Kevin Wickman - US grants

DESCRIPTION (provided by applicant): Neuronal G protein-gated inwardly rectifying K+ (GIRK) channels 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 shape behaviors associated with specific neurotransmitter signaling pathways. To accomplish this objective, we need to understand the functional implications of native GIRK channel diversity, and we need to develop the means to perturb GIRK channels with molecular precision. Accordingly, the goal of this proposal is to determine how subunit composition influences interactions between GIRK channels and related signaling molecules. Our working hypothesis is that subunit composition dictates interactions between GIRK channels and neuronal proteins that facilitate coupling with appropriate G protein-coupled receptors. The working hypothesis will be tested with cell biological, biochemical, electrophysiological, and behavioral experiments that examine the robust coupling between GIRK channels and the GABAB receptor. We will exploit the existence of a powerful set of mouse knockout lines and custom-derived antibodies to identify structural elements promoting robust functional interactions between GIRK2-containing channels and GABAB receptors (AIM #1), to determine how GIRK channel subunit composition influences interactions with endogenous neuronal proteins (AIM #2), and to examine how subunit composition influences the contribution of GIRK channels to the behavioral effects of GABAB receptor activation (AIM #3). While addressing issues related to GIRK channels and G protein signaling that have been refractory to standard recombinant approaches, the proposed research will also suggest novel points of intervention for the selective perturbation of signaling involving GABAB receptors and/or GIRK channels. In the context of GABAB-dependent signaling, such progress could translate into novel therapies for the treatment of epilepsy, pain, and addiction. Relevance to public health. All drugs used to treat human disease or distress produce both beneficial and untoward effects. The premise of the work presented in this application is that the beneficial effects of many drugs could be emphasized, or the negative effects suppressed, if we understood better the molecular mechanisms underlying the complex physiological and behavioral responses to drug administration.

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): Adjustments to cardiac output are made on a beat-to-beat basis in response to internal and external stimuli, and are mediated by the changing balance of input from the sympathetic and parasympathetic branches of the autonomic nervous system. While parasympathetic input normally tempers the pro-arrhythmic influence of sympathetic activation, too much parasympathetic influence can predispose to atrio-ventricular block or atrial fibrillation (a key risk factor for stroke). Conversely, too little parasympathetic influence is associated with heart failure, and is an independent predictor of increased morbidity and mortality in patients with coronary artery or congenital heart disease, and following myocardial infarction. Collectively, these observations suggest that altered parasympathetic regulation of the heart likely contributes to a broad spectrum of cardiac dysfunction, while simultaneously highlighting the inherent therapeutic promise of manipulations designed to predictably alter parasympathetic tone in the heart. The premise of this project is that the diagnostic and therapeutic potential associated with the parasympathetic regulation of the heart cannot be fully-realized without a clearer understanding of the signaling pathway(s) mediating its influence. A critical signaling pathway mediating the parasympathetic influence on the heart consists of the type 2 muscarinic acetylcholine receptor (M2R) and the G protein-gated potassium channel IKACh; the M2R-IKACh signaling pathway is a major determinant of cardiac rhythmicity, and dysregulation of this pathway has been implicated in both atrial and ventricular arrhythmias. Work in the initial project period established that the RGS6/Gß5 complex accounts for much of the RGS- dependent negative modulation of the M2R-IKACh signaling pathway, and that loss of this inhibitory influence yields enhanced parasympathetic signaling in the heart. The goals for the next project period are articulated in two Specific AIMs that are designed to: (1) Identify molecules and mechanisms underlying the RGS modulation of parasympathetic signaling, and (2) Develop targeted approaches to manipulate parasympathetic signaling in the heart. The proposed studies employ innovative and complementary approaches that leverage the unique strengths and reagents of two research labs, along with the expertise of an extended group of collaborators with overlapping research interests. Successful completion of the proposed work will yield a detailed understanding of the RGS-dependent modulation of parasympathetic signaling in the heart, along with tangible insights into the therapeutic potential associated with direct pharmacologic manipulation of the IKACh channel. Collectively, these efforts will result in a sophisticated understanding of the molecular and cellular mechanisms that shape the parasympathetic regulation of the heart, knowledge that can be translated into improved diagnostic and therapeutic approaches for arrhythmias.

PROJECT SUMMARY Drugs of abuse share an ability to enhance dopamine (DA) neurotransmission from the ventral tegmental area (VTA) to downstream targets, including the medial prefrontal cortex (mPFC) and nucleus accumbens (NAc). Direct stimulation of VTA DA neurons is reinforcing and sufficient to trigger the array of molecular, cellular, and behavioral adaptations that define addiction. The actions of drugs of abuse are opposed by inhibitory G protein signaling pathways in the reward circuitry. Psychostimulants can weaken inhibitory G protein signaling in VTA DA neurons and layer 5/6 pyramidal neurons of the prelimbic cortex (PLC), via a selective reduction in the cell surface expression of G protein-gated inwardly rectifying K+ (GIRK) channels. Genetic suppression of GIRK channel activity in drug-naïve mice evokes some of the cellular adaptations and behavioral outcomes typically associated with repeated drug exposure, supporting the outlook that GIRK channels are critical and exploitable contributors to innate addiction barriers. The goals of this project are to use new tools and approaches to gain refined insights into the mechanisms mediating the recruitment of GIRK-dependent signaling by drugs of abuse, and to investigate the therapeutic potential associated with enhancing GIRK channel activity in a neuron- and/or channel subtype-specific fashion. There are two specific aims: (1) To probe the recruitment and therapeutic potential of GIRK-dependent feedback to VTA DA neurons. Loss-of-function mutants, including mice lacking GIRK channels in DA neurons, suggest that the unique GIRK channel subtype found in VTA DA neurons is a key regulator of behavioral sensitivity to drugs of abuse. This premise will be tested using intersectional viral manipulations to enhance or suppress GIRK-dependent signaling selectively in VTA DA neurons, followed by behavioral assessments in non-contingent and response-contingent tests involving cocaine. In parallel, the therapeutic potential of novel activators of the unique VTA DA neuron GIRK channel subtype will be evaluated. Lastly, an in vivo optogenetic approach will be used to test whether phasic VTA DA activity is sufficient to engage and suppress GIRK-dependent signaling in VTA DA neurons. (2) To reveal the mechanisms and relevance of a GIRK-dependent feedforward inhibitory circuit in the PLC. Although work in the initial project period established that GIRK-dependent signaling in layer 5/6 PLC pyramidal neurons is an addiction barrier, how and when this barrier is engaged is unclear. Optogenetic and chemogenetic approaches will be used to test the working model that phasic activation of VTA DA neurons evokes a feedforward inhibitory circuit involving the D1R-dependent activation of layer 5/6 PLC GABA interneurons, which tempers the parallel DA-dependent activation of adjacent pyramidal neurons. The proposed studies also test the predictions that repeated engagement of this feedforward circuit is sufficient to trigger the suppression of GIRK channel activity in layer 5/6 PLC pyramidal neurons, and that strengthening GIRK-dependent signaling in these neurons confers resilience to the addictive effects of cocaine.

? 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.