The current study investigates the effects of vascular disease on white matter health by comparing participants with atherosclerotic vascular disease (AVD) to healthy control participants (HC). The comparison between groups will help elucidate the differences between early-stage mild vascular disease and normal aging processes in terms of their effects on white matter health as measured by diffusion tensor imaging (DTI). Relationships between white matter health and depression, attention, and processing speed are studied by the application of a variety of DTI neuroimaging techniques, which will allow investigation of these relationships at the levels of global, lobe-wise, and subregional analysis. The specific subregion of interest in the depression study is Brodmann Area 25, which has shown significant relationships with depressive symptomatology in patients with treatment refractory depression, but has not been studied in the context of aging, vascular disease, or subthreshold depressive symptoms. Results indicate that there are significant differences between AVD and HC participants in global and regional FA measures. Within the AVD group, significant relationships of FA with depressive symptoms and attentional function have been observed in the current study. Several unexpected findings emerged, most important of which was the observation that there is a significant relationship between FA in Brodmann Area 25 and depressive symptoms in AVD participants which is specific to the right hemisphere. These findings have implications for the treatment of depressive symptoms in older adults and participants with vascular disease.

Exercise has numerous health benefits. Yet, exercise can exacerbate pain for individuals with chronic musculoskeletal pain conditions such as myofascial pain syndrome (MPS) and fibromyalgia (FM). The exacerbation is out of proportion to the activity performed and lasts for long periods of time even after the cessation of activity. This pain acts as a barrier to healthy exercise and physical rehabilitation, which, when applied consistently, are effective treatments for MPS and FM--two diseases that produce substantial suffering and disability. The goal of the proposed studies is to determine the underlying peripheral mechanisms that contribute to enhanced pain following exercise. A better understanding of these mechanisms will lead to better pain management and prevention for these diseases. Previous data show that two hours of running wheel activity lowers the threshold necessary to induce muscle pain by acidic saline injection, producing robust pain behaviors to normally innocuous stimuli. Muscle activity that produces fatigue is associated with extracellular increases in protons, lactate, and ATP. These fatigue metabolites can directly activate muscle nociceptors and, when combined, produce a potentiated effect. Acid sensing ion channels (ASICs) are non-selective cation channels that open in response to increased proton concentrations, a response that is enhanced when lactate binds at a separate location. Ionotropic purinergic receptors (P2X) similarly produce an inward current in response to elevated ATP. Evidence suggests certain ASIC and P2X subtypes are capable of a physical interaction that allows ASIC activation at lower proton concentrations in the presence of ATP. This suggests that ATP, lactate, and protons released during exercise could activate ASIC and P2X receptors on muscle nociceptors, exciting the nociceptors and sensitizing them to subsequent muscle insult. However, the limitations of these experiments leave several gaps. First, the running wheel task fails to produce measurable increases in fatigue metabolites, possibly due to the fact that there was minimal fatigue (10%) or that their levels quickly return to baseline. Further, the running wheel task depends on central nervous system (CNS) activity and volitional running, which may introduce confounding factors upstream of muscle activation and result in large variation in the rate and duration of running. Second, it is unclear whether ASICs are necessary for the development of mechanical hyperalgesia induced by muscle activity, nor is it understood which ASIC subtypes might be required for such an effect. Finally, the molecules necessary for the induction of mechanical hyperalgesia after exercise are not known. Protons, lactate, and ATP have been suggested, but it is not known if these compounds are themselves sufficient or if they interact in an additive or synergistic manner. We address these concerns by developing an electrically-stimulated muscle fatigue paradigm that reliably fatigues a single muscle independent of the CNS, allowing for metabolite measurement during muscle activity and in vivo study of molecular mechanisms of muscle pain in the peripheral tissue. We then use genetic and pharmacologic approaches to test the role of ASIC subtypes in the development of mechanical hyperalgesia after exercise. Finally, we test the effectiveness of by-products of muscle activity in recapitulating the effects of the exercise-enhanced pain model.

Thermogenic brown and beige adipocytes convert chemical energy to heat by metabolizing glucose and lipids via uncoupling protein 1 (Ucp1), a process known as non-shivering thermogenesis. Serotonin (5-HT) neurons in the ventral medulla are known to regulate sympathetic efferent neurons in the intermediolateral nucleus (IML) necessary to maintain brown adipose tissue (BAT) activity. Previous studies show that mice lacking central 5-HT neurons are incapable of maintaining body temperature in cold, ambient conditions. Due to this direct linkage between 5-HT and thermoregulation, we hypothesized that central 5-HT neurons may be vital to the regulation of brown and beige adipocyte activity. Given that BAT consumes large amounts of substrate when active, we also hypothesized that inactivation of BAT due to deletion of the regulatory neural circuitry (5-HT neurons) would cause metabolic dysregulation. To test this, we generated mice in which the human diphtheria toxin (DT) receptor was selectively expressed in central 5-HT neurons under control of a Pet-1 promoter. Pet-1 is a transcription factor selectively located in mature, central 5-HT neurons. Coincidentally, some cells within pancreatic islets also express Pet-1, and contain adequate machinery to produce, release, and uptake 5-HT. Systemic treatment with DT eliminated 5-HT neurons and caused loss of thermoregulation, BAT steatosis, and a >50% decrease in Ucp1 expression in BAT and beige fat, indicative of reduced thermal production. In parallel, blood glucose increased 3.5-fold, free fatty acids 13.4-fold and triglycerides 6.5-fold. Intracerebroventricular (ICV) treatment with 1/30th the systemic dose of DT induced an even greater thermoregulatory impairment. The metabolic deficits following systemic DT treatment indicate that central 5-HT neurons are essential for proper metabolic regulation. However, such high levels of glucose and lipids also indicate failure of the pancreatic endocrine program following systemic treatment, likely due to moderate destruction of β-cells expressing Pet-1 and the DT receptor. Because ICV treatment caused even greater thermoregulatory and metabolic deficits, where little, if any, of the toxin would spread systemically, central 5-HT neurons are clearly essential for normal central regulation of metabolism. Interestingly, similar BAT and beige fat defects occurred in Lmx1bf/f/p mice, in which 5-HT neurons fail to develop in utero. Assessment of systemically treated animals using a euglycemic/hyperinsulinemic clamp showed extensive fasting hyperglycemia and systemic insulin resistance, coinciding with reduced glucose uptake in skeletal muscle and BAT. The hyperinsulinemic clamp failed to suppress hepatic glucose and fatty acid production, leading to the conclusion that loss of central 5-HT neurons disrupts central hepatic regulation. In attempts to induce BAT thermogenesis and metabolism, we optogenetically stimulated 5-HT neurons in the rostral raphe pallidus and measured BAT and body temperature along with blood glucose. Unfortunately, these stimulations were incapable of increasing BAT temperature and lowering blood glucose, perhaps limiting therapeutic potential of these 5-HT neurons. We conclude that 5-HT neurons are major players in central regulation of metabolic homeostasis, in part through recruitment and activation of brown and beige adipocytes and hepatic substrate production. Data also suggest that 5-HT neurons regulate glucose homeostasis via undefined neural mechanisms independently of BAT activity and pancreatic insulin secretion. Cumulative data on central 5-HT neurons indicate they are master regulators of whole-body metabolism.

Philosophical and scientific investigations into the nature of belief and knowledge are ancient, extending back to the beginnings of rational thought. It is not until the last few decades that we have been able to peer into and examine the organ of belief, the brain. Neuroanatomical perspectives have begun to address the long-standing questions of epistemology by identifying specific neural regions that are critical for the storage and evaluations of beliefs. Here, a novel neuroanatomical model of belief and doubt is presented, where post-rolandic association cortices are critical for the storage of beliefs and the prefrontal cortex is necessary for the doubt and evaluation process. It is proposed that the singular function of the prefrontal cortex is "false tagging" (the neuroanatomical essence of doubt) to mental representations in post-rolandic cortices. Individuals that have dysfunction to the prefrontal cortex, such as patients with explicit damage to the prefrontal cortex, from tumor resections or cerebral vascular events, should show a "doubt deficit", accompanied by a general increased belief to information. Evidence is presented indicating that deficiencies in the "false tagging" function may explain a wide assortment of abnormalities in neurological and psychiatric patients. Several experiments in various populations (neurological, developmental, and psychiatric) were conducted to examine the role of specific brain regions in the believing and doubting process. First, two studies gave participants explicitly-labeled false beliefs and measured the ability of the participants to falsify these beliefs. It was predicted that participants with dysfunction to the prefrontal cortex would be poor at falsifying novel beliefs. Results confirmed the predictions. Second, participants were given pairs of statements that represented opposite opinions on some issue and responded by agreeing or disagreeing with each statement. Participants with dysfunction to the prefrontal cortex, who, theoretically, have a "doubt deficit," should show compartmentalized minds, where cognitions are easily believed but rarely doubted against other extant mental information. Results suggested that participants with prefrontal cortex dysfunction were more likely to agree to opposing statements. Third, individuals with dysfunction to the prefrontal cortex should lack a dissonant state that can change attitudes, when two cognitions are in conflict. Using a free-choice paradigm, it was found that participants with prefrontal dysfunction showed either extreme attitude change after choice or no attitude change after choice, which is consistent with a "no dissonance" state. Finally, individuals with compartmentalized minds tend evince an authoritarian personality. A psychometric scale and a behavioral measure of authoritarianism were examined in the participants. Results indicated that participants with prefrontal cortex dysfunction showed increased authoritarianism on the psychometric scale, but decreased authoritarian behavior, reflecting a dissociation between knowledge and behavior. In conclusion, the results support the theoretical assertions that the prefrontal cortex is critical for "false tagging" or doubting cognitive representations. Data from neurological, developmental, and psychiatric populations are broadly consistent with the theory and offer strong external validity.

There is a limited research base on low voltage brain conditions, which are characterized by electrical activity being measured at below 20 microvolts. The purpose of this study was to examine the relationship between saliva cortisol levels and voltage using an EGG in a college student population. Illuminating this relationship is important to inform how low voltage conditions can affect daily memory and cognitive functioning of undergraduate college students that may be a result of stress. The college student population may be vulnerable to the low voltage condition because of stress from the transition between teenage and adult life and related social and academic pressures. Sapolsky's theory of stress, which hypothesized that high cortisol levels will manifest as a low voltage condition, guided this study. The sample included 60 undergraduate students recruited by flyers distributed on the campus of a liberal arts college. A multiple regression analysis was used to examine the relationship between explain the variables. Although no low voltage was found in this study sample, the study results contribute to positive social change by providing a better understanding for students and staff of brain functioning when exposed to chronic stress and encourage the implementation of programs for managing stress and prevention of stress before it reaches a chronic state and negatively impacts brain functioning.

Alzheimer’s disease (AD) is a devastating neurodegenerative condition in which a patient’s cognitive functioning, memory, and physical health progressively deteriorate. In order to treat physiological deterioration in AD, a neuroprotective recombinant human- erythropoietin (EPO) fusion protein was used. In addition to its ability to target amyloid beta (Aβ) aggregation, EPO has been shown to reduce inflammation, oxidative stress and synaptic loss. Recombinant human-erythropoietin (EPO) was combined with a chimeric transferrin receptor (TfR) monoclonal antibody (cTfRMAb) to form a fusion protein (cTfRMAb-EPO) that is able to cross the blood-brain barrier (BBB) by binding to the TfR expressed on the luminal side of the BBB. Thirty eight male APPswePSEN1dE9 (APP/PS1) mice were separated into four treatment groups (wildtype (WT) treated with saline, APP/PS1 treated with saline (TG), APP/PS1 treated with cTfRMAb-EPO (cTfRMAb-EPO), and APP/PS1 treated with rHu-EPO alone (rhu-EPO)) and were subcutaneously injected with their respective treatments twice a week for six weeks. Recognition memory and locomotive behavior were tested through the novel object recognition (NOR) task and open field (OF) test when the mice were 8 months old and again at 11 months old (after 8 weeks of treatment) to determine treatment effects. Both behavioral tests demonstrated a clear age effect in mice between 8- and 11-months old. In the NOR task, no significant differences in recognition memory were observed in TG, cTfRMAb-EPO, or rHu-EPO groups. Lastly, the OF test demonstrated no significant behavioral differences among treatment groups.

The corticospinal tract (CST), which carries commands from the cerebral cortex to the spinal cord, is vital to fine motor control. Spinal cord injury (SCI) often damages CST axons, causing loss of motor function, most notably in the hands and legs. Our preliminary work in rats suggests that CST circuitry is complex: neurons whose axons project to the lower cervical spinal cord, which directly controls hand function, also send axon collaterals to other locations in the nervous system and may engage parallel motor systems. To inform research into repair of SCI, we therefore aimed to map the entire projection pattern, or “connectome,” of such cervically-projecting CST axons. In this study, we mapped the corticospinal connectome of the Rhesus macaque - an animal model more similar to humans, and therefore more clinically relevant for examining SCI. Comparison of the Rhesus macaque and rat CST connectome, and extrapolation to the human CST connectome, may improve targeting of treatments and rehabilitation after human SCI. To selectively trace cervically-projecting CST motor axons, a virus encoding a Cre-recombinase-dependent tracer (AAV-DIO-gCOMET) was injected into the hand motor cortex, and a virus encoding Cre-recombinase (AAV-Cre) was injected into the C8 level of the spinal cord. In this intersectional approach, the gCOMET virus infects many neurons in the cortex, but gCOMET expression is not turned on unless the nucleus also contains Cre-recombinase, which must be retrogradely transported from axon terminals in the C8 spinal cord. Thus, gCOMET is only expressed in neurons that project to the C8 spinal cord, and it proceeds to fill the entire neuron, including all axon collaterals. Any gCOMET-labeled axon segments observed in other regions of the nervous system are therefore collaterals of cervically-projecting axons. gCOMET-positive axons were immunohistochemically labeled, and axon density was quantified using a fluorescence microscope and Fiji/ImageJ software. Specific regions of interest were chosen for analysis because of their known relevance in motor function in humans, and for comparison to results of a similar study in rats. Results in the first monkey have revealed both similarities and differences between the monkey and rodent CST connectome. Analyses of additional monkeys are ongoing. The final results will provide detailed information about differences between rodent and primate CST, will serve as a baseline for examining changes in the CST connectome after SCI, and will provide guidance for studies targeting treatment and functional recovery after SCI.

Autism Spectrum Disorder (ASD) refers to a heterogeneous neurological condition characterized by repetitive and restrictive behaviors and social communication deficits. ASD diagnoses are at a record high, at approximately 1 in 59 children according to the US Center for Disease Control. Currently, there are no available interventions that effectively treat the core symptoms of ASD. All pharmaceutical options address comorbid side effects of ASD but not core deficits and are particularly associated with negative side effects. Additionally, there are economic and geographic barriers that can prevent families of individuals with ASD from seeking or receiving effective interventions. Many of the available interventions are extremely costly, time-consuming, and age dependent. These factors, as well as others, have led to an increase in families independently utilizing complementary and alternative interventions. Due to the large amount of misinformation available on the Internet, families have become more susceptible to trying alternative forms of interventions that have not been scientifically proven as effective, and in some cases, are significantly detrimental. Thus, the need for accessible and inexpensive evidence-based nonpharmaceutical interventions is critical and must be addressed. Fortunately, recent groundbreaking research has discovered two strains of probiotics, Bacteroides fragilis and Lactobacillus reuteri, that have been shown to ameliorate behavioral and social deficits respectively, in validated ASD mouse models in a non-age-dependent manner. Probiotic intervention with a combination of these specific strains would effectively target both repetitive behaviors and social deficits, core ASD symptoms, and provide families with an accessible and inexpensive form of intervention. The mechanisms underlying the efficacy of these probiotics are thought to be associated with the gastrointestinal (GI) system and the oxytocin pathway. This study seeks to examine the necessity of accessible nonpharmaceutical interventions and to provide an effective intervention that is neither expensive or age dependent. This study also aims to provide greater insight into the pathways and systems in which these probiotics operate.

Mitochondrial dysfunction is a phenomenon observed in models of Traumatic Brain Injury (TBI). Loss of mitochondrial bioenergetics can result in diminished cellular homeostasis leading to cellular dysfunction and possible cellular death. Consequently, the resultant tissue damage can manifest as functional deficits and/or disease states. Therapeutic strategies to target this mitochondrial dysfunction have been investigated for models TBI and have shown promising effects. For this project, we tested the hypothesis that mitoNEET, a novel mitochondrial membrane protein, is a target for pioglitazone mediated neuroprotection. To test this, we used a severe Controlled Cortical Impact (CCI) injury model in mitoNEET null and wild-type mice. We then dosed these animals with pioglitazone or NL-1, which is a compound that has a similar structure to pioglitazone allowing us to hone in one the importance of mitoNEET binding. Wild-type animals treated with the mitoNEET ligands, both pioglitazone and NL-1, had improved mitochondrial function, tissue sparing and functional recovery, compared to mitoNEET null animals. In addition to this specific hypothesis tested, our experiments provided insight casting doubt on the central dogma that mitochondrial dysfunction following TBI is the result of vast oxidative damage and consequential irreversible mitochondrial loss. The data from these studies show that when mitoNEET is targeted with pioglitazone at 12 hours’ post-injury, mitochondrial dysfunction can be reversed. Additionally, when bypassing proteins upstream of Complex I with an alternative biofuel, such as beta-hydroxybuterate (BHB), TBI related mitochondrial dysfunction is once again reversed. This leads to novel hypothesis for future work which posits mitoNEET as a redox sensitive switch; when mitoNEET senses changes in redox, as seen in TBI, it inhibits mitochondrial respiration. When targeted with an agonist/ligand or bypassed with a biofuel TBI mitochondrial dysfunction can be reversed. These studies support the role of mitoNEET in the neuropathological sequelae of brain injury, supporting mitoNEET as a crucial target for pioglitazone mediated neuroprotection following TBI. Lastly, these studies propose a mechanism of TBI related mitochondrial dysfunction which can reversed with pharmacological agents.

Cerebrovascular disease is a major cause of dementia in elderly individuals, especially Black/African Americans. Within my dissertation, we focused on two vascular morphologies that affect small vessels: brain arteriolosclerosis (B-ASC) and multi-vascular profiles (MVPs). B-ASC is characterized by degenerative thickening of the wall of brain arterioles. The risk factors, cognitive sequelae, and co-pathologies of B-ASC are not fully understood. To address this, we used multimodal data from the National Alzheimer’s Coordinating Center, Alzheimer’s Disease Neuroimaging Initiative, and brain-banked tissue samples from the University of Kentucky Alzheimer’s Disease Center (UK-ADC) brain repository. We analyzed two age at death groups separately: < 80 years and ≥ 80 years. Hypertension was a risk factor in the < 80 years at death group. In addition, an ABCC9 gene variant (rs704180), previously associated with aging-related hippocampal sclerosis, was associated with B-ASC in the ≥ 80 years at death group. With respect to cognition as determined by test scores, severe B-ASC was associated with worse global cognition in both age groups. With brain-banked tissue samples, we described B-ASC’s relationship to hippocampal sclerosis of aging (HS-Aging), a pathology characterized by neuronal cell loss in the hippocampal region not due to Alzheimer’s disease. We also studied MVPs, which are characterized by multiple small vessel lumens within a single vascular (Virchow-Robin) space. Little information exists on the frequency, risk factors, and co-pathologies of MVPs. Therefore, we used samples and data from the UK-ADC, University of Kentucky pathology department, and University of Pittsburgh pathology department to address this information. We only found MVPs to be correlated with age. Lastly, given the high prevalence of cerebrovascular disease and dementia in Black/African Americans, we discussed the challenges and considerations for studying Blacks/African Americans in these contexts.