Kevin Wickman - US grants

DESCRIPTION: The long-term goal of my research is to understand how G protein-gated potassium channels (GIRK or KG channels) contribute to inhibitory signaling throughout the central nervous system, and how the cellular consequences of KG function translate into the establishment or modification of complex behaviors such as pain perception, addiction, and learning and memory. KG channels are formed by heteromultimeric assembly of members of the GIRK channel subunit family. The four mammalian GIRK subunits are distributed throughout the central nervous system, heart, pancreas, and testis. While the importance of neurotransmitter activation of KG channels to cardiac function is well understood, little is known regarding their contribution to inhibitory signaling and behavioral modification in the central nervous system. The work detailed in this proposal seeks to reveal how KG channels work in concert with other G protein-coupled effectors to elicit synaptic inhibition in the brain. First, however, considerable effort will be devoted to delineating precisely where each GIRK subunit is expressed in the brain. GIRK mRNAs will be localized in the mouse central nervous system by in situ hybridization. A novel transgenic strategy will be used to complement and extend the localization studies by revealing the distribution of the GIRK3 subunit proteins at the subcellular level. In addition, potential molecular mechanisms underlying their distribution will be explored. We will also evaluate the contribution of KG channels and other ion currents to the acute and chronic effects of opiates in the locus coeruleus. Currently, there is disagreement in the field concerning the contribution of KG to the acute effects of opiates in the locus coeruleus. In addition, our understanding of the mechanisms underlying the chronic effects of opiate administration (tolerance) is incomplete. Mice lacking KG in the locus coeruleus will be used to address these gaps in our knowledge. Completion of this work will constitute a first step toward a comprehensive understanding of KG regulation by neurotransmitters in the central nervous system. In addition, it will provide a basis for evaluating the impact of this particular ion channel class on a variety of behaviors at the cellular and whole-animal levels.

G protein-gated inwardly-rectifying potassium ion channels (GIRK) mediate the postsynaptic inhibitory effect of many neurotransmitters and related drugs of abuse. The long-term goal of my research is to understand how GIRK channels influence behaviors associated with the modulation of inhibitory neuretransmitter signaling pathways. Recent findings from both forward and reverse genetic studies have suggested that the GIRK3 subunit influences the sensitivity of mice to key behavioral effects of opiates, including analgesia, reward, and dependence. Though the GIRK3 cDNA was cloned more than a decade ago, the precise function of this subunit remains controversial. The goal of this proposal is to understand how and where GIRK3 influences the sensitivity of mice to the behavioral effects of opiates. Our current working hypothesis is that GIRK3 assembles with other GIRK subunits to form functional channels that are relatively insensitive to GABA(B)-dependent inhibition. Indeed, preliminary studies show the loss of GIRK3 renders dopamine neurons of the VTA more sensitive to GABA(B) receptor activation. The observed decreased sensitivity of mice lacking GIRK3 to the behavioral effects of opiates could reflect, therefore, an increased sensitivity of VTA dopamine neurons to the tonic GABA(B)-dependent inhibition provided by local GABAergic interneurons. Consequently, relatively high levels of opiates would be required to disinhibit VTA dopamine neurons, a process thought to underlie the motor stimulatory and reinforcing effects of opiates such as morphine. This working hypothesis and conceptual framework will be tested using multi-disciplinary approaches described in two specific aims: #1) To measure the contribution of GIRK3 to GABA(B)- dependent inhibition in neurons. The function of GIRK3 will be evaluated by measuring GABA(B)-dependent GIRK currents in cultured neurons following multiple genetic manipulations designed to perturb the level and/or function of GIRK3. #2) To probe the contribution of GIRK3 and the VTA to opiate-induced behaviors. Stereotaxic methods to deliver drugs and genetic reagents to the mouse VTA will be employed, followed by assessments of opiate-induced behavior in an established testing paradigm.

DESCRIPTION (provided by applicant): Opioid-based drugs are mainstays for pain management despite their significant side effects and addictive liability. Abuse of opioid drugs such as heroin is linked to many serious health problems, including fatal overdose, spontaneous abortion, infectious disease such as hepatitis and HIV/AIDS, and cardiovascular and pulmonary problems. Given both their clinical significance and adverse impact on public health, it is imperative that we understand mechanisms underlying the physiological and behavioral effects of opioids. The work proposed herein centers on a key neural substrate of opioid reward - the ventral tegmental area (VTA) - and challenges conventional wisdom concerning the signaling pathway mediating the opioid-induced disinhibition of dopaminergic (DA) neurons, a key mechanism of opioid reward. The opioid-induced disinhibition of DA neurons in the VTA involves the direct hyperpolarization of VTA GABA neurons. G protein-gated inwardly- rectifying K+ (GIRK/KIR3) channels are widely-considered to mediate the opioid-induced hyperpolarization of GABA neurons, due largely to their documented roles in metabotropic postsynaptic inhibition in many neuron populations. Our recent attempt to validate this paradigm failed, however, revealing that GIRK channels do not mediate the inhibition of VTA GABA neurons, the disinhibition of VTA DA neurons, or reward-related behavioral effects of opioids. Instead, our findings suggest that the acute inhibitory actions of opioids on VTA GABA neurons are mediated by the inhibition of adenylyl cyclase and consequent activation of an ion channel exhibiting the unique regulatory and biophysical signature of the Trek subfamily of 2-pore (K2P) K+ channels. The goal of this study is to test the hypothesis that opioids indirectly stimulate VTA dopamine neurons, and evoke reward-relevant behaviors, by activating Trek channels in VTA GABA neurons. At present, there are scant data concerning Trek expression in the VTA and no reports of Trek channel involvement in opioid signaling. As such, we will begin in AIM 1 by determining whether it is Trek1 or Trek2 that carries the MOR- activated K+ current in VTA GABA neurons. Well-characterized function-blocking antibodies directed against Trek1 and Trek2, as well as single-cell RT-PCR, will be applied to electrophysiological studies involving VTA GABA neurons in slices. In AIM 2, we will use available Trek knockout mice to measure the impact of Trek ablation on opioid signaling in the VTA, and on the motor-stimulatory and reinforcing effects of morphine. In AIM 3, we will seek a better understanding of the novel observation that the MOR-activated K+ current in VTA GABA neurons is significantly enhanced in Girk knockout mice. Electrophysiological and behavioral approaches will be used to probe the relationship between the MOR-activated K+ current in VTA GABA neurons and the complex adaptations linked to chronic drug administration. The proposed work will reframe our understanding of signaling downstream from opioid receptors and as such, may have significant implications for diagnostic or therapeutic strategies relevant to pain management and addiction. PUBLIC HEALTH RELEVANCE: Opioid-based drugs target neural circuitry important for pain processing and reward, actions that explain both their beneficial (analgesic) and untoward (addictive) effects. This proposal challenges conventional wisdom concerning the molecular details of opioid signaling in a key neuron population involved in reward. A clear understanding of the molecular mechanisms of opioid reward is crucial to our understanding of addiction and to the design of more selective and effective therapeutic approaches to pain management.

DESCRIPTION (provided by applicant): Cardiac output adjusts on a beat-to-beat basis due to the changing balance of parasympathetic and sympathetic input to the heart. G protein signaling pathways are fundamental to this important process. Indeed, the prototypical signaling pathway consisting of the type 2 muscarinic acetylcholine receptor (m2R) and the G protein-gated atrial potassium channel IKACh mediates in large part the inhibitory effects of parasympathetic activity on the heart. Too much or too little parasympathetic influence on the heart can trigger arrhythmias, often with fatal consequences. The long-term goal of our research is to identify and characterize molecular mechanisms that control or impact m2R-IKACh signaling, and consequently, the parasympathetic regulation of the heart. This application capitalizes on the recent discovery that IKACh associates physically with a regulatory complex consisting of the sixth member of the Regulator of G protein Signaling protein family (Rgs6) and the fifth member of the G protein beta subunit family (G¿5). The interaction between Rgs6/ G¿5 and IKACh has clear functional implications, as temporal aspects of m2R-IKACh signaling are altered in atrial myocytes from mice lacking Rgs6. Together, these preliminary data suggest the central hypothesis of this proposal, namely that Rgs6/ G¿5 serves as a negative regulator of m2R-IKACh signaling in the heart, with a corresponding and predictable influence on the parasympathetic control of cardiac output. This hypothesis will be tested by pursuing three complementary Specific Aims: (1) To understand the molecular organization and functional significance of the Rgs6/ G¿5 -IKACh complex, (2) To define the impact of Rgs6/ G¿5 on m2R-IKACh signaling in atrial myocytes, and (3) To understand the significance of the Rgs6/ G¿5 complex to cardiac physiology. The strategy proposed to address these aims will entail a synergistic combination of biochemical, molecular biological, electrophysiological, and physiological approaches, each exploiting the existence of a powerful array of reagents and animal models. Successful completion of these studies will yield detailed insights into the molecular determinants of Rgs6/ G¿5 -IKACh complex assembly and a clear understanding of the functional correlates of complex formation on m2R-IKACh signaling as assessed in the well-controlled expression system, the native context of the atrial myocyte, and the whole animal. By leveraging the unique strengths and research infrastructures of two laboratories, this project is poised to reveal novel insights into the organization and regulation of G protein signaling pathways and the parasympathetic regulation of cardiac output. As such, this information could prove useful for the development of novel therapeutic interventions designed to prevent or treat certain types of arrhythmia.

DESCRIPTION (provided by applicant): Drug addiction is a progressive disorder characterized by compulsive drug-taking behavior and high rates of relapse, even after prolonged periods of abstinence. The costs associated with drug addiction, factoring in lost productivity, health problems, and crime, are estimated at $600 billion per year in the United States alone. Our limited understanding of the neurochemical, molecular, and cellular mechanisms underlying drug reward, craving, and relapse has impeded our ability to confront this major public health issue effectively. The premise of this proposal is that a better understanding of the signaling pathways that mediate the cellular and behavioral effects of drugs of abuse will improve our ability to combat addiction. The focus of this proposal is on a form of inhibitory signaling and its relevance to the cellular and behavioral effects of acute and repeated cocaine exposure. Our work over the last decade has revealed that the behavioral effects of many drugs of abuse, including cocaine, are dependent on G protein-gated inwardly-rectifying K+ (Girk/KIR3) channels. More recently, we have found that in vivo cocaine exposure suppresses Girk signaling in dopamine (DA) neurons of the ventral tegmental area (VTA) and glutamatergic output neurons of the medial prefrontal cortex (Layer 5/6 mPFC pyramidal neurons), neuron populations instrumental to the reward-related behavioral effects of acute and repeated cocaine exposure. The goals of this project are to understand how cocaine suppresses Girk signaling in VTA DA and Layer 5/6 mPFC pyramidal neurons, and how these adaptations influence reward- related behavior and excitatory neurotransmission in the mesocorticolimbic reward circuitry. The novel conceptual framework is that Girk signaling in VTA DA and Layer 5/6 mPFC pyramidal neurons is an early addiction barrier that is overcome by cocaine exposure, paving the way for enduring adaptations linked to craving and relapse. Proposed studies will combine slice electrophysiological and behavioral assessments, with both approaches utilizing a novel array of mutant mouse lines and exploiting recent progress in our ability to perturb Girk signaling with unprecedented molecular, anatomic, and temporal precision. Efforts will center on three specific aims: 1) To understand the acute cocaine-induced suppression of Girk signaling in VTA DA neurons, 2) To understand the repeated cocaine-induced suppression of Girk signaling in mPFC pyramidal neurons, and 3) To probe the relevance of Girk signaling in the VTA and mPFC to reward-related behavior. Successful completion of this project will yield novel insights into the relevance of Girk signaling to reward related behavior, while also highlighting the role of such signaling in the cocaine-induced neuroadaptations that underlie key facets of addiction, including craving and relapse. Accordingly, this project targets multiple strategic goals of the National Institute on Drug Abuse, including prevention and treatment objectives that hinge on expanding our understanding of basic neurobiology as it relates to circuitry underlying addiction.

? DESCRIPTION (provided by applicant): G protein-gated Inwardly-Rectifying K+ (GIRK) channels are critical mediators of cell excitability in the brain and heart. GIRK channels are homo- and heterotetrameric complexes formed by four subunits (GIRK1-4). While evidence from gene ablation studies in mice suggests that specific GIRK channel subtypes make discrete contributions to organ physiology and behavior, the lack of subtype-selective GIRK channel probes has prevented rigorous evaluation of the consequences, and therapeutic potential, associated with GIRK channel activation. Recently, we identified a promising GIRK channel activator scaffold using a high-throughput screening approach. This scaffold was used to develop ML297, the first subtype-selective, small-molecule GIRK channel activator. ML297 is potent and strongly-selective for GIRK1-containing relative to GIRK1-lacking channels. ML297 reduces anxiety-related behavior in a GIRK1-dependent manner in mice, and suppresses seizures in rat epilepsy models. However, ML297 suffers from poor aqueous solubility, as well as low brain penetration and rapid clearance following systemic administration. Moreover, ML297 exhibits only modest selectivity for the predominant neuronal GIRK channel (GIRK1/2) relative to other GIRK1-containing subtypes, including the cardiac GIRK channel (GIRK1/4). These features of ML297 limit its utility as an in vivo probe for elucidating the relevance and therapeutic potential associated with GIRK1/2 channel activation. Accordingly, the goal of this project is to use the ML297 scaffold to develop new GIRK1/2 activators with an improved channel subtype selectivity profile and pharmacokinetic properties required of a brain-penetrant, in vivo probe. The project combines state-of-the-art approaches in medicinal chemistry and compound characterization, and will be conducted by an investigative team with complementary expertise in GIRK channel biology and in vivo probe development. The work is framed around three Specific AIMs: 1) To develop potent and selective GIRK1/2 channel activators, 2) To develop GIRK1/2 channel activators with optimized pharmacokinetic properties, and 3) To characterize the pharmacodynamics properties of GIRK1/2 channel activators in vivo. Work in AIMs 1 and 2 will involve an iterative parallel medicinal chemistry synthesis and testing strategy, and will yield GIRK1/2 activators with pharmacodynamic and pharmacokinetic properties superior to those of ML297. In AIM 3, these compounds will be characterized for in vivo efficacy, selectivity, and potency in the stress-induced hyperthermia (SIH) test, a paradigm with strong face validity for anxiolytic compounds. A small set of optimized probes exhibiting GIRK1/2-dependent efficacy in this test will undergo further evaluation in another anxiety model, and tests that explore the effects of the GIRK1/2 probes on motor activity and coordination. Completion of this research project will yield selective and effective in vivo probes for GIRK1/2 channels. These probes will facilitate future research into the relevance and therapeutic potential of GIRK1/2 channels, including more rigorous evaluation of their utility for treatment of anxiety-related disorders.

PROJECT SUMMARY Repeated cycles of alcohol intoxication and withdrawal foster adaptations in brain regions that regulate mood, learning, and goal-directed behavior. These adaptations are thought to promote heightened anxiety, cognitive deficits, and craving ? hallmarks of alcohol use disorder (AUD) that collaborate to promote compulsive drug- seeking behavior, increase relapse susceptibility, and impede the development of adaptive behaviors that could support abstinence. Although treatment options for AUD are limited, preclinical and clinical data have generated interest in the GABAB receptor (GABABR) as a potential target for therapeutic interventions aimed at diminishing craving and reducing alcohol intake. The focus of this project is on an ethanol-induced adaptation in GABABR-dependent signaling in the basal amygdala ? a key substrate of anxiety as well as learning related to rewards and aversive experiences. Using two distinct ethanol exposure models that yield repeated cycles of intoxication, we found that somatodendritic GABABR-dependent signaling is suppressed in principal neurons of the mouse BA, as measured 3-4 days after the last ethanol exposure. The adaptation is not seen in principal neurons of the lateral amygdala or pyramidal neurons of the medial prefrontal/prelimbic cortex, nor is it evoked by repeated cocaine. The adaptation is attributable to a suppression of G protein-gated inwardly rectifying K+ (GIRK) channel activity, a known determinant of anxiety-related behavior and associative learning. The goal of this project is to understand the salient features and mechanisms, as well as neurophysiological and behavioral implications, of the ethanol-induced suppression of GIRK channel activity in BA principal neurons. The two interrelated AIMs are to: (1) Elucidate mechanisms underlying the ethanol- induced suppression of GIRK channel activity. Proposed studies will employ techniques in ex vivo electrophysiology, immunoelectron microscopy, and neuron-specific viral manipulations to test the hypothesis that the ethanol-induced suppression of GIRK channel activity in BA principal neurons is mediated by the GIRK3 subunit-dependent internalization of GIRK channels. (2) Understand the downstream neurophysiological and behavioral implications of GIRK channel plasticity. Proposed studies will probe the implications of the suppression of GIRK channel activity in BA principal neurons, testing the hypothesis that it is sufficient to provoke adaptations in glutamatergic neurotransmission in discrete BA projections. In parallel, the consequences of the adaptation to anxiety-related behavior, associative (fear) learning, and voluntary ethanol consumption will be evaluated in ethanol-naïve mice, following viral genetic suppression of GIRK channel activity in BA principal neurons. Summary: This project leverages the complementary expertise of an experienced team, and the availability of custom research tools, to investigate a previously undescribed ethanol-induced neuroadaptation involving a known influence on anxiety-related behavior and associative learning. Successful completion of this project may yield new targets for interventions designed to treat AUD.

PROJECT SUMMARY: Viral Innovation Core Contemporary research on the neuroscience of addiction utilizes a broad set of genetically encoded tools that can be used to control neuronal excitability, highlight connectivity between neurons that form microcircuits, and report cellular activity states in behaving animals. Viral vectors exploiting the beneficial features of the Adeno- Associated Virus (AAV) backbone have proven invaluable for delivering genetically encoded tools to specific cell types and circuits in the nervous system, and are critical tools used by the addiction neuroscience community at the University of Minnesota (UMN). With the recent investments made in hiring to grow addiction-related research at UMN, the need for high-quality and efficient AAV vector production will increase substantially over the next decade. The Viral Innovation Core (VIC) seeks to meet the AAV vector needs of the UMN addiction research community, providing Center Investigators and Affiliates with access to advanced and experimental AAV production services, as well as a rigorous set of quality control and product evaluation processes that will inform the optimal design of future AAV-based investigations. The mission of the VIC is encapsulated in two Specific Aims: 1) Generation and advanced characterization of AAV vectors. The VIC will support the generation of AAV vectors ? including custom vectors ? for the UMN addiction research community. The VIC will employ a stringent process of product evaluation and quality control that, in aggregate, will represent a comprehensive profile of virus quality that can be used to help optimize vector production and purification approaches. This effort, combined with application-specific feedback, will help the VIC best advise investigators on the design, use, and storage of these tools. Providing this labor-intensive service through a centralized entity with skilled staff represents a critical efficiency for the VIC user base, and will facilitate the centralized examination, evaluation, and interpretation of data from a large and broad array of vector tools. 2) Engineering tropism of AAV. The VIC will also direct a research and development (Special Projects) program with the goal of developing new methodologies to improve the delivery of AAV vectors, oriented around the specific needs of the UMN addiction research community. The VIC will investigate whether ?arming? tropism-null AAV vectors with antibodies or other non-immunoglobulin scaffolds can redefine their tropism in a user-specified manner. Engineered tropism, which will enable viral gene delivery based on one or more surface receptors or markers, would represent a powerful approach to achieving precise manipulation of neural circuits relevant to addiction. Summary/impact. Tools generated by the VIC will promote engagement with the Structural Circuits Core and Imaging Cells during Behavior Core, fueling insights that will be consolidated within the Addiction Connectome Core. The efforts of the VIC will also yield synergies that expand the scope of supported projects and increase the impact of UMN research in the area of addiction. Furthermore, new multi-modal AAV targeting paradigms will represent a substantial benefit to the broader internal and external neuroscience research communities.