Major depressive disorder (MDD) represents an enormous societal burden that will directly affect nearly one fifth of Americans within their lifetime. Despite its high prevalence and contribution to disability, current treatment strategies primarily SSRIs have low efficacy and take weeks to months of treatment before alleviation of symptoms occurs. Recently, the noncompetitive N-methyl D-aspartate (NMDA) receptor antagonist ketamine was shown to induce rapid and long lasting antidepressant effects in both rodent models of depression and MDD patients. However, the mechanism underlying ketamines effects are not completely known. The bed nucleus of the stria terminalis (BNST) is a part of the extended amygdala and sits at the nexus of higher order processing, reward circuitry, and stress circuitry and has been implicated in depression-like behavior. We found that antagonism of the GluN2B subunit of the NMDAR, through both global pharmacology as well as genetically restricted to the BNST, resulted in reduced depression-like behavior in the Novelty-Induced Hypophagia task (NIH). We also found that ketamine was able to reduce depression-like behavior induced by forced abstinence from a six week two bottle choice ethanol (EtOH) drinking paradigm. Female singly housed mice displayed high preference for EtOH, and drinking seemed to become habit-driven over the course of the paradigm. Protracted forced abstinence was required to induce long-lasting depression-like behaviors, which were ameliorated by ketamine, but not another NMDAR antagonist memantine. Additionally, we found that amelioration of depression-like behaviors could also be achieved by augmenting the endocannabinoid (eCB) 2-AG through inhibition of its degradation enzyme monacylglycerol (MAG) lipase by JZL-184. Antidepressant-like effects of JZL-184 were completely blocked by low dose rimonabant, a cannabinoid receptor 1 (CB1) antagonist. We found that eCB levels were altered during EtOH drinking and abstinence in the basolateral amygdala. Future studies will aim to examine the mechanisms underlying antidepressant effects eCBs and ketamine within the BNST both basally and following EtOH administration.

Cannabinoid receptors have been examined as potential targets to alleviate the negative consequences of anxiety, trauma-related, and stress-related disorders. However, in preclinical animal studies, synthetic cannabinoids can produce adverse motoric and cognitive effects. Thus, pharmacological strategies that augment endocannabinoid levels in the brain, with the aim of enhancing signaling through cannabinoid receptors, are being investigated for their ability to modulate anxiety and stress responses. Previously, we have demonstrated that either genetic removal of prostaglandin-endoperoxide synthase 2 gene, which codes for the cyclooxygenase-2 (COX-2) enzyme that degrades the endocannabinoids, anandamide and 2-arachidonylglycerol, or pharmacologically inhibiting COX-2 activity with a substrate-selective COX-2 inhibitor (SSCI), LM-4131, can increase brain anandamide levels. These elevations in endocannabinoid levels in the rodent brain resulted in enhanced endocannabinoid signaling through the cannabinoid type 1 receptor and, subsequently, reduced anxiety-like behaviors in mice under basal conditions. Using the novelty-induced feeding suppression assay, elevated plus maze, and in vivo electrophysiology, we tested the hypothesis that endocannabinoid augmentation via SSCIs may have the potential to counteract stress-induced anxiety-like behaviors. We have found that the SSCIs, LM-4131 and lumiracoxib, and the selective COX-2 inhibitor, celecoxib, can reduce anxiety-like behaviors in mice subjected to footshock stress. In contrast, these inhibitors had little effect in non-stressed mice. The anxiolytic action of the SSCI, LM-4131, was mediated through the cannabinoid type 1 receptor under non-stressed (control) conditions, but mediated through the small conductance calcium-activated potassium (SK) channels when mice were subjected to footshock stress. Also, we have found that the anxiolytic effects of SSCIs in stressed mice may be due to a decrease in excitatory cell firing in the amygdala.

Despite the presence of a cytosolic fatty acid synthesis pathway, mitochondria have retained their own means of creating fatty acids via the mitochondrial fatty acid synthesis (mtFASII) pathway. The reason for its conservation has not yet been elucidated. Therefore, to better understand the role of mtFASII in the cell, we used a variety of methods to characterize the consequences of changes in mtFASII functionality in whole cells, isolated mitochondria, and mitochondrial secretions. We altered mtFASII functionality by knockdown of acyl carrier protein (ACP) or overexpression of mitochondrial trans-2-enoyl-CoA reductase (MECR). As a control for known respiratory deficits in mtFASII knockdowns, we also knocked down a component of complex I of the electron transport chain. We found that loss of mtFASII function disturbs metabolism and bioactive lipid regulation at the whole-cell and mitochondrial levels. We found that the mitochondrial secretome may contain bioactive lipids and small peptides, and that knockdown of the mtFASII pathway results in increased levels of dipeptides, among other metabolites, in the mitochondrial secretome. These data indicate that the mtFASII pathway may have a role in mitochondrial signaling in a manner not linked to mtFASIIâs effects on the electron transport chain.

Dopamine is a critical neurotransmitter used across phylogeny to regulate many aspects of behavior. Synaptic control of dopamine signaling is vital for normal nervous system function in humans, and dysregulation of this signaling is associated with many disease states, including addiction, attention-hyperactivity deficit disorder (ADHD), schizophrenia, and Parkinsonâs disease. The model organism Caenorhabditis elegans is a useful system in which to dissect nervous system function, including the synaptic regulation of dopamine signaling. Our lab has employed a forward genetic screen in C. elegans based on the dopamine-related behavior Swimming-induced paralysis (Swip) to identify swip-13, a novel genetic regulator of dopamine signaling. Genetic analysis of swip-13 mutants has revealed a role for this gene in dopamine neurons to positively regulate the activity of the presynaptic dopamine transporter DAT-1. swip-13 encodes an ortholog of the mammalian atypical MAP kinase ERK7/8, and work in human cell lines revealed a conserved role for human ERK8 in regulating the human dopamine transporter DAT. Furthermore, recent evidence supports a role for the small GTPase Rho in mediating the regulation of DAT by ERK8.

Migraine is a prevalent and potentially debilitating neurological disorder with complex pathophysiology. Previous studies have provided compelling evidence for a role of Cortical Spreading Depression (CSD) in the symptomatology of migraine, most importantly the auras experienced by a subset of migraineurs. However, the precise role of CSD in the development of other symptoms of migraine, most notably pain, is less certain. Limitations of previous animal models of CSD have proven problematic in our ability to elucidate the role of these events in the development of migraine pain. In the newly developed animal model described here, electrical activity was recorded from multiple sites on the cerebral cortex in freely-moving animals. This model allows for recording bioelectrical changes in the cortex while concurrent behavioral changes are being measured. In contrast to other studies that employ invasive measures to evoke CSD, recordings described here are from animals that have not undergone any cortical insult, but alternatively received prolonged treatment with sumatriptan, an agent demonstrated to contribute to the development of medication-overuse headache when administered chronically. The studies described here show that animals pretreated with sustained sumatriptan have a significant increase in frequency of spontaneous CSDs. Moreover, this increase in frequency is augmented following exposure of animals to a bright light stress. These findings demonstrate a lowered threshold of cerebral cortex for development of spreading depression. These findings also suggest a dissociation between development of CSD and activation of nociceptive pathways responsible for migraine headache. Additionally, these findings show that this is a viable platform for further study of CSD in freely-moving animals and their function in the development of migraine headache. The implications of these findings on the understanding of the role of CSD in migraine headache pathogenesis are significant.

The essential micronutrient manganese is enriched in brain, especially the basal ganglia. We sought to identify neuronal signaling pathways responsive to neurologically relevant manganese levels, as previous data suggested manganese alterations occur in Huntingtons disease (HD). We found that p53 phosphorylation is highly responsive to manganese levels in human and mouse striatal-like neuroprogenitors. The Ataxia Telangiectasia Mutated (ATM) kinase is responsible for this manganese-dependent phosphorylation of p53. Activation of ATM-p53 by manganese was severely blunted by pathogenic alleles of Huntingtin. HD neuroprogenitors exhibited a highly manganese selective deficit in ATM kinase activation, since DNA damage and oxidative injury, canonical activators of ATM, did not show similar deficits. Manganese was previously shown to activate ATM kinase in cell-free assays. We found that human HD neuroprogenitors have reduced intracellular manganese with neurologically relevant manganese exposures. Pharmacological manipulation to equalize manganese between HD and control neuroprogenitors rescued the ATM-p53 signaling deficit. The compound that normalized these levels was the small molecule, KB-R7943, a known inhibitor sodium/calcium exchanger (NCX) inhibitor. However, the mechanism by which KB-R7943 corrects manganese accumulation does not seem to be via direct inhibition of the NCX transporters. We also demonstrated a severe deficit in NCX1 expression in HD cells that may also play a key role in the HD manganese deficiency. Huntingtons disease cells also show increased genomic instability and DNA damage signaling under basal conditions. Manganese is known to be an important cofactor for several enzymes involved in DNA repair and replication, and we found that the manganese deficiency was most severe in the nucleus compared with other compartments. Manganese supplementation reduced the elevated DNA damage signaling to those found in non-HD cells suggesting that manganese deficiency underlies this phenotype In short, the ATM-p53 signaling pathway is a manganese responsive signaling pathway. Manganese is an important cofactor with diminished accumulation in HD cell models. These reduced levels may be the reason for observed increases in DNA damage and genomic instability. Further experimentation is needed to elucidate the mechanism of manganese accumulation deficiency mechanism in HD and the KB-R7943 rescue.

Nearly 30% of the global burden of nonfatal disease is due to affective disorders. Stress is a potent risk factor for affective disorders including major depression and posttraumatic stress disorder. The neurobiological mechanisms by which stress influences psychopathology are poorly understood, however, it is clear that stress does not affect every individual equally. Understanding the mechanisms conferring resilience to adverse consequences of stress could have broad implications for the treatment of affective disorders. Endogenous cannabinoid (eCb) signaling is implicated in modulating affective behavior, but the role of the most abundant eCb, 2-arachidonoylglycerol (2-AG), has not been adequately investigated. We hypothesize that 2-AG signaling is a critical regulator of basal and stress-induced affective responses. In support of this, herein we demonstrate a necessity for 2-AG in regulating basal anxiety. We then develop a novel model of stress-resilience and show that 2-AG signaling is critical for resilience to stress-induced anxiety. Germline depletion of brain 2-AG produces a striking anxiety-like phenotype and acute, systemic 2-AG depletion increases susceptibility to stress-induced anxiety-like phenotypes in previously resilient mice. Conversely, acute, systemic 2-AG augmentation enhances resilience in previously susceptible mice. Human imaging studies strongly suggest an association between affective disorders and hyperactivity of the amygdala, a well-established regulator of responses to aversive stimuli. Additionally, rodent studies indicate that stress exposure increases excitatory drive to the basolateral amygdala (BLA). eCbs are implicated in regulating the effects of stress and inhibit glutamate inputs to the BLA. We hypothesize that the anxiogenic effects of global 2-AG depletion are due to impaired BLA eCb signaling. Using our model of stress-resilience, we show that amygdala-specific 2-AG depletion impairs adaptation to repeated stress and that stress-resilience is associated with increased BLA 2-AG signaling. These data demonstrate that BLA 2-AG signaling promotes resilience to stress. Altogether these data strongly support the suggestion that eCb deficiency could promote susceptibility to affective disorders and pharmacological enhancement of 2-AG signaling could be an effective treatment for stress-related affective dysfunction.

Body weight is tightly controlled by the homeostatic feeding system, which is primarily reliant upon communication between the endocrine system and the brain. The endocrine system and brain are highly complex systems with multiple components that can present with altered function. Decreased neural insulin or leptin sensitivity and altered neural underpinnings of disgust are associated with obesity. As such, it is important to explore new factors that are associated with obesity and to continually expand upon existing methodologies to increase our understanding of this multi-faceted disorder. Our results across two studies reveal the structural and functional underpinnings of Disgust Proneness, as well as how Disgust Proneness is altered in obesity. Obese individuals have lower levels of Disgust Sensitivity, as measured by the Disgust Propensity and Sensitivity Scale â Revised (DPSS-R). Using an fMRI-optimized task designed to elicit disgust and food-related disgust, we found that obese individuals have less insula BOLD activation than the lean group. This is the first identification of altered levels of BOLD activation in obese individuals within the insula. Furthermore, the self-reported measures of Disgust Sensitivity were positively correlated with insula activation extracted from the lean group, but negatively correlated with insula activation extracted from the obese group. This finding suggests that there is a functional dissociation between self-report of Disgust Sensitivity and neural activation in obese individuals. Our study did not reveal between-group differences in insula grey matter volume. Diet- and obesity-induced parenchymal density changes have been documented in the rodent mediobasal hypothalamus using immunohistochemistry. Emerging MRI techniques are being developed to quantify these changes in parenchymal density in living humans. Our study sought to explore the viability of using single echo T1 MRI scans to identify parenchymal density changes in human subjects before and after weight loss and insulin detemir intervention. Our null results suggest that single echo T1-weighted MRI is not a suitable alternative to single or multi echo T2-weighted MRI.

Synaptic circuits are dynamically refined during development as synapses are either stabilized or eliminated. This process requires both neuronal activity and genetic programming; however, the molecules that mediate this interaction are poorly understood. Here, I identify a Degenerin/Epithelial Sodium Channel (DEG/ENaC) protein, UNC-8, as a regulator of synapse removal in C. elegans. UNC-8 is transcriptionally-regulated to promote synapse disassembly in an activity-dependent pathway that requires calcium influx through voltage-gated calcium channels and activation of the neuronal phosphatase calcineurin. Activation of the canonical apoptotic protein CED-4 also promotes removal of the presynaptic density through the UNC-8 pathway. We propose a model in which voltage-gated calcium channels activate calcineurin to promote UNC-8 channel activity. Sodium influx through UNC-8 may act as a molecular trigger, depolarizing the presynaptic membrane to enhance activity of the local calcium channels. We propose that intracellular calcium then exceeds a critical threshold that activates a downstream pathway including the cell death pathway components CED-3 and CED-4 and the F-actin severing protein, gelsolin. Previous work has shown that the apoptotic pathway stimulates gelsolin to physically dismantle an actin network that stabilizes the presynaptic active zone. <p> In addition to defining a mechanism for the remodeling role of UNC-8, this work demonstrates that efficient elimination of remodeling GABAergic synapses also depends on a parallel-acting pathway regulated by the homeodomain transcription factor IRX-1/Iroquois. Removal of the synaptic vesicle priming protein UNC-13 is dependent on IRX-1 activity, but does not require UNC-8 function, suggesting that these pathways can differentially regulate the turnover of specific active zone components. Additionally, we find that GABAergic signaling is required for proper synapse removal in the UNC-8 pathway, and that ionotropic and metabotropic GABA receptors adopt opposing roles in presynaptic disassembly. <p> Our results provide the first example of a presynaptic DEG/ENaC protein that promotes synapse elimination. Neurotransmission and genetic programming both converge on the UNC-8-dependent pathway; therefore, providing a link between transcriptional regulation and neuronal activity. This work advances our understanding of synapse disassembly, and thus may eventually reveal therapeutic targets against diseases that arise from synaptic dysfunction.

Neuroinflammation is defined as inflammation that occurs in the central nervous system (CNS) and is characterized by cytokine release and glial reactivity. Previous efforts indicate that neuroinflammation is involved in many facets of the CNS, including development, homeostatic maintenance, and degeneration in response to injury and disease. Elucidating how specific cytokines are involved in these facets is important for the development of new therapies as well as expanding our knowledge of how the CNS develops, functions, and responds to stress. Using the optic projection as a model of the CNS, this thesis investigates how the cytokine interleukin-6 (IL-6): 1) contributes to the constitutive development and function of retinal ganglion cells (RGCs) and 2) impacts degeneration of RGCs in response to glaucoma-related stressors (i.e. ocular hypertension). Our data indicate that IL-6 signaling is necessary for proper development and function of RGC axons, yet facilitates axon degeneration and vision loss in response to ocular hypertension. The apparent switch of IL-6 from a constructive signal to a destructive signal correlates with elevations in the soluble isoform of the IL-6 receptor (sIL-6Rα), an isoform that is linked to degeneration within and outside the CNS.