Thesis (Ph.D.)--Ohio University, March, 2005. / Includes bibliographical references (p. 152-159)

Thesis (Ph.D.)--Ohio University, March, 2005. / Title from PDF t.p. Includes bibliographical references (p. 152-159)

Stroke is a leading cause of death and morbidity worldwide, and leaves stroke survivors with chronic disabilities. One of the key mechanisms that the brain triggers during stroke recovery is the sprouting of new axons and the formation of new neuronal connections. Meanwhile, studies have evidenced this phenomenon with methods using unspecific cell/axon markers. The dopamine (DA) system is thought to be implicated in stroke recovery. However, the specific contribution and remodeling of this system to enhance stroke recovery, and whether D1- class receptors play a role in this process, remain unclear. Using a mouse photothrombosis stroke model, immunohistochemical methods, imaging analysis of axonal fiber density and branching in the motor cortex, we demonstrated a specific dopaminergic axon remodeling in the periinfarct region, with or without DA agonist administration. Axonal remodeling of noradrenergic fibers was subtle. In mice subjected to saline IP injection and physical rehabilitation (running wheels), we observed an increase of only DA fiber density in the periinfarct area as compared to the contralateral (intact) side. However, mice treated with DHX for 7 days followed by physical rehabilitation did not show difference between the two hemispheres. Our results suggest a modulatory effect of DHX on axonal remodeling mainly in the contralateral side. Interestingly, treatment of naïve mice with DHX had no effect of DA axon remodeling suggesting that D1- mediated axonal remodeling is stroke-dependent. We also established the temporal profile of post-stroke DA axon remodeling in the absence of DHX and physical rehabilitation. At 4 days poststroke, there was a significant decrease in DA fiber density and a significant recovery was measured after 28 days relative to the contralateral side. Altogether, our data highlight a major remodeling of DA axons in motor cortex following stroke, and a potential role for D1-class receptors in improving post-stroke recovery. Understanding adaptations of the DA system following stroke will have a great impact on stroke recovery research. Aida Said Thesis submitted to the Faculty of Graduate and Postdoctoral Studies in partial fulfillment of the requirements for the Master of Science degree in Neuroscience Department of Cellular and Molecular Medicine Faculty of Medicine University of Ottawa August 30, 2019 © Aida Said, Ottawa, Canada, 2019   Abstract Stroke is a leading cause of death and morbidity worldwide, and leaves stroke survivors with chronic disabilities. One of the key mechanisms that the brain triggers during stroke recovery is the sprouting of new axons and the formation of new neuronal connections. Meanwhile, studies have evidenced this phenomenon with methods using unspecific cell/axon markers. The dopamine (DA) system is thought to be implicated in stroke recovery. However, the specific contribution and remodeling of this system to enhance stroke recovery, and whether D1-class receptors play a role in this process, remain unclear. Using a mouse photothrombosis stroke model, immunohistochemical methods, imaging analysis of axonal fiber density and branching in the motor cortex, we demonstrated a specific dopaminergic axon remodeling in the periinfarct region, with or without DA agonist administration. Axonal remodeling of noradrenergic fibers was subtle. In mice subjected to saline IP injection and physical rehabilitation (running wheels), we observed an increase of only DA fiber density in the periinfarct area as compared to the contralateral (intact) side. However, mice treated with DHX for 7 days followed by physical rehabilitation did not show difference between the two hemispheres. Our results suggest a modulatory effect of DHX on axonal remodeling mainly in the contralateral side. Interestingly, treatment of naïve mice with DHX had no effect of DA axon remodeling suggesting that D1-mediated axonal remodeling is stroke-dependent. We also established the temporal profile of post-stroke DA axon remodeling in the absence of DHX and physical rehabilitation. At 4 days post-stroke, there was a significant decrease in DA fiber density and a significant recovery was measured after 28 days relative to the contralateral side. Altogether, our data highlight a major remodeling of DA axons in motor cortex following stroke, and a potential role for D1-class receptors in improving post-stroke recovery. Understanding adaptations of the DA system following stroke will have a great impact on stroke recovery research.

This thesis provides insights into the mechanism by which thalamocortical axons (TCAs) approach the cortex from their origin in the thalamus. Previous studies suggested that the reciprocal projections from the prethalamus and the ventral telencephalon guide TCAs to descend through the prethalamus and cross the diencephalic-telencephalic boundary (DTB), after which TCAs navigate through permissive corridor cells in the ventral telencephalon and cross the pallial-subpallial boundary (PSPB) before reaching their final targets in the cortex. The ‘Handshake Hypothesis’ proposed that pioneer axons from cortical preplate neurons guide TCAs into corresponding cortical areas. However, there is a lack of convincing evidence on whether TCAs need any guidance to cross the PSPB. In the current study, Adenomatous polyposis (Apc) gene is conditionally deleted from the cortex, by using Emx1Cre-APCloxP recombination technology. Apc is widely expressed in the nervous system including the cortical plate of the cortex and regulates axonal growth and neuronal differentiation. Deleting Apc may block neurite extension and/or affect the formation of attractive or repulsive cues in the cortex. By using DiI tracing as well as L1 immunohistochemistry techniques, I showed that in the Apc mutants cortical axons are absent and that TCAs initially navigate into the ventral telencephalon normally but fail to complete their journey into the cortex. They stop as they approach the PSPB, although the PSPB doesn’t seem to be directly affected by the mutation of Apc in the cortex. Additionally, Ig-Nrg1 (Neuregulin-1), the secreted protein that was suggested to play long-range roles in attracting TCAs towards the cortex, is present in the Apc mutant. This implies that Ig-Nrg1 is not sufficient for guiding TCAs into the cortex, and that additional guidance factors are needed. Moreover, my in vitro explant culture experiments show that the mutant cortex neither repel nor inhibit thalamic axonal outgrowth, indicating that the failure of TCAs in reaching the cortex is not due to the change of repulsive cues secreted by the mutant cortex. It rather indicates that the guidance factors for TCAs are likely to function through cell-cell contact mediated mechanisms. The Apc mutant cortex lacks these guidance factors, which might be the cortical axons. In conclusion, my data reveal a choice point for TCAs at the PSPB. Guidance factors from the cortex are needed for TCAs to cross the PSPB, which are absent in the Apc mutant. TCAs may need the direct contact with cortical axons and use them as an axonal scaffold to navigate into the cerebral cortex.

Promising strategies for treating diseases and conditions like cancer, tissue necrosis from injury, congenital abnormalities, etc., involve replacing pathologic tissue with healthy tissue. Strategies devoted to the development of tissue to restore, maintain, or improve function is called tissue engineering. Engineering tissue requires three components, cells that can proliferate to form tissue, a microenvironment that nourishes the cells, and a tissue scaffold that provides mechanical stability, controls tissue architecture, and aids in mimicking the cell’s natural extracellular matrix (ECM). Currently, there is much focus on designing scaffolds that recapitulate the topology of cells’ ECM, in vivo, which undoubtedly wields structures with nanoscale dimensions. Although it is widely thought that sub-microscale features in the ECM have the greatest vii impact on cell behavior relative to larger structures, interactions between cells and nanostructures surfaces is not well understood. There have been few comprehensive studies elucidating the effects of both feature dimension and geometry on the initial formation and growth of the axons of individual neurons. Reconnecting the axons of neurons in damaged nerves is vital in restoring function. Understanding how neurons react with nanopatterned surfaces will advance development of optimal biomaterials used for reconnecting neural networks Here, we investigated the effects of micro- and nanostructures of various sizes and shape on neurons at the single cell level. Compulsory to studying interactions between cells and sub-cellular structures is having nanofabrication technologies that enable biomaterials to be patterned at the nanoscale. We also present a novel nanofabrication process, coined Flash Imprint Lithography using a Mask Aligner (FILM), used to pattern nanofeatures in UV-curable biomaterials for tissue engineering applications. Using FILM, we were able to pattern 50 nm lines in polyethylene glycol (PEG). We later used FILM to pattern nanowells in PEG to study the effect of the nanowells on the behavior preadipocytes (PAs). Results of our cell experiments with neurons and PAs suggested that incorporating micro- and nanoscale topography on biomaterial surfaces may enhance biomaterials’ ability to constrain cell development. Moreover, we found the FILM process to be a useful fabrication tool for tissue engineering applications. / text

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Axons are directed along stereotypic pathways to their targets by cues arrayed in the extracellular environment. Identifying the cellular and molecular nature of these signals is of high interest and the developing optic pathway is a useful model system for achieving this. Although previous studies have identified several molecules essential for optic pathway formation, in vivo only subsets of retinal axons rely on them. I focused on the Dscam (Down’s syndrome cell adhesion molecule) and Sidekick (Sdk) cell adhesion molecules for potentially playing crucial roles in this system. In situ hybridisation in the embryonic mouse visual system showed Dscam and Sdk-1 expression in the RGC layer of the retina, along the optic pathway and in the visual targets. Sdk-2 was detected in the glia of the optic nerve and optic chiasm, marking the pathway that RGC axons follow, but not in RGCs. No DscamL1 was detected in RGCs or the optic pathway at the stages investigated and it was discounted from future analysis. In vitro, DSCAM promoted RGC axon outgrowth, whereas SDK 1 was inhibitory. SDK 2 had no effect on RGC axon outgrowth, suggesting it does not play a direct role in their pathfinding. Repeating this assay using retinal explants from the Dscamdel17 mouse mutant, showed that DSCAM enhanced retinal axon outgrowth, at least in part, through homophilic interactions. Analysis of visual system development in Dscam mutants showed DSCAM involvement in RGC axon fasciculation and in enhancing their growth, particularly within the ipsilateral optic tract. Retinal cell counts revealed that DSCAM played diverse roles in controlling cell number. Pre- and postnatal retinas lacking DSCAM contained more RGCs and mitotic cells. Postnatally, Dscam-/- retinas also show decreased cell death. In many cases, defect severity was dose-dependent, with an intermediate phenotype in the heterozygous mice, implicating DSCAM in the neurological defects of Downs’ Syndrome patients.

Potential differences in developmental trajectory were investigated in autism at both the macro- and micro-scopic scale, using regional volumetric measurements from in-vivo scans and measurements of minicolumnar organisation of the cortex in post-mortem tissue. In addition, a study was carried out to investigate the sensitivity of measures of cortical diffusion to cortical architecture. Three key regions of interest were studied throughout this thesis, orbital frontal cortex (BA11), primary auditory cortex (BA41) and part of the inferior parietal lobe (BA40). Subjects with ASD showed increases in grey matter in left parietal cortex and decreases in left BA11 compared to controls. In addition, subjects with ASD showed increased grey matter volume with age in both BA41 and the inferior parietal lobe, whereas controls only showed a negative correlation between grey matter volume in BA41 and age. Wider minicolumns were found in ASD in all regions, suggesting pathology is not restricted to higher order association areas. Differences seemed more pronounced at younger ages suggesting an altered developmental trajectory in ASD. Such an increase in minicolumnar width arguably underlies the feature-based processing style seen in ASD. A pilot study using post-mortem DTI scans of MS brains revealed a relationship between measures of the directionality of diffusion and the width of axonal bundles in the cortex, an aspect of the minicolumnar arrangement. When extending this investigation to a set of ASD and control brains, evidence was found for different relationships between axon bundle width and measures of the directionality of diffusion in the cortex, suggesting that although differences in axon bundle width were not seen between groups, there may be differences in the composition of the axon bundles between ASD and control groups.

Amyotrophic Lateral Sclerosis (ALS) is a debilitating and fatal neurodegenerative disease affecting upper and lower motor neurons. Though studied for over two decades since the first ALS-associated genetic mutation was discovered, researchers have yet to uncover the pathological processes that lead to progressive degeneration of motor neurons in ALS, or to develop effective treatments. One prominent hypothesis proposes that excitotoxicity caused by increased motor neuron firing plays a role in ALS pathogenesis. While prior studies reported increased action potential firing in early postnatal ALS-model motor neurons in vivo, it remains unknown whether the increased activity stems from increased intrinsic excitability of ALS motor neurons or from increased excitatory drive, and whether these changes are transient or persist into adulthood, when ALS symptoms emerge. In this thesis, I circumvented the difficulties in standard measurement of electrophysiological properties of adult spinal motor neurons in vivo by relying on the visualization of the axon initial segment, a subcellular structure known to undergo compensatory structural changes in response to perturbations in excitatory input. I discovered that cultured motor neurons derived from stem cells of the SOD1G93A mouse model of ALS display shortened axon initial segments and hypoexcitable electrophysiological properties. The shortening of the axon initial segment is compensatory, as ALS motor neurons receive increased numbers of excitatory inputs and manifest increased spontaneous activity. Remarkably, similar shortening of the axon initial segment was detected in early presymptomatic spinal motor neurons in vivo. The shortened axon initial segment persists into the symptomatic stages and is particularly pronounced in motor neurons containing p62 immunoreactive aggregates and neurons exhibiting swollen mitochondria, two signs of stress and neurodegeneration in the disease. Based on these observations I propose that early in the presymptomatic stages of the disease, spinal motor neurons recruit excessive excitatory inputs, resulting in their increased activity that is in part compensated by shortening of the axon initial segment. This state persists and becomes even more pronounced in motor neurons exhibiting biochemical changes preceding neurodegeneration. While these observations support the potential role for excitotoxic stress in spinal ALS motor neurons, I paradoxically observed the opposite phenotype in ALS-vulnerable cranial motor neurons in the brainstem of the SOD1G93A animals, raising the possibility that the cellular stress that drives the neurodegeneration in ALS is motor neuron subtype specific.