Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2015-09-17T21:10:57Z No. of bitstreams: 1 AnnieDaCostaSouza_DISSERT.pdf: 4581835 bytes, checksum: 75b58eb36554ceea210d1f40fcbb7f5e (MD5) / Approved for entry into archive by clediane guedes (clediane@bczm.ufrn.br) on 2015-10-27T15:25:21Z (GMT) No. of bitstreams: 1 AnnieDaCostaSouza_DISSERT.pdf: 4581835 bytes, checksum: 75b58eb36554ceea210d1f40fcbb7f5e (MD5) / Made available in DSpace on 2015-10-27T15:25:21Z (GMT). No. of bitstreams: 1 AnnieDaCostaSouza_DISSERT.pdf: 4581835 bytes, checksum: 75b58eb36554ceea210d1f40fcbb7f5e (MD5) Previous issue date: 2014-10-15 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / O ritmo teta consiste em uma oscila??o eletrofisiol?gica hipocampal presente em v?rias esp?cies de mam?feros (4-12 Hz, com varia??es entre esp?cies). Essa oscila??o est? presente durante a vig?lia ativa de ratos e tamb?m ? predominante no PCL desta esp?cie durante o sono de movimento r?pido dos olhos (sono REM). V?rios trabalhos demonstraram que o ritmo teta ? importante em tarefas cognitivas. O septo medial ? uma regi?o importante na gera??o do ritmo teta hipocampal. Possui proje??es colin?rgicas, GABA?rgicas e glutamat?rgicas para o hipocampo, que por sua vez, possui proje??es de feedback para o septo. Al?m do septo, outras regi?es est?o envolvidas na regula??o do teta, formando uma rede complexa de intera??o e coordena??o entre ?reas que resultam no ritmo. A optogen?tica ? uma ferramenta desenvolvida recentemente que tem sido amplamente utilizada em pesquisas de diversas ?reas. Ela nos permite manipular a atividade el?trica de neur?nios atrav?s de estimula??o luminosa. A t?cnica consiste em, atrav?s de um vetor viral, induzir a express?o neuronal de canais i?nicos associados a opsinas (ex.: ChR2), que uma vez infectados passam a ser sens?veis a luz de determinado comprimento de onda. O presente trabalho de pesquisa de mestrado teve como objetivo implantar a optogen?tica em animais em livre comportamento pioneiramente no Brasil, atrav?s de experimentos com implantes cr?nicos de eletrodos e fibras ?ptica em animais infectados com vetor viral para express?o de ChR2. Foram realizadas cirurgias de inje??es de v?rus no septo medial; resultados histol?gicos confirmaram a express?o de ChR2 atrav?s da marca??o da prote?na rep?rter eYFP no septo e tamb?m em processos hipocampais. Al?m disso, foram realizados experimentos agudos com estimula??o luminosa do septo medial e registro de potenciais de campo local (PCL) no pr?prio septo e hipocampo em animais anestesiados. Ainda nesses experimentos foi poss?vel registrar potenciais de a??o no septo. Nesses experimentos observamos aumento da taxa de disparo dos neur?nios septais durante estimula??o luminosa (n=300 est?mulos). Al?m disso, encontramos uma resposta evocada no PCL do hipocampo no in?cio do pulso luminoso. Tamb?m foram realizados experimentos cr?nicos com estimula??o luminosa do septo medial e registro de PCL do hipocampo em animais em livre comportamento. Atrav?s de an?lise do PCL, verificamos se a estimula??o luminosa do septo ? capaz de induzir ritmo teta no hipocampo. / Theta rhythm consists of an electrophysiological hippocampal oscillation present in mammalian species (4-12 Hz with variations across species). This oscillation is present during active waking and is also prevalent in local field potentials (LFP) during rapid eye movement sleep (REM sleep). Several studies have shown that theta rhythm is important in cognitive tasks and that the medial septum is a key region for its occurrence. The septum sends cholinergic, GABAergic and glutamatergic projections to the hippocampus, which in turn projects axons to the septum. Besides the septum, other regions are involved in regulating theta rhythm, forming a complex network of interactions among brain areas that result in theta rhythm. Optogenetics is a recently developed method that has been widely used in various research areas. It allows us to manipulate the electrical activity of neurons through light stimulation. One of the existing techniques consists in using a viral vector to induce the neuronal expression of ion channels associated with the light-sensitive molecule rhodopsin (e.g. ChR2). Once infected, the neurons become sensitive to light of a particular wavelength. The present M. Sc. research aimed to perform luminous stimulation of the brain in anesthetized and freely behaving animals using chronically implanted electrodes and optical fibers in animals infected with a viral vector for ChR2 expression. Surgical viral injections were performed in the medial septum; histological results confirmed the expression of ChR2 by way of the presence of the eYFP reporter protein in the septum and also in hippocampal processes. Moreover, we performed acute experiments with luminous stimulation of the medial septum and LFP recordings of the septum and hippocampus of anesthetized animals. Action potentials were recorded in the septum. In these experiments we observed a significant increase in the firing rates of septal neurons during luminous stimulation (n = 300 trials). Furthermore, we found an early light-evoked response in the hippocampal LFP. Chronic experiments with luminous stimulation of the medial septum and hippocampus in freely behaving animals were also performed in combination with LFP recordings. We found that the luminous stimulation of the septum is able to induce theta rhythm in the hippocampus. Together, the results demonstrate that the luminous stimulation of the medial septum in optogenetically-modified animals causes relevant electrophysiological changes in the septum and the hippocampus.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2016-04-09T00:26:04Z No. of bitstreams: 1 StefanoPupeJohann_TESE.pdf: 36592987 bytes, checksum: f44357f3110776cf9f7cb628b2d65a95 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2016-04-09T00:36:11Z (GMT) No. of bitstreams: 1 StefanoPupeJohann_TESE.pdf: 36592987 bytes, checksum: f44357f3110776cf9f7cb628b2d65a95 (MD5) / Made available in DSpace on 2016-04-09T00:36:12Z (GMT). No. of bitstreams: 1 StefanoPupeJohann_TESE.pdf: 36592987 bytes, checksum: f44357f3110776cf9f7cb628b2d65a95 (MD5) Previous issue date: 2015-03-20 / Os neurocientistas tem uma diversidade de perspectivas com as quais podem classificar diferentes partes do c?rebro. Com o surgimento de t?cnicas baseadas na gen?tica, como a optogen?tica, se torna cada vez mais importante identificar se um grupo de c?lulas, definidas atrav?s de morfologia, fun??o ou posi??o anat?mica possui um padr?o caracter?stico de express?o de um ou mais promotores gen?ticos. Isso permitiria melhores formas de estudar essas popula??es de neur?nios definidas geneticamente. Neste trabalho, eu apresento uma discuss?o te?rica e tr?s estudos experimentais nos quais essa foi a principal quest?o sendo abordada. O Estudo I discute as quest?es envolvidas em selecionar um promotor para estudar estruturas e subpopula??es na ?rea Tegmental Ventral. O Estudo II caracteriza uma subpopula??o de c?lulas na ?rea Tegmental Ventral que compartilha a express?o de um promotor, que ? anatomicamente muito restrita, e que induz avers?o quando estimulada. O Estudo II utiliza uma estrat?gia similar para investigar a subpopula??o no n?cleo subtal?mico que expressa PITX2 e VGLUT2 que, quando inativada, causa hiperlocomo??o. O Estudo IV explora o fato de que um grupo de c?lulas previamente identificadas no Hipocampo Ventral expressa CHRNA2, e indica que essa subpopula??o pode ser necess?ria e suficiente para o estabelecimento do ritmo teta (2-8 Hz) no Hipocampo Ventral de camundongos anestesiados. Todos esses estudos foram guiados pela mesma estrat?gia de identificar um promotor gen?tico capaz de permitir o controle de uma popula??o de neur?nios identificada geneticamente, e eles demonstram as diferentes formas em que essa abordagem pode generar novas descobertas. / Neuroscientists have a variety of perspectives with which to classify different parts of the brain. With the rise of genetic-based techniques such as optogenetics, it is increasingly important to identify whether a group of cells, defined by morphology, function or anatomical location possesses a distinct pattern of expression of one or more genetic promoters. This would allow for better ways to study of these genetically defined subpopulations of neurons. In this work, I present a theoretical discussion and threeexperimental studies in which this was the main question being addressed. Paper I discusses the issues involved in selecting a promoter to study structures and subpopulations in the Ventral Tegmental Area. Paper II characterizes a subpopulation of cells in the Ventral Tegmental Area that shares the expression of a promoter and is anatomically very restricted, and induces aversion when stimulated. Paper III utilizes a similar strategy to investigate a subpopulation in the subthalamic nucleus that expresses PITX2 and VGLUT2 which, when inactivated, causes hyperlocomotion. Paper IV exploits the fact that a previously identified group of cells in the ventral hippocampus expresses CHRNA2, and indicates that this population may be necessary and sufficient for the establishment of the theta rhythm (2-8 Hz) in the Local Field Potential of anesthetized mice. All of these studies were guided by the same strategy of characterizing and studying the role of a genetically defined subpopulation of cells, and they demonstrate the different ways in which this approach can generate new discoveries.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2016-05-03T22:56:48Z No. of bitstreams: 1 HeltonMaiaPeixoto_TESE.pdf: 4738937 bytes, checksum: ea09c631840db43949ce6fbc2d32045c (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2016-05-05T20:05:06Z (GMT) No. of bitstreams: 1 HeltonMaiaPeixoto_TESE.pdf: 4738937 bytes, checksum: ea09c631840db43949ce6fbc2d32045c (MD5) / Made available in DSpace on 2016-05-05T20:05:06Z (GMT). No. of bitstreams: 1 HeltonMaiaPeixoto_TESE.pdf: 4738937 bytes, checksum: ea09c631840db43949ce6fbc2d32045c (MD5) Previous issue date: 2015-07-31 / As prote?nas fluorescentes constituem uma ferramenta fundamental em v?rios campos da biologia, pois permitem enxergar o desenvolvimento de estruturas e processos din?micos de c?lulas em tecido vivo, com o aux?lio da microscopia de fluoresc?ncia. A Optogen?tica ? outra t?cnica que atualmente ganha destaque na Neuroci?ncias e que, de forma geral, permite ativar/desativar neur?nios a partir da irradia??o luminosa de certos comprimentos de onda sobre as c?lulas que possuem canais i?nicos sens?veis ? luz, e ainda pode ser utilizada concomitantemente com as prote?nas fluorescentes. Esta tese possui dois objetivos principais. Inicialmente, s?o estudados os efeitos da intera??o da luz e o c?rebro de camundongos para aplica??es em experimentos de Optogen?tica. Nesta etapa, s?o modelados, a partir de caracter?sticas do c?rebro de camundongos e utilizando a teoria de Kulbelka-Munk, os efeitos de absor??o e espalhamento da luz, em comprimentos de onda espec?ficos, em fun??o da dist?ncia de penetra??o no tecido cerebral. Al?m disso, s?o modeladas as varia??es de temperatura utilizando o m?todo dos elementos finitos na resolu??o da equa??o de bioaquecimento de Pennes, com o aux?lio do COMSOL Multiphysics Modeling Software 4.4, onde s?o simulados protocolos de estimula??o luminosa, tipicamente utilizados em Optogen?tica. Posteriormente, s?o desenvolvidos algoritmos computacionais capazes de reduzir a exposi??o das c?lulas nervosas ? irradia??o luminosa necess?ria ? visualiza??o da fluoresc?ncia emitida por elas. Nesta etapa, s?o descritas as t?cnicas de processamento digital de imagens desenvolvidas para uso em microscopia de fluoresc?ncia, com o intuito de reduzir a exposi??o das amostras de c?rebro ? luz cont?nua, respons?vel pela excita??o dos fluorocromos. As t?cnicas de processamento de imagens desenvolvidas e utilizadas s?o capazes de rastrear, em tempo real, uma regi?o de interesse (ROI) e substituir a fluoresc?ncia emitida pelas c?lulas por uma m?scara virtual, como resultado da sobreposi??o da ROI que est? sendo rastreada e a informa??o de fluoresc?ncia previamente armazenada, mantendo a localiza??o das c?lulas independentemente do tempo de exposi??o ? luz fluorescente. Em resumo, esta tese pretende entender os efeitos da irradia??o luminosa no c?rebro, no contexto da Optogen?tica, al?m de fornecer uma ferramenta computacional que possa auxiliar certos experimentos em microscopia de fluoresc?ncia na redu??o do desvanecimento (bleaching) das amostras e dos danos (photodamage) causados ao tecido devido ? intensa exposi??o das estruturas fluorescentes ? luz. / The fluorescent proteins are an essential tool in many fields of biology, since they allow us to watch the development of structures and dynamic processes of cells in living tissue, with the aid of fluorescence microscopy. Optogenectics is another technique that is currently widely used in Neuroscience. In general, this technique allows to activate/deactivate neurons with the radiation of certain wavelengths on the cells that have ion channels sensitive to light, at the same time that can be used with fluorescent proteins. This dissertation has two main objectives. Initially, we study the interaction of light radiation and mice brain tissue to be applied in optogenetic experiments. In this step, we model absorption and scattering effects using mice brain tissue characteristics and Kubelka-Munk theory, for specific wavelengths, as a function of light penetration depth (distance) within the tissue. Furthermore, we model temperature variations using the finite element method to solve Pennes? bioheat equation, with the aid of COMSOL Multiphysics Modeling Software 4.4, where we simulate protocols of light stimulation tipically used in optogenetics. Subsequently, we develop some computational algorithms to reduce the exposure of neuron cells to the light radiation necessary for the visualization of their emitted fluorescence. At this stage, we describe the image processing techniques developed to be used in fluorescence microscopy to reduce the exposure of the brain samples to continuous light, which is responsible for fluorochrome excitation. The developed techniques are able to track, in real time, a region of interest (ROI) and replace the fluorescence emitted by the cells by a virtual mask, as a result of the overlay of the tracked ROI and the fluorescence information previously stored, preserving cell location, independently of the time exposure to fluorescent light. In summary, this dissertation intends to investigate and describe the effects of light radiation in brain tissue, within the context of Optogenetics, in addition to providing a computational tool to be used in fluorescence microscopy experiments to reduce image bleaching and photodamage due to the intense exposure of fluorescent cells to light radiation.