiction. This program provides a strongly interactive, dynamic, and supportive environment for trainees to develop into outstanding researchers. It serves as the focus for drug abuse training and research within the University and the surrounding area.

The Administrative Core is the nexus of the NIDA P30 Center. It fosters and promotes thematic integration, synergy, and interdisciplinary research. The Administrative Core advances a collaborative research environment by providing a variety of avenues to stimulate the exchange of scientific ideas. The Administrative Core is necessary for the overall cohesiveness of the NIDA P30 Center. It provides the framework and administrative support to conduct innovative multidisciplinary research, to foster collaborations, and to plan and execute the Center Activities. Recruitment of new investigators, mentoring and educational activities are centered in the Administrative Core. This Core oversees the progress and operation of the Research Support Cores through monthly meetings. It organizes yearly reviews by an External Advisory Board to provide critical feedback about the effectiveness of the individual Cores and overall NIDA P30 Center.

DESCRIPTION (provided by applicant): Cocaine addiction is established and maintained by several interrelated factors. The strong positive reinforcing properties of cocaine are clearly necessary for both its initial use and its repetitive self- administration. With prolonged use, other factors play a role in maintaining cocaine dependence including long-lasting neuroadaptations. As such, it is critical for the design of therapeutics to treat cocaine addiction to identify the mechanisms underlying cocaine-induced reward and plasticity. One molecule that recently has received attention for its role in psychiatric disorders and the therapeutics used to treat them, as well as in synaptic plasticity, is glycogen synthase kinase-3 (GSK3). Evidence suggests that GSK3 is uniquely situated to modulate neuronal function and plasticity. Our published and preliminary data demonstrate that GSK3 activity is necessary for cocaine-induced behaviors including reward, hyperlocomotion, and behavioral sensitization, and that the activity of GSK3 is regulated during cocaine exposure in a brain-region specific manner. Our data also demonstrate that agents that inhibit GSK3 are effective at blocking not only cocaine-induced reward, but also the reconsolidation of cocaine contextual memories, thus attenuating cocaine-seeking behaviors. The following specific aims are proposed to address the central hypothesis that GSK3 is a critical molecular mediator of cocaine-induced actions including drug reward and drug-seeking behaviors. In Specific Aim 1, the contribution of GSK3 to the rewarding effects of cocaine will be established using the place preference procedure. The role of GSK3 in reconsolidation of cocaine contextual memories and reinstatement to cocaine-seeking behavior will be determined. In Specific Aim 2, changes in GSK3 activity will be measured following acute and repeated cocaine administration and following expression of cocaine conditioned place preference. The receptor systems activated by cocaine that engage GSK3 signaling will be elucidated. Regulation of the upstream kinase, Akt, will also be measured. Immunohistochemical analyses are proposed in Aim 3 to determine the specific neuronal pathways where regulation of GSK3 occurs. Specific Aim 4 will elucidate sites of action of GSK3 inhibitors in modulating cocaine reward and reconsolidation. Taken together, these results will provide critical information about the molecular mechanisms driving cocaine addiction-related behaviors and have the potential of establishing GSK3 as a novel target for therapeutics to treat cocaine dependence. PUBLIC HEALTH RELEVANCE: The goal of this proposal is to determine the role of the GSK3 signaling pathway in the actions of cocaine as related to addiction. These studies will determine if modulation of GSK3 can interfere with processes involved in cocaine addictive behaviors and how exposure to cocaine alters this important intracellular pathway in brain regions critically involved in mediating the rewarding and conditioned stimulus effects of drugs of abuse. These studies aim to provide preclinical data to establish GSK3 as a novel target for the treatment of cocaine addiction which could be a major public health advancement and significantly reduce the costs of addiction to society.

Project Summary/Abstract The Drugs of Abuse Training Program prepares predoctoral students and postdoctoral fellows for successful independent careers in the field of substance abuse research. This program has been the cornerstone of interdisciplinary basic science training in addiction, drugs of abuse, and HIV/AIDS at Temple University since 1988. This application is requesting continuation of the program at the current level of support, 6 predoctoral students and 4 postdoctoral fellows. The Drugs of Abuse Training Program is administered through the Center for Substance Abuse Research (CSAR) at the Lewis Katz School of Medicine. CSAR brings together faculty from multiple disciplines who share a common interest in researching topics related to the neurobiology of addiction, pharmacology of drugs of abuse, and the intersection of drugs of abuse with immune function including HIV/AIDS. This common goal creates a rich, highly collaborative environment for trainees who?s research often extends beyond a single laboratory. The Drugs of Abuse Training Program is supported by a cadre of dedicated faculty mentors who are devoted to the education and training of the next generation of scientists. These faculty are highly successful researchers as evidenced by their excellent extramural research funding and strong publication records. The program provides rigorous training in scientific methods, experiment design, data analysis, and research ethics. It includes didactic instruction through a set of core courses and seminars focusing on the pharmacology of drugs of abuse, neurobiology of addiction, and neuroimmunology. Excellence in scientific communication is achieved through coursework and practical opportunities for written and oral presentations. Participation in seminars, meetings with visiting scientists, journal club, scientific retreats, annual self-assessments and faculty evaluations of progress results in trainees who are exceedingly well-prepared for the next step in their scientific careers. The program promotes the highest level of scientific integrity and ethics, along with personal and public accountability in the conduct of science. The Drugs of Abuse Training Program at Temple University has been effective in recruiting outstanding trainees including individuals from diverse backgrounds and has a near perfect record of retention. Trainees complete the program with excellent credentials and go on to successful careers in academia, industry, government and other science- related employment, thus fulfilling the NIH directive to improve human health. The Drugs of Abuse Training Program is a critical resource for supporting students and postdoctoral fellows and advancing the field of substance abuse research.

Compulsive drug-seeking and drug-taking behaviors are the hallmark of addiction, and these behaviors continue despite intense negative consequences to the individual. Maladaptive conditioned learning processes, together with molecular and cellular plasticity, play major roles in the development of this compulsive behavior. Addictive drugs including cocaine engage molecular signaling pathways that are involved in associative learning processes. Once learned, exposure to cues previously associated with cocaine can lead to conditioned physiological responses that are accompanied by intense drug craving which can trigger relapse. Hence, a goal of addiction treatment is to break the associations between previously learned positive subjective effects of cocaine and environmental cues that signal cocaine availability. In order to achieve this goal, detailed information is needed about the circuitry and molecular signaling within that circuitry that serves to reinforce and strengthen cocaine reward contextual memories. Glycogen synthase kinase-3beta (GSK3b) is uniquely positioned to regulate neuronal function and plasticity. It is important in dopamine-mediated behaviors and in memory processes, and GSK3 activity is required for cocaine-induced conditioned reward, hyperactivity, and locomotor sensitization. Relevant to relapse, GSK3b is activated by exposure to an environment previously paired with cocaine and, importantly, inhibition of GSK3b activity after reactivation of cocaine memories can abolish a previously established cocaine place preference. This project will address the overall hypothesis that GSK3b signaling is necessary for cocaine-induced reward, neuroplasticity and the reconsolidation of cocaine-associated contextual memories. Using a conditional genetic deletion mouse model, the anatomical and cellular substrates where GSK3b mediates cocaine-induced conditioned reward will be elucidated. Components of the GSK3b signaling pathway that are necessary for cocaine reward and reconsolidation of cocaine memories will be investigated including the downstream effector of GSK3b, the mTORC1 complex. The anatomic substrates of importance for maintenance of cocaine memories will be studied with the focus on a circuit consisting of the dorsal hippocampus to basolateral amygdala to nucleus accumbens. An additional aim will be to extend the studies into a cocaine iv self-administration model in order to investigate if cocaine seeking behaviors can be abolished by inhibition of GSK3 after recall of cocaine memories. Little is known about how the processes governing maintenance of cocaine memories may differ between males and females and hence, investigation of sex differences is another goal of the project. Using these multidisciplinary approaches, we are positioned to reveal molecular mechanisms and pathways underlying cocaine reward, contextual memories, craving and relapse, with the potential of establishing GSK3b as a novel target for therapeutics to prevent relapse to cocaine seeking.

Post-Traumatic Stress Disorder (PTSD) is a debilitating anxiety disorder that negatively affects the lives of men and women worldwide. The diagnosis of PTSD requires exposure to a traumatic or life- threatening event with persistent symptoms of hyperarousal, re-experiencing, avoidance, and negative cognition and mood. Although a large portion of the population is exposed to a traumatic event sometime during their lifetime, only about 15-30% develop PTSD. Among those that do, rates of co-occurring substance use disorder and/or alcohol use disorder are 3-4 times higher than in the general population. Despite the appreciation that PTSD increases susceptibility to substance and alcohol use, little is known about the biological basis of this co-morbidity. If the mechanism of the increased susceptibility to substance use is identified, then treatment approaches could be applied to help prevent or reduce the incidence of substance abuse and addiction in persons with PTSD. Several excellent rodent models of traumatic stress have been developed, however they have been inadequate to model the elevated drug- seeking behaviors that occurs in humans. This has restricted the ability of researchers to elucidate underlying biological mechanisms that increase susceptibility to drug and alcohol use in the setting of PTSD. We hypothesize that this is due to the failure to consider individual responses to the traumatic stress. Similar to the clinical situation where only a subpopulation of trauma-exposed people develop PTSD, only a subpopulation of trauma-exposed animals shows extreme behavioral phenotypes reminiscent of PTSD. Thus, the goal of this project is to establish methods that recapitulate the behavioral phenotypes of both PTSD and drug and alcohol use disorders, which is critical to determine the causative neurobiology of this co-morbidity and for its eventual treatment. The goal will be achieved by behavioral profiling of out-bred rats exposed to the single prolonged stress (SPS) model of PTSD, in order to select those subjects with a phenotype of persistent high anxiety and exaggerated trauma cue responses. The Aims of this project will determine if the SPS-susceptible rats show high rates of cocaine self-administration and voluntary ethanol consumption as compared with SPS-resilient and non-stress controls, thus establishing a robust model of co-occurring PTSD and substance/alcohol abuse. Potential sex differences in the prevalence of co-occurring high stress reactivity and high cocaine- and alcohol- seeking behaviors will be investigated.

PROJECT SUMMARY We propose to investigate how the chemokine CXCL12 (SDF-1?), and its main receptor, CXCR4, are influenced by exposure to the synthetic cathinone MDPV (methylenedioxypyrovalerone), and, in turn, how CXCL12/CXCR4 antagonism influences behavioral and neurochemical correlates of synthetic cathinone (ab)use. Different cathinones are found in bath salts products, but MDPV and its next-generation analogs appear more apt to cause life-threatening medical consequences including hypertension, tachycardia, aggression and suicide. Part of the problem with MDPV, as well as more traditional illicit psychostimulants such as cocaine, is a highly-addictive phenotype that perpetuates ongoing drug taking and relapse to drug taking. MDPV, similar to cocaine, blocks cellular monoamine reuptake but with enhanced potency at DAT and NET. Little is known about how non-monoamine systems in the brain reward pathway are affected by MDPV and contribute to its addictive effects. Since there is still no approved medication available for cocaine abuse, let alone for synthetic cathinones, it is anticipated that a new target, a new approach or both will lead to the first approved pharmacotherapy. Our proposed target is a chemokine, specifically CXCL12, and its receptor CXCR4. CXL12 is one of the few chemokines found in the brain. It also has a FDA-approved, commercially- available antagonist (AMD3100) to investigate a CXCR4-receptor mechanism. Notably, of all the chemokines, CXCL12 is the one most linked to psychostimulant addiction. Mice exposed to acute cocaine display increased plasma levels of CXCL12. In human cocaine abusers, CXCL12 is the only chemokine correlated to the history of pathological cocaine use and severity of dependence. Recently, we took the critical next step of linking the CXCL12/CXCR4 system in the mesolimbic dopamine circuit with psychostimulant reward by showing that repeated cocaine exposure increases CXLC12 gene expression in the midbrain ventral tegmental area (VTA) and produces place preference in rats that is reduced by a CXCR4 antagonist (AMD3100). The efficacy of CXCR4 antagonism extends to MDPV-induced behaviors, as our data show that AMD3100 reduces MDPV place preference, locomotor activation, and acquisition (self-administration) in rats. We propose behavioral, cellular and neurochemical experiments to test the hypotheses that CXCL12 and CXCR4 in the mesolimbic DA circuit are dysregulated by MDPV exposure and abstinence and that genetic or pharmacological antagonism of CXCR4 receptors in the VTA reduces MDPV acquisition, reinforcement and relapse by decreasing mesolimbic dopamine (DA) output. By providing information about how interplay between chemokine and mesolimbic brain reward systems impact psychostimulant addiction, we expect to identify CXCL12/CXCR4 as a chemokine- based therapeutic target for countering adverse effects of both new and established psychostimulant drugs.

The Animal Core for Addiction Related Behaviors of the NIDA P30 Center at Temple University will support collaborative NIH-funded research projects using in vivo rodent models in order to advance scientific knowledge on drugs of abuse, addiction, pain and the intersection of drugs of abuse with HIV/AIDS. One aim of the Core is to provide the scientific expertise, personnel, equipment and other resources that are necessary to assess drug reinforcement, reward, reinstatement, and related endpoints of cognition and emotional function in rats and mice as needed to rigorously evaluate potential new therapeutic compounds and targets, as well as to elucidate neurobiological mechanisms underlying addiction. Another aim of the Core is to provide mutant rodent models to P30 collaborating investigators in order to enable study of specific molecules and pathways in vivo. Breeding animals in a central facility reduces the expense and promotes access of these valuable models to multiple researchers. Core personnel will work with investigators to design experiments including an emphasis on appropriate controls, data analysis, and result interpretation. If requested by collaborators, the Animal Core will provide training in behavioral methods, or Core staff will perform the tests independently. The Core offers the following approaches: intravenous and oral drug and ethanol self- administration, operant responding for food or sucrose, conditioned place preference and aversion, intracranial self-stimulation, learning and memory assessments, anxiety- and depression-like behavioral tests, use of stress models and other methods as needed. New innovative behavioral assessments and animal models are proposed in this renewal application in order to enhance the impact of the research supported by the Animal Core for Addiction Related Behaviors. These include the application of behavioral economic methods and intermittent access paradigms to drug self-administration studies, measurements of ultrasonic vocalization to assess positive and negative affect during behavioral testing, breeding of transgenic Cre rats, and support of humanized mouse models of HIV-1 infection. The Core serves as a national resource by providing the knowledge, skills and equipment necessary to enhance research programs and to promote extension of NIH-funded projects to include behavioral endpoints and animal models relevant to substance abuse research.