The survival of several neuron populations during development, including sympathetic neurons, is strictly regulated by neurotrophins such as nerve growth factor (NGF) released from innervation targets. NGF activates its receptor, TrkA, at axon terminals, to generate signals that are transmitted retrogradely to cell bodies to induce signaling cascades regulating survival. A general view of this process is that NGF generates retrograde survival signals that, when delivered to cell bodies, induce downstream survival signaling that prevents apoptosis. A retrograde survival signal proposed to be necessary for sympathetic neuron survival consists of endosomes containing NGF and phosphorylated TrkA. For this signal, phosphorylated TrkA arriving at cell bodies is required to initiate survival signaling. Studies have tested the necessity of TrkA phosphorylation in the cell bodies for survival: results from different studies contradict each other. Moreover, the Trk inhibitor, K252a, used in these studies, has reported non-specific effects. Using an alternate Trk inhibitor, Gö6976, data presented in this thesis demonstrates that NGF can promote survival by retrograde signaling that does not require TrkA phosphorylation in the cell bodies. These retrograde signals may be composed of signaling molecules activated downstream of TrkA in axons since pro-survival molecules downstream of TrkA, Akt and CREB, were found activated in the cell bodies/proximal axons. Data presented in this thesis also reveals a fundamentally different mechanism for how NGF promotes sympathetic neuron survival: a retrograde apoptotic signal that is suppressed by NGF. NGF withdrawal from axons induced the “axon apoptotic signal” that was retrogradely transmitted to cell bodies to activate a key pro-apoptotic molecule, c-jun. The axon apoptotic signal, which was blocked by the kinase inhibitors rottlerin and chelerythrine, was necessary for apoptosis in response to NGF deprivation. Evidence GSK3 is involved in generation or transmission of the axon apoptotic signal was provided by experiments with GSK3 inhibitors and siRNA. The axon apoptotic signal discovery refutes the previous view that NGF acting on axon terminals supports survival exclusively by generating retrograde survival signals. The axon apoptotic signal has broad implications for understanding nervous system development and other conditions where neuronal apoptosis occurs, such as neurotrauma and neurodegenerative diseases.

The thesis presents a new paradigm for studying the importance of interactions between an organism and its environment using a combination of biology and technology: embodying cultured cortical neurons via robotics. From this platform, explanations of the emergent neural network properties leading to cognition are sought through detailed electrical observation of neural activity. By growing the networks of neurons and glia over multi-electrode arrays (MEA), which can be used to both stimulate and record the activity of multiple neurons in parallel over months, a long-term real-time 2-way communication with the neural network becomes possible. A better understanding of the processes leading to biological cognition can, in turn, facilitate progress in understanding neural pathologies, designing neural prosthetics, and creating fundamentally different types of artificial cognition. Here, methods were first developed to reliably induce and detect neural plasticity using MEAs. This knowledge was then applied to construct sensory-motor mappings and training algorithms that produced adaptive goal-directed behavior. To paraphrase the results, most any stimulation could induce neural plasticity, while the inclusion of temporal and/or spatial information about neural activity was needed to identify plasticity. Interestingly, the plasticity of action potential propagation in axons was observed. This is a notion counter to the dominant theories of neural plasticity that focus on synaptic efficacies and is suggestive of a vast and novel computational mechanism for learning and memory in the brain. Adaptive goal-directed behavior was achieved by using patterned training stimuli, contingent on behavioral performance, to sculpt the network into behaviorally appropriate functional states: network plasticity was not only induced, but could be customized. Clinically, understanding the relationships between electrical stimulation, neural activity, and the functional expression of neural plasticity could assist neuro-rehabilitation and the design of neuroprosthetics. In a broader context, the networks were also embodied with a robotic drawing machine exhibited in galleries throughout the world. This provided a forum to educate the public and critically discuss neuroscience, robotics, neural interfaces, cybernetics, bio-art, and the ethics of biotechnology.

The key to understanding any system, including physiologic and pathologic systems, is to obtain a truly comprehensive view of the system. The purpose of this dissertation was to develop foundational analytical and modeling tools, which would enable such a comprehensive view to be obtained of any physiological or pathological system by combining experimental, clinical, and theoretical viewpoints. Specifically, we focus on the development of analytical and modeling techniques capable of predicting and prioritizing the mechanisms, emergent dynamics, and underlying principles necessary in order to obtain a comprehensive system understanding. Since physiologic systems are inherently complex systems, our approach was to translate the philosophy of complex systems into a set of applied and quantitative methods, which focused on the relationships within the system that result in the system's emergent properties and behavior. The result was a set of developed techniques, referred to as relational modeling and analysis that utilize relationships as either a placeholder or bridging structure from which unknown aspects of the system can be effectively explored. These techniques were subsequently tested via the construction and analysis of models of five very different systems: synaptic neurotransmitter spillover, secondary spinal cord injury, physiological and pathological axonal transport, and amyotrophic lateral sclerosis and to analyze neurophysiological data of in vivo cat spinal motoneurons. Our relationship-based methodologies provide an equivalent means by which the different perspectives can be compared, contrasted, and aggregated into a truly comprehensive viewpoint that can drive research forward.

Le sujet de thèse porte sur la maîtrise et la mesure des formes neuronales, “maîtrise” du fait de l’emploi de micropatterns adhésifs permettant un contrôle des formes cellulaires en deux dimensions, “mesure” du fait de notre volonté d’accéder au volume ainsi qu’à la masse sèche de la cellule par l’emploi de deux techniques complémentaires faisant appel à l’interférométrie ou à des mesures de fluorescence en espace confiné.La question biologique au cœur de cette thèse est celle de la régulation par le neurone de diverses caractéristiques morphologiques comme sa longueur, son volume en lien avec l’établissement de la polarité axo-dendritique. Ces aspects sont développés et approfondis au cours de cette thèse des points de vue expérimentaux mais aussi théoriques (coll. Nir Gov, Institut Weizmann).Ce sujet de thèse multidisciplinaire porte ainsi des aspects de biologie et d’instrumentation physique. / The thesis deals with the control and the measurement of neuronal shapes, "control" by using adhesive micropatterns allowing to constrain cells shape in two dimensions, "measurement" by using either interferometry or fluorescence measurements in confined spaces to gain knowledge on cell dry mass and volume.The biological question at the heart of this thesis is the regulation by the neuron of its various morphological characteristics such as length, volume, in association with the establishment of the axo-dendritic polarity. These aspects are developed and deepened in the course of this thesis on experimental but also theoretical (coll. Nir Gov, Weizmann Institute) point of views.This multidisciplinary thesis topic thus builds on biological aspects and physical instrumentation.

Non linear dynamical systems can present a diversity of unconventional features when perturbed by an external noise, such as an enhancing of transport properties, stabilization of spatial patterns and noise induced phase transitions. In particular, the external noise can improve the system s response to weak external periodic pulses, a phenomenon termed as stochastic resonance due to its similarity with the resonance phenomena displayed by deterministic dynamical systems. Stochastic resonance ideas have been widely applied to better understand the behavior of many physical, chemical and biological systems, such as optical, electronic and magnetic systems, chemical reactions, as well as several features regarding neuro-physiological aspects of sensory systems. In this work, we study the stochastic resonance phenomenon in the integrate-fire model for the neuronal response by a sub-threshold periodic signal. In the traditional approach the threshold level is reached by superposing a gaussian noise to the periodic drive. As non gaussian noises have been shown to be quite overspread in natural systems, we investigate the sensitivity of the stochastic resonance condition upon the noise s probability distribution function. To generate a power-law distributed noise, we considered a stochastic process with both additive and multiplicative noises which allows for a fine tuning of the asymptotic power-law decay exponent. We employed both analogical and computational solutions of the stochastic differential equations which produced similar results. The dependence of the optimal noise intensity for the stochastic resonance condition on the power-law exponent of the non gaussian noise is reported. Our main finding is that the stochastic resonance condition is achieved with a minimum intensity of the input noise when it has a probability distribution with a finite decay exponent. Therefore, neural systems can explore the non gaussian character of the input noise to improve the ability to identify sub-threshold signals. Instituto de F´ısica - UFAL / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Sistemas dinâmicos não lineares podem apresentar uma diversidade de características não convencionais quando perturbados por ruídos externos, tais como uma otimização das propriedades de transporte, estabilização de padrões espaciais e transições de fase. Em particular, o ruído pode melhorar a resposta do sistema a pulsos periódicos externos fracos, um fenômeno conhecido como ressonância estocástica devido a sua similaridade com o fenômeno de ressonância mostrado por sistemas dinâmicos determinísticos. A idéia de ressonância estocástica foi largamente aplicada para se entender o comportamento de muitos sistemas físicos, químicos e biológicos, tais como sistemas magnéticos, ópticos, eletrônicos, reações químicas, assim como vários aspectos neurofisiológicos de sistemas sensoriais. Nesta dissertação, nós estudamos o fenômeno de ressonância estocástica em um modelo integra-dispara para resposta neuronal estimulada por um sinal periódico sub-limiar. No enfoque tradicional, o nível de limiar de disparo é alcançado por uma superposição de um ruído gaussiano com um estímulo periódico. Como ruídos não gaussianos surgem em sistemas naturais com elevada freqüência, nós investigamos a sensibilidade da condição de ressonância estocástica em relação à função distribuição de probabilidade do ruído. Para gerarmos um ruído distribuído tipo lei de potência, nós consideramos um processo estocástico com ruído multiplicativo e aditivo que permite o ajuste fino do expoente de decaimento assintótico da lei de potência. Utilizamos tanto solução analógica quanto digital de equações diferenciais estocásticas que produzem resultados similares. A dependência da intensidade ótima de ruído para a condição de ressonância estocástica com o expoente da lei de potência de um ruído não gaussiano é relatada. Em particular, obtivemos que a condição de ressonância é atingida com o mínimo ruído possível para ruídos que apresentam um expoente da lei de decaimento finito. Portanto, a natureza não gaussiana do ruído pode ser explorada para otimizar a identificação de sinais sub-limiares por sistemas neuronais.

Pesquisas in vivo e in vitro, têm estabelecido uma correlação entre alterações na morfologia dendrítica e a epilepsia. No entanto, ainda não se conhecem em detalhe as consequências dessas modificações, sobre a eletrofisiologia e o padrão de disparo. Também existe um fenômeno que não tem sido completamente explicado, conhecido como o paradoxo do dendrito epiléptico, no qual neurônios piramidais, mesmo com a diminuição dramática do principal lugar de inervação glutamatérgica (como consequência de, por exemplo, uma redução do diâmetro e comprimento das árvores dendríticas), inesperadamente apresentam um estado de hiperexcitabilidade crônica. Nesta pesquisa foram aproveitadas as vantagens de uma abordagem neurocomputacional, para induzir sistematicamente alterações na arquitetura dendrítica do mesmo tipo às observadas na epilepsia, e avaliar os seus efeitos sobre a eletrofisiologia e o padrão de disparo. Para isso foi construído um modelo computacional biologicamente realista, de um neurônio piramidal do neocórtex. O código-fonte do modelo está na linguagem do NEURON, e foi baseado em dados eletrofisiológicos (i.e. propriedades da membrana e condutâncias iônicas) e morfométricos, obtidos in vitro previamente por outros pesquisadores. A análise foi feita com base em parâmetros eletrofisiológicos do padrão de disparo. O nosso modelo sugere uma influencia muito forte da morfologia dendrítica sobre a eletrofisiologia, a geração de potencias de ação e o padrão de disparo. Os resultados obtidos mostram que, mesmo mantendo constantes todos parâmetros biofísicos (que têm a ver com as dinâmicas elétricas dos canais iônicos), é possível induzir um aumento grande no comportamento elétrico e na geração de potenciais de ação, a partir da redução do diâmetro e comprimento das ramificações das árvores dendríticas. Estes resultados, também permitem contribuir no fornecimento de uma explicação para o paradoxo mencionado. / In vivo and in vitro studies had found a correlation between dendritic morphology alterations and epilepsy. Nevertheless, it has not been established in detail the consequences of those modifications, over the electrophysiology and firing pattern. There is also a phenomenon still not completely understood, known as the epileptic dendrite paradox, in which pyramidal neurons with a dramatic reduction in the principal place of glutamatergic innervation (due to, for example, a loss in dendritic trees\' diameter and length), unexpectedly present a chronic hyperexcitable state. In this study we took advantage of a neurocomputational approach, to systematically induce dendritic alterations of the same type as observed in Epilepsy, and evaluate their effect over the electrophysiology and firing behavior. With that purpose in mind, we constructed a biologically realistic computational model of a pyramidal neuron of the neocortex. For this model, it was implemented the programming language (hoc) of the NEURON software, and was elaborated based on electrophysiological data (i.e. membrane properties and ionic conductances), and morphological measurements, taken in vitro previously by other investigators. The analysis was done from electrophysiological parameters of the firing pattern. Our model suggests a great influence of dendritic morphology over the electrophysiology, spike generation and firing pattern. The results obtained show that, even when all the biophysical parameters involved in ion channel dynamics are maintained constant, it is possible to induce a strong increase in electric behavior and spike firing, from a reduction in the length and diameter of the dendritic trees\' ramifications. These results, also contribute to a explanation of the mentioned paradox.

Submitted by Helmut Patrocinio (hell.kenn@gmail.com) on 2017-11-09T01:14:16Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Eduardo_Sequerra_2008_TESE.pdf: 15865584 bytes, checksum: fcfa610e8add1f0dd217541746ae3a44 (MD5) / Approved for entry into archive by Ismael Pereira (ismael@neuro.ufrn.br) on 2017-11-09T11:57:09Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Eduardo_Sequerra_2008_TESE.pdf: 15865584 bytes, checksum: fcfa610e8add1f0dd217541746ae3a44 (MD5) / Made available in DSpace on 2017-11-09T11:57:29Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Eduardo_Sequerra_2008_TESE.pdf: 15865584 bytes, checksum: fcfa610e8add1f0dd217541746ae3a44 (MD5) Previous issue date: 2008 / A zona subventricular (SVZ) ? um s?tio de cont?nua neurog?nese em mam?feros p?s-natos e adultos. Ao longo de toda a vida, os progenitores neuronais gerados destinam-se ao bulbo olfat?rio (BO) para onde migram atrav?s da via migrat?ria rostral (RMS). Uma vez no BO, os novos neur?nios se diferenciam em neur?nios GABA?rgicos que integram-se ? circuitaria local. A express?o de GABA inicia ainda na zona germinativa. Essa express?o precoce poderia levar a hip?tese de que estes progenitores j? estariam comprometidos com o fen?tipo GABA?rgico. Por?m, para demonstrar seu comprometimento GABA?rgico, um dos passos necess?rios ? mostrar que a descarboxilase do ?cido glut?mico (GAD), a enzima que sintetiza GABA em neur?nios maduros, est? presente nestas c?lulas. Nesta tese mostramos que a express?o e atividade enzim?tica de GAD, s?o muito baixas na SVZ. Revelamos que o GABA presente em neur?nios imaturos da SVZ prov?m de uma via de s?ntese alternativa, a via da putrescina. Para analisar a import?ncia do GABA proveniente de putrescina para estas c?lulas realizamos a inibi??o farmacol?gica de sua s?ntese atrav?s da administra??o de DFMO. Observamos que o tratamento com DFMO regula positivamente a express?o de GAD na SVZ e RMS. Mostramos tamb?m que os neuroblastos da SVZ que expressam GABA s?o realmente pl?sticos quanto a sua escolha de fen?tipo neuroqu?mico. Quando explantes de SVZ s?o co-cultivados com fatias de telenc?falo embrion?rio dorsal, s?tio de gera??o de neur?nios glutamat?rgicos, uma subpopula??o se diferencia em neur?nios GABA?rgicos e outra menor em glutamat?rgicos. Sugerimos, portanto, que a via da putrescina permite que neur?nios imaturos sintetizem GABA sem, no entanto, haver comprometimento com o fen?tipo GABA?rgico. Esta produ??o de GABA parece ser importante para a migra??o de neuroblastos da SVZ, embora n?o tenhamos tido sucesso em mostrar um papel na prolifera??o com o decr?scimo na produ??o do precursor putrescina. Mostramos que a libera??o de GABA de putrescina parece ter um papel em inibir a express?o de GAD nestes neuroblastos. Em contrapartida, a subregula??o desta sinaliza??o levaria ao comprometimento pelo fen?tipo GABA?rgico. Se mudarmos os sinais apresentados ?s c?lulas da SVZ, como ?queles presentes na VZ do telenc?falo embrion?rio, pelo menos uma de suas subpopula??es ? capaz de mudar seu destino fenot?pico, e diferenciar-se em neur?nios glutamat?rgicos piramidais. / The subventricular zone (SVZ) is proliferative epithelium that continuously gives rise to new neurons in postnatal and adult mammals. The neurons generated in the SVZ migrate through the rostral migratory stream (RMS) where they differentiate in GABAergic interneurons. A characteristic of these neuron precursors is that they start to express GABA while they are still in the SVZ. This fact can lead to the conclusion that at this time they are already commited to the GABAergic phenotype. However, to affirm this one has to show that the origin of GABA in these cells is the same as in mature neurons. One of the most important steps to define GABAergic commitment in neurons is to demonstrate the expression of glutamic acid decarboxylase (GAD), the synthetic enzyme for GABA in mature neurons. Here we show that SVZ cells display low levels of GAD immunocytochemistry and enzyme activity as compared with the olfactory bulb. We also show that these cells are able to synthesize GABA using an alternative source, the putrescine pathway. To test the importance of putrescine made GABA in vivo, we pharmacolgically inhibited putrescine synthesis through DFMO administration. We observed that this treatment lead to an increase of GAD expression in the SVZ and RMS. We also show here that SVZ cells can display phenotypic plasticity. Co-culturing SVZ explants and dorsal telencephalic slices, a spot of glutamatergic neurogenesis, we observed that a subpopulation of SVZ derived neurons differentiated into GABAergic neurons and another into glutamatergic pyramidal neurons. Our working hypothesis is that the putrescine pathway is a mechanism to synthesize GABA without commitment to the GABAergic phenotype. The release of putrescine derived GABA inhibits GAD expression leaving these neuroblasts in an undifferentiated state. The inhibition of putrescine synthesis caused an upregulation of GAD expression which would lead to GABAergic commitment. If we present these neuroblasts with different signals, as those present in the embryonic dorsal telencephalon, they would show plasticity in their phenotypic fate and differentiate into other neurochemical and morphological phenotypes, one of which is the glutamatergic pyramidal neuron.

Introdução: A urocortina 3 (UCN 3) é um neuropeptídeo pertencente a família CRF, com seletividade de ligação a receptor CRF2. Em roedores as células UCN 3 se localizam principalmente em hipotálamo e amígdala, mas o mapeamento não foi realizado em modelo de primata não-humano. Objetivo: realizar o mapeamento da distribuição da UCN 3 no sistema nervoso central na espécie Cebus apella. Material e métodos: cortes de encéfalo de animais machos foram submetidos aos métodos de imuno-histoquímica e hibridização in situ para UCN 3. Séries adjacentes foram coradas pelo método de Nissl e hematoxilina-eosina. Resultados: Células UCN 3 se localizam principalmente em regiões hipotalâmicas, amigdalóides e límbicas. Há colocalização de UCN 3/CRF no núcleo paraventricular do hipotálamo e UCN 3/insulina em células <font face=\"Symbol\">b do pâncreas. Conclusão: a distribuição de UCN 3 em primata não-humano é semelhante à de roedores. / Introduction: Urocortin 3 (UCN 3) is a neuropeptide with 38-aa and member of the CRF peptide family, it is a selective agonist for the CRF2 receptor. UCN 3 cells in rodents showed containing- neurons found mainly in hypothalamic and amygdaloid regions. However, such mapping was not done in a non-human primate model. Objective: study the UCN 3 distribution in the brain of a monkey. Material and methods: frontal sections (40<font face=\"Symbol\">mm) were subjected to immunohistochemistry technique and in situ hybridization, Nissl and Hematoxylin-eosin staining. Results: UCN 3 cells were found mainly in the amygdaloid, limbic and hypothalamic regions. Double-labeled cells (CRF/UCN 3) were found in the PaMD and, in <font face=\"Symbol\">b cells (UCN 3/insulin) of pancreas. Conclusion: the distribution of UCN 3 in non-human primate is similar to the rodents distribution.

Neste trabalho as equações fenomenológicas (tetra-dimensionais) de Hodgkin-Huxley [5], para a membrana da fibra do neurônio, são estudadas mediante transformações não-lineares de variáveis. As transformações de variáveis visam estabelecer um processo controlado de redução de variáveis até chegar a um modelo bidimensional com o menor prejuízo quantitativo possível. O objetivo primordial é aprofundar o entendimento da aparente relação das equações de Hodgkin-Huxley com uma versão da equação de 2ª ordem de van der Pol, conhecida na literatura pelos nomes de equação de FitzHugh-Nagumo [83], equação de Nagumo [84] ou equação Bonhoeffer-van der Pol [7]. É proposta também uma nova formulação matemática para o modelo da corrente de potássio. Estas modificações possibilitam a elaboração de uma remodelagem do aspecto e funcionamento interno do circuito equivalente da membrana. Este circuito, além de facilitar as simplificações para comparar as novas equações em relação ao modelo tipo van der pol, apresenta também potencial teórico mais desenvolvido do que o circuito equivalente original de Hodgkin-Huxley, já que ao contrário deste os elementos do novo circuito podem ser mais facilmente reconhecidos e manipulados dentro da teoria usual de circuitos elétricos. Uma primeira conseqüência da concepção do novo circuito, aqui explorada, é a formulação do modelo da membrana na linguagem da mecânica analítica. / The phenomenological four-variable equations of Hodgkin and Huxley [5] for the neuron fiber membrane are studied by means of nonlinear transformations of variables . The purpose is gradually reduce the number of variables to a three and then to a two-dimensional model, with smallest possible deviations from the quantitative properties of the original model. The primary aim is to get better insights into the apparent connect ion between the Hodgkin-Huxley equations and a version of the second order equation of van der Pol, usually called FitzHugh-Nagumo equation [83], or Nagumo equation [84], or Bonhoeffer- van der Pol equation [7]. An alternative formulation for the potassium current is also proposed. The above modifications lead to an alternative circuit model for the nerve membrane. Such circuit helps the comparison with the van der Pol-type model. It exhibits also better theoretical appeal than the original circuit of Hodgkin and Huxley in the sense that the circuit elements are now properly defined in terms of usual electrical circuit theory. An application of the proposed equivalent circuit i s a description of the Hodgkin-Huxley membrane model according to the formalism of analytical mechanics.

O sistema olfatório é notável por sua capacidade de discriminar odores muito similares, mesmo que estejam misturados. Essa capacidade de discriminação é, em parte, devida a padrões de atividade espaço-temporais gerados nas células mitrais, as células principais do bulbo olfatório, durante a apresentação de um odor. Tais padrões dinâmicos decorrem de interações sinápticas recíprocas entre as células mitrais e interneurônios inibitórios do bulbo olfatório, por exemplo, as células granulares. Nesta tese, apresenta-se um modelo do bulbo olfatório baseado em modelos pulsados das células mitrais e granulares e avalia-se o seu desempenho como sistema reconhecedor de padrões usando-se bases de dados de padrões artificiais e reais. Os resultados dos testes mostram que o modelo possui a capacidade de separar padrões em diferentes classes. Essa capacidade pode ser explorada na construção de sistemas reconhecedores de padrões. Apresenta-se também a ferramenta denominada Nemos, desenvolvida para a implementação do modelo, que é uma plataforma para simulação de neurônios e redes de neurônios pulsados com interface gráfica amigável com o usuário. / The olfactory system is a remarkable system capable of discriminating very similar odorant mixtures. This is in part achieved via spatio-temporal activity patterns generated in mitral cells, the principal cells of the olfactory bulb, during odor presentation. Here, we present a spiking neural network model of the olfactory bulb and evaluate its performance as a pattern recognition system with datasets taken from both artificial and real pattern databases. Our results show that the dynamic activity patterns produced in the mitral cells of the olfactory bulb model by pattern attributes presented to it have a pattern separation capability. This capability can be explored in the construction of high-performance pattern recognition systems. Besides, we proposed Nemos a framework for simulation spiking neural networks through graphical user interface and has extensible models for neurons, synapses and networks.