Neurons are polarized cells with dendrites and an axon projecting from their cell body. Due to this polarized structure a major challenge for neurons is the transport of material to and from the cell body. The transport that occurs between the cell body and axons is called Axonal transport. Axonal transport has three major components: molecular motors which act as vehicles, microtubules which serve as tracks on which these motors move and microtubule associated proteins which regulate the transport of material. Axonal transport maintains the integrity of a neuron and its dysfunction is linked to neurodegenerative diseases such as, Alzheimer’s disease, Frontotemporal dementia linked to chromosome 17 and Pick’s disease. Therefore, understanding the process of axonal transport is extremely important. Single particle tracking is one method in which axonal transport is studied. This involves fluorescent labelling of molecular motors and microtubule associated proteins and tracking their position in time. Single particle tracking has shown that both, molecular motors and microtubule associated proteins exhibit motion with multiple components. These components are directed, where motion is in one direction, diffusive, where motion is random, and static, where there is no motion. Moreover, molecular motors and microtubule associated proteins also switch between these different components in a single instance of motion. We have developed a MATLAB program, called MixMAs, which specializes in analyzing the data provided by single particle tracking. MixMAs uses a sliding window approach to analyze trajectories of motion. It is capable of distinguishing between different components of motion that are exhibited by molecular motors and microtubule associated proteins. It also identifies transitions that take place between different components of motion. Most importantly, it is not limited by the number of transitions and the number of components present in a single trajectory. The analysis results provided by MixMAs include all the necessary parameters required for a complete characterization of a particle’s motion. These parameters are the number of different transitions that take place between different components of motion, the dwell times of different components of motion, velocity for directed component of motion and diffusion coefficient for diffusive component of motion. We have validated the working of MixMAs by simulating motion of particles which show all three components of motion with all the possible transitions that can take place between them. The simulations are performed for different values of error in localizing the position of a particle. The simulations confirm that MixMAs accurately calculates parameters of motion for a range of localization errors. Finally, we show an application of MixMAs on experimentally obtained single particle data of Kinesin-3 motor.

Mobility is essential for the independent lifestyle. However, as the US population ages, challenges to mobility start to arise, among them just the aging itself which leads to decreased postural stability, falls and the second most common neurodegenerative disease, that is Parkinson’s disease (PD). We decided to investigate step initiation as it is crucial to mobility: walking is not possible without the first step. Step initiation is impaired in PD. However, the impact of PD on the neural mechanisms of step initiation when some of the step parameters are unpredictable remains unexplored. Cortical preparation for step initiation can be assessed by beta event-related desynchronization (ERD) derived from electroencephalography (EEG) recordings. We hypothesized that subjects with PD would exhibit less cortical modulation between conditions of forward step initiation with and without prior knowledge of limb choice. Further, we hypothesized that decreased cortical modulation in PD would associate with a higher impairment of motor performance. Results identified that the group with PD exhibited decreased beta ERD amplitudes that were similar regardless of condition, whereas control subjects modulated beta ERD amplitudes between conditions, particularly in early stages of pre-movement processing in areas overlying sensory cortex. Subjects with PD presented with delayed and reduced postural preparation with increased step target error across both conditions and exhibited a greater incidence of multiple anticipatory postural adjustments (APAs) in the predictable relative to the unpredictable condition. Delayed postural preparation significantly correlated with lower amplitudes of beta ERD. We concluded that diminished early pre-movement processing over sensory cortex was concomitant with poor pre-selection of the stepping limb in predictable conditions and that a generally diminished amplitude of cortical pre-movement processing relates to delayed step initiation in people with PD. Furthermore, impaired mobility accompanies healthy aging, but there is a need for deeper understanding of how aging changes central control of motor behavior. Using previous study’s method, we compared cortical preparation for step initiation using beta ERD in young and older healthy subjects performing forward steps with and without prior knowledge of limb choice. Our results show that older subjects exhibited increased beta ERD amplitudes before the step regardless of whether they were informed of limb choice or not. Moreover, older subjects exhibited early increases in beta ERD in the “sensory” cluster of electrodes, but only when full limb-choice information was available. Behaviorally, the older subjects also exhibited shortened and increased anticipatory postural adjustments which led to earlier step initiation and similar swing-foot velocities but was also accompanied by greater target step placement errors and decreased postural stability. For the older group, condition-related increases in beta ERD amplitudes and stability correlated with condition-related prolongation of APA durations. We conclude that older subjects exhibited a spectrum across two strategies: (1) a “fast” strategy associated with decreased neural preparation that trades shortened step preparation and higher swing-foot velocity for target step errors and lowered postural stability; and (2) an “accurate” strategy associated with greater neural preparation, longer step-preparation time, and higher stability during step execution. In conclusion, this thesis provides more support for beta ERD as a useful tool for studying cortical preparation non-invasively. We have also established the importance of the signals recorded by “sensory” clusters: in subjects with PD the absence of beta ERD similar to the control group was associated with impaired motor behavior even when conditions were predictable. Similarly, a part of the older group seemed to pre-potentiate its cortex lying beneath the cluster of “sensory” electrodes which was associated with more safe and accurate steps. Further investigations should focus on the importance of sensorimotor integration and its’ changes due to PD or healthy aging and beta ERD may be an excellent tool for this task.

ABSTRACT A multitude of complex cellular changes are required throughout development in order for a single cell to transform into a fully functioning organism. Cellular events including proliferation, migration, and differentiation have to be carefully controlled in order for development to proceed correctly. In order to study such dynamic processes, in vivo models are often utilized. Using the zebrafish (Danio rerio) as a model system, we have investigated the role of an axon guidance signaling pair, Semaphorin6A (Sema6A) and PlexinA2 (PlxnA2), in neurodevelopment. A previous investigation into the developmental expression patterns of sema6A and plxnA2 in zebrafish, revealed overlapping expression in the developing eye. At this early stage, the cells in the optic vesicles are undifferentiated retinal precursor cells (RPCs) and therefore do not require Sema/Plxn signaling for their canonical axon guidance role. To understand what the function of this early expression was, we knocked down both sema6a and plxna2 and observed 1) a loss of cohesion of RPCs within optic vesicles, and 2) a decrease in RPC proliferation (Ebert et al., 2014). Because these phenotypes were seen at an early stage and given that many developmental processes are dependent on genetic regulation, we hypothesized that Sema6A/PlxnA2 signaling could be regulating transcription of downstream target genes. To investigate this, we performed a microarray experiment and uncovered 58 differentially regulated genes (Emerson et al., 2017a). Prior to our study, it was not known that Sema/Plxn signaling led to changes in gene transcription. In an effort to understand the contribution of identified candidate genes to early sema6A/plxnA2 knockdown phenotypes, candidate genes with predicted functions in proliferation and migration were investigated. First, we show that rasl11b is important for regulation of RPC proliferation in the developing optic vesicles. Second, we show that shootin-1 is important in optic vesicle migration, retinal pigmented epithelium formation and optic tract patterning. Furthermore, PlxnA2 regulation of shootin-1 levels is important in sensory and motor axon patterning and branching in the peripheral nervous system. Belonging to a large family of proteins with the ability to cross talk, Semas and Plxns rely on spatially and temporally differential expression patterns to perform their tissue-specific roles. Here, we used in situ hybridization to comprehensively uncover the neuronal expression patterns of the PlxnA family in the early developing zebrafish (Emerson et al., 2017b). In addition, we present for the first time that zebrafish have two genes for PlxnA1, A1a and A1b, which show divergent expression patterns. Semas and Plxns are critical for many aspects of development and together, this body of work provides further insight into the downstream signaling mechanisms and roles of these essential developmental signaling proteins.

During embryonic development, cells respond to extracellular signals to establish proper tissue organization. Semaphorins (Semas) are a large class of secreted and transmembrane proteins that signal through Plexin (Plxn) receptors to guide migrating cells to their correct position and thus play critical roles in the development of various tissues including the nervous and cardiovascular systems. We have previously shown that Sema6A-PlxnA2 signaling is essential for visual system development, as decreasing endogenous Sema6A or PlxnA2 in zebrafish results in decreased cohesion of the early eye field, impaired retinal lamination, and smaller eye size. However, the molecular mechanisms governing these phenotypes are unknown. This dissertation describes the elucidation of functionally-relevant mechanisms of Sema6A-PlxnA signaling during eye development using biochemical and proteomic approaches in cell culture systems and the zebrafish as an in vivo vertebrate model of eye development. We first describe our investigations on the receptor-proximal mechanisms of Sema6A-PlxnA signaling. The Src-family tyrosine kinase Fyn was known to bind to and phosphorylate PlxnA receptors. However, the specific sites of phosphorylation and their function were unknown. Using mass spectrometry, we identified highly-conserved, Fyn-induced PlxnA tyrosine phosphorylation sites. Mutation of these tyrosines to phenylalanine nearly eliminated Fyn-dependent PlxnA phosphorylation. Furthermore, unlike mRNA encoding wild type human PlxnA2, mRNA encoding the tyrosine-to-phenylalanine mutant PlxnA2 could not rescue the smaller eye size phenotype caused by endogenous PlxnA2 knockdown in zebrafish. This suggests that Fyn-dependent PlxnA2 phosphorylation is critical for proper vertebrate eye development. Next, we report the discovery and functional characterization of a naturally-released soluble ectodomain of Sema6A (sSema6A). We show that sSema6A production is increased by PKC activity. The identification of several PKC-dependent phosphorylation sites in the intracellular region of Sema6A suggests a mechanism for PKC-dependent release of sSema6A. Importantly, we show that sSema6A is functional as it promotes the cohesion of zebrafish early eye field explants. This is the first report of a soluble ectodomain of the Sema6 class and suggests that Sema6A can have regulated, long-range signaling capacity in addition to its canonical contact-mediated functions. Finally, we present our findings characterizing the role in eye development of CRMP2, a downstream effector of Sema-Plxn signaling. CRMP2 is known to be critical for lamination of the cerebral cortex, leading us to hypothesize that CRMP2 could also be involved in the lamination of the retina. Using morpholino-based knockdown of endogenous zebrafish Crmp2, we show that Crmp2 has a critical function in visual system development. Crmp2 knockdown results in smaller eye size, impaired retinal lamination and a weakened optic tract. Together, this dissertation describes important novel Sema6A-PlxnA signaling mechanisms and places them in the context of vertebrate eye development.

Cannabis use initiated during adolescence may precipitate lasting consequences on the brain and behavioral health of the individual. However, research on the risk factors for cannabis use during adolescence has been largely cross-sectional in design. Despite the few prospective studies, even less is known about the neurobiological predictors. This dissertation improves on the extant literature by leveraging a large longitudinal study to uncover the predictors of cannabis use in adolescent samples collected prior to exposure. All data were drawn from the IMAGEN study and contained a large sample of adolescents studied at age 14 (N=2,224), and followed up at age 16 and 19. Participants were richly characterized using psychosocial questionnaires, structural and functional MRI, and genetic measurements. Two hypothesis-driven studies focused on amygdala reactivity and two data-driven studies across the feature domains were completed to characterize cannabis use in adolescence. The first study was cross-sectional and identified bilateral amygdala hyperactivity to angry faces in a sample reporting cannabis use by age 14 (n=70). The second study determined this amygdala effect was predictive of cannabis use by studying a sample of cannabis-naïve participants at age 14 who then used cannabis by age 19 (n=525). A dose-response relationship was observed such that heavy cannabis users exhibited higher amygdala reactivity. Exploratory analyses suggested amygdala reactivity decreased from age 14 to 19 within the cannabis sample, although statistical significance was not found. In the third study, data-driven machine learning analyses predicted cannabis initiation by age 16 separately for males (n=207) and females (n=158) using data from all feature domains. These analyses identified a sparse set of shared psychosocial predictors, whereas the identified brain predictors exhibited sex- and drug-specificity. Additional analyses predicted initiation by age 19 and identified a sparse set of psychosocial predictors for females only (n=145). The final study improved on drug-specificity by performing differential prediction analyses between matched samples of participants who initiated cannabis+binge drinking vs. binge drinking only by age 16 (males n=178; females n=148). A sparse subset of psychosocial predictors identified in the third study was reproduced, and novel brain predictors were identified. Those analyses were unique as they compared two machine learning algorithms, namely regularized logistic regression and random forest analyses. These studies substantiated the use of both hypothesis- and data-driven prediction analyses applied to large longitudinal datasets. They also addressed common issues related to human addiction research by examining sex-differences and drug-specificity. Critically, these studies uncovered predictors of use in samples collected prior to cannabis-exposure. The identified predictors are therefore disentangled from consequences of use. Results from all studies inform etiological mechanisms influencing cannabis use in adolescence. These findings can also be used to stratify risk in vulnerable adolescents and inform targets for interventions designed to curb use.

Traumatic Brain Injury (TBI) is an increasingly diagnosed constellation of injuries derived from acute mechanical trauma to the brain. With the rise of advanced neuroimaging techniques recent focus has oriented primarily towards the mild-moderate range of TBI which previously was missed diagnostically. Characteristically, these advances have shown increasing areas of micro-hemorrhage in susceptible areas of the brain and to date there are no treatment modalities targeting micro-hemorrhages or their sequelae. This dissertation explores the effects of the resulting heme processing response in the days following injury with a particular focus on inducing early heme clearance from the parenchyma using a rat central fluid percussion injury model in the mild-moderate injury range. Since heme is released ~24-48 hours post-injury and is known to be cytotoxic we observed there may be a critical window for treatment to clear heme before it is spontaneously released and to increase the buffering capacity of the tissue. We targeted heme clearance by using drugs known to increased expression of Nrf2, an upstream transcriptional regulator of the canonical heme processing protein heme oxygenase 1 (HO-1), and tracking expression of HO-1, the iron sequestration/storage proteins Lipocalin 2 (LCN2) and Ferritin (FTL), as well as the activity of matrix metalloproteinases 2 and 9 (MMP2, MMP9). We examined both tissue known to be frankly hemorrhagic (the neocortex) as well as tissue lacking any identifiable bleed (the hippocampus). We demonstrated that using the HO-1 inducers Hemin and Sulforaphane in a single dose paradigm given 1 hour post-injury heme clearance was accelerated in the neocortex with the majority of heme pigment processed by 24 hours post-injury. Further there was significant attenuation of protein expression in HO-1 and ferritin as well as the enzyme activity of MMP2 and MMP9 in both the neocortex and the hippocampus. Behavioral attenuation was also seen in both rotarod and Morris water maze tests. While we intended to target hemorrhagic processing after injury, and indeed demonstrated improved clearance of heme from post-injury hemorrhagic regions of the brain, in both tissues studied we observed remarkably similar responses to the drugs utilized in protein expression, enzyme activity, and behavioral improvement which may suggest a globally improved pathologic state or that there are unidentified pathologic micro-hemorrhages or leaky vessels which extend further into the brain parenchyma than currently identified.

A growing body of evidence implicates the importance of the endogenous cannabinoid 2-arachidonyl glycerol (2-AG) in memory regulation. The biosynthesis of 2-AG occurs primarily through the diacylglycerol lipases (DAGL-α and -β), with 2-AG serving as a bioactive lipid to both activate cannabinoid receptors and as a rate limiting precursor for the production of arachidonic acid and subsequent pro-inflammatory mediators. Gene deletion of DAGL-α shows decrements in synaptic plasticity and hippocampal neurogenesis suggesting this biosynthetic enzyme may be important for processes of normal spatial memory. Additionally, 2-AG is elevated in response to pathogenic events such as traumatic brain injury (TBI), suggesting its regulatory role may extend to conditions of neuropathology. As such, this dissertation investigates the in vivo role of DAGL-α and -β to regulate spatial learning and memory in the healthy brain and following neuropathology (TBI). The first part of this dissertation developed a mouse model of learning and memory impairment following TBI, using hippocampal-dependent tasks of the Morris water maze (MWM). We found modest, but distinct differences in MWM performance between left and right unilateral TBI despite similar motor deficits, histological damage, and glial reactivity. These findings suggest that laterality in mouse MWM deficit might be an important consideration when modeling TBI-induced functional consequences. The second part of this dissertation work evaluated DAGL-β as a target to protect against TBI-induced learning and memory deficit given its selective expression on microglia and the role of 2-AG as a precursor for eicosanoid production. The gene deletion of DAGL-β did not protect against TBI-induced MWM or motor deficits, but unexpectedly produced a survival protective phenotype. These findings suggest that while DAGL-β does not contribute to injury-induced memory deficit, it may contribute to TBI-induced mortality. The third and final set of experiments investigated the role of DAGL-α in mouse spatial learning and memory under physiological conditions (given the predominantly neuronal expression of DAGL-α). Complementary pharmacological and genetic manipulations produced task specific impaired MWM performance, as well as impaired long-term potentiation and alterations to endocannabinoid lipid levels. These results suggest that DAGL-α may play a selective role in the integration of new spatial information in the normal mouse brain. Overall, these data point to DAGL-α, but not DAGL-β, as an important contributor to hippocampal-dependent learning and memory. In contrast, DAGL-β may contribute to TBI-induced mortality.

Self-concept is strongly influenced by beliefs about one’s personal psychological attributes, and these beliefs are held with varying degrees of confidence and consequence. Hence, it is investment in self-views of those attributes that helps to regulate and maintain stable self-concept. Self-view investment is relevant to numerous self-related functions, but high self-view investment can also contribute to maladaptive self-views. Theory suggests that mindfulness cultivates a less personal, more objective perception of one’s thoughts, emotions and behaviors, and training in mindfulness has been shown to alter self-referential processing. The current pilot study (N=21) investigates the possible role of dispositional mindfulness in two forms of self-view investment, epistemic certainty and emotive importance, as indicated by self-reported and neural (functional magnetic resonance imaging-based) indicators of investment. Results indicated that dispositional mindfulness was positively associated with self-reported epistemic certainty but not emotive importance. Trait mindfulness was associated with activity in the amygdala and parahippocampal gyrus during judgements of both epistemic certainty and emotive importance. Caudate activity was positively associated with trait mindfulness specifically for judgements of emotive importance.

According to the Center for Disease Control’s (CDC) report to the Congress, there are 2.2 million emergency department visits; 80,000 hospitalizations; and 50,000 deaths each year due to traumatic brain injury. Adults 65 years and older account substantially for the majority of the hospitalization and deaths. Over 70% of the traumatic brain injuries of the older adults are classified as mild to moderate; however, even with these milder injuries, older adults present with a significantly higher morbidity and mortality compared to all other age groups (LeBlanc et al., 2006). With that in mind, it seems essential to develop a deeper understanding of the causes behind higher mortality and morbidity of traumatic brain injury in the elder population. It is well documented that increased age is accompanied by increased CNS inflammation. Recently, our laboratory showed that inflammation drives brain pathology. Specifically, we reported that the axon initial segment of cortical neurons was structurally and functionally compromised in an inflamed CNS environment. With this in mind, we proposed that age-related inflammation predisposes that brain to exacerbated pathologic consequence. To test this hypothesis, we administered a mild to moderate central fluid percussion brain injury in aged and young adult mice. Using immunocytochemical labeling against the axon initial segment protein ankyrinG combined with laser scanning confocal microscopy, we quantitatively compared axon initial segment number and length between age groups and within age groups with and without injury. Additionally, we also quantified global axonal pathology by immunolabeling for amyloid precursor protein (APP) positive swelling as an indicator of compromised axonal transport. We proposed that ankyrinG labeling will be both reduced in the aged injured mice compared against aged uninjured, young adult injured and young adult non-injured. We observed a significant increase in APP accumulations due to injury independent of aging, and due to aging independent of injury. No significant changes in the effect of injury between young and aged injured mice were observed. Although AIS length was not altered between age groups following injury, our results demonstrate that the elderly population presents with significantly shorter initial segments. The consequence of this shortening is not clear but may reflect compensatory changes in the brain to maintain homeostasis.

Linking cognition and behavior has long been an area of interest to the field of psychology in its endeavors to understand what innate factors influence human behavior. To date, the majority of research linking emotional reactivity to cognition has focused on single areas of intellectual functioning on specific diagnostic profiles or learning disorders rather than a comprehensive comparison to cognitive profile typology. Nearly all the research conducted to date continues to define cognition and emotion as disparate entities, rather than exploring a more integrated view of emotion and cognition. The purpose of this quantitative study was to examine cognitive profile differences among children with internalizing versus externalizing profiles of emotional reactivity in terms of Cattell-Horn-Carroll theory constructs as measured by the Wechsler Intelligence Test for Children, Fourth Edition [WISC-IV]. A cognitive-behavioral approach was used in conducting a secondary analysis of BASC2 and WISC-IV composite scores from a limited data set of 128 male and female students 6-16 years of age obtained from a local public school district. Results of paired-sample t tests indicated that the VCI was significantly higher for the BASC2 internalizing group (t =3.063, p < .05, two-tailed), suggesting the existence of distinct verbal cognitive skillsets among groups. This study contributes to social change by providing information to researchers and practitioners about cognitive differences among children with internalizing and externalizing behaviors that may lead to more effective cognitive-behavioral research and intervention strategies.