The Reelin signaling pathway is critical for neuronal migration during development and the function of excitatory synapses in the adult forebrain. Despite a growing body of evidence implicating impaired Reelin signaling in the pathogenesis of numerous neuropsychiatric and neurodegenerative disorders, including Schizophrenia and Alzheimer's disease, little is known still regarding the specific molecular and cellular mechanisms whereby Reelin signaling modulates the function of synapses to enable normal learning and memory. In this dissertation, we addressed these knowledge gaps by identifying mechanisms of Reelin proteolysis following synaptic potentiation (Chapter 2) and dissociated the synaptic function of Reelin signaling at excitatory (Chapter 3) and inhibitory synapses (Chapter 4). In the adult brain, Reelin is secreted by GABAergic interneurons into the extracellular space, after which it is cleaved by unknown proteases to generate active fragments that signal downstream. In Chapter 1, we demonstrate that tissue plasminogen activator (tPA) and its major in vivo substrate, plasminogen, cleave Reelin under cell-free conditions to generate major Reelin fragments found in vivo. Since manipulation of tPA levels under basal conditions had no effect on Reelin processing, we hypothesized that synaptic activity may be required to render Reelin susceptible to proteolysis by tPA. Indeed, the modulation of Reelin processing by synaptic potentiation of ex vivo hippocampal slices required the presence of tPA. These data are the first to demonstrate a specific context in which Reelin signaling may be initiated in the intact brain and further emphasize that extracellular proteolysis of Reelin by tPA and other yet-to-be identified proteases is important to consider when trying to understand how altered Reelin processing and/or expression contribute to cognitive impairments associated with disease states. In Chapters 2 and 3 of this dissertation, we describe recent attempts by our lab to elucidate cellular mechanisms of Reelin signaling in the adult brain. To do this, we generated two conditional knockout mutants that lack the obligate downstream adaptor protein, Disabled-1 (Dab1), specifically in postnatal excitatory neurons (eKO) or GABAergic interneurons (iKO). Despite some overlap of Reelin and Dab1 in a subset of GABAergic interneurons, we found that their expression was generally juxtaposed, with Dab1 being primarily expressed by principle neurons and a more widespread population of Reelin-negative GABAergic interneurons. While eKO mice exhibited normal forebrain lamination, dendritic architecture, and dendritic spine density, they did have reductions in spine volume and a loss of basal and activity-dependent Akt and MAPK activation. These changes culminated in impairments in short-term and long-term synaptic plasticity, as well as impairments in associative learning and spatial memory. Taken together, our observations in the eKO mice are the first to definitively establish a synaptic function of Reelin signaling in the adult hippocampus. While characterizing the eKO mice, we also observed that GABAergic interneurons expressed Dab1, which motivated us to explore the inhibitory synapse as a novel locus of Reelin signaling (Chapter 4). Although loss of Dab1 in GABAergic interneurons did not affect forebrain development or the overall patterning of inhibitory synapses, iKO mice presented with an ataxic gait, resting tremor and cerebellar hypoplasia. Interestingly, loss of Dab1 in interneurons led to altered expression of some major glutamatergic synapse proteins (i.e. NMDA receptor subunits NR1 and NR2B), while other excitatory and inhibitory synapse proteins were normal (e.g. NR2A, GAD67/65, and gephyrin). Hippocampal field recordings further demonstrated that even partial loss of Dab1 expression in iHET mice, led to enhanced presynaptic activation and impaired theta-burst induced LTP. These data establish the inhibitory synapse as a novel locus of Reelin signaling in the developing and adult brain. Taken together, data discussed herein should prove useful for understanding and treating disorders associated with Reelin signaling impairments (e.g. AD and Schizophrenia).

Many experimental therapies have been used in the search for effective approaches to improve recovery after spinal cord injury (SCI). One of the most promising approaches is the augmentation of spontaneously occurring plasticity in uninjured neural pathways. Acute intermittent hypoxia (AIH-brief exposures to reduced O2 levels alternating with normal O2 levels) elicits plasticity in respiratory and non-respiratory spinal systems in experimental animals. AIH treatment has also been shown to improve walking abilities in persons with chronic incomplete SCI. In this thesis, I first examined the effect of AIH treatment, alone or in combination with motor training, on functional recovery in a rat model of incomplete cervical SCI. Second, I examined the effect of AIH on the expression of plasticity- and hypoxia-related proteins in the spinal cords of SCI rats. In a randomized, blinded, normoxia-controlled study, rats were trained to cross a horizontal ladder and footslip errors were measured before surgery for SCI, 4 wks post-surgery, each day of daily AIH treatment, and 1, 2, 4 and 8 weeks after treatment. dAIH treatment consisted of 10 episodes of AIH: (5 min 11% O2: 5 min 21% O2) for 7 days beginning at 4 wks post-SCI. AIH-treated rats made fewer footslips on the ladder task compared to normoxia-treated control rats after 4 days of treatment and this improvement was sustained for 8 wks post-treatment. Importantly, daily ladder training was required for AIH treatment to facilitate recovery. AIH treatment + motor training also increased the expression of Hypoxia-inducible factor-1α (HIF-1α), Vascular endothelial growth factor (VEGF), Brain-derived neurotrophic factor (BDNF), tyrosine kinase B receptors (trkB) and phospho-trkB in spinal motor neurons in SCI rats compared to normoxia-treated SCI rats. In particular these hypoxia- and plasticity-related proteins were differentially expressed both temporally and spatially in the spinal cord during AIH treatment. These findings demonstrate that AIH + motor training can augment neural plasticity and improve motor recovery in an animal model of SCI. Taken together with the promising findings from human SCI studies, the results of this thesis suggest that AIH has potential as an effective therapy to restore motor function after nervous system injury.

The causes and consequences of variation in aposematic signals during immature stages are not clearly understood. This thesis explores the effects of early environment on the expression of aposematic signals in the green and black poison frog (Dendrobates auratus), and the consequences of variation in such components in the wild. It also explores how aposematic expression relates to levels of chemical defences in immature froglets. Embryos and larvae of poison frogs in the genus Dendrobates are known to be darkly pigmented. This thesis reports for the first time polymorphism in egg pigmentation in D. auratus and ontogenetic colour change through development reverting to a normally pigmented phenotype; however whether this pigmentation results from constraints or has adaptive consequences remains unclear. Evidence on how immature individuals allocate resources to growth and warning signalling is scarce. Experimental results in this thesis show that food supply during early environment affected body size and signal luminance in post-metamorphic froglets. Therefore the relative importance of these traits in relation to predation risk was further tested, using artificial prey in a field experiment. The results indicated that rates of attack by birds correlated negatively with body size, and on the contrary survival of artificial prey was independent of signal luminance. I therefore tested the hypothesis that in the wild larger, relatively well-nourished juvenile frogs are chemically better defended. I found that in fact larger juveniles are at a selective advantage conferred by their greater foraging efficiency and their superior levels of chemical defences. Overall, these results shows plasticity in aposematic traits in relation to early environmental nutrition in D. auratus; and suggests that acquiring large body size and similar integument colour as to adults are key determinants for survival during the early stages of their terrestrial life.

Fatigue of metals is a problem that affects almost all sectors of industry, from energy to transportation, and failures to account for fatigue or incorrect estimations of service life have cost many lives. To mitigate such fatigue failures, engineers must be able to reliably predict the fatigue life of components under service conditions. Great progress has been made in this regard in the past 40 years; however one aspect of fatigue that is still being actively researched is the behavior of microstructurally small cracks (MSCs), which can diverge significantly from that of long cracks. The portion of life spent nucleating and growing a MSC over the first few grains/phases can consume over 90% of the total fatigue life under High Cycle Fatigue (HCF) conditions and is the primary source of the scatter in fatigue lives. Therefore, the development of robust fatigue design methodologies requires that the MSC regime of crack growth can be adequately modeled. The growth of microstructurally small cracks is dominated by influence of the local heterogeneity of the microstructure and is a highly complex process. In order to successfully model the growth of these microstructurally small cracks (MSCs), two computational frameworks are necessary. First, the local behavior of the material must be modeled, necessitating a constitutive relation with resolution on the scale of grain size. Second, a physically based model for the nucleation and growth of microstructurally small fatigue cracks is needed. The overall objective of this thesis is best summarized as the introduction these two computational frameworks, a crystal plasticity constitutive model and fatigue model, specifically for aluminum alloy 7075-T6, a high-strength, low density, precipitation hardened alloy used extensively in aerospace applications. Results are presented from simulations conducted to study the predicted crack growth under a variety of loading conditions and applied strain ratios, including uniaxial tension-compression and simple shear at a range of applied strain amplitudes. Results from the model are compared to experimental results obtained by other researchers under similar loading conditions. A modified fatigue crack growth algorithm that captures the early transition to Stage II growth in this alloy will also be presented.

Many species-specific sensorimotor behaviors, such as speech in humans, emerge from the interplay between genetically defined developmental programs and sensory experience. How these processes interact during learning to shape motor circuits is not well understood. The zebra finch (Taeniopygia guttata), an oscine bird that learns to imitate the song of its tutor (usually the father), provides a uniquely tractable model for exploring this question. Song learning in zebra finches takes place during a discrete three-month period during which male juveniles progress from producing highly variable rudimentary sounds that are noisy and unstructured, to a highly stereotyped imitation of their tutor's song. Here I characterize learning-related changes in the functional connectivity within a motor cortex-analogue brain area (RA) that control song production.

No description available.

Divergent natural selection for use of locally abundant resources can lead to diversification within and across species. However, the consequences of divergent selection for phenotypic evolution also depend on the development of variation. Because relationships among traits such as shared developmental timing or common involvement in an organismal function can channel variation generated during development, these relationships strongly influence the direction of evolution.During development of the mammalian mandible multiple tissues of distinct developmental origins interact with inputs from the functioning of attached muscles to produce a cohesive and well integrated trait. In soricid shrews, part of the mandible matures late in ontogeny, coinciding with the onset of foraging. In this case, foraging-linked muscle activity should influence the development of the late maturing mandibular region. Here, I show that variation in this late ossifying region reveals the local functional requirements of the jaw and results in an opportunity to decouple internal and external sources of variation (developmental and environmental respectively) in the mandible. Capitalizing on this feature of the Sorex system, I empirically examined the historical persistence of internal and external patterns of variation, the consequences of variation patterning for ecological and morphological diversification across taxa, and differences between early and late ossifying regions in their contribution to local adaptation in mandible morphology.I found that the functional requirements of diet directed mandible development and determined species similarity in both mandible morphology and function. Timing of bone maturation determined the morphological effects of foraging-linked muscle activity, resulting in differential expression of adaptive variation in the late maturing region. Further, I found higher levels of interspecific variation in the late maturing region of the mandible, and showed that interspecific divergence in foraging morphology occurs along the lines delineated by epigenetic inputs of muscle on bone formation during late ontogeny within species. These findings indicate that differences in functional requirements are critical for divergence among taxa in this system. Further, these results suggest that, when external inputs into trait development are indicative of local functional requirements, the same epigenetic mechanism of development can generate diversity both within and among taxa.

Damage to motor areas of the brain, caused by stroke, can produce devastating motor deficits, including aberrant control of force. After stroke, reorganization of the brain’s motor system has been identified as one of the fundamental mechanisms involved in recovery of motor control after stroke. Yet, few studies have investigated how force production and modulation are encoded in the brain after stroke and how this relates to motor outcome. Thus, the purpose of this study was to (1) understand how past neuroimaging literature has contributed to establishing common patterns of brain reorganization during both relative and absolute force production after stroke, (2) examine how brain function is reorganized during force production and modulation in individuals with stroke, and (3) relate this task-related reorganization of brain function to the amount of paretic arm use after stroke. In the second chapter, we systematically reviewed all relevant literature examining brain activation during force production after stroke. The following chapters (chapters 3 and 4) applied functional magnetic resonance imaging (fMRI) to examine the neural correlates of force production and modulation after stroke. Chapter 2 supports differences in task-related brain activation dependent on features of stroke, such as severity and chronicity, as well as influence of rehabilitation. In addition, results suggest that activation of common motor areas of the brain during force production can be identified in relation to functional outcome after stroke. Results from the subsequent two chapters (3 and 4), demonstrate that brain function reorganizes in terms of absolute, and not relative force production after stroke. Specifically, stroke participants exhibit greater activation of motor areas than healthy controls when matched for absolute force production. Moreover, there is a relationship between paretic arm usage and brain activation, where stroke participants having less paretic arm use, as measured using wrist accelerometers, exhibit higher brain activation. Results of this thesis suggest that during absolute force production, brain activation may approach near maximal levels in stroke participants at lower forces than healthy controls. Furthermore, this effect may be amplified even further in subjects with less paretic arm usage, as increased activation in motor areas occurs in participants with less arm use after stroke. Ultimately, the results from this thesis will contribute to research relevant to brain reorganization in individuals with stroke and may lead to the development of new, beneficial therapeutic interventions that optimize brain reorganization and improve functional recovery after stroke.

This report extends findings (Duffy & Mitchell, 2013) of a dramatic recovery of vision in the deprived eye of amblyopic kittens following a short ‘dark-pulse’ (a 10 day period of darkness) to situations of clinical relevance. To this end, the initial deprivation began at post-natal day 7 rather than post-natal day 30. As before, the dark-pulse was imposed either immediately after the initial monocular deprivation, or was delayed several weeks after stable amblyopia was establsihed. In some animals, this dark-pulse was shortened, or disrupted by short periods of daily binocular visual experience. The effects on the visual acuity and alignment acuity of the two eyes were documented as well as the effects on binocular depth perception. The benefits of a short dark-pulse were identical to those of the prior study. A dark-pulse of 5 days was ineffective as was a dark-pulse interrupted daily by light for 30-minutes.

In most organisms the cellular response to hypoxia is mediated by the master regulator hypoxia-inducible factor-1 (HIF-1). Zebrafish embryos can also arrest development (suspended animation) to tolerate low oxygen. I tested the hypothesis that induction of HIF-1 and associated target genes (eg. erythropoietin) during embryonic development would alter the hypoxia tolerance phenotype of larval and adult fish. I exposed zebrafish embryos at 3 developmental stages to acute (4 h) bouts of hypoxia (5% dissolved oxygen, DO) or anoxia (<0.5% DO). I found that embryos that mount a HIF-1 response have a greater hypoxia tolerance as larvae. Additionally, populations that experienced embryonic HIF-1 induction show an increase in the proportion of males (~70% male), that are more hypoxia tolerant than female fish, compared to control populations (~45% male). Overall, induction of HIF-1 during ontogeny alters the larval and adult zebrafish phenotype to better tolerate future hypoxic bouts. / NSERC