The sexually dimorphic nucleus of the preoptic area (SDN-POA) was first discovered in rats and is one of the most famous and best studied sex differences in the field of neuroscience. Though well documented in rats (larger in males than females), this sex difference was only recently able to be observed in mice due to the discovery of the protein calbindin-D28k as a marker. Recent studies have shown a larger, more distinct calbindin-immunoreactive (ir) cell cluster in male mice compared to females. However, the exact location of the cluster and whether the sex difference is one of total cell number or cell distribution remains unclear. In this study, we use defined contours to demonstrate that male mice have more calbindin-ir cells than females both in the central cell cluster and areas surrounding the cluster. We also report a full masculinization of these characteristics in females given a single injection of estradiol benzoate (EB) on the day of birth. The potential role of cell death in the development of this sex difference was tested using mice with a deletion of the bax gene, which codes for a pro death factor required for the establishment of other sex differences in the mouse brain. We demonstrate that bax knockout (KO) mice have more cells in the POA region in general, but eliminating cell death does not affect the development of the sex difference in calbindin-ir cell number, nor does it affect calbindin-ir cell spread. Taken together, this suggests that cell death is not a significant underlying mechanism in the establishment of the sex difference in the calbindin-ir cell cluster in the mouse POA.

A diagnosis of Autism Spectrum Disorder (ASD) can impact the entire family. Parents of children with ASD reportedly have greater stress levels, family conflict, financial concerns, and poor health habits than parents of neurotypical (NT) children. While many parent-focused interventions have been developed, these interventions focus on parent training and child behavior outcomes rather than the health and well-being of the parents. Therefore, this study aimed to examine the effects of a 15-week family judo program on physical and psychosocial health in parents of children with ASD. A total of 18 parents of children with ASD participated in a weekly judo program, with each session lasting 45 minutes. Parents completed online surveys that asked about sociodemographic information and parental stress and wore wrist accelerometers that measured their physical activity and sleep quality. Paired samples t-tests were used to examine parental stress, physical activity, and sleep quality differences in parents of children with ASD pre- and post-judo program. Both a decrease in parental stress (47.77 vs. 41.61, p High-stress levels can also negatively impact physical health and have been linked to poor sleep and low physical activity levels. This is particularly concerning as research suggests that parents of children with neurodevelopmental disorders already report worse sleep quality and lower levels of physical activity than parents of NT children. Furthermore, the treatment of sleep disorders in children with ASD has been related to reductions in parental stress. Additionally, child engagement in health behaviors has been correlated with parent behaviors. The majority of these studies, however, have utilized self-report measures of sleep and physical activity, which are prone to bias. Furthermore, while there are several components that make up sleep quality, the majority of studies have primarily focused on sleep duration rather than other aspects of sleep, such as sleep efficiency. Parental stress can also spill over into the parent-child relationship, resulting in diminished communication quality and decreased optimism about the future. Furthermore, evidence suggests a bidirectional relationship exists between parent/caregiver stress and child ASD symptoms. In other words, increased stress levels of the parent/caregiver may exacerbate the child's ASD symptoms, further worsening parent/caregiver stress. The immediate need for interventions to ease parents' stress and improve the quality of life for both parents and children is apparent. Physical activity has been deemed an intervention to reduce stress and is associated with improved well-being and mental health for both neurotypical and ASD populations. Several studies have demonstrated the benefits of physical activity interventions incorporating mind-body interaction, such as yoga or martial arts, for children with ASD. Martial arts training, such as judo, benefits participants and their families by encompassing mindfulness, balance, strength, and coordination, emphasizing social interaction. Despite the increasing prevalence of ASD, the reported stress on families of children with ASD, and the negative effects of parent stress on both parents and their children with ASD, there are no studies that have examined the benefits of a family-based, mind-body physical activity program on stress and health behaviors in parents of children with ASD. The current study will address this gap by examining the effects of a family judo intervention on parent stress and sleep patterns of parents of children with ASD. There is a significant decrease in parent-reported stress post-judo program. Parents also have increased levels of physical activity. Finally, we see parents reporting decreased stress and improved self-confidence with their children during the semi-structured interviews at the end of the program. These findings may be used to explore further whether a family judo program may lead to better parent and family outcomes, such as increased parental efficacy, improved parent-child bonding, and strengthened family resiliency.

<p>This thesis presents research investigating structural neural correlates of major depressive disorder (MDD). Although there are clear clinical differences between early- and late-onset MDD, they are still subject to the same diagnostic criteria and treatment strategy. Whether these differences translate into differences in cortical structure was examined in this study. By directly comparing early-onset (EOD) and late-onset (LOD) patients, we test whether age-of-onset results in changes in the extent or spatial pattern of cortical thinning.</p> <p>Chapter 1 provides a general background on the cerebral cortex, followed with a focus on cortical thickness. Chapter 2 presents a comprehensive review of the clinical and neurobiological literature on major depressive disorder as it pertains to age-of-onset. Three working hypotheses regarding the differences between early- and late-onset depression are presented and discussed.</p> <p>The results presented in this thesis show that there are both differences and similarities in cortical thickness between patients with EOD and LOD, with differences reflecting spatial extent, region-specificity, and magnitude of thickness differences. We confirmed the hypothesis of greater thinning in the dorsal lateral prefrontal cortex in depressed patients compared to healthy controls. We also correlated cortical thickness with clinical variables, which resulted in the finding of a positive correlation in the posterior cingulate cortex with illness severity.</p> <p>Few studies have used age-of-onset as a factor, which may account for some of the heterogeneity and inconsistent results seen in studies of MDD. We found that depression onset in early life is associated with greater disturbances in cortical thickness than LOD, possibly reflecting atypical development. These results provide novel insights into vulnerability and how development of depression is differentially affected by age.</p> / Master of Science (MSc)

In recent years, the development of new genomic technologies has allowed for the investigation of many regulatory epigenetic marks besides expression levels, on a genome-wide scale. As the price for these technologies continues to decrease, study sizes will not only increase, but several different assays are beginning to be used for the same samples. It is therefore desirable to develop statistical methods to integrate multiple data types that can handle the increased computational burden of incorporating large data sets. Furthermore, it is important to develop sound quality control and normalization methods as technical errors can compound when integrating multiple genomic assays. DNA methylation is a commonly studied epigenetic mark, and the Infinium HumanMethylation450 BeadChip has become a popular microarray that provides genome-wide coverage and is affordable enough to scale to larger study sizes. It employs a complex array design that has complicated efforts to develop normalization methods. We propose a novel normalization method that uses a set of stable methylation sites from housekeeping genes as empirical controls to fit a local regression hypersurface to signal intensities. We demonstrate that our method performs favorably compared to other popular methods for the array. We also discuss an approach to estimating cell-type admixtures, which is a frequent biological confound in these studies. For data integration we propose a gene-centric procedure that uses canonical correlation and subsequent permutation testing to examine correlation or other measures of association and co-localization of epigenetic marks on the genome. Specifically, a likelihood ratio test for general association between data modalities is performed after an initial dimension reduction step. Canonical scores are then regressed against covariates of interest using linear mixed effects models. Lastly, permutation testing is performed on weighted correlation matrices to test for co-localization of relationships to physical locations in the genome. We demonstrate these methods on a set of developmental brain samples from the BrainSpan consortium and find substantial relationships between DNA methylation, gene expression, and alternative promoter usage primarily in genes related to axon guidance. We perform a second integrative analysis on another set of brain samples from the Stanley Medical Research Institute.

In zebrafish, CoPA (Commissural Primary Ascending) is the first among ascending commissural axons to pathfind anteriorly and form the spinal commissure. One pathway that guides their anterior growth is the planar cell polarity (PCP) signaling pathway, but it is not fully known how PCP signaling regulates anterior guidance. We examined CoPA pathfinding in various PCP mutants to determine if anterior-posterior (A-P) guidance of CoPAs is dependent on PCP signaling. We found that certain PCP mutants exhibited anterior pathfinding defects, with approximately half of all affected CoPAs migrating incorrectly posteriorly. By using a translation-blocking DCC (Deleted in Colorectal Cancer) morpholino to prevent CoPA midline crossing, we discovered that CoPA axons in Fzd3a and Scribble mutants show severe defects in A-P guidance, which suggest that PCP influences A-P guidance of CoPAs prior to and after midline crossing.

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.

The steroid hormone ecdysone plays vital roles during Drosophila development. Pulses of 20E during Drosophila life cycle function as temporal cues, signaling the onset of metamorphic processes, including the stage specific programmed cell death of larval tissues. Ecdysone is the critical developmental cue orchestrating the metamorphic reformation of CNS, resulting in the formation of adult-specific neural circuitry. Ecdysone signaling is transduced by a heterodimeric receptor complex formed between two nuclear receptors: EcR and Ultraspiracle (USP). There are 18 nuclear receptors known in Drosophila and EcR is the only receptor whose functions in neuronal PCD have been well recognized. Therefore, the current study is aimed to define the role of nuclear receptors in neuronal cell death mechanisms in Drosophila. Here, I examine the function of nuclear receptors in PCD of two groups of peptidergic neurons: vCrz and CCAP. EcR and USP receptor complex on activation results in the coordinated transcriptional regulation of a host of transcription factors regulating genes essential for PCD. USP plays a dual role in ecdysone response, as its function is necessary for both activation and repression of ecdysone primary response genes. I have developed a possible dominant-negative mutant USP (usp3), and expressed it in flies using the GAL4-UAS system to illustrate the role of USP in ecdysone mediated PCD of vCrz neurons. Targeted expression of usp3 in corazonin neurons results in a complete blockage of PCD pathway. Another interacting partner of USP, Drosophila Hormone Receptor 38, however shows no involvement in PCD of vCrz neurons. I have also designed an ecdysone sensor to monitor the developmental timing of EcR activation in vCrz neurons. Further, I investigate the survival factors required for preventing the untimely PCD of these two groups of neurons. The study reveals that DIAP1 is required for the survival of larval vCrz and CCAP neurons. Also, the nuclear receptor E75 is shown to be critical for preventing premature PCD of CCAP neurons.

Human genetics has identified essential roles for many centriole- and cilia-related proteins during human development. Mutations in centrosome-associated genes commonly cause microcephaly, or "small brain," and mutations in cilia-associated genes cause a diverse spectrum of diseases termed "ciliopathies." However, the functional relationships between these two crucial organelles are less well studied. The activities of centrosome-related proteins during mitosis and cytoskeletal remodeling are well-characterized, but their in vivo functions are incompletely understood. Here, we identify novel human mutations in a centrosomal gene which encodes a regulatory subunit of a microtubule interacting protein, and uncover unexpected pathways during vertebrate development. Human mutations cause severe microlissencephaly, reflecting defects in cerebral cortical neurogenesis, and loss of function in mice and zebrafish confirm essential roles in embryonic development, neurogenesis, and cell survival. Surprisingly, null mutant embryos display hallmarks of aberrant Sonic hedgehog signaling, including holoprosencephaly. Deficient induced pluripotent stem cells and lymphoblasts show defective proliferation and spindle structure, while deficient fibroblasts also demonstrate a remarkable excess of centrioles, including excessive maternal centrioles, with supernumerary cilia but deficient Hedgehog signaling. Our results reveal novel roles for this protein in regulating overall centriole number, mother centriole and cilia number, and as an essential gene for normal Hedgehog signaling during neocortical development.

Synaptic connectivity is highly plastic in early development and undergoes extensive remodeling in response to changes in neuronal activity and sensory experience. Microglia, the resident central nervous system macrophages, participate in shaping mature neuronal circuits by dynamically surveying the brain parenchyma and pruning away less active synaptic connections. However, it is unknown how changes in neuronal activity regulates microglial pruning within circuits and whether this activity-dependent pruning is necessary to achieve plasticity. Using the rodent somatosensory circuit, I identified that microglia engulf and eliminate synapses in the cortex following early postnatal (P4) unilateral removal of mouse whiskers. I found this early life microglial synaptic remodeling requires specific chemokine signaling between neurons and microglia. Mice that lack expression of either the neuronal chemokine CX3CL1 (fractalkine), or its microglial receptor CX3CR1, have significantly reduced microglial synapse engulfment and fail to eliminate synapses following whisker removal. To gain more insight into how this signaling is regulated, I performed both single-cell RNA sequencing of the primary somatosensory cortex as well as microglia-specific Translating Ribosome Affinity Purification (TRAP) sequencing. I identified that the majority of central nervous system (CNS) cell populations in the somatosensory cortex, including microglia, undergo transcriptional changes following whisker removal. Further, the transcriptional changes in microglia after whisker cauterization require expression of the receptor CX3CR1. Importantly, I also found that Adam10, a gene encoding the metalloprotease known to post-translationally cleave CX3CL1 into a soluble chemokine, is upregulated in the deprived cortex after whisker ablation. Pharmacological inhibition of ADAM10 inhibits microglia-mediated removal of synapses in the deprived cortex. These data support a mechanism by which cleavage of membrane-bound CX3CL1 by ADAM10 is necessary for neuronal signaling to microglia via CX3CR1 to induce transcriptional changes within microglia upstream of synaptic engulfment and elimination following sensory deprivation.

In the nervous system, billions of neurons undergo a multistep process to establish functional circuits. This entails accurate extension of dendritic and axonal processes and coordinated efforts of pre- and postsynaptic neurons to form synaptic connections. Although many axon guidance molecules and synaptic organizers have been identified, the molecular redundancy and the vast number of synapses in the brain has complicated attempts to define their precise roles. In order to understand the molecular mechanisms that encompass these processes, my studies utilize the genetic strengths and cellular precision available in Caenorhabditis elegans for in vivo investigations of nervous system development. In this work, I unravel cell-specific requirements for the transmembrane receptor integrin in regulating developmental axon guidance of GABAergic motor neurons. Furthermore, I address important questions about mechanisms of synapse formation and maintenance using a novel dendritic spine model in C. elegans. Using high resolution microscopy, I find that the formation of immature presynaptic vesicles and postsynaptic receptors are established prior to the outgrowth of dendritic spines at nascent synapses. During this early period of synapse formation, the kinesin-3 family protein UNC-104/KIF1A transports a transsynaptic adhesion molecule neurexin/NRX-1 to developing active zones, in order to maintain postsynaptic receptors and dendritic spines in the mature circuit. In the absence of nrx-1, spines initially form normally but collapse following their extension. These findings demonstrate that presynaptic NRX-1 is required to maintain postsynaptic structures. Together my work provides new insights into molecular mechanisms that define spatiotemporal characteristics of nervous system development and the maintenance of connectivity.