Alcoholism results from an interaction between genetic and environmental factors. However, the neurobiological mechanisms mediating the propensity to consume excessive amounts of alcohol are still not well understood. Using genetically selected alcohol-preferring rats, a well-established animal model of alcoholism, we demonstrate that central administration of a peptide antagonist for the pituitary adenylate cyclase-activating polypeptide receptor 1 (PAC1), the cognate receptor for the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP), blocks excessive alcohol drinking as well as motivation to drink. On the other hand, the PAC1 antagonist does not significantly affect water intake, saccharin intake, or responding for ethanol in non-selected outbred Wistar rats. In addition, the antagonist significantly reduced responding maintained by alcohol-associated incentive stimuli (alcohol seeking behavior). Using immunohistochemistry, a significant reduction in the number of PAC1 positive cells was observed selectively in the Nucleus Accumbens (NAcc) Core of alcohol-preferring compared to Wistar rats. Proving the functional relevance of these changes, excessive drinking in alcohol-preferring rats was markedly reduced following microinfusion of the PAC1 antagonist into the Core, but not the Shell, of the NAcc. Finally, using retrograde tracing techniques coupled with immunofluorescence, we show that the dopaminergic neurons of the VTA which project to the NAcc core co-express PACAP. Altogether, our findings demonstrate that the dysregulation of the PACAP/PAC1R system, specifically in the NAcc core, promotes excessive drinking and alcohol-seeking behavior, indicating that blockade of the PACAP/PAC1R system may represent a novel target for alcohol addiction.

INTRODUCTION: Alcohol Use Disorder (AUD) is a chronic relapsing condition characterized by compulsive, uncontrolled consumption of alcohol. AUD is also characterized by impairments in decision making, driven by dysregulation of prefronto-cortical regions, including the anterior cingulate cortex (ACC) and medial prefrontal cortex (mPFC). Little is known about the neurotransmitter systems involved in both the excessive, compulsive drinking and the cognitive deficits observed in alcohol addiction. The Sigma 1 Receptor (Sig-1R) system has been suggested as a novel target for the treatment of addictive disorders, due to its ability to modulate the rewarding and reinforcing effects of multiple drugs of abuse, including alcohol. OBJECTIVE: The goal of the present study was to examine the role of Sig-1Rs in prefronto-cortical regions in alcohol addiction-relevant behaviors, including alcohol self-administration, motivation to work to obtain alcohol, compulsive alcohol seeking, and cognitive flexibility. METHODS: Male Wistar rats were microinfused with a viral vector containing either a Sig-1R knockdown shRNA (Sig-1R-knockdown) or GFP-Control in prefrontal regions, which encompassed the mPFC and the ACC. Rats were trained to self-administer alcohol under increasing fixed-ratio (FR) schedules of reinforcement, as well as a progressive-ratio (PR) schedule of reinforcement, a measure of motivation to work to obtain alcohol. Animals were then trained to seek and take alcohol on a chained-schedule of reinforcement; in addition, to test compulsive alcohol seeking in the face of aversive consequences, a footshock punishment was introduced following the completion of a seeking response cycle. To test whether prefrontal Sig-1R-knockdown affected anxiety-like behavior, rats were subject to a light/dark box test, which involves allowing rats to move freely between a light and a dark compartment. The latency to leave the dark compartment and the amount of time spent in the light compartment were used as measures of anxiety-like behavior. A secondary aim of the study was to start investigating the effects of prefrontal Sig-1R-knockdown on cognitive flexibility. Alcohol naïve rats were tested in an operant attentional set-shifting paradigm, where rats were initially trained to lever press for a reward using a visual-cue strategy. Rats were then subsequently trained to lever press for a reward using a spatial response strategy. Thus, during this “set-shift,” rats were required to extinguish the use of the visual-cue and return to using the spatial response strategy for obtaining a reward. Finally, the spatial strategy rule was inverted in a reversal task. In both the attentional set-shifting and reversal tasks, a greater number of previously reinforced errors (e.g. perseverative responding) was considered to reflect lower cognitive flexibility. RESULTS: Prefrontal Sig-1R-knockdown resulted in significantly higher responding for alcohol under high-effort conditions (FR3 and FR5) and higher motivation for alcohol (PR). In the compulsive alcohol seeking task, while GFP-Control animals decreased seeking lever responses after the addition of the aversive footshock consequence, Sig-1R-knockdown animals showed no significant changes in lever responses. In the light/dark test, Sig-1R-knockdown animals displayed decreased latencies to enter the light compartment, possibly indicating lower anxiety-like behavior as compared to the GFP-Controls. In an attentional set-shifting task, Sig-1R-knockdown animals committed a greater number of perseverative errors during the shift to response discrimination strategy than GFP-Controls, indicating some deficits in cognitive flexibility. CONCLUSION: Prefrontal Sig-1R-knockdown resulted in greater alcohol responding, motivation, and compulsive seeking behavior. Additionally, prefrontal Sig-1R-knockdown reduced cognitive flexibility in an operant attentional set-shifting task in alcohol-naïve rats. Results from these experiments support a key role for prefrontal Sig-1Rs in alcohol addiction and cognitive flexibility. / 2020-07-03T00:00:00Z

Obesity and eating disorders represent a severe problem in Western societies. Both the increased availability of highly palatable foods and dieting are major risk factors contributing to the epidemic disorders of feeding. The purpose of this study was to characterize an animal model of maladaptive feeding induced by intermittent access to a palatable diet alternation in mice. In this study, mice were either continuously provided with standard chow food (Chow/Chow), or provided with standard chow for 2 days, with 1 day of access to a high-sucrose, palatable food (Chow/Palatable). Following stability of intake within the cycling paradigm, we investigated the effects of several pharmacological treatments: Naltrexone, an opioid antagonist, SR141716A (rimonabant), a type 1 cannabinoid receptor antagonist, and BD-1063, a type 1 sigma receptor antagonist. Over successive cycles, Chow/Palatable mice showed an escalation of palatable food intake within the first-hour of renewed access to palatable diet, and displayed hypophagia upon its removal. Naltrexone, SR141716A, and BD-1063 reduced overconsumption of palatable food during this first hour. Here we provide evidence of strong face and convergence validities in a palatable diet alternation model in the mouse, confirming multiple shared underlying mechanisms of pathological eating across species, and thus making it a useful therapeutic development tool. / 2019-07-11T00:00:00Z

The vast array of causes, mechanisms, and interventional strategies for pain has created an extensive field of research spanning a variety of disciplines. This thesis aims to describe the multidimensional factors leading to pain, how pain can be assessed, and how we can best target these pain pathways to improve acute pain management. Although opioids have been used for centuries in many analgesic therapies, new research and public concern are increasingly deterring clinicians from prescribing them. This thesis will discuss opioid's mechanism of action, risk of adverse effects, and limitations. Furthermore, the 'opioid crisis' will be examined from its beginning to where we are now. Alternative pharmacological and nonpharmacological therapeutic options are presented in the hope of exposing opioid-sparing improvements to analgesia; their mechanism of action, efficacy, and limitations are described where applicable. Beyond individual analysis and evaluations of reputable, highly cited studies for each therapeutic option, this thesis also examines multimodal analgesia and how it is changing acute pain management. Multimodal analgesia allows multiple dimensions of the pain pathway to be targeted by using multiple drugs, leading to greater pain relief, decreased doses of medications, and reduced side effects. Multidisciplinary advantages are also discussed, including dynamic clinician involvement, individualization, organizational procedures, and patient education interventions. The complexities of pain management therapy and suggestions for future directions presented in this thesis are intended to expose additional options or techniques to ultimately improve surgical outcomes, increase patient satisfaction, and decrease public health risks.

Dopamine D2 receptor (D2R) is a G protein-coupled receptor (GPCR) that activates G protein and arrestin signaling molecules. D2R antagonism has been a hallmark of antipsychotic medications for more than half a century. However, this drug-class is associated with substantial side effects that decrease quality of life and medication compliance. The development of novel antipsychotic medications with superior therapeutic and side effect profiles has been hampered in part due to a poor understanding of the specific D2R populations and downstream signaling molecules that must be blocked to confer therapeutic efficacy. It has been proposed that antipsychotic medications confer their effects through the blockade of arrestin but not G protein signaling downstream of D2R, and thus substantial efforts have gone towards the development of ligands that selectively block arrestin signaling. However, this approach suffers from several major limitations, namely that blockade of G protein signaling may also be important in conferring antipsychotic effects. Moreover, currently available pharmacological and genetic tools that have been used to probe G protein and arrestin signaling downstream of D2R in vivo suffer from on- and off-target effects that add substantial confounds to our understanding of these processes. Herein, we describe the development of several tools that can be used to probe G protein and arrestin-mediated processes in vivo with high specificity, as well as mechanisms by which these processes are activated.

Pathological cardiac remodeling results from myocardial stresses including pressure and volume overload, neurohumoral activation, myocardial infarction, and hypothyroidism. These pathological conditions converge on altered cell signaling and subsequent transcriptional cardiac remodeling that is thought to initially be an adaptive response but ultimately drives progression to heart failure (HF). Mediator complex is a transcriptional co-activator that coordinates signal dependent transcription factors with basal transcriptional machinery. This thesis specifically addresses the role of two Mediator kinase submodule proteins, cyclin-dependent kinase 8 (Cdk8) and Mediator subunit 13 (Med13), in regulating pathological transcriptional remodeling in response to cardiac hypertrophy and hypothyroidism, respectively. Cdk8 kinase activity regulates transcriptional responses to pathological cardiac hypertrophy Pathological cardiac hypertrophy represents a major risk factor for HF. The hypertrophic response is orchestrated in part through transcriptional alterations that ultimately modify cardiac function. Mediator is a multiprotein complex that bridges signal dependent transcription factors with basal transcriptional machinery. Cdk8 is a Mediator kinase demonstrated to have a complex role in transcriptional regulation through mechanisms involving both transcriptional activation and inhibition. We have previously demonstrated that cardiac-specific overexpression of Cdk8 results in eccentric cardiac hypertrophy resulting in HF. The studies presented herein demonstrate that Cdk8 activity is induced in numerous cardiac hypertrophy models. To specifically assess the role of Cdk8 kinase activity in regulating hypertrophic transcriptional programs, Cdk8 kinase inhibitors, Senexin A and CCT251545, were utilized in an in vitro neonatal rat cardiomyocyte (NRCM) hypertrophy model. Inhibition of Cdk8 activity resulted in blunted cardiomyocyte hypertrophy and altered regulation of hypertrophic transcriptional programs. These studies demonstrate a role for Cdk8 activity in transcriptional regulation of gene programs associated with pathological cardiac remodeling that ultimately drives HF. Regulation of cardiac transcription by thyroid hormone and Med13 Thyroid hormone (TH) is a key regulator of transcriptional homeostasis in the heart. While hypothyroidism is known to result in adverse cardiac effects, the molecular mechanisms that modulate TH signaling are not completely understood. Mediator complex protein, Med13, was previously demonstrated to repress numerous thyroid receptor (TR) response genes in the heart. Further, cardiac-specific overexpression of Med13 in mice that were treated with propylthiouracil (PTU), an inhibitor of the biosynthesis of the active TH, triiodothyronine (T3), resulted in resistance to PTU-dependent decreases in cardiac contractility. Therefore, these studies aimed to determine if Med13 is necessary for the cardiac response to hypothyroidism. Here we demonstrate that Med13 expression is induced in the hearts of mice with hypothyroidism. To elucidate the role of Med13 in regulating gene transcription in response to TH signaling in cardiac tissue, we utilized an unbiased RNA sequencing approach to define the TH-dependent alterations in gene expression in wild-type mice or those with a cardiac-specific deletion in Med13 (Med13cKO). Mice were fed a diet of PTU to induce a hypothyroid state or normal chow for either 4 or 16 weeks, and an additional group of mice on a PTU diet were treated acutely with T3 to re-establish a euthyroid state. Echocardiography revealed that wild-type mice had a decreased heart rate in response to PTU with a trend toward a reduced cardiac ejection fraction. Furthermore, loss of cardiac Med13 resulted in upregulation of genes associated with cardiac fibrosis and inflammation following PTU treatment. Notably, cardiomyocyte-specific deletion of Med13 exacerbated cardiac dysfunction. Collectively, these studies reveal cardiac transcriptional pathways regulated in response to hypothyroidism and re-establishment of a euthyroid state and define molecular pathways that are regulated by Med13 in response to TH signaling.

Postoperative pain constitutes a common form of acute and persistent pain. The current approaches for postoperative pain control are inadequate and problematic. A better understanding of mechanisms for postoperative pain is necessary to improve pain management. Pain at rest is a major complaint of patients after surgery. Such ongoing pain is transmitted by increased spontaneous activity (SA) in nociceptive pathways. Using the rodent plantar incision model, guarding behavior after incision has been found as a correlate to pain at rest in patients after surgery. Similar to the time course change in pain at rest after operation, guarding behavior is the greatest immediately after plantar incision and then gradually resolves over several days. When SA in nociceptive pathways was examined previously, however, SA in both dorsal horn neurons (DHNs) and primary nociceptors was not evident immediately after plantar incision. One day after incision, great SA was present. The underlying reason for the disagreement between ongoing guarding behavior and SA in nociceptive pathways is still unknown. Here we hypothesize that incised skin has a minimal effect on the ongoing pain genesis, whereas incised deep muscle tissue produces strong ongoing pain and SA in nociceptive pathways after incision. In the first experiment, we found that skin incision induced moderate guarding on the day of incision only, whereas skin plus deep tissue incision caused guarding for 5 days. Mechanical and heat hyperalgesia were similar in both incised groups. On postoperative day (POD) 1, greater SA was present in DHNs in the skin plus deep tissue incision group than in the skin incision group. Seven days after skin plus deep tissue incision, the amount of SA in DHNs was the same as the sham control. In the second experiment, we found that on POD1 skin incision produced a similar prevalence of SA in nociceptors as sham control, whereas skin plus deep tissue incision generated a greater prevalence of SA. Seven days after skin plus deep tissue incision, the SA was similar as sham control. In the third experiment, we found that under normal conditions group III and IV muscle afferents have little SA in vitro. Incision increased the prevalence of SA. Incision also caused a greater percentage of lactic acid-responsive fibers and reduced heat and mechanical response threshold. In the fourth experiment, anti-nerve growth factor (NGF) and capsaicin were found to be effective in inhibiting ongoing pain after skin plus deep tissue incision. Taken together, we propose that deep muscle tissue rather than skin plays a central role in the genesis of ongoing pain and SA in nociceptive pathways after incision, whereas incised skin is sufficient to induce mechanical and heat hyperalgesia. We further propose increased lactic acid in incised muscle tissue may contribute to the development of SA in muscle afferents.

Tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis, is stimulated by N-terminal phosphorylation on its regulatory domain and inhibited by protein serine/threonine phosphatase 2A (PP2A). PP2A comprising of an AC core dimer composed of catalytic (C) and scaffolding (A) subunit is complexed to a variable regulatory subunit derived from three gene families (B, B', B"). My thesis work was focused on studying the regulation of TH by PP2A. I found out that in catecholamine-secreting PC12 cells, inducible expression of PP2A/B'β decreased both N-terminal Ser phosphorylation and in situ TH activity, whereas inducible silencing of endogenous B'β had the opposite effect. Furthermore, PP2A/ B'β directly dephosphorylated TH in vitro. B'β was highly expressed in dopaminergic cell bodies in the rat substantia nigra, however it was excluded from TH-positive terminal fields in the striatum and failed to colocalize with presynaptic markers in general. I detected higher TH phosphorylation in processes than in somata of dopaminergic neurons. This is consistent with a model in which B'β enrichment in neuronal cell bodies helps confine catecholamine synthesis to axon terminals. I further investigated the mechanism of substrate specificity by B'β holoenzyme. Using the PP2A/ B'γ crystal structure as a guide, I identified Glu153 of B'β as a critical residue for dephosphorylation of TH in PC12 cells. PC12 cell lines expressing B'β/ E153R mutant were unable to dephosphorylate TH and this mutant demonstrated reduced pSer40-TH phosphatase activity in vitro. By site directed mutagenesis, I demonstrated that Arg37 and Arg38 within the PKA-Ser40 {part of a PKA consensus sequence (-RRXS-)} sequence of TH was critical for dephosphorylation by B'β. I also showed that PC12 cell line expressing B'β/E153R mutant exhibited reduced ERK phosphorylation in response to NGF treatment as compared to increase observed in wild type B'β expressing cells. Taken together, these results show that B'β recruits PP2A to modulate TH activity in a tissue- and cell compartment specific fashion and that the Glu153 residue of B'β is crucial for dephosphorylation of TH.

Renin (REN) expression is required to maintain blood pressure and electrolyte homeostasis. However, the mechanisms by which REN is transcriptionally regulated remain elusive. We reported a functional role for several nuclear receptors (NRs) on REN gene transcription. To identify other candidate NRs that regulate REN, we analyzed a publicly available microarray dataset (GUDMAP Developing Kidney ST1) to compare the expression pattern of REN and the 48 NRs across different kidney cell types. Our analysis revealed 14 NRs exhibiting a similar pattern as REN. We hypothesized that these NRs are co-regulated with REN and can regulate REN transcription. To test this hypothesis, we set up 2 cohorts of mice in which REN expression was either high or low compared to control mice and measured expression of REN and NRs in renal cortex by qPCR. The high-REN cohort was given the ACE inhibitor captopril (100g/kg/day) for 10 days, and the low-REN group was implanted subcutaneously with a deoxycorticosterone acetate pellet (50mg) and received 0.15 M NaCl in drinking water for 21 days (DOCA-salt) in addition to water. Captopril increased REN and reduced NR2F6 expression relative to vehicle treatment (REN: 16±1, p

The renin-angiotensin system (RAS) greatly contributes to energy homeostasis through opposing actions in the brain and adipose. We hypothesize that site- and receptor-specific effects of the RAS may represent a novel therapeutic target for obesity, a concept which is fully reviewed in chapter 1. Transgenic “sRA” mice exhibit brain-specific RAS hyperactivity, and a suppressed circulating RAS presumably secondary to the chronic hypertension exhibited by these animals. In chapter 2, we demonstrated that the hypertension observed with elevated brain RAS is mediated by increased vasopressin signaling. In chapter 3, we investigated how suppressed circulating RAS activity contributes to the elevated resting metabolic rate (RMR) of sRA mice. Despite having no effect upon energy intake, chronic angiotensin II type 2 (AT2) receptor activation suppressed energy expenditure and caused weight gain in sRA mice. The AT2 receptor alters inguinal white adipose tissue to contribute to obesity through the suppression of RMR. Lastly, in chapter 4, we documented moderately-improved glycemic control with elevated brain RAS/reduced circulating RAS activity, though the mechanism behind this remains unknown. Taken together, we determined that low circulating RAS activity is metabolically beneficial due to reduced activation of the AT2 receptor. Thus, specific inhibition of the systemic RAS may ultimately stimulate energy expenditure and thus may be a viable anti-obesity pharmaceutical approach. Overall, our data highlight the importance of site-specific effects of the RAS on energy homeostasis.