Alcohol use disorders (AUDs) are a common problem with few effective treatment options. There are only three FDA-approved drugs for the treatment of AUDs, all of which show limited treatment efficacy and none alleviate comorbid anxiety and/or depression. Thus, there is a significant need to identify novel targets for the treatment of AUDs, and the delta opioid receptor (DOR) could represent such a target. DOR agonists have been demonstrated to reduce EtOH consumption in rodent models and have well-established roles in motivation and anxiety. Importantly, the expression and function of DORs are known to change dynamically with a variety of perturbations including EtOH exposure, stress, and pain. This manuscript examines some ways in which the DOR regulation of reward and anxiety behaviors changes with behavioral state. We examined the preference to the DOR-1 agonist DPDPE and the DOR-2 agonist deltorphin in the conditioned place preference paradigm in animals that were either naïve or ethanol consuming, as well as either stressed or un-stressed. While neither DPDPE nor deltorphin induced a place preference in naïve unstressed animals, deltorphin induced a significant place preference in unstressed ethanol consuming animals. In stressed animals, DPDPE does not induce a place preference whether or not the animals were consuming ethanol. In contrast, deltorphin induces a place preference in ethanol naïve stressed animals, however not in animals that were both stressed and ethanol consuming. We also examined the effects of the DOR-1 agonist TAN 67 and the DOR-2 agonist SNC 80 on anxiety-like behavior measured by the marble burying task in both naïve and ethanol consuming animals. We found that while SNC 80 showed anxiolytic-like effects in naïve animals, TAN 67 did not. However, neither DOR agonist showed an anxiolytic-like effect in ethanol consuming animals. This is in contrast to previous findings with the light/dark transition test in which DOR agonists increased anxiolytic efficacy with ethanol consumption. Together these studies examine DOR effects on reward and anxiety behaviors and further our understanding of how the effects of DOR agonists are dynamically changed by physiological state to help to refine targets for therapeutic development.

In 2020, 254 people died because of opioid overdose every day in the United States. Opioid analgesics are still regarded as the gold standard for alleviating pain clinically but present significant risks. Their analgesic utility (the effect) is limited by multiple side effects, including respiratory depression, constipation, and the development of an Opioid Use Disorder (OUD). A lack of consensus on the cellular signaling events responsible for these negative side effects remains a major limitation to the development of an opioid with a reduced effect/side effect profile.All opioids exert their analgesic effects through the activation of the Gi-coupled μ- Opioid receptor (MOR). Acute action at MOR results in the reduction of neuronal excitability, reducing pain signal transmission. However, chronic use of opioid analgesics results in cellular adaptations. A central theory, biased agonism, seeks to explain the manifestation of these adaptations by comparison to endogenous signaling patterns. When MOR is activated by its endogenous ligand, a regulatory molecule called Arrestin-3 (Arr3) is recruited to the receptor, causing titration of signal transduction and subsequent internalization and recycling of the receptor. Morphine and other clinically used therapeutics do not effectively recruit Arr3. The lack of Arr3 recruitment results in receptors remaining present on the membrane without being internalized and recycled back to the membrane. Therefore, recruitment of Arr3 could be beneficial in preventing counter adaptations to chronic morphine. However, a competing theory states that Arr3 may mediate the negative side effects seen with continued opioid use. There is no consensus on the role of Arr3 in mitigating or causing the negative side effects seen in chronic opioid use. To investigate the effect of Arr3 on drug-seeking behavior, this study utilizes three different genotypes of mice with varying Arr3 recruitment profiles in response to morphine: 1) WT(poorly recruits Arr3), 2) RMOR (strongly recruits Arr3), 3) Arr3 vii -KO (does not recruit Arr3). Chapter 2 utilizes a novel oral operant self-administration paradigm for 17 weeks to model the transition from impulsive to compulsive drug use, difficulties in ceasing drug-seeking behavior, and relapse. This model shows that 38% of animals in WT and Arr3-KO genotypes exhibit compulsive drug-seeking behavior, but 0% of RMOR mice become compulsive drug-seekers. In Chapters 3-4, I investigate what underlying environmental causes explain why a subset of genetically homologous mice develop compulsive drug-seeking behavior. About 10-30% of persons prescribed opioids will develop an OUD. The explanation for why some individuals transition to misuse of opioids, placing themselves at higher risk for overdose, remains unclear. To model how individual behavior can predispose a subject to transition to compulsive drug use, a battery of anxiety measures was collected pre- and post-study to determine if baseline behavior or changes in behavior in response to drug exposure predict compulsive drug use. I determine that individual anxiety state does not influence compulsive drug-seeking behavior. In Chapter 5, I determine how changes in gut microbiome composition in response to morphine vary among individuals and how we can leverage these individual differences to develop microbial biomarkers of compulsive drug-seeking behavior.

The increasing prevalence of type 2 diabetes (T2D) and obesity poses substantial concerns and economic challenges to the healthcare system. Type 2 Diabetes is characterized by disruption of glucose homeostasis. Insulin is the primary driver of glucose homeostasis and released by pancreatic -cells in response to nutritional and endocrine signals. Glucose stimulated insulin secretion is augmented by the actions of incretin hormones including Glucagon-Like peptide 1 (GLP-1), which activates the GLP-1 receptor (GLP-1R) in islets producing the “incretin effect”. Because of their insulinotropic properties, incretin drugs, such as GLP-1 receptor agonists are used as therapeutic agents to maintain glucose homeostasis in type-2 diabetes. However, concerns regarding the long-term effects of these drugs and development of tolerance to GLP-1R agonist persist. In this study, we investigate the role of G protein-coupled receptor-associated sorting protein 1 (GASP1), a critical regulator involved in post-endocytic trafficking of GLP-1R, on development of tolerance to GLP-1R agonists. By combining CRISPR-Cas9 technique, Homogenous Time Resolved Fluorescence (HTRF) biochemical assay, transgenic mice and in vivo and ex vivo animal studies, we found the following:

• GASP1 is expressed in both human embryonic kidney (HEK 293) and rat insulinoma derived insulin producing INS-1 cells.

• Following acute treatment with the GLP-1R agonist Exendin-4 (Ex-4), both HEK 293 and INS-1 cells demonstrate a dose-dependent increase in intracellular cyclic adenosine monophosphate (cAMP) levels.

• Prolonged Ex-4 pretreatment of HEK 293 and INS-1 cells with Ex-4 results in loss of responsiveness of the receptor (i.e “Tolerance”).

•CRISPR-Cas9-mediated removal of GASP1 in both HEK 293 and INS-1 cells did not impact acute GLP-1R signaling. However, GASP1 knockout prevented the development of tolerance in response to prolonged Ex-4 pretreatment, indicating that GASP1 plays a role in regulating the post-endocytic trafficking of GLP-1R.

• Similarly, in INS-1 cells, deletion of GASP1 has no effect on incretin-mediated insulin secretion in response to Ex-4. However, upon prolonged Ex-4 pretreatment wild-type (WT) INS-1 cells show reduced incretin response, whereas GASP1 knockout (KO) INS-1 cells retained the incretin effect.

• Furthermore, in a longitudinal mouse islet insulin secretion assay, both GASP1 wild-type and beta-cell specific GASP1 deleted (β-GASP1-KO) islets show robust incretin effect and increase in insulin secretion when stimulated with Ex-4 acute treatment.

• Interestingly, after an Ex-4 pretreatment, GASP1-WT islets show reduced insulin secretion indicating development of tolerance while β-GASP1-KO islets maintained their incretin effect.

• Importantly, both GASP1-WT and β-GASP1-KO islets displayed a substantial incretin effect after a 24-hour recovery period, suggesting that the observed tolerance effect is not due to any inherent unhealthiness of the islets.

• In WT mice which are chronically treated with Ex-4 for six weeks show development of tolerance to glucose-stimulated insulin secretion effect of Ex-4.

• Furthermore, in WT mice treated chronically with Ex-4, there is not only the development of tolerance to exogenous Ex-4 treatment but also to their endogenous incretins.

• In mice with selective disruption of GASP1 in pancreatic beta cells (β-GASP1-KO mice), the acute treatment of oral glucose and Ex-4 are indistinguishable from WT mice.

• However, chronic Ex-4 treatment of β-GASP1-KO mice with Ex-4 does not develop tolerance to either exogenous Ex-4 or endogenous incretins.

This study highlights the pivotal role of GASP1 in regulating the post-endocytic trafficking of GLP-1R in pancreatic β-cells and its impact on receptor function during prolonged drug administration. These findings also emphasize the critical role of GASP1-mediated GLP-1R trafficking in the development of tolerance to incretin drugs and offers potential novel strategies for improving therapeutic efficacy. Therefore, gaining a deeper understanding of the molecular mechanisms governing GASP1-mediated GLP-1R trafficking could help in the development of improved therapies utilizing GLP-1R agonists to effectively combat type 2 diabetes and obesity.

In recent years, opioids have become a household name as America has faced down an unprecedented wave of overdose death. The fallout of the emergent public health crisis known as “the opioid epidemic” has a storied past stretching well into the last century. Despite a long history of human interest and scientific study of opioid drugs, the current state of biological opioid research has little to offer in service to reducing the scope of this ongoing tragedy. The intent of this work is to enhance the perspective of the fields of opioid pharmacology and neuroscience, both by encouraging routes of investigation which break from tradition, and casting sharper focus on studies with high relevance to real world scenarios. We present data from our own studies which employ novel strategies to examine behavioral and neural signaling changes that arise with acute and chronic opioid exposure. Additionally, we conduct extensive review and analysis of the domains of the opioid literature that inspired these studies, prioritizing the resolution of key controversies and illuminating scholarly gaps that necessitate further inquiry.

The biological actions of morphine and other opioid narcotics are mediated primarily by the mu opioid receptor (MOR). In response to endogenous opioids, the signaling of the MOR is regulated by a conserved endocytic mechanism, involving both the desensitization of signaling by receptor phosphorylation and arrestin recruitment and the subsequent resensitization by endocytosis and recycling. In response to morphine, however, this regulatory mechanism is only weakly engaged, resulting in persistent MOR desensitization without resensitization in some cells and in persistent MOR activation without desensitization in others. We hypothesized that this could have multiple consequences for morphine's physiological effects. First, persistent MOR desensitization, arising within minutes to days of morphine administration, could contribute to acutely diminished sensitivity to morphine and acute tolerance. Second, compensatory adaptations in response to persistent MOR activation, arising within hours to weeks of morphine administration, could contribute to chronic tolerance, physical dependence, and possibly addiction. To test this hypothesis, we generated a mutant Recycling MOR (RMOR) that desensitizes, internalizes, and recycles in response to morphine. Here, we describe the phenotype of knock-in mice expressing the mutant RMOR in place of the wild-type receptor. Consistent with our hypothesis, RMOR mice exhibited increased analgesia and reward selectively in response to morphine. Furthermore, they developed reduced tolerance and physical dependence following chronic morphine treatment, and they were less likely to progress from controlled to compulsive drug seeking when given the opportunity to freely self-administer morphine. These results suggest that promoting agonist-induced MOR desensitization and endocytosis preserves the therapeutically desirable effects of opioid drugs while preventing their negative side effects, particularly tolerance, physical dependence, and addiction.

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