The most prevalent histological risk factor for Parkinson’s disease is the development of alpha-synuclein clumps, known as Lewy Bodies, in the substantia nigra portion of the midbrain. However, the etiology of the disease remains unknown. Neurons are heavily dependent on aerobic respiration for ATP due to their high energy demands. In neurons, mitochondria are transported throughout axons and dendrites for facilitating subcellular functions, and are critically important for membrane excitability and neurotransmission. Past evidence indicates that mitochondrial dysfunction plays a significant role in the progression of degenerative pathologies in the brain. In this review, genetic factors and biochemical mechanisms representative of healthy mitochondrial structure and function are examined to determine the role of mitochondrial dysfunction in the onset and progression of Parkinson’s disease. Treatments and therapies targeting mitochondrial dysfunction for the purposes of improving Parkinson’s disease pathologies are also explored. Published data examined in this review has shown that mitochondrial dysfunction plays a significant role in the development and progression of Parkinson’s disease. Through interactions with alpha-synuclein protein aggregates and through facilitation of general dopaminergic neurodegeneration, mitochondrial dysfunction provides a pathway for the progression of Parkinsonian pathologies. In addition, genes involved with mitochondrial biogenesis, as well as genes involved in the onset of Parkinson’s disease show overlap and interactions indicative of an association between defective mitochondria and parkinsonism. With a focus on improving mitochondrial function and reducing Parkinson’s disease pathology, a number of potential drug treatments and therapies have proven to be of interest. While there is currently no cure for Parkinson’s disease, evidence consolidated in this review supports the need for investigation into Parkinson’s disease treatments that target mitochondrial dysfunction and oxidative stress. Subsequent research studies and treatments should focus on genes that play a regulatory role in mitochondrial biogenesis, with the goal of determining more transcriptional pathways that overlap between mitochondrial dysfunction and parkinsonism. Drug compound screens for improving mitochondrial biogenesis and reducing alpha-synuclein aggregation should be explored as well.

Neuromodulation plays important roles in adjusting our nervous system to produce behaviors. The same neuromodulator could have different effects on different targets, or the same target could be modulated by multiple neuromodulators. In the first project of my dissertation I investigated differential modulation of mitral cells (MCs) contained in the main (MOB) and accessory (AOB) olfactory bulb by serotonin (5-HT) using an in vitro, brain slice approach in postnatal (P15-30) day mice. In the MOB, 5-HT elicited three types of responses in 94% of 158 cells tested. Cells were either directly excited (73%, n = 115), inhibited (9%, n = 15), or showed a mixed response −first inhibition followed by excitation (12%, n = 19). In the AOB, 83% of 115 cells were inhibited with 17% of cells showing no response. Albeit located in parallel partitions of the olfactory system, 5-HT largely elicited excitation of MOB MCs while it evoked two different kinetic rates of inhibition in MCs of the AOB. Using a combination of pharmacological agents, I found that the excitatory responses in MOB MCs were mediated by 5-HT2A receptors through a direct activation. In comparison, 5-HT-evoked inhibitory responses in the AOB arose due to a polysynaptic, slow-onset inhibition attributed to 5-HT2 receptor activation exciting GABAergic interneurons. The second type of inhibition had a rapid onset as a result of direct inhibition mediated by the 5-HT1 class of receptors. The distinct serotonergic modulation of MCs between the MOB and AOB could provide a molecular basis for differential chemosensory behaviors driven by the brainstem raphe nuclei into these parallel systems. In the second project of my dissertation, I explored the modulation of glucagon-like peptide-1 (GLP-1) neurons in the olfactory bulb (OB). A population of GLP-1 neurons was recently discovered in the OB. The functions of these neurons remain incompletely understood. Herein, I used an in vitro, brain slice approach to investigate the modulations of GLP-1 neurons. Juvenile mice (P20 to P45) of both sexes were used to examine the involvement of centrifugal projections from higher brain areas including serotonergic, cholinergic, and noradrenergic afferents. Bath application of serotonin (40 µM, n = 4) and norepinephrine (100 µM, n = 4) had no effect on the evoked firing frequency. Acetylcholine (ACh; 100 µM), however, led to either inhibition or excitation of GLP-1 neurons. For inhibition, ACh induced a small outward current (5.1 ± 1.8 pA, n = 9) recorded by voltage-clamp when neurons were held at −70 mV. When recorded in current-clamp mode, ACh delayed the latency to first spike (control: 253 ± 30 ms, ACh: 396 ± 4 ms; n = 2). For excitation, bath application of ACh resulted in 1.9 ± 0.6-fold increase in firing frequency (n = 21). Previous evidence showed that GLP-1 neurons in the brainstem could be modulated by metabolic-related hormones such as leptin and cholecystokinin (CCK). I found that GLP-1 neurons could be modulated by CCK, but not by leptin. Bath application of CCK (0.8 µM) led to either cessation of firing (n = 10) or an increase in firing of 1.7 ± 0.4-fold (n = 11). Lastly, mice were injected intraperitoneally with the GLP-1 analogue Exendin-4 (0.4 µM /kg) or control saline and tested 30 minutes post injection in a habituation-dishabituation odor test. Mice receiving Exendin-4 failed to show significant dishabituation, demonstrating impaired ability to discriminate a novel odor from a familiar odor. One primary target of neuromodulation is ion channels. Depending on which group of neurons and in which brain region it is expressed, the same type of ion channel can contribute to multiple functions. In the third project of my dissertation I examined the consequences of loss of function of voltage-gated potassium channel Kv1.3. It has long been recognized that olfaction and emotion are linked. My study aimed to investigate the roles of olfaction in modulating anxiety. Kv1.3 knockout mice (Kv1.3-/-), which have heightened olfaction, and wild-type (WT) mice were examined for anxiety-like behaviors. Because Kv1.3-/- mice have also been observed to show increased locomotor activity, which is one behavior reported in animal models of attention-deficit/hyperactivity disorder (ADHD), inattentive behavior was quantified for both genotypes. Kv1.3-/- mice showed increased anxiety levels compared to their WT counterparts and administration of methylphenidate (MPH) via oral gavage alleviated their increased anxiety. Object-based attention testing indicated Kv1.3-/- mice had attention deficits and treatment with MPH also ameliorated this condition. My data suggest that heightened olfaction does not necessarily lead to decreased anxiety levels, and that Kv1.3-/- mice may be used as a behavioral model of the inattentive subtype of ADHD. / A Dissertation submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Fall Semester 2017. / November 16, 2017. / Includes bibliographical references. / Debra Ann Fadool, Professor Directing Dissertation; Timothy M. Logan, University Representative; David M. Gilbert, Committee Member; Lisa C. Lyons, Committee Member; Zuoxin Wang, Committee Member.

Both aging and hypertension are significant risk factors for the development of vascular pathology, the underlying cause of vascular cognitive impairment (VCI), which is the second most common cause of dementia. Cerebral microbleeds (CMBs) are small blood degradation products found in brain tissues accompanying aging, dementia, and cerebrovascular diseases (Lee et al., 2018), and are associated with a decrease in cognitive performance. Evidence has shown that in rodent brains CMBs can develop with aging and inflammation exacerbates the development. However, few studies have investigated the interactions among aging, hypertension, and cerebrovascular pathology. Previous studies conducted in our laboratory have shown a sex-dependent increase in blood pressure in male Sprague Dawley (SD) rats. Based on these findings, the goals of the current study are to define the relationship between age and the presence of 1) sub-acute cerebral microhemorrhage and/or 2) acute cerebral microhemorrhage, in spontaneously hypertensive male SD rats, in an effort to better understand the links between hypertension and VCI. SD rats at ages of 3 months, 8 months, and 16 months were acquired and their blood pressure levels were recorded. Brains were extracted, sectioned and stained to reveal any microbleeds in the hippocampus. The results support the hypothesis that there is an age-dependent increase in sub-acute CMBs in the hippocampus, but no relationship with acute CMBs could be drawn since there was no acute CMBs observed. This study demonstrates the positive relationship between sub- acute CMBs and age in an age-dependent hypertension animal model, and such understanding will further elucidate the pathological mechanisms involved in hypertension and VCI, and contribute to the discovery of future therapeutics in treating hypertension, vascular cognitive impairment and relate pathologies.

Extracellular vesicles derived from mesenchymal stem cells (MSC-EVs) have demonstrated to be neuroprotective and an excellent candidate for the treatment of cortical injury. In our laboratory, it was demonstrated the enhancement of motor recovery after cortical motor injury on a rhesus monkey model. This study will build on those findings and provide an in-depth examination of the nature and rate of recovery function specifically looking at the slope of recovery comparing the vehicle and MSC-EVs treated group. The monkeys were trained on the Hand-Dexterity Test (HDT), a fine motor task, for four weeks prior to induced cortical injury on hand representation on the contralateral primary motor cortex of the dominant hand. The monkeys received vehicle or MSC-EVs treatment 24 hours and 14 days after injury. The post-operative HDT test was performed to analyze the recovery of motor function of both impaired dominant-hand and non-impaired non-dominant hand on both large and small wells of the testing apparatus. Both the vehicle and MSC-EVs group demonstrated a positive recovery slope. The non-dominant hand for the large well also showed to be significantly different (p = 0.01) when comparing in between MSC-EVs treated and vehicle groups. These results support previous findings from our lab, reinforce that MSC-EVs to be potentially used as a clinical treatment, and demonstrate an important approach to the non-impaired hand recovery to be analyzed in future cell therapy studies.

Normal aging, even in the absence of neurodegenerative disease such as Alzheimer’s disease (AD), is still characterized by cognitive decline in the areas of learning, memory, processing speed, and executive function. Though initially believed to be due to neuronal loss, the root of cognitive decline in normal aging is now appreciated to be due to loss of white matter volume and accruing damage to insulating myelin sheaths that impairs axon conduction and leads to cortical disconnection. The causes of white matter disruption appear to be multifactorial and include oligodendrocyte dysfunction and increasing white matter neuroinflammation that together impede myelin homeostasis (i.e. maintenance and repair). Myelin damage leads to an accumulation of interstitial myelin debris that is normally phagocytosed by microglia. With age, microglia become ineffective at phagocytosis and clearance of myelin debris and become chronically reactive, secreting pro-inflammatory cytokines that perturb the processes underlying myelin maintenance and repair. However, it is unknown how these neuroinflammatory signals may call upon T cells of the adaptive immune system, which may also exacerbate myelin degradation and affect age-related cognitive decline. The overall goal of this dissertation work was to characterize the role that T cells may play in normal aging white matter dysfunction. To study the processes underlying normal aging, the rhesus monkey serves as a gold standard model due to similarity to humans in their extended lifespan, gray:white matter ratio, behavioral testing abilities, and development of age-related cognitive decline, in the absence of the confounds of AD pathology. Here, using immunohistochemistry on brains from behaviorally characterized monkeys, we demonstrate that CD3+ T cells in the perivascular space and within the parenchyma increase with age in the white matter but not the gray matter. In situ hybridization experiments showed that T cells express RNA for tissue entry (LFA1) but not for tissue egress (CCR7) suggesting that T cells actively enter the brain parenchyma. Further, T cell infiltration into the brain is correlated with the degree of microglial reactivity measured by morphologic density analysis of LN3-staining. These infiltrating T cells were predominantly CD8+ cytotoxic T lymphocytes, with a smaller percentage of CD4+ helper T cells present. The distribution of CD8+ T cells correlated with the distribution of CD4+ T cells as well as CD4+ microglia, suggesting a means for T cell reactivation upon entry into the white matter parenchyma. Single cell RNA sequencing in young versus aged white matter show that T cells in the old brain exhibit increased expression of genes involved in T cell activation and production of proinflammatory cytokines. The subset of proinflammatory microglia enriched in the aged brain are also enriched for gene pathways involved in activating T cells and inflammatory signaling. To explore the interaction between these two cell types, receptor ligand analyses were performed showing evidence that in the young brain, microglia are able to suppress T cell activation, but lose this interaction with age and instead gain an interaction which leads to the activation of T cells. Finally, beyond the myelin damage that cytotoxic CD8+ T cells can inflict directly, ligand receptor analysis revealed that in the old brain, oligodendrocytes may be promoting T cell activation via cytokine secretion. T cells may suppress myelination by blocking the FGFR2 receptor on oligodendrocytes. Together, these data provide convincing evidence that T cells enter the white matter parenchyma of the aging brain where they may be contributing to neuroinflammation, myelin damage, and attenuate myelin repair, and thus may be a novel therapeutic target with the goal of preventing or slowing cognitive decline associated with normal aging.

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The hippocampus plays a central role in episodic memory and spatial navigation. The activity of individual neurons and ensembles of cells encodes location within an environment, the spatial context, and non-spatial behavioral task demands, creating unique codes for these features. The hippocampus plays a role both during the initial encoding of memory representations, and also at extended intervals in tasks which require flexible retrieval or self-localization. While behavior and memory can be stable for long periods of time, many studies have shown that their neural basis is more dynamic than expected. In the studies presented here, I used single-photon calcium imaging in freely behaving mice to track hippocampal single-unit activity over many recording sessions to test how circuit instability interacts with ongoing behavioral demands. In the first study, I asked whether the representation of multiple task demands remained stable alongside an animal’s behavior. Previous work has indicated that hippocampal activity will change as an animal’s performance in a task improves. Additionally, drift, the inactivation and replacement of neuron membership within the active population, may affect neurons that code for different aspects of a task at different rates. I tested this hypothesis by recording hippocampal activity in an alternation task which animals performed stably for multiple weeks. I found that the population code separating each task dimension was highly stable in spite of cell turn over, but that the distribution of task dimensions encoded by single neurons changed as a function of time. This change in distribution of task dimensions encoded by single neurons was not driven by different levels of stability among the different coding populations, as indicated by previous reports, but instead was driven by changing rates with which newly active neurons encoded task dimensions. In the second study, I looked at how new learning affected a previously-encoded task representation. Mice performed two different tasks in a plus-shaped maze in the sequence A-B-A over a nine-day sequence. One group performed the entire sequence on a single maze, while another group performed the second rule on a second maze. This allowed me to test the hypothesis that new learning in a single environment would cause greater change in the hippocampal representation for that environment than can be accounted for alone by time between recordings. This hypothesis is confirmed by multiple measures of single unit activity, and in the population code. Together, these results demonstrate that the instability observed in long term patterns of neuronal activity does not impair behavior, and that it may have a role in the ongoing refinement of the organization of hippocampal memory representations.

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