Characterizing the mechanisms of kappa opioid receptor signaling within mesolimbic dopamine circuitry Katie Reichard A dissertation submitted in partial fulfillment of the degree requirements for the degree of: Doctor of Philosophy University of Washington 2020 Reading Committee: Charles Chavkin, Chair Paul Phillips Larry Zweifel Program Authorized to Confer Degree: Neuroscience Graduate Program TABLE OF CONTENTS Summary/Abstract………………………………………………………………………….……..6 Dedication……………………………………………………………………………….………...9 Chapter 1 The therapeutic potential of the targeting the kappa opioid receptor system in stress- associated mental health disorders……………………………….………………………………10 Section 1.1 Activation of the dynorphin/kappa opioid receptor system is associated with dysphoria, cognitive disruption, and increased preference for drugs of abuse…………………..13 Section 1.2 Contribution of the dyn/KOR system to substance use disorder, anxiety, and depression………………………………………………………………………………………..15 Section 1.3 KORs are expressed on dorsal raphe serotonin neurons and contribute to stress- induced plasticity with serotonin circuitry……………………………………………………….17 Section 1.4 Kappa opioid receptor expression in the VTA contributes to the behavioral response to stress……………………………………………………………………………………....…..19 Section 1.5 Other brain regions contributing to the KOR-mediated response to stress…………23 Section 1.6 G Protein signaling at the KOR …………………………………………………….25 Chapter 2: JNK-Receptor Inactivation Affects D2 Receptor through both agonist action and norBNI-mediated cross-inactivation Introduction………………………………………………………………………………………32 Methods…………………………………………………………………………………..………34 Results……………………………………………………………………………………………35 Discussion………………………………………………………………………………………..40 Chapter 3: Dopamine neurons of the VTA are heterogenous and there is a sex difference in their responses to KOR agonists 2 Introduction………………………………………………………………………………………43 Methods………………………………………………………………………………………......46 Results……………………………………………………………………………………………47 Discussion ……………………………………………………………………………………….52 Chapter 4: Regulation of kappa opioid receptor inactivation depends on sex and cellular site of antagonist action Introduction………………………………………………………………………………………56 Methods……………………………………………………………….………………………….58 Results...…………………………………………………………….……………………………63 Discussion…………………………………………….………………………………………….75 Chapter 5: Evaluating the Contribution of Pre- and Postsynaptic Potassium Channels to KOR Regulation of Dopamine Neurons Introduction ……………………………………………………………………………………..80 Methods …………………………………………………………………………………………83 Results.…………………………………………… …………………………………………….86 Discussion ………………………………………………………………………………………94 Thesis Summary and Conclusions…………………….……………………………………….106 Curriculum Vitae……………………………………………………………………………….107 Bibliography………………………….………………………………………………………...110 3 LIST OF FIGURES # Title Page # 2.1 NorBNI pretreatment shifts potency of quinpirole at inhibition of dopamine 37 release 2.2 D2 Receptor stimulation by quinpirole results in MJ33-dependent ROS 39 production in HEK293 cells expression both the short and long isoform of DRD2 3.1 Characterization of optically evoked dopamine release from subpopulations 50 of dopamine neurons genetically defined by neuropeptide genes 3.2 Sex Differences in U69,593 inhibition of dopamine release 52 4.1 Long-acting norBNI antagonism in females is effective if given in repeated 69 doses or after CMPD101 4.2 norBNI does not cause a long-lasting block of KOR inhibition of dopamine 71 release in the nucleus accumbens (NAc) core, whereas b-CNA receptor inactivation persists at least a week in NAc terminals 4.3 KOR Agonist U69,593 Activates a GIRK current in VTA dopamine neurons 73 that can be blocked by norBNI pretreatment 4.4 KOR agonist, nalfurafine, generates reactive oxygen species in VTA 74 Dopamine cell bodies, but not dopamine terminals in the nucleus accumbens, as measured by HyPerRed. 5.1 Non-specific potassium channel blockers attenuate the inhibition of dopamine 97 release by KOR agonist U69,593 5.2 Selective knockdown of potassium channels in dopamine neurons using 99 AAV-FLEX-SaCas9-U6-SgRNA. 5.3 Assessment of the KCNA2 knockdown on the regulation of evoked dopamine 101 release and dopamine-mediated behaviors 5.4 Genetic and functional validation of AAV1-FLEX-SaCas9-sgRNA(Kcnj6) 102 5.5 Impact of KCNJ6 knockdown on KOR signaling and U50,488 Conditioned 104 Place Aversion 4 Abstract & Public Summary An essay is due, your rent check is late, the national guard has been deployed to your city, you are experiencing violence within your own home: humans experience stress in varying degrees and durations throughout their lives, and the severity and proportionality often align with societal inequities. As stress neurobiologists, we play a role in describing the ways this stress manifests in our bodies, how it forever alters the pathways in our brains and shifts our own reactions to the world around us. In part, we do this work to understand the basic science of stress and how it increases risk for diseases like heart disease, stroke, hypertension, anxiety, depression, and substance use disorders. We also seek to find treatments, so that while society inflicts unearned stressors on its most vulnerable citizens, we may use pharmacology to ameliorate a fraction of its harms. Our bodies use neuropeptides to modulate our behavioral and emotional responding to stimuli, like stress. These small proteins act on receptors in our brain cells, or neurons, to promote certain behaviors and make long-lasting changes in neural connectivity. The opioid peptide dynorphin is produced in multiple brain regions, including those involved in decision making and reward learning, and plays an important role in the stress response. When released in response to drug-taking or stress, dynorphin (dyn) binds to a receptor called the kappa opioid receptor (KOR). Years of research from the Chavkin lab and others have shown that signaling at the kappa opioid receptor contributes to increased depression, anxiety, and substance use after the experience of behavioral stress. Researchers have also shown that molecules which can block dynorphin from binding to its receptor can reduce these negative psychological consequences. This dissertation digs into two big questions surrounding the effects of kappa opioid receptors: (1) How do the antagonists, or compounds which disrupt KOR signaling, work and how does 5 this knowledge help us better design therapeutic compounds in the future and (2) How do KORs signal within dopamine neurons, a population of neurons involved in decision making & reward that is altered by stress. In Chapter 1, I review the literature evaluating the therapeutic potential for KOR antagonists in treating mood disorders and substance use disorders. The chapter describes studies that demonstrated the role of dyn/KOR in mood and substance use disorders, reviews the brain regions critical for these behaviors, and discusses the importance of biased agonism and development of functionally selective ligands. In Chapter 2, I assess the role of c-Jun Kinase (JNK) signaling both by KOR antagonist norbinaltorphimine (norBNI) and Dopamine D2 Receptor agonist quinpirole. Both agonists and antagonists of the KOR can stimulate JNK signaling, producing reactive oxygen species and disrupting signaling. We found that Quinpirole can stimulate JNK-dependent ROS at both D2 receptor isoforms. We also found that NorBNI pretreatment shifts the dose response to Quinpirole at inhibiting dopamine release, indicating that JNK-ROS stimulated by the KOR can disrupt signaling at neighboring Gi-coupled receptors. In Chapter 3, I present findings from two collaborations assessing the regulation of presynaptic dopamine release in the nucleus accumbens. We found that KOR-mediated inhibition of dopamine release is less potent in female mice, and that this effect is blocked by the GRK inhibitor CMPD101. In a collaboration with the Zweifel lab, I also helped demonstrate that different neuropeptide marker genes, CCK and CRHR1, segregate populations of dopamine 6 neurons which project to the NAc Shell and NAc Core, respectively. In Chapter 4, I present findings which discuss the mechanism and limitations of KOR antagonist norBNI. We found that there are sex-differences in the long-lasting nature of norBNI, which are prevented by pretreatment with GRK inhibitor CMPD101. We also found that norBNI is not long-lasting at dopamine terminals, an effect that may be due to the lack of ROS production downstream of norBNI in NAc terminals. In Chapter 5, results from a series of experiments assessing the role of potassium channels in the kappa opioid receptor regulation of dopamine neurons. We used a CRISPR-Cas9 based system to knockdown genes for KV1.2, KV3.4, and Kir3.2 or GIRK2 (KCNA2, KCNC4, KCNJ6). We then assess the effect of those knockdowns on basal dopamine physiology and KOR signaling in dopamine neurons. 7 Dedication I dedicate this work, with gratitude & love, to my family: Mat, Jess, Sandell, Steve, and Fika. This dissertation was written in the midst of the 2020 COVID-19 Pandemic. I also dedicate this work to the first responders, essential workers, medical professionals, basic scientists, virologists, epidemiologists, and vaccinologists fighting for humanity. To the souls we’ve lost and their families and those still battling this virus. 8 Chapter 1 The therapeutic potential of the targeting the kappa opioid receptor system in stress-associated mental health disorders Introduction The stress response