A distrofia muscular do Golden Retriever é uma miopatia hereditária, recessiva e fatal. Achados clinicopatológicos no trato gastrintestinal desses cães, como atrofia muscular, megaesôfago, dilatação gástrica são relatados, com possíveis alterações nos plexos entéricos. Este trabalho tem como objetivo analisar os neurônios colinérgicos e nitrérgicos do plexo mioentérico, a expressão do receptor P2X7 e a morfologia do íleo de cães afetados pela distrofia muscular comparados aos de cães não afetados. Os tecidos foram preparados por métodos imunohistoquímicos de marcação do Óxido Nítrico Sintase (NOS), Acetilcolina Transferase (ChAT), do pan-neuronal anti-HuC/D e do receptor P2X7. As análises qualitativas e quantitativas das contagens das marcações, das densidades neuronais e da área dos perfis foram obtidas dos Microscópios de Fluorescência, de Confocal de Varredura à Laser, e Microscópio Eletrônico de Transmissão. Os resultados qualitativos demonstraram que neurônios NOS-ir e ChAT-ir apresentaram morfologia Dogiel Tipo I com fibras que colocalizam com o receptor P2X7 e a musculatura intestinal com núcleos picnóticos e maior quantidade de fibras colágenas no grupo distrófico. Os dados quantitativos demonstraram: a) diminuição na área do perfil neuronal dos neurônios NOS-ir e ChAT-ir no grupo distrófico b) maior densidade de neurônios NOS-ir no grupo distrófico. O presente estudo adicionou informações sobre o código químico do plexo mioentérico de cães que podem facilitar o entendimento de desordens intestinais nesses animais. / The Golden Retriever muscular dystrophy is a hereditary myopathy, recessive and fatal. Clinical and pathological findings in the gastrointestinal tract of these dogs as muscle atrophy, megaesophagus, and gastric dilatation are reported, with possible changes in the enteric plexus. This work aims to analyze the cholinergic and nitrergic neurons of myenteric plexus, the P2X7 receptor expression and morphology of the ileum of dogs affected by muscular dystrophy compared to those of unaffected dogs. Tissues were prepared by immunohistochemical methods of labeling Nitric Oxide Synthase (NOS), acetylcholine transferase (ChAT), the panneuronal anti-HuC / D and P2X7 receptor. Qualitative and quantitative analyzes of scores of labeling, density and of neuronal profiles area were obtained from Fluorescence Microscopes, Confocal Laser Scanning, and Transmission Electron Microscope. The results showed that NOS-(immunoreactive)ir and ChAT-ir neurons present morphology Dogiel Type I, their fibers colocalize with the P2X7 receptor and the intestinal muscles present pyknotic nuclei with increased amount of collagen fibers in the dystrophic group. The quantitative data showed: a) decrease in the neuronal area profile of NOS-ir and ChAT-ir neurons in the dystrophic group b) higher density of NOS-ir neurons in the dystrophic group. The present study added information about the chemical code of the myenteric plexus of dogs that can facilitate the understanding of intestinal disorders in these animals.

In hippocampal CA1 and CA3 regions, various properties of neuronal activity follow skewed, lognormal-like distributions, including average firing rates, rate and magnitude of spike bursts, magnitude of population synchrony, and correlations between pre- and postsynaptic spikes. In recent studies, the lognormal features of hippocampal activities were well replicated by a multi-timescale adaptive threshold (MAT) neuron network of lognormally distributed excitatory-to-excitatory synaptic weights, though it remains unknown whether and how other neuronal and network properties can be replicated in this model. Here we implement two additional studies of the same network: first, we further analyze its burstiness properties by identifying and clustering neurons with exceptionally bursty features, once again demonstrating the importance of the lognormal synaptic weight distribution. Second, we characterize dynamical patterns of activity termed neuronal avalanches in in vivo CA3 recordings of behaving rats and in the model network, revealing the similarities and differences between experimental and model avalanche size distributions across the sleep-wake cycle. These results show the comparison between the MAT neuron network and hippocampal readings in a different approach than shown before, providing more insight into the mechanisms behind activity in hippocampal subregions. / Nas regiões CA1 e CA3 do hipocampo, várias propriedades da atividade neuronal seguem distribuições assimétricas com características lognormais, incluindo frequência de disparo média, frequência e magnitude de rajadas de disparo (bursts), magnitude da sincronia populacional e correlações entre disparos pré- e pós-sinápticos. Em estudos recentes, as características lognormais das atividades hipocampais foram bem reproduzidas por uma rede de neurônios de limiar adaptativo (multi-timescale adaptive threshold, MAT) com pesos sinápticos entre neurônios excitatórios seguindo uma distribuição lognormal, embora ainda não se saiba se e como outras propriedades neuronais e da rede podem ser replicadas nesse modelo. Nesse trabalho implementamos dois estudos adicionais da mesma rede: primeiramente, analisamos mais a fundo as propriedades dos bursts identificando e agrupando neurônios com capacidade de burst excepcional, mostrando mais uma vez a importância da distribuição lognormal de pesos sinápticos. Em seguida, caracterizamos padrões dinâmicos de atividade chamados avalanches neuronais no modelo e em aquisições in vivo do CA3 de roedores em atividades comportamentais, revelando as semelhanças e diferenças entre as distribuições de tamanho de avalanche através do ciclo sono-vigília. Esses resultados mostram a comparação entre a rede de neurônios MAT e medições hipocampais em uma abordagem diferente da apresentada anteriormente, fornecendo mais percepção acerca dos mecanismos por trás da atividade em subregiões hipocampais.

Motor neuron disease (MND) is a set of neuromuscular diseases that affect the upper and/or lower motor neurons, resulting in progressive disability. Amyotrophic lateral sclerosis (ALS) and Primary lateral sclerosis (PLS) are two forms of MND that both involve upper motor neuron degeneration, which can also accompany extra-motor changes in cognitive, behavioral, and/or emotional functioning for some individuals. Characterization of the cognitive profile of MND is still evolving, with growing interest in cognitive subtypes. The development of cognitive screens targeted to the MND cognitive profile aim to provide efficient and accurate brief assessments. However, empirical evaluation of tailored MND cognitive screens is needed for cross-validation independent of tests’ original developers. The present study addresses the cognitive profile of MND and the utility of brief cognitive screens with a focus on impairments in the language domain. The two primary aims include: (1) comprehensive assessment and characterization of language dysfunction in MND, and (2) empirical evaluation of brief cognitive screens with regard to detecting language impairments. Forty-one patients with MND (ALS n = 36; PLS n = 5) were administered a comprehensive language battery to classify cognitive impairment (MND/ALSci; Strong et al., 2017) in the language domain and/or verbal fluency. Patients also completed two tailored cognitive screens [ALS Cognitive Behavioral Screen (ALS-CBS), Edinburgh Cognitive and Behavioral ALS Screen (ECAS)] and one general screen (Montreal Cognitive Assessment; MoCA). The current preliminary results suggest language dysfunction in MND is characterized by prominent difficulties with word retrieval (confrontation naming) and/or syntax comprehension. However, evidence of reduced word production resembling nonfluent/agrammatic aphasia was not found. In total, 19.5% of the sample met criteria for MND/ALSci in the language domain (n = 8, all ALS); 22.0% met criteria for MND/ALSci in the verbal fluency domain (n = 9). Patients were classified into three subgroups, those with broad language impairments (ALSci-L n = 4, 9.8%), phonemic fluency impairments (MNDci-VF n = 5, 12.2%), or both impairments (ALSci-L+VF n = 4, 9.8%). Results also revealed existing challenges in accurately classifying patients with language dysfunction using brief cognitive screens. The ECAS Language subscore offered limited classification of broad language impairments in the present MND sample (sensitivity 50%, specificity 70%). Among the broader cognitive screens, sensitivities to language impairments were: ALS-CBS (100%), ECAS ALS-Specific Score (75%), and MoCA (71%). Convergent validity was demonstrated between outcomes on the ALS-CBS and ECAS ALS-Specific Score (rФ = .59). Discriminant validity was also demonstrated between outcomes on ALS-CBS compared to the MoCA (rФ = .11). Future research is needed to assess whether language dysfunction reflects a distinct MND cognitive phenotype(s) and potential relationships with disease prognosis. Naming and syntax comprehension may be fruitful language screening targets for future research.

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.

<p>Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with an incidence of 1.5-2.7/100000 people/year. Today there is no cure for the disease and only symptomatic treatments are available. ALS progresses rapidly and only 50% of the patients are alive three years after the symptom debut. In ALS, the upper and lower motor neurons undergo degeneration in a process resembling apoptosis. This leads to muscle atrophy and paralysis. The causes of neuronal death are however unknown. In this thesis we have studied transgenic mice carrying human mutant superoxide dismutase, as a model for familial ALS. These mice develop ALS-like symptoms after four months of age with degeneration of the motor neurons. Our results show an involvement of endoplasmic reticulum stress, caspase-12, -9, -3 and procaspase-7 in the ALS mice spinal cord. Overexpression of the antiapoptotic protein XIAP in spinal cord neurons inhibited the activation of caspase-12 and reduced caspase-3 and calpain activity. Calpastatin, the regulator of calpain activity, was kept intact in the ALS-XIAP mice. These mice showed a 12% increase in the mean survival suggesting a beneficial effect of XIAP in ALS. The reason for the ultimate cell death of motor neurons in the ALS-XIAP mice may be due to the activation of additional cell death pathways. Thus, we observed that lysosomal proteases particularly, cathepsinB, -D, and -L were activated in the ALS mice spinal cord together with a less marked upregulation of the inhibitors, cystatinB and -C. We also found activation of astrocytes and microglial cells in the spinal cord of ALS mice indicating their involvement in the disease. The results show that both caspase-dependent and -independent pathways are activated during neuronal degeneration in the ALS spinal cord. The results obtained may help to identify novel drug targets for future treatments of ALS.</p>

Mammalian central nervous system (CNS) neurons lose their ability to regenerate their axons after injury during development. For example, optic nerve injury studies in hamsters have shown that optic nerve axons injured around the time of birth retain the ability to regenerate to their target, but this ability is lost during development (So et al., 1981). The development of an inhibitory CNS environment has been implicated in the inability of the adult CNS to regenerate, however there is also support for this loss being a result of changes in developmental programs intrinsic to the neurons themselves (Goldberg et al., 2002a; Goldberg, 2004). While some molecules have been identified as being involved in intrinsic mechanisms controlling axon growth, there is still much to be discovered. Using genes shown to be regulated in retinal ganglion cells (RGCs) during development (Wang et al., 2007), I performed an overexpression screen in embryonic primary neurons measuring changes in neurite growth. Of these genes, the most significant effect in neurite growth was seen with overexpression of Krüppel-like factor 4 (KLF4), resulting in a greater than 50% decrease in growth. KLF4 is a member of the KLF family of transcription factors which all possess a DNA binding domain containing 3 zinc finger motifs. Outside of the nervous system, KLF4 has been implicated in cancer (Black et al., 2001; Rowland and Peeper, 2006), mitotic growth arrest (Shields et al., 1996) and most recently in the induction of pluripotency (Yamanaka, 2008; Zhao and Daley, 2008). In the CNS, KLF4 has recently been implicated in increasing the sensitivity of cortical neurons to NMDA insult (Zhu et al, 2009), though no effect of KLF4 on neurite growth or regeneration has yet been described. I found that KLF4 overexpression in RGCs results in decreased neurite growth and neurite initiation. KLF4 overexpression also leads to decreases in polarity acquisition in hippocampal neurons, though even when they acquire polarity, they still display decreased neurite growth. Additionally, KLF4 knockout targeted to RGCs leads to an increased neurite growth ability and increased neurite initiation in vitro. In vivo, KLF4 knockout increases RGC axon regeneration after optic nerve injury. Interestingly, KLF4 is one of 17 members of the KLF family, known for their ability to act redundantly and competitively amongst family members for their binding sites. Therefore, we looked to see if other KLFs could affect neurite growth ability. 15 of 17 KLF family members are expressed in RGCs, and their overexpression results in differential effects on neurite growth in both cortical neurons and RGCs. Additionally, many of the family members are developmentally regulated in a manner that typically correlates with their ability to affect neurite growth. For example, KLF6 and -7, whose expression decreases during development, when overexpressed, increase neurite growth, whereas KLF9, whose expression increases developmentally, when overexpressed, decreases neurite growth. Surprisingly, there are multiple KLFs expressed in RGCs that are neurite growth-suppressors, and further study has revealed that the combination of KLF growth enhancers with KLF growth suppressors results in a suppressive or neutral phenotype (Moore et al., 2009), suggesting that to further enhance regeneration after injury in vivo, we will need to additionally remove the growth suppression from other KLF family members. Taken together, these data suggest that KLFs may play an important role in the intrinsic loss of axon growth and regeneration seen during development. Further characterization of downstream targets of KLF4 and other KLF family members may reveal specific neuronal gene targets that could mediate the phenotypic effects of these transcription factors. It is my hope that by determining the developmental programs that underlie the loss of intrinsic axon growth ability of CNS neurons, we may ultimately determine how to revert adult CNS neurons to their embryonic axon growth ability.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with an incidence of 1.5-2.7/100000 people/year. Today there is no cure for the disease and only symptomatic treatments are available. ALS progresses rapidly and only 50% of the patients are alive three years after the symptom debut. In ALS, the upper and lower motor neurons undergo degeneration in a process resembling apoptosis. This leads to muscle atrophy and paralysis. The causes of neuronal death are however unknown. In this thesis we have studied transgenic mice carrying human mutant superoxide dismutase, as a model for familial ALS. These mice develop ALS-like symptoms after four months of age with degeneration of the motor neurons. Our results show an involvement of endoplasmic reticulum stress, caspase-12, -9, -3 and procaspase-7 in the ALS mice spinal cord. Overexpression of the antiapoptotic protein XIAP in spinal cord neurons inhibited the activation of caspase-12 and reduced caspase-3 and calpain activity. Calpastatin, the regulator of calpain activity, was kept intact in the ALS-XIAP mice. These mice showed a 12% increase in the mean survival suggesting a beneficial effect of XIAP in ALS. The reason for the ultimate cell death of motor neurons in the ALS-XIAP mice may be due to the activation of additional cell death pathways. Thus, we observed that lysosomal proteases particularly, cathepsinB, -D, and -L were activated in the ALS mice spinal cord together with a less marked upregulation of the inhibitors, cystatinB and -C. We also found activation of astrocytes and microglial cells in the spinal cord of ALS mice indicating their involvement in the disease. The results show that both caspase-dependent and -independent pathways are activated during neuronal degeneration in the ALS spinal cord. The results obtained may help to identify novel drug targets for future treatments of ALS.

The prevalence of spasticity after first-ever stroke is approximately 20%, but there are no data on the prevalence of disabling spasticity.The reported prevalence of pain after stroke varies between 19% and 74%, whether pain is associated with spasticity is not known. Until now, there is no health economic analysis of patients with spasticity after stroke. Methods: Two groups of patients were studied. Cohort I was a cross-sectional survey. A representative sample of 140 patients was investigated 1 year after their first-ever stroke. Spasticity was defined as ≥ 1 score on the modified Ashworth scale, disabling spasticity was defined as spasticity having such an impact that intervention, e.g. intensive physiotherapy, orthoses or pharmacological treatment, should be offered. Pain was assesed with the Visual Analogue Scale. All direct costs during one year were identified and converted into Purchasing Power Parities US dollar (PPP$). Cohort II was a prospective cohort study. Forty-nine patients were examined at day 2–10, at one month, and at six months after their first-ever stroke. Assessment and definitions were similar as for cohort I. Results: Spasticity occurs within 1 month and disabling spasticity occur within 6 months. After one year, the prevalence of spasticity was 17% and that of  disabling spasticity 4%. Disabling spasticity was more frequent in the upper extremity. There was an independent effect of severe upper extremity paresis (OR 22, CI 3.9–125) and age below 65 years (OR 9.5, CI 1.5–60). The prevalence of stroke-related pain was 21% after one year. Stroke-related pain was associated with paresis (OR 3.1, 95% CI 1.2–7.7), sensory disturbance (OR 3.1, 95% CI 1.1–8.9) and depression (OR 4.1, 95% CI 1.4–13), but not with spasticity as an independent variable. The majority of the direct costs for one year (78%) were associated with hospitalization, whereas 20% was associated with municipality services. Only 1% of all direct costs were related to primary health care and 1% to medication. The mean (median, inter-quartile range) direct cost for stroke patients with spasticity was PPP$ 84 195 (72 116, 53 707) compared to PPP$ 21 842 (12 385, 17 484) for stroke patients without spasticity (P &lt; 0.001).

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.

Noise-like activity (NLA) refers to spontaneous subthreshold fluctuations in membrane potential. In this thesis, we examine the role that synaptic channel fluctuations play in contributing to NLA by comparing a detailed biophysical model to experimental data from whole-intact hippocampal interneurons. To represent the contribution from synaptic channel fluctuations, we switch the synapses in the model from traditional to Markovian formalisms and demonstrate statistically relevant increases the standard deviation; power-law scaling exponent; and power spectral density in the 5-100 Hz and 1-5 kHz ranges. However, while synaptic channel fluctuations have a definite effect, we found that they were significantly more subtle than the synaptic response to network activity. This indicates that synaptic channel fluctuations do indeed play a significant role in subthreshold noise, but, overall, synaptic NLA is dominated by the synaptic response to presynaptic network activity.