Sara R. Jones - US grants

[unreadable] DESCRIPTION (provided by applicant): The purpose of this application is to examine the effects of ethanol dependence and withdrawal on dopamine (DA) systems in genetically defined mice. Using a repeated ethanol vapor exposure and withdrawal-stress protocol shown to increase drinking in a two-bottle choice paradigm, we have documented changes in DA uptake, biosynthesis and D2 receptor function that are consistent with reductions in extracellular DA levels in C57BI/6J and DBA/2 inbred mice. We will examine these parameters in INIA mutant mice such as the 22TNJ and others with extreme ethanol-related phenotypes as well as potentially an inducible 5-HT1A receptor knockout as an anxious mouse model. We will extend our studies into the effects of two or more cycles of ethanol exposure and withdrawal on dopamine systems, because greater increases in drinking have been shown following two cycles. Changes in DA system function during extended withdrawal will be studied; to date all of our studies have been on mice sacrificed immediately after ethanol exposure, and there is evidence that the dopamine system is low-functioning for weeks after chronic exposure to ethanol. Overall, our goal is to challenge the DA system with a combination of stress and ethanol and document the adaptations and recovery profiles that result. We hope to establish specific molecular relationships between stress and alcohol on dopamine systems in the brain. Relevance to public health: Alcoholism is a pervasive disorder, and this research will address some of the fundamental mechanisms that may lead to alcohol addiction. Finding relationships between stress and alcohol on adaptations produced in dopamine systems may increase our understanding of why alcoholics relapse during abstinence. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The cellular mechanisms that mediate the intoxicating and reinforcing effects of ethanol are not clearly understood. One of the many neuronal targets of ethanol is the NMDA subtype of excitatory glutamate receptors. Many studies have demonstrated that ethanol acutely inhibits NMDA receptor function. This inhibition may be involved in behavioral sensitivity to ethanol effects. In addition, inhibition of NMDA receptor function has been shown to elevate dopamine (DA) system activity. Considerable evidence supports the hypothesis that increased mesolimbic DA system activity is one of the important components of the reinforcing effects of many drugs of abuse, including ethanol. Inhibition of NMDA receptors leads to DA system activation in several brain areas. Interestingly, few studies have demonstrated that ethanol inhibition of NMDA receptors may play a central role in elevating DA levels. The overall hypothesis guiding this research is that ethanol inhibition of NMDA receptor activity may represent an important mechanism through which ethanol elevates DA in the nucleus accumbens. The main approaches to be used to test this hypothesis include microdialysis and voltammetry studies of DA function following acute and chronic administration of MK-801 and ethanol to normal (wild-type) mice and mice with a genetic deficit in NMDA receptors. These models of reduced NMDA receptor function will be used to evaluate dopamine system function in an addiction-related brain area, the nucleus accumbens. From these studies we hope to gain insights into the receptor mechanisms involved in alcohol action.

DESCRIPTION (provided by applicant): Advances in molecular biology, including the creation of genetically altered mice, have had a tremendous impact on addiction research. Many mouse strains show altered responsiveness to cocaine and other abused drugs, providing a source of information on initial sites of action and downstream contributors to the effects of drugs. A powerful biosensor technique, fast-scan cyclic voltammetry (voltammetry), has recently been applied to the measurement of dopamine (DA) and other monoamine neurotransmitters in freely moving rats, and we propose to establish this technique for use in freely moving mice. This will allow monitoring of DA in mice with intact brain circuitry without the interference of anesthesia. In addition, the newest, state-of-the-art types of voltammetric recordings are of spontaneous DA release without any electrical stimulation. These measurements are true reflections of DA system activity that will be immensely helpful in determining how DA function is altered by cocaine and other abused drugs. DA "spikes", or rapid fluctuations of DA, are thought to be due to synchronous firing of DA neurons and provide a less artificial way of examining DA system activity than using electrical stimulation. The focus of this application is on two technical innovations: 1) use of voltammetry in freely moving mice and 2) recording spontaneous DA spikes. There are many applications of these measurements to addiction research in the future, including, for example, monitoring other neurotransmitters and using genetically altered or recombinant inbred mouse strains.

DESCRIPTION (provided by applicant): The neurobiological consequences of cocaine self-administration vary according to the experimental protocol used and are highly dependent upon the dose and frequency of administration. We have recently found that high-dose "binge" cocaine self-administration and deprivation in rats produces dramatic decreases in the potency of cocaine to alter dopamine (DA) neurotransmission. Using microdialysis to get an overall view of DA levels, we found that an i.v. administered dose of cocaine (1.5 mg/kg) no longer significantly elevated extracellular DA levels in the nucleus accumbens (NAc). To examine cocaine effects on the DA transporter, we used voltammetry in brain slices to measure DA uptake directly. The ability of cocaine to inhibit DA uptake was severely limited in rats that had self-administered cocaine compared to drug naive animals. In addition, the maximal rate of DA uptake was either increased or decreased, depending on the protocol, but the potency of cocaine was consistently decreased. This suggested a dissociation between DA uptake and inhibition of uptake by cocaine, although binding studies show that cocaine still binds to the transporter with unaltered affinity. This is a unique situation, caused by self-administration of high doses of cocaine. The implication of these findings is that cocaine effects can be manipulated independently of DA uptake, which may ultimately have relevance for the design of pharmacotherapies for cocaine addiction which would block cocaine inhibition but leave DA uptake intact. These are early days, however, in the documentaion of the dissociation between DA uptake and cocaine effects. The goal of this proposal is to provide a thorough investigation of the reduction in the potency of cocaine following self-administration. Relevance: The overall aim of this proposal is to understand how the dopamine transporter, the main target of cocaine in the brain, becomes insensitive to cocaine after several days of high-dose treatment with cocaine. The results will provide a new direction in the design of drugs to treat cocaine addiction. Potentially, the new drugs could change the dopamine transporter so that cocaine has no addictive effects and would not be abused.

DESCRIPTION (provided by applicant): The clinical use of methylphenidate (MPH, Ritalin) for treatment of attention-deficit/hyperactivity disorder is widespread, and there is a growing problem of MPH abuse, especially in college-age adults. College students use MPH non-medically, mainly to enhance performance, stay up late to study or to get high. In addition, adults of all ages are using high doses of MPH off-label for energy and cognitive enhancement. The abused dosages of MPH are 2-10 times those recommended for clinical use, however, little is known about the neurobiological effects of chronic exposure to these MPH doses. Our laboratory has recently discovered an unexpected consequence of chronic high-dose MPH treatment in mice. We have found that the behavioral and neurochemical responses to fluoxetine are qualitatively transformed. Fluoxetine is a serotonin (5-HT) transporter inhibitor which normally reduces extracellular nucleus accumbens (NAc) dopamine (DA) levels in control animals and fails to act as a reinforcer. Remarkably, however, we found that following chronic MPH treatment, fluoxetine takes on the characteristics of a psychostimulant drug, exhibiting rewarding effects as well as DA-elevating effects. Given that serotonergic drugs often suppress the reinforcing effects of DA agonists, these and other data suggest the possibility of a fundamental alteration in 5-HT-DA interactions whereby activation of the 5-HT system leads to elevated DA levels in limbic brain areas and activation of reward-related processes. We hypothesize that chronically elevated DA levels causes 5-HT1B receptors in the VTA to become supersensitive and their activation stimulates DA release into the NAc and other DA terminal regions. We hypothesize that this change could specifically increase the reinforcing effects of drugs with strong 5-HT activity such as MDMA, potentially leading to enhanced risk of polydrug abuse in people taking MPH. To explore the impact of chronic MPH treatment in mice on specific interactions between the 5-HT and DA systems, and to explore the neurochemical and behavioral consequences of MPH self-administration in rats, we propose to examine 1) 5-HT alterations in response to i.p. MPH treatment in mice, 2) Sites of 5-HT action, using dual probe microdialysis in mice 3) MPH self-administration in rats 4) 5-HT alterations in response to MPH self-administration.

DESCRIPTION (provided by applicant): The overall strategy of the INIA-Stress consortium is to take a cross-species approach to define adaptations of the cortical-limbic-HPA axis produced by excessive alcohol exposure as a platform for studying the reciprocal relationship between stress and excessive drinking. This project proposes to define the status of monoamine neurotransmission and GABAergic neuroactive steroid responsivity in key brain structures involved in stress, motivation, reinforcement and ultimately the regulation of excessive ethanol drinking. It will serve as one node for comparison of monkey and mouse tissues, with microarray, genetic, electrophysiology and HPA axis measures serving as other nodes. The overarching hypothesis of this project is that chronic ethanol exposure produces monoamine system adaptations which reduce activity during abstinence, leading to a state of low monoamine function which would produce dysphoria and potentially a drive to consume alcohol. Further, we predict that these adaptations are exacerbated by subsequent exposure to stress. Of particular interest is the novel finding that KORs become supersensitive during chronic ethanol exposure, especially given the efficacy of naltrexone, a non-selective opioid receptor antagonist, in treating alcoholism. Four aims are proposed, including 1) DA changes after chronic intermittent ethanol (CIE) exposure in mice and monkeys 2) CIE followed by forced swim stress in mice 3) Monoamine tissue levels in mice and monkeys and 4) GABAergic neuroactive steroids response to CIE and stress. These aims will provide a comprehensive assessment of monoamine changes induced by chronic ethanol exposure and the differential impact of a stress challenge on a background of chronic ethanol adaptations.

Breath alcohol estimation (BrACE) training, which emphasizes biofeedback and education about the effects of alcohol, is an intervention that may be valuable in the context of prevention programs. Preliminary data in our laboratory have identified enduring post-training changes in approaches to drinking and driving. The overarching goal of this Project is to extend quantification of the feasibility and benefits of BrACE training to adults who report early life stress (ELS). ELS, defined as experience of abuse or neglect through age 18, is a risk factor for alcoholism and has been associated with memory deficits in adulthood. Individuals with ELS are particularly prone to anxiety, an additional risk factor for alcohol use disorders. Thus, identification of an intervention that can minimize the potential for excess alcohol use among these individuals is of paramount importance. This Project will recruit three groups of nondependent alcohol drinkers: individuals with no ELS, individuals with ELS, and individuals with ELS plus high trait anxiety. The Project will differentiate baseline BrACE accuracy (Aim 1), efficacy of BrACE training (Aim 2), the influence of BrACE training on alcohol use and related behaviors (Aim 3), and sensitivity to the impairing effects of alcohol on memory, balance, and simulated driving (Aim 4) among these groups. Adults in these three groups will be randomized to Intervention and Control groups that will attend pretraining, training, and testing sessions. During these sessions, subjects will receive the equivalent of four standard drinks in a two-hour period. A cognitive/behavioral test battery will be completed before and after alcohol drinking, and subjects will estimate breath alcohol concentration at multiple time points. During the training session, the Intervention group, but not the Control group, will receive BrACE training. One and three months after the testing session, all subjects will return to the laboratory and complete self-report questionnaires regarding alcohol intake and alcohol-related risk-taking behaviors, including driving under the influence of alcohol. We hypothesize that BrACE errors and alcohol-related memory impairments will be highest in the ELS+Anxiety group, followed by the ELS group, then the no ELS group. We also hypothesize that BrACE training will be efficacious in all three groups. RELEVANCE (See instructions): Relevance of this Research to Public Health: The Project will identify feasibility of an educational intervention in two populations at significant risk for alcohol abuse and dependence: individuals with ELS with and without concurrent trait anxiety. The Project will also measure the effectiveness of this training in altering alcohol use and driving while intoxicated in these populations. Successful completion of this proposal's Specific Aims will clarify the utility of breath alcohol estimation training as a viable intervention for binge drinkers.

DESCRIPTION (provided by applicant): The clinical literature has emphasized that the abuse potential of cocaine is related to its speed of onset and that each time a person experiences a rapid and intense cocaine rush there is an increased risk of further drug taking and an increased likelihood of addition. While it is well known that cocaine intake in rats shows a fast- rising loading phase, most rodent models of cocaine addiction instead have focused on the maintenance phase and have explored the effects of long access sessions. The general assumption is that more drug exposure and high intake produces an addicted phenotype. This grant challenges that premise. Our hypothesis is that brief episodes (eg. 5 min) of intense drug use are sufficient to cause a transition from recreational to binge-like patterns of intake. Our data show that self-administration procedures that engender spiking drug levels produce a more robust escalation of drug intake and a dramatic increase in the motivation to self- administer cocaine. Our hypothesis is that the number of 'spikes' (or loading phases) have a much greater impact on the addiction process than the maintenance phase or total drug intake. The experiments proposed in this grant are designed to confirm, extend and validate our initial findings. Specific Aim 1 will test the hypothesis that the number of spikes, the change in spike height and the rise time of each spike is an important determinant of escalation of drug intake and the motivation to response (as measured by a progressive ratio schedule). Specific Aim 1 will also test the hypothesis that cocaine self-administration is regulated by an endogenous circadian influence and that spiking cocaine levels can dysregulate this important physiological control mechanism. Our theoretical viewpoint draws heavily on modeling of cocaine concentrations in brain and it becomes important for us to examine and validate the assumptions underlying the model. Specific Aim 2 will validate the kinetic model using self-administration procedures and will examine the effects of cocaine consumption on extracellular cocaine and dopamine parameters. A method for separating appetitive and consummatory responding will be developed in Specific Aim 3. A two lever procedure will allows us to study cocaine consumption on one lever and the motivation to gain access to cocaine on the other lever. By using a PR schedule and manipulating timeouts and time of day the procedure will enable us to test hypotheses regarding the relationship between brain levels cocaine, drug seeking and drug taking.

? DESCRIPTION (provided by applicant): The overall goal of the current application is to understand the neurobiological mechanisms that help confer pathological behaviors like enhanced negative affect following ethanol physical dependence. Recent studies suggest that intrinsic lateral/basolateral amygdala (BLA) GABAergic neurons tightly control the expression of negative emotions including those expressed during withdrawal following chronic ethanol exposure. Dopaminergic inputs from the ventral tegmentum/substantia nigra pars compacta have been shown to regulate this GABAergic system and disinhibit BLA principal neurons which drive the expression of anxiety-like behaviors. Based on strong preliminary evidence, our proposed experiments will test the central hypothesis that ethanol dependence dis-inhibits BLA output by dysregulating dopaminergic modulation of these GABAergic neurons. We will test our central hypothesis and accomplish our overall goal by utilizing a well- established rat model of chronic ethanol exposure and by integrating optogenetic, pre- and post-synaptic dopamine neurophysiology, and behavioral experimental approaches. The BLA has been extensively implicated as an important regulatory component of the neural circuitry controlling both anxiety-like behavior during withdrawal from chronic ethanol exposure as well as reward-seeking in drug-naïve and -exposed animals. Specific Aim 1 will test our central hypothesis hypothesis by examining presynaptic dopamine function during withdrawal from ethanol dependence. We will directly measure DA release and reuptake in vitro by integrating in vitro fast-scan cyclic voltammetry with optogenetic control of DA release and chronic ethanol exposure. We hypothesize that, based on our previous publications, chronic ethanol exposure will differentially modulate basal or `tonic' DA levels and phasic DA release to ultimately enhance DA signaling. Specific Aim 2 will examine how postsynaptic DA receptor function is altered in the BLA using in vitro whole cell patch clamp electrophysiology with innovative optogenetic approaches to measure postsynaptic DA receptor signaling. Our working hypothesis is that ethanol physical dependence will increase postsynaptic D1- and D2-like DA receptor signaling such that DA-mediated inhibition of GABAergic function is up-regulated. Specific Aim 3 will place the detailed cellular effects of dependence on DA signaling within a whole-animal context by integrating optogenetic control of DA release with in vivo measures of BLA-dependent behaviors. Our working hypothesis is that dependence-related changes in dopamine neurotransmission and signaling ultimately control withdrawal-dependent anxiety-like behavior. The proposed work employs a unique and highly integrated experimental approach to provide unparalleled insight into the neurobiological mechanisms governing the negative reinforcing effects of chronic ethanol exposure. Ultimately, these studies will provide insight into potential cellular mechanisms governing abuse and relapse in human alcoholics.

Center for the Neurobiology of Addiction Treatment Pilot Studies Core Summary Dr. Sara Jones, Core Director The Center for the Neurobiology of Addiction Treatment (CNAT) Pilot Studies Program has proven to be an important mechanism for evolution of the Center by incorporating new and innovative project areas and technical expertise, and providing seed funding to junior investigators to help them obtain independent funding on their own. We are proposing a program with $30,000 available each year to fund one or two Pilot Projects. The selection process involves 1) a call for proposals and 2) analysis of the proposals by two non-Center reviewers, either from the External Advisory Board or ad-hoc scientists with particular technical or topical expertise relevant to the project. 3) The reviewers assign priority scores to the projects, and the highest scoring projects, usually 3-4, are 4) distributed to all Center investigators for evaluation, are discussed and rank- ordered by voting at one of the monthly Center meetings. This process provides the opportunity for open critique of Pilot Studies by all members of the Center, and often develops productive collaborative projects between CNAT and Pilot Study PI's. 5) Based on the rankings, final decisions for funding are made by the Pilot Studies Program Director. Rankings of pilot applications will be made according to the innovation and excellence of the research proposed, as well as its likely impact in the area of substance abuse research. Priority will be given to Studies that could have high impact, those most closely related to the theme of the Center, and those that may offer new technologies and research for future incorporation into CNAT.

Stress is a well-known factor in promoting heavy drinking and relapse to drinking in alcoholics, and avoidance of the negative affective symptoms of ethanol withdrawal (e.g. anxiety) is also believed to play a critical role in relapse to heavy ethanol use. Further, anxiety disorders may predispose individuals to drink heavily and these high intakes may exacerbate the interaction of heavy drinking and anxiety. Negative affective behaviors are modulated by neuropeptides such as dynorphin, and its target, kappa opioid receptors (KORs). We will use a mouse model of chronic intermittent ethanol (CIE) exposure combined with forced swim stress to understand the role that KORs play in the maladaptive neurobiological changes induced by stress/ethanol interactions. Our previous work revealed that the function of the dynorphin/KOR system in the nucleus accumbens (NAc) was robustly up-regulated by chronic ethanol exposure and withdrawal in a time/exposure dependent manner in both the CIE model in mice and in a long-term voluntary ethanol drinking model in monkeys. In addition, in mice we found that systemic administration of a KOR antagonist reduced anxiety/compulsive behaviors (marble burying) and withdrawal-induced excessive drinking. Because norepinephrine (NE) and dopamine (DA) are both involved in negative affective responses to stress, and are known targets of KOR activity, we will examine KOR regulation of DA signaling in the NAc, which regulates motivation to drink alcohol, and NE signaling in the basolateral amygdala (BLA), which regulates stress/anxiety-related behaviors. We have established that KOR inhibition of DA signaling in the NAc is up- regulated by chronic intermittent ethanol (CIE) exposure, and we will expand these findings to include activation of the NE system by forced swim stress (FSS) and CIE, regulation of this system by KORs, and interactions with DA that may exacerbate negative affective responses and subsequent ethanol drinking. The overall goal is to define FSS and CIE-induced adaptations in stress-responsive limbic circuitry nodes that are responsible for withdrawal-associated negative affect and that can be targeted for the treatment of alcoholism.

NEUROBIOLOGY OF DRUG ABUSE TRAINING PROGRAM SUMMARY One of the keys to understanding the actions of drugs of abuse is a systematic study of the neurobiological basis of drug abuse. Such studies must involve multi-disciplinary approaches to examine the effects of drugs of abuse at several different levels of brain function. This is the goal of the current application, the Neurobiology of Drug Abuse Training Program, which proposes to continue a successful tradition of NIDA training at Wake Forest School of Medicine (WFSM). This program requests funding to train four predoctoral students in a multi-disciplinary program in the neurobiology of drug abuse. The program consists of 15 faculty members at WFSM, with research interests including molecular biology, receptor pharmacology, brain imaging techniques in humans and non-human primates, electrophysiology, and behavioral analysis of drug self- administration. The research of the faculty is supported by a significant number of federally-funded grants related to the field of substance abuse. A central focus of research for the training program is the NIDA-funded Center for the Neurobiology of Addiction Treatment, which offers highly integrated collaborative research projects among a number of faculty. The program is organized around three principal areas of research: Molecular/Cellular Neurobiology, Neurobiological Systems, and Behavioral Neurobiology. The training program offers a specific course in drug abuse related to each of these three areas. Predoctoral students have a choice of two different Ph.D. degree tracks: Integrated Physiology/Pharmacology and Neuroscience. Although these programs have their own individual requirements, specific drug abuse-related topics are integrated into the standard programs. The training program offers specific seminars and journal clubs for trainees. The program also contains specialized components dealing with grant writing, rigor and transparency in research, and ethics in scientific research. Recruitment of students will be aided by the fact that the field of neuroscience is one of the fastest growing disciplines in the biological sciences. In addition, recruitment of applicants from under- represented minorities will be a high priority, including a bridge program with North Carolina Central University. In summary, the Neurobiology of Drug Abuse Training Program not only offers students outstanding opportunities for education and research in the neurobiology of drug abuse, but is also a valuable resource for the field of drug abuse by providing trained young investigators capable of independent scientific careers.

SUMMARY The Wake Forest Translational Alcohol Research Center (WF-TARC) Pilot Core will provide a mechanism for evolution of the Center by incorporating new and innovative research areas and technical expertise, and provide seed funding to junior investigators to help them obtain independent funding of their own. We are proposing a program with $100,000 available each year to fund four Pilot Projects. The selection process involves 1) a call for proposals, 2) analysis of the proposals by two non-Center reviewers, either from the External Advisory Board or ad-hoc scientists with particular technical or topical expertise relevant to the project, and 3) the reviewers assign priority scores to the projects, and the highest scoring projects, usually 5- 7, are 4) distributed to all Center investigators for evaluation, and are discussed and rank-ordered by voting at one of the monthly Center meetings. This process provides the opportunity for open critique of Pilot Projects by all members of the Center, and often develops productive collaborative projects between WF-TARC and Pilot Project PI's. Rankings of pilot applications will be made according to the innovation and excellence of the research proposed, as well as its likely impact in the area of alcohol use disorder research. Priority will be given to studies that could have high impact, those most closely related to the theme of the Center, and those that may offer new technologies and research for future incorporation into WF-TARC.

Project Summary/Abstract This R13 application requests support for the Gordon Research Conference (GRC) and associated GRS on Alcohol and the Nervous System that will take place in March 2020 and 2022 at the historic Hotel Galvez in Galveston, TX. The inaugural GRC on this topic took place in 2014 and was considered a tremendous success by all attendees. This application seeks to build on the success of the first three meetings by bringing together young and established alcohol researchers, as well as preeminent scientists from related fields, to discuss the latest groundbreaking developments in neuroscience research on alcohol-related health issues. The central goal will be to foster deep and open discussion of research developments, build new scientific collaborations, and propel the next generation of scientific advances in alcohol neuroscience research. The following four specific aims will ensure that we accomplish this goal: 1) To provide an international forum that promotes open discussion of cutting-edge research at the forefront of alcohol effects on the nervous system and related neuroscience areas. 2) To introduce breakthrough neuroscience techniques that facilitate the understanding of brain mechanisms driving alcohol drinking and abuse, as well as mechanisms involved in fetal alcohol spectrum disorders. 3) To promote interaction between young and senior investigators and the exchange of ideas that will shape the future directions of the alcohol neuroscience community. 4) To foster the development of the next generation of alcohol researchers by encouraging the participation of students and postdoctoral fellows, both in alcohol and related research fields. The Alcohol and the Nervous System GRC will focus on presentations and discussions at the forefront of our field. The scope of the research presented will be restricted to basic and clinical studies involving alcohol and the nervous system as well as groundbreaking new neuroscience techniques that can be brought to bear on the greatest challenges facing our field. Talks will address questions centered on the neural substrates that contribute to alcohol-related health disorders across the entire lifespan. The unique GRC format is ideal for breaking down barriers to progress and includes formal talks interspersed with ample discussion time, poster sessions, and informal discussion periods designed to stimulate communication, planning, and collaboration. To foster the growth of the next generation of alcohol researchers, we will put concerted efforts into encouraging and supporting participation of students, postdoctoral fellows and young investigators, both in the GRC and GRS. Thus, this R13 will foster communication of frontier alcohol research, opportunities for cutting edge scientific collaboration, and entry of new and younger investigators into the alcohol research field. We envision the GRC and associated GRS on Alcohol and the Nervous System will significantly advance our current understanding of the neurobiology of alcohol addiction and stimulate the development of effective therapeutic approaches for the treatment of alcohol use disorders.

Biased Kappa Opioid Agonists as Non-addictive Analgesics. Prescription opioid abuse has increased dramatically over the last decade and now comprises the largest growing substance abuse problem in the United States. These abused medications are mu opioid agonists, and alternatives for pain treatment are needed. Kappa opioid receptor (KOR) agonists are analgesic in humans and have low abuse liability, however the dysphoric and sedating properties of these compounds have precluded their clinical utility and development. Recently several groups have discovered that the analgesic properties are mediated through G- protein signaling, while the sedating and dysphoric effects are mediated through non-G-protein pathways, likely either ßarrestin2 or ERK1/2. Interestingly, several groups have described KOR agonists that preferentially activate G-protein pathways over ßarrestin or ERK1/2 pathways, termed biased KOR agonists. These compounds are promising candidates as non-addictive analgesics that will be devoid of the sedating and dysphoric effects of other typical KOR agonists. We have recently demonstrated that one such compound produces antinociception in mice and rats without evidence of sedation and dysphoria. Further this compound does not interfere with forebrain dopamine neurotransmission, thought to underlie the untoward effects of typical KOR agonists. The extent and nature of signaling bias is determined in vitro, using cell-based assays. As such, it is not readily apparent how this signaling bias in vitro translates into improved pharmacology in vivo. These compounds have been explored as analgesics primarily using non-clinically relevant pain assays, such as tail flick or hot plate tests in rodents, and these assays do not generally predict clinical efficacy. In this application we propose to assess a wide profile of signaling bias for KOR agonists developed using two distinct chemical scaffolds, and to determine the relationship between intracellular signaling bias across multiple pathways in native neuronal tissue (striatum and dorsal root ganglia) with in vivo efficacy in dysregulation of dopamine neurochemistry and production of antinociception, sedation/dysphoria, and abuse liability using clinically relevant rodent models of inflammatory, post-operative, and neuropathic pain, and drug self- administration. Additionally we will assess the role of gender in signaling bias efficacy, as females have been shown to be more sensitive to the analgesic effects of KOR agonists and less sensitive to the dysphoric effects. We hope to determine a profile of signaling bias in vitro that translates into an improved pharmacological profile for KOR agonists in vivo that can be used for further drug development and mechanistic studies.