Parkinson’s disease (PD) is a common neurodegenerative disorder that is characterized by the degeneration of dopaminergic (DA) neurons in the substantia nigra and motor deficits. Although the majority of PD cases are sporadic, several genetic defects in rare familial cases have been identified. Animal models of these genetic defects have been created and have provided unique insights into the molecular mechanisms of the pathogenesis of PD. However, the etiology of PD is still not well understood. Here, taking advantage of the unique features offered by zebrafish, I characterized the functions of PINK1 (PTEN-induced kinase 1) gene, which is associated with recessive familial PD, in the development and survival of DA neurons. In zebrafish, antisense morpholino knockdown of pink1 did not cause a large loss of DA neurons in the ventral diencephalon (vDC), but the patterning of these neurons and their projections were perturbed. The pink1 morphants also showed impaired response to touch stimuli and reduced swimming behaviour. Moreover, the pink1 knockdown caused a significant reduction in the number of mitochondria, as well as mitochondrial morphological defects such as smaller size or loss of cristae, thus affecting mitochondrial function. These results suggest that zebrafish pink1 plays conserved important roles in the development of DA neurons and in the mitochondrial morphology and function. To better follow DA neurons after injury or administration of toxins, I generated a transgenic zebrafish line, Tg(dat:EGFP), in which the green fluorescent protein (GFP) is expressed under the control of cis-regulatory elements of dopamine transporter (dat). In Tg(dat:EGFP) fish, all major groups of DA neurons are correctly labeled with GFP, especially the ones in the vDC, which are analogous to the ascending midbrain DA neurons in mammals. In addition, we observed that the DA neurons in the vDC could partially be replaced after severe laser cell ablation. This suggests that zebrafish may have the unique capacity of regenerating DA neurons after injury. Taken together, my studies suggested that zebrafish could be a useful alternative animal model for the study of the molecular mechanisms underlying PD and for the screening of potential therapeutic compounds for PD.

This thesis focuses on the development of a robotic device to be used in parallel with observational learning techniques for facilitating the recovery of the upper limb in post-stroke patients. It has been shown in the existing observational learning literature that observational practice for the execution of goal-directed single arm movements can engage the mirror neuron system and motor areas involved in learning motor actions. On the other hand, robotic-based therapy protocols have proven successful in which participants are able to learn the required perception-action skill. However, robotics have not been overly successful in the generalization of learning to other tasks and this is an essential aspect on improving performance on Activities of Daily Life (ADL). Observational learning of motor skills has been shown to produce transfer across limbs and generalization across muscle groups in the same limb, as well as transfer to perceptual tasks. Therefore, our long-term hypothesis is that a combination of interactive robotics and action observation techniques might offer a greater benefit regarding transfer to ADLs in comparison to pure robotic training. The results from this research broaden the theoretical understanding of observational learning and drive the future development of rehabilitation protocols using the combination of robotic and observational learning techniques. We hypothesize that if the application of these techniques, for non-stroke individuals, yield benefits for the learning of motor/skill actions, then such paradigm will serve as a foundation in the future development of methods for facilitating the recovery of upper limb function after stroke.

Amyotrophic lateral sclerosis (ALS) is a devastating motor neuron disease which results from the degeneration of upper and lower motor neurons in the brainstem, spinal cord and motor cortex. Tragically there is no treatment to prevent ALS. The drug Riluzole acts to delay progression, but only by a month or so in this disease that has a survival length of three to five years. The identification of genes that are mutated in patients with ALS would help devise novel therapeutic strategies as much remains to be discovered about the genetics of ALS. Familial forms of the disease account for only 5-10% of patients. Among these familial cases, about 15-20% are caused by mutations in the zinc/copper superoxide dismutase gene, but the genetic basis of the remaining familial cases and the many sporadic cases continues to be largely unknown. / Altogether, the results presented in this thesis came from the use of several strategies to establish the genetic cause of ALS and the related motor neuron disorders like hereditary spastic paraplegia (HSP) and primary lateral sclerosis (PLS). A concerted and collaborative effort was put forth to identify the gene causative for ALS3 on chromosome 18. In addition, a recently reported locus has been confirmed on chromosome 9p for patients that present both ALS and frontotemporal dementia. The major finding involves the discovery of eight mutations in the TARDBP gene in nine patients with sporadic and familial ALS. Furthermore, a large association study evaluated the role of common polymorphisms in the paraoxonase gene cluster in susceptibility to the development of ALS. In the analysis of upper motor neuron diseases, mutations in a novel gene, KIAA0196, were identified for the HSP locus SPG8 on chromosome 8. Finally, the first locus for PLS was discovered on the p-arm of chromosome 4 following genome scan analysis of a large Quebec family with PLS. / These genetic discoveries all contributed novel advances to the field of motor neuron disorders. As more is elucidated regarding the biochemical function of these the proteins encoded by these genes, a more comprehensive picture of ALS and other motor neuron disorders will hopefully emerge.

Several decades of physiology, imaging and psychophysics research on attention has generated an enormous amount of data describing myriad forms of attentional effects. A similar breadth of theoretical models have been proposed that attempt to explain these effects in varying amounts of detail. However, there remains a need for neurally detailed mechanistic models of attention that connect more directly with various kinds of experimental data -- behavioural, psychophysical, neurophysiological, and neuroanatomical -- and that provide experimentally testable predictions. Research has been conducted that aims to identify neurally consistent principles that underlie selective attentional processing in cortex. The research specifically focuses on describing the functional mechanisms of attentional routing in a large-scale hierarchical model, and demonstrating the biological plausibility of the model by presenting a spiking neuron implementation that can account for a variety of attentional effects. The thesis begins by discussing several significant physiological effects of attention, and prominent brain areas involved in selective attention, which provide strong constraints for developing a model of attentional processing in cortex. Several prominent models of attention are then discussed, from which a set of common limitations in existing models is assembled that need to be addressed by the proposed model. One central limitation is that, for many existing models, it remains to be demonstrated that their computations can be plausibly performed in spiking neurons. Further, few models address attentional effects for more than a single neuron or single cortical area. And finally, few are able to account for different forms of attentional modulation in a single detailed model. These and other limitations are addressed by the Attentional Routing Circuit (ARC) proposed in this thesis. The presentation of the ARC begins with the proposal of a high-level mathematical model for selective routing in the visual hierarchy. The mathematical model is used to demonstrate that the suggested mechanisms allow for scale- and position-invariant representations of attended stimuli to be formed, and provides a functional context for interpreting detailed physiological effects. To evaluate the model's biological plausibility, the Neural Engineering Framework (NEF) is used to implement the ARC as a detailed spiking neuron model. Simulation results are then presented which demonstrate that selective routing can be performed efficiently in spiking neurons in a way that is consistent with the mathematical model. The neural circuitry for computing and applying attentional control signals in the ARC is then mapped on to neural populations in specific cortical laminae using known anatomical interlaminar and interareal connections to support the plausibility of its cortical implementation. The model is then tested for its ability to account for several forms of attentional modulation that have been reported in neurophysiological experiments. Three experiments of attention in macaque are simulated using the ARC, and for each of these experiments, the model is shown to be quantitatively consistent with measured data. Specifically, a study by Womelsdorf et al. (2008) demonstrates that spatial shifts of attention result in a shifting and shrinking of receptive fields depending on the target's position. An experiment by Treue and Martinez-Trujllo (1999) reports that attentional shifts between receptive field stimuli produce a multiplicative scaling of responses, but do not affect the neural tuning sensitivity. Finally, a study by Lee and Maunsell (2010) demonstrates that attentional shifts result in a multiplicative scaling of neural contrast-response functions that is consistent with a response-gain effect. The model accounts for each of these experimentally observed attentional effects using a single mechanism for selectively processing attended stimuli. In conclusion, it is suggested that the ARC is distinguished from previous models by providing a unifying interpretation of attentional effects at the level of single cells, neural populations, cortical areas, and over the bulk of the visual hierarchy. As well, there are several advantages of the ARC over previous models, including: (1) scalability to larger implementations without affecting the model's principles; (2) a significant increase in biological plausibility; (3) the ability to account for experimental results at multiple levels of analysis; (4) a detailed description of the model's anatomical substrate; (5) the ability to perform selective routing while preserving biological detail; and (6) generating a variety of experimentally testable predictions.

The goal of this thesis is to investigate the biophysical basis underlying the nonlinear relationship between firing response and current stimulation in single motor neurons. After reviewing the relevant motoneuron physiology and theories that describe complex dendritic signaling properties, I hypothesize that at least five passive electrical properties must be considered to better understand the physiological input-output properties of motor neurons in vivo: input resistance, system time constant, and three signal propagation properties between the soma and dendrites that depend on the signal direction (i.e. soma to dendrites or vice versa) and type (i.e. direct (DC) or alternating (AC) current). To test my hypothesis, I begin with characterizing the signal propagation of the dendrites, by directly measuring voltage attenuations along the path of dendrites of the type-identified anatomical neuron models. Based on this characterization of dendritic signaling, I develop the novel realistic reduced modeling approach by which the complex geometry and passive electrical properties of anatomically reconstructed dendrites can be analytically mapped into simple two-compartment modeling domain without any restrictive assumptions. Combining mathematical analysis and computer simulations of my new reduced model, I show how individual biophysical properties (system input resistance, time constant and dendritic signaling) contribute to the local excitability of the dendrites, which plays an essential role in activating the plateau generating membrane mechanisms and subsequent nonlinear input-output relations in a single neuron. The biophysical theories and computer simulations in this thesis are primarily applied to motor neurons that compose the motor neuron pool for control of movement. However, the general features of the new reduced neuron modeling approach and important insights into neuronal computations are not limited to this area. My findings can be extended to other areas including artificial neural networks consisting of single compartment processors. / Medical Sciences – Biomedical Engineering

Movement is central for life, and all animals depend on accurate regulation of movement for purposeful behavior. There is great diversity of movements, ranging between simple and vital breathing movements to minute and subtle movements of the face used to communicate emotions. Consequently, motor neurons, which are the only route of central nervous system output, are essential for all motor behaviors. To control the many motor behaviors expressed by an animal, motor neurons are exposed to a large number and variety of modulating synaptic inputs and have evolved into subtypes with specific functions. In this thesis, motor neuron subtypes and the synaptic input to motor neurons from Renshaw cells and Ia afferents have been studied. Novel molecular markers that identify subtypes of motor neurons are described. Three markers, Chodl, Calca and ERRβ, have been used to study the degeneration of subtypes of motor neurons in a mouse model of the neurodegenerative disease amyotrophic lateral sclerosis (ALS). Another marker, 5-ht1d, has been used to record the electrophysiological character of gamma motor neurons. In mice that lack 5-ht1d, motor neurons develop with reduced proprioceptive input. Remarkably, these mice had fewer foot faults than control animals when challenged to cross a narrow beam suggesting that the amplitude of monosynaptic proprioceptive input to motor neurons is not essential for motor coordination. In a final set of experiments, genetic removal of vesicular transport of neurotransmitter from Renshaw cells suggest that Renshaw cells are not integral for motor circuit function or motor behaviors. However, they are involved in the development of motor circuits in the spinal cord. Together, this thesis provides novel molecular tools for studies of motor neuron subtypes and novel data regarding the development and function of spinal motor circuits.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative syndrome of unknown etiology that most commonly affects people in middle and high age. The hallmark of ALS is a progressive and simultaneous loss of upper and lower motor neurons in the central nervous system that leads to a progressive muscle atrophy, paralysis and death usually by respiratory failure. ALS is not a pure motor neuronal syndrome; it extends beyond the motor system and affects extramotor areas of the brain as well. The majority of the patients suffer from a sporadic ALS disease (SALS) while in at least ten percent the disease appears in a familial form (FALS). Mutations in the gene encoding the antioxidant enzyme superoxide dismutase-1 (SOD1) are the most common cause of FALS. More than 165 SOD1 mutations have been described, and these confer the enzyme a cytotoxic gain of function. Evidence suggests that the toxicity results from structural instability which makes the mutated enzyme prone to misfold and form aggregates in the spinal cord and brain motor neurons. Recent studies indicate that the wild-type human SOD1 protein (wt-hSOD1) has the propensity to develop neurotoxic features. The aim of the present study was to investigate if wt-hSOD1 is involved in the pathogenesis of SALS and FALS patients lacking SOD1 mutations and to evaluate the neurotoxic effect of misfolded wt-hSOD1 protein in vivo by generating a transgenic wt-hSOD1 mice model. We produced specific SOD1-peptide-generated antibodies that could discriminate between the misfolded and native form of the enzyme and optimized a staining protocol for detection of misfolded wt-hSOD1 by immunohistochemistry and confocal microscopy of brain and spinal cord tissue. We discovered that aggregates of misfolded wt-hSOD1 were constitutively present in the cytoplasm of motor neurons in all investigated SALS patients and in FALS patients lacking SOD1 gene mutations. Interestingly, the misfolded wt-hSOD1 aggregates were also found in some motor neuron nuclei and in the nuclei of the surrounding glial cells, mainly astrocytes but also microglia and oligodendrocytes, indicating that misfolded wt-hSOD1 protein aggregates may exert intranuclear toxicity. We compared our findings to FALS with SOD1 mutations by investigating brain and spinal cord tissue from patients homozygous for the D90A SOD1 mutation, a common SOD1 mutation that encodes a stable SOD1 protein with a wild-type-like enzyme activity. We observed a similar morphology with a profound loss of motor neurons and aggregates of misfolded SOD1 in the remaining motor neuron. Interestingly, we found gliosis and microvacuolar degeneration in the superficial lamina of the frontal and temporal lobe, indicating a possible frontotemporal lobar dementia in addition to the ALS disorder. Our morphological and biochemical findings were tested in vivo by generating homozygous transgenic mice that over expressed wt-hSOD1. These mice developed a fatal ALS-like disease, mimicking the one seen in mice expressing mutated hSOD1. The wt-hSOD1 mice showed a slower weight gain compared to non-transgenic mice and developed a progressive ALS-like hind-leg paresis. Aggregates of misfolded wt-hSOD1 were found in the brain and spinal cord neurons similar to those in humans accompanied by a loss of 41 % of motor neurons compared to non-transgenic litter mates. In conclusion, we found misfolded wt-hSOD1 aggregates in the cytoplasm and nuclei of motor neurons and glial cells in all patients suffering from ALS syndrome. Notable is the fact that misfolded wt-hSOD1 aggregates were also detected in FALS patients lacking SOD1 mutations indicating a role for SOD1 even when other genetic mutations are present. The neurotoxicity of misfolded wt-hSOD1 protein was confirmed in vivo by wt-hSOD1 transgenic mice that developed a fatal ALS-like disease. Taken together, our results support the notion that misfolded wt-hSOD1 could be generally involved and play a decisive role in the pathogenesis of all forms of ALS.

Glutamate is the essential neurotransmitters in pain pathways. The discovery of the vesicular glutamate transporters (VGLUT1-3) has been a fundamental step on the way to describe glutamate-dependent pain pathways. We used the Cre-lox system to construct conditional knockouts with deficient Vglut2 transmission in specific neuronal populations. We generated a Vglut2f/f;Ht-Pa-Cre line to selectively delete Vglut2 from the peripheral nervous system. These Vglut2 deficient mice showed decreased acute nociceptive responses and were less prone to develop an inflammatory state. They did not develop cold allodynia, or heat hyperalgesia and were less hypersensitive to mechanical stimuli in the PSNL chronic pain model. Further analyses of genes with altered expression after nerve injury, revealed candidates for future studies of chronic pain biomarkers. Interestingly, the Vglut2f/f;Ht-Pa-Cre mice developed an elevated itch behavior. To investigate more specific neuronal populations, we analyzed mice lacking Vglut2 in the Nav1.8 population, as inflammatory hyperalgesia, cold pain, and noxious mechanosensation have been shown to depend upon Nav1.8Cre positive sensory neurons. We showed that deleting Vglut2 in Nav1.8Cre positive neurons abolished thermal hyperalgesia in persistent inflammatory models and responses to noxious mechanical stimuli. We also demonstrated that substance P and VGLUT2-dependent glutamatergic transmission are co-required for the development of formalin-induced inflammatory pain and heat hyperalgesia in persistent inflammatory states. Deletion of Vglut2 in a subpopulation of neurons overlapping with the vanilloid receptor (TRPV1) primary afferents in the dorsal root ganglia resulted in a dramatic increase in itch behavior accompanied by a reduced responsiveness to thermal pain. Substance P signaling and VGLUT2-mediated glutamatergic transmission in TRPV1 neurons was co-required for the development of inflammatory pain states. Analyses of an itch phenotype uncovered the pathway within TRPV1 neurons, with VGLUT2 playing a regulatory role and GRPR neurons, which are to plausible converge the itch signal in the spinal cord. These studies confirmed the essential role of VGLUT2-dependent glutamatergic transmission in acute and persistent pain states and identified the roles of specific subpopulations of primary afferent neurons. Additionally, a novel pain and itch transmission pathway in TRPV1/VGLUT2 positive neurons was identified, which could be part of the gate control of pain.

Brain tumours comprise about 25% of all cancers in children. Medulloblastoma – which arise in the cerebellum – are the most common and severe malignant pediatric brain tumour and the leading cause of cancer-related deaths in children under the age of 9. Treatment of medulloblastoma remains conventional, with surgery followed by chemotherapy and radiation. These measures are successful in about 60-80% of cases but treatment results in severe side effects due to its toxicity to the central nervous system. Therefore it is of utmost importance to define the signalling pathways and genetic changes involved in the formation of medulloblastoma in order to allow for better diagnosis and treatments with higher efficiency and decreased toxicity. The cell of origin for medulloblastoma is thought to be the granule neuron progenitor, a cell type arising from cerebellar stem cells of the ventricular zone. After birth granule neuron progenitors differentiate into mature granule neurons which populate the majority of the cerebellum and are crucial for its cognitive functions and motor coordination. The Hedgehog signalling pathway plays an important role in medulloblastoma generation and murine models with activated Hedgehog signalling develop medulloblastoma at high frequencies. In addition, the Notch pathway has been implicated in the generation of medulloblastoma, and interaction between the two pathways has been suggested. Inhibitors of both Hedgehog and Notch are currently in clinical trials however knowledge of possible interactions between them could lead to more effective treatment strategies. The aim of this project was to investigate the interaction of Hedgehog and Notch signalling in normal brain development and medulloblastoma. Two mouse models allowed activation of Hedgehog and inactivation of Notch signalling in granule neuron progenitors and cerebellar ventricular zone stem cells. In granule neuron progenitors canonical Notch signalling is not required and the layering and cell types of RBP-Jlox/lox;Math1-Cre cerebella appear identical to control brains. In contrast, Notch inactivation in ventricular zone stem cells with GFAP-Cre resulted in increased differentiation of stem cells into progenitor cells accompanied by an overall developmental delay in neuronal differentiation. Medulloblastoma generated by Hedgehog activation (through inactivation of the negative Hedgehog regulator Ptc1) in both cell types cannot be blocked by Notch inactivation. Furthermore, medulloblastoma of Ptc1lox/lox;RBP-Jlox/lox;GFAP-Cre and those of Ptc1lox/lox;RBP-Jlox/lox;Math1-Cre mice are identical in incidence as well as histology to the tumours in which only Hedgehog signalling is activated. This implies that even though Notch signalling plays an important role in cerebellar stem cells it is not required for the initiation and development of Hedgehog induced medulloblastoma. Therefore it may be crucial to consider the Hedgehog status of patients in order to interpret clinical data of Notch pathway inhibitors and even more importantly these results suggest that determining the Hedgehog status might be crucial before treatment of medulloblastoma patients with Notch pathway inhibitors.

Doctor of Philosophy / Abstract Neurodegenerative disorders are a diverse group of disorders that affect specific subsets of neurons. Motor neuron diseases, neurodegenerative disorders of motor neurons, are seen commonly as sporadic cases and less frequently as familial disease forms. The familial forms show genetic and phenotypic heterogeneity. Clinically motor neuron diseases may be seen as rapidly progressive disorders like amyotrophic lateral sclerosis, ALS or slowly progressive disorders like hereditary motor neuropathies, HMN. The only proven causes for motor neuron diseases are gene mutations that lead to motor neuron degeneration in familial disease forms. Only some of these genes have been identified and have contributed greatly to our understanding of the neurobiology of familial and sporadic disease forms. Identification of additional disease causing genes would help enhance our knowledge of the pathophysiological mechanisms underlying all forms of motor neuron disorders, which would lead to early diagnoses, effective prophylaxis and efficient therapies for these disorders. This study aimed to find gene mutations that cause rapid and slowly progressive familial motor neuron disorders in Australian families and to determine their relevance to sporadic forms of motor neuron disease. The familial forms of ALS show reduced disease penetrance, that is, not all gene mutation carriers manifest the disease. This study examines ALS penetrance in a group of Australian families. The most frequently observed mutations in ALS families are cytosolic superoxide dismutase/SOD1 gene mutations. In a collection of ALS families in our centre, families without the common SOD1 gene mutations were genotyped for other ALS genes and loci and studied using genetic linkage and haplotype analyses. Studies in a large Australian ALS family further confirmed genetic heterogeneity in non-SOD familial ALS, all known autosomal dominant ALS genes and chromosomal loci were excluded as cause of disease in this family. Such families can be studied further to identify additional disease genes and loci mapped in other ALS families. These families represent powerful resources for identification of additional ALS genes. Identifying the pathogenic genes in families with reduced disease penetrance may be more relevant to sporadic forms of disease. dHMN is a chronic neurodegenerative disorder predominantly affecting motor neurons. In a large Australian dHMN family, all the known dHMN genes and chromosomal loci were excluded as cause of disease. A genome wide microsatellite screen was performed in this family and genetic linkage was established to a novel 12.98 Mb locus on chromosome 7q34.2-q36. Candidate genes in this large interval will be screened based on their function and expression profile. Identification of a new dHMN locus provides the basis for future identification of a novel gene involved in motor neuron degeneration. Genes in dHMN have been shown to be pathogenic in ALS and Charcot Marie Tooth syndromes. The new locus for dHMN mapped in this project would lead to identification of a novel dHMN gene, which may elucidate the pathogenesis underlying a wide range of neurodegenerative disorders.