Major depressive disorders affect 350 million people globally and are the leading cause of disability worldwide. Chronic or prolonged stress can trigger development of depression. Key symptoms of depression are anhedonia, helplessness, and decreased socialization. These behavioral outcomes suggest a dysfunction within the brain’s reward system, the mesolimbic system. The nucleus accumbens (NAc) is regarded as the brain’s reward hub, integrating signals from multiple brain regions to influence motivated behavioral output. The NAc consists of medium spiny neurons (MSNs) which represent 95% of the cellular landscape. These neurons can be separated into two distinct groups, dopamine receptor-1 (DR1 or D1) and dopamine receptor-2 (DR2 or D2). Differentiating between these two cell types is ideal as activation results in opposing outcomes. One protein of interest sirtuin-1 (SIRT1) has been found to alter dendritic morphology in brain regions involved in stress. Discovery that SIRT1, a histone deacetylase (HDAC), has cell-type-specific action in the NAc in a mouse model of depression and resulting behavioral changes suggest possible underlying morphological changes. Neuronal morphology includes measurement of the dendritic arbor and dendritic spines, small protrusions from the dendritic shaft. These studies seek to elucidate morphological changes following knockout or overexpression of SIRT1 in either D1-or D2-MSNs in both male and female mice. Results show that SIRT1 overexpression in male D1-MSNs results in a significant increase in stubby spines and a decrease in mushroom spines. Conversely, in female mice with SIRT1 OVEXP in D1-MSNs, there was found a significant increase in mushroom spines accompanied by a significant decrease in stubby spines. The D2-targeted mice also showed significant changes across spine types. In both treatment types, D2- males had a significant increase in stubby spines, filopodia, and thin spines. Females with SIRT1 knocked out had a significant decrease in filopodia and thin spines. SIRT1 overexpression in D2- females showed a significant decrease in stubby spines. These results suggest SIRT1 has a regulatory role in the density of spine type and possibly the maturation of spines. This discovery of an increase in stubby spines in male D1-MSNs overexpressing mice establishes a role for SIRT1 in stubby spine formation.

Substance use disorders (SUDs) are difficult to treat, in part because drug craving can be elicited by exposure to drug-associated environments and cues within the environment. Furthermore, this craving becomes more pronounced as abstinence progresses and it can take months to years for cue-elicited craving to finally wane. This important hallmark of addiction is modeled in rodents by exposing them to light/tone cues associated with the self-administration (SA) of cocaine. Cue exposure results in drug-seeking behavior, an animal analogue for drug craving. The overarching goal of this dissertation was to use the rodent SA model to explore the nucleus accumbens (NAc), a key brain region in the neural pathway of craving, and examine ribonucleic acid (RNA) expression that may underlie cocaine-seeking behavior. This includes messenger RNAs (mRNAs), which encode directly for proteins, and non-coding RNAs, which are important regulators of mRNA expression and cellular function. My first experiment aimed to identify non-coding microRNAs, which directly target and suppress mRNA expression, that are differentially expressed in animals with high or low cocaine-seeking behavior. In the second study, I compared RNA-sequencing (RNA-seq) datasets from rodent models of cocaine abstinence and developed a novel workflow to narrow candidate genes. In the final experiment, I utilized RNA-seq and reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR) to identify and explore non-coding, circular RNAs that may influence gene regulatory networks and impact drug-seeking behavior. Overall, these studies promote our understanding of the neurogenetic mechanisms of craving and they suggest recommendations for improving the experimental design of future neurogenomic studies.

Cocaine use disorders (CUDs) and human immunodeficiency virus (HIV) are a common comorbidity, although it is largely unknown whether HIV interacts with cocaine abstinence to uniquely alter neuroimmune function and whether HIV may modulate the efficacy of medications intended to treat CUDs. My dissertation research demonstrates using preclinical rodent models of drug self-administration and craving that systemic exposure to the HIV protein gp120 produces a unique profile of neuroimmune changes within the nucleus accumbens core (NAc core) that is distinct from early cocaine abstinence alone. After a protracted period of abstinence, gp120 exposure abolished the effect of the dopamine D3 receptor (D3R) partial agonist MC-25-41, which successfully attenuated cue-induced cocaine seeking in non-exposed rats. Further probing the role of downstream, intracellular neuroimmune function on cue-induced cocaine seeking, I examined the role of the nuclear factor kappa B (NF-κB) signaling pathway within the NAc core on cue-induced cocaine seeking after a period of protracted abstinence across sex and reinforcer type. I demonstrated that knockdown of the p65 subunit of NF-κB results in a decrease in cue-induced cocaine seeking in males, but not in females. This effect was specific to cocaine, as p65 knockdown did not affect cue-induced sucrose seeking in either males or females. Moreover, I examined expression levels of the extracellular matrix enzyme MMP-9 within the NAc core, as it is regulated by NF-κB and is an important mediator of cue-induced cocaine seeking and associated synaptic plasticity. I demonstrated that males express higher levels of MMP-9 within the NAc compared to females, and that p65 knockdown decreases NAc core MMP-9 in males but not females among cocaine cue-exposed animals. Altogether, these results suggest that immunotherapeutic medications may be useful tools in the treatment of CUDs, particularly among males that are disproportionately impacted by HIV.

Schizophrenia, a debilitating neuropsychiatric disorder, affects 1% of the population. This multifaceted disorder is comprised of positive (hallucinations/psychosis), negative (social withdrawal/anhedonia) and cognitive symptoms. While treatments for schizophrenia have advanced over the past few years, high economic burdens are still conferred to society, totaling more than $34 billion in direct annual costs to the United States of America. Thus, a critical need exists to identify the factors that contribute towards the etiology of schizophrenia. This research aimed to determine the interactions between environmental factors and genetics in the etiology of schizophrenia. Specifically, this research shows that the immediate early gene, early growth response 3 (EGR3), which is upregulated in response to neuronal activity, resides at the center of a biological pathway to confer risk for schizophrenia. While schizophrenia-risk proteins including neuregulin 1 (NRG1) and N-methyl-D-aspartate receptors (NMDAR’s) have been identified upstream of EGR3, the downstream targets of EGR3 remain relatively unknown. This research demonstrates that early growth response 3 regulates the expression of the serotonin 2A-receptor (5HT2AR) in the frontal cortex following the physiologic stimulus, sleep deprivation. This effect is translated to the level of protein as 8 hours of sleep-deprivation results in the upregulation of 5HT2ARs, a target of antipsychotic medications. Additional downstream targets were identified following maximal upregulation of EGR3 through electroconvulsive stimulation (ECS). Both brain-derived neurotrophic factor (BDNF) and its epigenetic regulator, growth arrest DNA-damage-inducible 45 beta (GADD45B) are upregulated one-hour following ECS in the hippocampus and require the presence of EGR3. These proteins play important roles in both cellular proliferation and dendritic structural changes. Next, the effects of ECS on downstream neurobiological processes, hippocampal cellular proliferation and dendritic structural changes were examined. Following ECS, hippocampal cellular proliferationwas increased, and dendritic structural changes were observed in both wild-type and early growth response 3 knock-out (Egr3-/-) mice. Effects in the number of dendritic spines and dendritic complexity following ECS were not found to require EGR3. Collectively, these results demonstrate that neuronal activity leads to the regulation of schizophrenia risk proteins by EGR3 and point to a possible molecular mechanism contributing risk for schizophrenia.

Intermittent social defeat stress induces psychostimulant cross-sensitization, as well as long-lasting social avoidance behavior. Previous data reveal heightened expression of AMPA receptor (AMPAR) GluA1 subunits in rat ventral tegmental area (VTA), which occurs concurrently with social stress-induced amphetamine (AMPH) cross-sensitization. These studies described herein examined whether VTA GluA1 AMPARs are important for the behavioral consequences of social stress and investigated the role of the infralimbic (IL) to VTA pathway in the induction of these responses. Functional inactivation of GluA1 in VTA DA neurons prevented stress-induced AMPH sensitization without affecting social avoidance behavior, while GluA1 overexpression in VTA DA neurons mimicked the effects of stress on AMPH sensitization. Female rats were more sensitive to the effects of stress on AMPH administration than males, specifically during proestrus/estrus, which is characterized by higher circulating estradiol. Fluorescent immunohistochemistry revealed that females expressed higher GluA1 in VTA DA neurons as a result of intermittent social defeat stress, independent of estrus stage; by contrast, females during proestrus/estrus displayed higher tyrosine kinase receptor type 2 (TrkB) expression, which is the receptor for brain derived neurotrophic factor (BDNF), in VTA DA neurons, independent of stress exposure. Functional inactivation of GluA1 in VTA DA neurons prevented stress-induced AMPH sensitization and overexpression mimicked the effects of stress on AMPH sensitization. This suggests that BDNF-TrkB signaling may work concomitantly with GluA1 signaling in the VTA to drive sex-dependent differences in stress-induced locomotor sensitization effects. Optogenetic inhibition of the IL-VTA pathway in male rats prevented stress-induced AMPH sensitization compared to control animals. In addition, fluorescent immunohistochemistry displayed less Fos labeling in the nucleus accumbens (NAc) of rats with IL-VTA light inhibition compared to control animals. This suggests that the IL-VTA pathway plays a critical role in the induction of stress-induced sensitivity to AMPH, and blocking this pathway prevents mesolimbic DA signaling to the NAc. We conclude that IL glutamate projections onto GluA1-homomeric AMPA receptors in VTA DA neurons play a critical role in driving the stress-induced sensitization response in males and females. Therefore, GluA1 VTA DA neurons could potentially be a therapeutic target to prevent stress-induced drug susceptibility in the future.