Michael R. Bruchas - US grants

DESCRIPTION (provided by applicant): The rationale of this study is to understand the mechanims underlying kappa opioid receptor (KOR) mediated stress-induced potentiation of cocaine conditioned place preference (CPP) from a signal transduction and anatomical perspective. This research proposal will examine the molecular and physiological mechanisms underlying the KOR-mediated stress response. Preliminary results suggest that this response is mediated by KOR-induced MAPK activation. Using striatal brain region homogenates, we will examine how stress (modified forced swim) changes reward circuitry in a KOR-dependent fashion. This proposed study will assess the role of G-protein coupled receptor kinase (GRK3) shown in preliminary studies to be necessary for the KOR-mediated stress response. In addition, these studies will examine the cell types and neuronal populations that these stress-induced KOR-mediated events occur by using antibodies specific for the the phosphorylated KOR (activated), MAPKs and neuronal markers. In doing so we will use tissue immunohistochemistry and primary culture techniques to understand the spatial and temporal relationship of KOR-mediated signal transduction following stress.

This proposal is an extention of a career development award (5K99-DA025182-02) for Dr. Michael R. Bruchas, who is trained as a pharmacologist, and has focused his research career studying the molecular, cellular, and behavioral components of stress and addiction. The proposed project, is to be conducted at Washington University-St. Louis, in the Departments of Anesthesiology & Anatomy/Neurobiology, a strong and enriching environment for neuroscience research. The proposal's overall goal is to better understand the neuronal relationship between stress and drug seeking. Specifically, it concerns Kappa opioid receptor (KOR) signaling and noradrenergic mechanims in stress and drug seeking. Recently, Kappa opioid receptors were shown to regulate stress-induced behavioral responses to drugs of abuse, including stress-induced reinstatement (termed relapse in humans), and potentiation of cocaine-conditioned place preference. We and others have also demonstrated that kappa opioid receptors couple to mitogen-activated protein kinase (MAPK) signaling cascade, that is required for these behaviors. In addition, it has been suggested that KOR systems interact with noradrenergic systems, although the behavioral consequences, molecular and cellular nature of these interactions are poorly understood. This proposal has 2 specific aims: 1) To determine the anatomical brain regions, subpopulations, and cell types where kappa opioid and noradrenergic circuits converge following behavioral (stress) and pharmacological stimuli. 2) To determine how stress and dynorphin/KOR activation regulate noradrenergic circuits to ultimately influence cocaine and methamphetamine reward. We will investigate noradrenergic systems, the mediation of KOR-dependent behavioral responses, and interaction between both systems. This project will investigate the anatomical relationships (receptors, cell types, regions) where opioid and noradrenergic circuits converge, and their respective roles in drug-seeking behavior. This project's goal is to further define the pharmacological and physiological mechanisms of stress-induced drug reward by dissecting the dynorphinergic and noradrenergic brain circuitry involved in the interactions between stress and drugs of abuse.

DESCRIPTION (provided by applicant): Activation of kappa opioid receptors (KOR) in humans elicits dysphoria, and KOR activation by agonists or by stress-induced dynorphin release in rodents produces reinstatement of drug seeking. The dysphoric/aversive effects of dynorphin/KOR system activity have been linked to increased cocaine self-administration, and cause reinstatement of cocaine seeking behaviors. While many reports of KOR dependent regulation of cocaine, morphine, and alcohol seeking exist, there are very few studies examining the role and mechanisms of stress-induced dynorphin-KOR activity on nicotine reinstatement. Despite recent efforts, nicotine use is at an all time high, is responsible for millions of deaths each year and remains one of the most difficult drugs to quit. The neural networks and cellular-molecular mechanisms responsible for KOR-dependent nicotine reinstatement are not understood. Given nicotine's diverse pharmacological profile and interaction within multiple circuits, understanding how dynorphin neural circuits and KOR activation causes nicotine reinstatement will provide novel, valuable, and important insights and suggest new therapeutic approaches to the treatment and prevention of stress-related nicotine relapse. While evidence shows a key role for KOR-dependent inhibition in ventral tegmental (VTA) nucleus Accumbens (NAc) circuits, recent evidence has strongly implicated dynorphin activation of p38 MAPK in the serotonergic dorsal raphe nucleus (DRN) as required for KOR-dependent reinstatement and aversion. The role of each pathway in nicotine reinstatement is not known, so we propose to methodically dissect how activation of KOR, either by stress-induced dynorphin, optogenetic modulation of dynorphin/CRF release, or systemic administration of a selective KOR agonist, results in reinstatement of nicotine conditioned place preference. To accomplish this we propose the following Aims using an array of multidisciplinary approaches: 1) to determine the role of dynorphin/KOR activity in serotonergic circuits as necessary and sufficient for stress-induced nicotine preference using viral rescue (gain of function), in vivo pharmacology, and mouse genetics to assess KOR in circuits mediating stress-induced and dynorphin/KOR-mediated reinstatement of nicotine preference. 2) to measure the effects of p38 MAPK activation in circuits required for stress-induced reinstatement of nicotine preference. 3) To use a novel mouse line coupled with optogenetic approaches to dissect dynorphinergic neural inputs into key sites of KOR action mediating reinstatement of nicotine preference. The proposed studies would test our central hypothesis that stress and KOR-induced reinstatement of nicotine reward is mediated as consequence of dynorphin-KOR-dependent activation of downstream signaling pathways in selected neural circuits.

DESCRIPTION (provided by applicant): Stress resulting from chronic uncontrollable pain has been directly linked to numerous mental health diseases. In patients exhibiting chronic pain the incidence of depression, anxiety, and addiction are a major concern and a growing health problem. The locus coeruleus (LC) noradrenergic (NE) system has been implicated in numerous affective disorders including stress-induced anxiety and depression-like behavioral states. Additionally, some have reported that the LC-NE system is also a critical component for integration of the pain neuraxis of ascending transmission and descending modulation. In this R21 proposal we focus on further dissection of this putative convergent role of noradrenergic neural circuits by examining the circuits and receptor-mediated signaling pathways involved in aversion, anxiety, and pain behavior using pharmacological and optogenetic approaches. The central hypothesis of this research proposal is that the LC-amygdala circuit is a key site of convergence for the coordination of the negative affect in response to pain. In order to better understand the role of this system in behavior we propose 2 aims. 1) To dissect the function of the locus coeruleus (LC) - Amygdala neural circuits in the control of aversive and pain-like behavioral responses. Using optogenetic, pharmacological, and behavioral approaches we will selectively express ChR2/NpH3.0 and stimulate and/or inhibit LC-noradrenergic and CeA-CRF neurons determine how cell-type specific activation or inhibition of these neurons are mediate aversion, anxiety, and chronic pain-like states in mice. 2) To determine whether noradrenergic activation of G-protein and/or arrestin-mediated signal transduction in the amygdala is sufficient for aversive and anxiety-like behaviors by using and engineering a novel optically sensitive biased G- protein coupled receptors. Together, this project and the experiments described could provide novel and important information about noradrenergic function and the intersection of negative affect and pain.

DESCRIPTION (provided by applicant): A major challenge in the field of neuroscience research on affective disorders is identifying the critical signaling pathways and neural circuits involved in complex behaviors that underlie stress-induced depression, anxiety, addiction and related psychiatric diseases. In basic neuroscience research, one major challenge in this area is modeling animal behavior such that we can predict human correlates effectively. The more complex the behavioral paradigm, and the more unique the neural circuit targeting approach, the more difficult this challenge becomes. Recent developments in the field of optogenetics have greatly improved our understanding of the functional neural circuits and behavioral responses in psychiatric disease, opening new avenues for treatment. However, one key limitation to these techniques is that animals are tethered, access to discrete subnuclei is limited, and control of multiple inputs simultaneously becomes cumbersome and challenging. As materials engineering and nanotechnology have developed the potential for the field of bioengineering and neuroscience to converge, has become more possible in solving these limitations and challenges. We have developed novel micro-ILED, biocompatible devices for completely wireless control of behavior including social defeat stress, home cage behavior and drug reinstatement. These micropolymeric devices could be used for the study and treatment of psychiatric diseases including depression, anxiety, and addiction. Recent evidence has implicated corticotropin-releasing factor and dynorphin as critical stress neuropeptides involved in social defeat stress, social interaction, and reinstatement of cocaine seeking. In this EUREKA proposal we combine our novel multimodal, optogenetic micro-LED devices with specific aims geared towards dissecting the role of stress neural circuits in affective behavior. We propose to: 1) Develop and refine micro-ILED devices by further miniaturization and adding additional functions to the semiconductor platform 2) to dissect hypothalamic and central amygalar CRF and dynorphin neural circuits in social defeat stress and reinstatement of drug seeking 3) develop and use optical GPCR signaling in a wireless context to assess how activation of downstream signaling for CRF and dynorphin ultimately influence behavioral responses and finally 4) to assess the heterogeneity of CRF and dynorphin inputs simultaneously using our wireless multimodal micro-ILED devices. This research will provide a foundation for the integration of cellular scale semiconductor devices deep within mammalian neural circuits, and will guide future efforts to interface and interact with selected neural circuits in psychiatric diseases.

Abstract: Opioid receptors have been targeted for the treatment of pain and related conditions for thousands of years. Three opioid receptors have been characterized, with a fourth opioid receptor called nociceptin (NOPR) or opioid-receptor like-1 being the newest member of the family. Recent evidence has shown that opioid receptors are potentially novel targets for addiction, anxiety, and depression. Furthermore, reports suggest that the NOPR - nociceptin system is involved in cocaine reward behavior. However, where in the brain NOPR receptors modulate these behaviors, how NOPR is regulated, and how it signals has not been defined. In order to better understand the role of this system in behavior we propose 2 aims: 1) We will determine how NOPR and NOPR -signaling mutants function in specific cells and dopaminergic neural circuits to control cocaine preference behavior using targeted viral rescue strategies, and behavioral models. Here we will employ viral-mediated cell type specific knock-in gain-of-function experiments in mice, using wild-type and mutant receptors to determine how NOPR receptors function in dopaminergergic circuits to control cocaine preference in vivo. 2) In aim 2 we will use a combination of mouse genetics, anatomical techniques with tDtomato-reporter mice, and optogenetic approaches to dissect the role of nociceptin containing neurons on cocaine place preference, reward, and aversion behavior. Together, this project and the experiments described could provide novel and important information about nociceptin-NOPR system function in drug abuse behaviors.

Abstract: Stress has been directly linked to numerous mental health diseases. In patients stress increases pain incidence of depression and anxiety, and are a major concern and a growing health problem. Anxiety disorders currently affect 18% of the US population. The locus coeruleus (LC) noradrenergic (NE) system has been implicated in numerous affective disorders including stress-induced anxiety. Additionally, some have reported that the LC-NE system is also a critical component for integration of stress-induced anxiety-like responses through its elevated activity and output to downstream circuits. In addition, recent evidence suggests that the central amygdala corticotropin-releasing factor positive cells may provide discrete input and regulation of the LC during stress. In this proposal we focus on further dissection of this putative convergent role of corticotropin-release factor and noradrenergic neural circuits in the LC, and its output to the basolateral amygdala by examining the circuits and receptor-mediated signaling pathways involved in aversion and anxiety. Here we use a multi-disciplinary approach that includes pharmacology, chemogenetics, optogenetics, and in vivo imaging approaches to define the specific cells, circuits, and receptors with the noradrenergic LC system that mediate stress-induced anxiety. The central hypothesis of this research proposal is that an LC- amygdala circuit is a key site of convergence for the coordination of the negative affective behavior in response to stress. In order to better understand the role of this system in behavior we propose 3 aims: 1) To dissect the function of the CeA - locus coeruleus (LC) ?neural circuit in the control of stress-induced aversion and anxiety- like behavioral responses; 2) to determine the necessity and sufficiency of the locus coeruleus (LC) ? BLA neural circuit, cell types, adrenergic receptors, and their signaling pathways in the control of stress-induced aversion and anxiety-like behaviors and 3) to determine and decode the network dynamics of specific BLA neuronal populations in response to stress, chemogenetic modulation upstream, and pharmacological disruption of receptor signaling. Together, this project and the experiments described could provide novel and important information about noradrenergic function and the intersection of negative affect and stress.

Project Summary/Abstract: The emerging field of optogenetics ? using light to engage biological systems ? holds tremendous promise for dissection of neural circuits, cellular signaling and manipulating neurophysiological systems in awake, behaving animals. However, the technological limits for implementing optogenetics in dissecting neuromodulators in awake, freely-moving behavior is clear while working with paradigms that require discrete spatiotemporal control of receptor signaling and when investigating neural circuits that have very small diverse, ?hard to reach? architecture, such as heterogeneous brain nuclei. To engage neuropharmacological receptor substrates, neuroscientists in nearly every field use cannulas (simple metal tubes) and have more recently adopted tethered fiber optics for in vivo optogenetics to control local release of neuromodulator monoamine or neuropeptides. Unfortunately, these current methods are rather limited and difficult to implement because they severely limit the spatiotemporal control over receptor signaling pathways in discrete cell types. Moreover, current technology lacks a full tool box for multiplexed, subcellular, spatiotemporal control of G protein coupled receptor signaling, the predominant means for neuromodulator communication in the brain. For these reasons, an innovative effort combining neuroscience with biochemistry and pharmacology was necessary in order to bring spatial-temporal in vitro and in vivo control over GPCR- neuromodulator signaling. Therefore, here we directly address the central goals of this RFA-NS-16-775 in the following manner. The central goal of this proposal is to develop a cutting-edge v2.0 Opto-XR receptors that spatially and temporally control neuromodulator signaling in vitro and in freely moving animals. We have proposed an uniquely integrated approach to achieve this goal that brings pharmacologists, physiologists, biochemists, and neuroscientists together in a unique parallel manner. In the two specific aims we will develop and test these novel tools in vitro and in vivo: 1) To develop mutant Gi and Gs, Opto-XR v2.0 receptors with greater signaling dynamics and altered color spectra and sensitivity using structure-function analyses and thorough in vitro characterization; and 2) To develop utility and characterize Gi and Gs versions of Opto-XR v2.0 constructs in vivo and in models of freely-moving behavior using both traditional and wireless optogenetic approaches. Successful completion of the proposal will provide the wider community of neuroscience with a long awaited spatiotemporal manipulation of GPCRs ? neuromodulator signaling within neural circuits in awake freely behaving animals. This new technology will also further widen the field for approaches that are capable of discrete control and optodynamic simulation of neuromodulator function in brain tissue.

Project Abstract The goal of this R21 proposal is to develop a robust, minimally invasive wireless photometry system for in vivo calcium measures in freely moving behavior. To achieve this, a miniaturized, wireless, `injectable' photometry platform (~300 mm wide, ~100 mm thick and several mm long) that enables quantitative measurements of fluorescence stimulated using a high performance microscale inorganic light emitting diode (µ-ILED) and captured using a co-located, sensitive microscale inorganic photodetector (µ-IPD) is proposed. These devices directly address current limitations in measuring calcium transient activity within any environment and facilitate sensing of genetically defined neural networks in more ethologically relevant behaviors -- central goal of the BRAIN initiative and RFA-EY-16-001. These small-scale device components will mount on thin, flexible filaments with overall dimensions significantly smaller than fiber optic cables. The resulting systems will greatly reduce motion artifacts, due to their direct integration at targeted regions of the brain; when implemented using wireless schemes for power delivery and data communication, they will allow complete freedom of motion of awake, behaving animals, suitable for use in complex, three dimensional environments and in socially interacting communities. Preliminary data from using hard-wired versions of these technologies and separate demonstrations of wireless implantable platforms establish feasibility of the foundational concepts. This proposal is divided into following two aims: Specific Aim 1: To examine brain activity in real-time on awake, behaving animals using a platform of injectable µ-ILEDs and µ-IPDs, with a test case measuring the fear conditioning responses. The thin, flexible, lithographically defined photometry probes will be insert into targeted regions of the deep brain, such as the basolateral amygdala (BLA), including lateral, basal, and accessory basal nuclei using stereotactic positioning hardware and surgical procedures adopted from those used in previous wireless, injectable systems for optogenetics. Specific Aim 2: To develop wireless schemes for power delivery and data communication for these systems, with demonstrations in fear conditioning and social interaction. Further size reductions and purely wireless modes of operation will greatly enhance the technology and the opportunities in neuroscience studies. Behavior experiments including fear conditioning (outlined in Aim 1) and social interaction (known to evoke BLA activity) will serve to demonstrate and optimize the fully wireless capabilities.

Project Summary The purpose of this application is to request a further five years of support for an Institutional National Research Service Award (years 26-30) which we have held for 25 years to support multidisciplinary, interdepartmental post-doctoral training focused on the neurobiology of substance abuse with a strong emphasis on neuroimaging, molecular and familial genetics, pharmacoepidemiology, behavior, neural circuits, and pharmacology. We request support for six postdoctoral fellows, as we have had for the past five years ? filling all slots consistently. The fellowship will usually last two years for PhDs and often three for MDs due to their lack of research training in pursuit of their medical degrees. Fellows with a wide variety of backgrounds will be recruited including: Psychology, Psychiatry, Genetics, Medicine, Anthropology, Sociology, Biology, Neuropharmacology and Neuroscience. The primary research training experience is the apprenticeship model ? conduct of supervised research under the tutelage of one or more preceptors who are well established researchers, and experienced mentors. The trainee's work is usually focused on a single but broad ranging research project developed jointly by the preceptor and trainee. Mentored research takes up approximately 70%-80% of the trainee's effort. The remainder of the training program is made up of lectures, seminars, reading courses, individual tutorials and, when appropriate, formal didactic course work available in our graduate and medical school. Tutorials in neurobiology and genetics are tailored for trainees with specific interests that may or may not fall into their major area of focus. To arrange these tutorials, the program coordinator and preceptor identify areas of specific interest and set up the tutorial for the trainees with one of our preceptors or tutors who best meets the training objective. The main research interests of the five Departments involved in this grant (Psychiatry [as the central, administrative department], Anesthesiology, Psychological & Brain Sciences, Neurology and Genetics) are broad yet all have a heavy focus on neurobiology in its broadest context and/or pain and pain management (i.e., anesthesiology). These Departments as a whole offer more than 30 hours of relevant seminars per week dealing with the subject matter of this grant and are open to any of our interested postdoctoral fellows, although to be sure we cannot envision any circumstances that would encourage attendance at all, or most, of these seminars. Some careful selection must be made to ensure that trainees focus on their main research and career objective, mentored research. None-the-less, it is reassuring to have that much ancillary training available to amplify or to broaden the perspectives of our trainees.

Project Summary/Abstract: The emerging field of optogenetics ? using light to engage biological systems ? holds tremendous promise for dissection of neural circuits, cellular signaling and manipulating neurophysiological systems in awake, behaving animals. However, the technological limits for implementing optogenetics in dissecting neuromodulators in awake, freely-moving behavior is clear while working with paradigms that require discrete spatiotemporal control of receptor signaling and when investigating neural circuits that have very small diverse, ?hard to reach? architecture, such as heterogeneous brain nuclei. To engage neuropharmacological receptor substrates, neuroscientists in nearly every field use cannulas (simple metal tubes) and have more recently adopted tethered fiber optics for in vivo optogenetics to control local release of neuromodulator monoamine or neuropeptides. Unfortunately, these current methods are rather limited and difficult to implement because they severely limit the spatiotemporal control over receptor signaling pathways in discrete cell types. Moreover, current technology lacks a full tool box for multiplexed, subcellular, spatiotemporal control of G protein coupled receptor signaling, the predominant means for neuromodulator communication in the brain. For these reasons, an innovative effort combining neuroscience with biochemistry and pharmacology was necessary in order to bring spatial-temporal in vitro and in vivo control over GPCR- neuromodulator signaling. Therefore, here we directly address the central goals of this RFA-NS-16-775 in the following manner. The central goal of this proposal is to develop a cutting-edge v2.0 Opto-XR receptors that spatially and temporally control neuromodulator signaling in vitro and in freely moving animals. We have proposed an uniquely integrated approach to achieve this goal that brings pharmacologists, physiologists, biochemists, and neuroscientists together in a unique parallel manner. In the two specific aims we will develop and test these novel tools in vitro and in vivo: 1) To develop mutant Gi and Gs, Opto-XR v2.0 receptors with greater signaling dynamics and altered color spectra and sensitivity using structure-function analyses and thorough in vitro characterization; and 2) To develop utility and characterize Gi and Gs versions of Opto-XR v2.0 constructs in vivo and in models of freely-moving behavior using both traditional and wireless optogenetic approaches. Successful completion of the proposal will provide the wider community of neuroscience with a long awaited spatiotemporal manipulation of GPCRs ? neuromodulator signaling within neural circuits in awake freely behaving animals. This new technology will also further widen the field for approaches that are capable of discrete control and optodynamic simulation of neuromodulator function in brain tissue.

PROJECT SUMMARY In 2019, abuse of prescription and illicit opioids resulted in an estimated over 47,000 deaths in the United States. The transition from therapeutic use to destructive opioid use disorder occurs through the maladaptive activation of mesocorticolimbic circuits. Despite decades of research linking these pathways with opioids, surprisingly little is understood about how opioids modulate the brain in vivo in space and time in freely moving animals. This is, in part, driven by the inability to detect and monitor opioids at sub-second timescales. Together, these issues highlight the need for significant advancements for ?in vivo precision pharmacology? as indicated specifically in this RFA-DA-20-019 NIDA program announcement. Recent developments using photoactivatable opioid compounds (optopharmacology) together with new optofluidic hardware devices show exciting promise for finally understanding the temporal characteristics of opioid signaling. However, further advances in opioid detection and activation are necessary for fully decoding how opioids modulate neural circuits in vivo. Here we address this challenge head on with a multi-disciplinary team of biochemists, neuroscientists and bioengineers. We will utilize a series of cutting-edge approaches to: 1) develop novel opioid sensors for in vivo, sub-second measures of fentanyl, morphine, and methadone, 2) demonstrate the utility of optopharmacological approaches for dissecting opioid action, and 3) apply the sensors and optopharmacological approaches to perform in vivo precision pharmacological experiments to modulate pain and reward circuits related to drug abuse.