Characterizing the mechanisms of kappa opioid receptor signaling within mesolimbic dopamine circuitry

Abstract

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 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. This dissertation demonstrates novel insights into the mechanisms of norBNI and advances in tool and technology use in its depiction of HyPerRed and application of the AAV1-FLEX-SaCas9 CRISPR constructs. It expands upon the mechanisms and constraints of long-acting kappa opioid receptor antagonists, such as norBNI, by characterizing sex differences in the JNK-ROS mechanism, exploring the transferability of this mechanism to other receptor systems, and determining the extent of receptor inactivation in different neuronal compartments of dopamine neurons. The lack of long-acting norBNI effect, in conjunction with the finding the nalfurafine inhibits dopamine release despite not being a dysphoric compound, are counter to the prevailing hypothesis that dynorphin release in the NAc causing dopamine inhibition is responsible stress and drug-withdrawal associated decreases in mood. We also utilized and optimized a new tool in neuroimaging, HyPerRed, to answer questions about localized ROS generation downstream of GPCR activation in dopamine neurons. This study both helped answer an experimental question about norBNI’s action in dopamine terminals, but also helped determine the timecourse and dynamics of KOR agonist-induced ROS in living brain tissue. Although inconclusive, the studies using CRISPR SaCas9 viral strategies to excise potassium channel subunits from dopamine neurons outline a workflow for using these tools and present a variety of follow up experiments. The KV1.2 (Kcna2) channel knockdown showed robust increases in dopamine release, and may be a helpful model for studying reward valuation. The GIRK2 (Kcnj6) channel knockdown was functionally and genetically validated and, perhaps in combination with a GIRK1 knockdown, will be helpful in elucidating the p38 MAP Kinase regulation of VTA dopamine neurons and DRN serotonin neurons.