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 2016-04-01T19:13:13Z No. of bitstreams: 1 JoseHenriqueTarginoDiasGois_DISSERT.pdf: 8111349 bytes, checksum: df4a2a7f9ab95c60e4e75a96288b99f9 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2016-04-06T00:26:58Z (GMT) No. of bitstreams: 1 JoseHenriqueTarginoDiasGois_DISSERT.pdf: 8111349 bytes, checksum: df4a2a7f9ab95c60e4e75a96288b99f9 (MD5) / Made available in DSpace on 2016-04-06T00:26:58Z (GMT). No. of bitstreams: 1 JoseHenriqueTarginoDiasGois_DISSERT.pdf: 8111349 bytes, checksum: df4a2a7f9ab95c60e4e75a96288b99f9 (MD5) Previous issue date: 2015-02-24 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico - CNPq / A administra??o de nicotina em seres humanos e roedores ? pensada como um melhorador de mem?ria e aten??o, tamb?m pelo seu efeito positivo na Doen?a de Alzheimer. O complexo do Septo Medial / Banda Diagonal de Broca (MS/DBB) ? um dos principais sistemas colin?rgicos ? massivamente projetado para o hipocampo atrav?s da F?mbria-F?rnix, via esta chamada de via septo-hipocampal. Foi demonstrado que o MS/DBB afeta diretamente o potencial de campo local (LFP) e a organiza??o r?tmica do hipocampo, especialmente na gera??o do ritmo teta - um LFP r?tmico intrinsecamente relacionado com a fun??o mnem?nica do hipocampo. Experimentos in vitro deram evid?ncias de que a nicotina aplicada no MS/DBB pode gerar um ritmo teta na rede local do MS/DBB. Assim, o presente estudo se prop?e a elucidar a fun??o da nicotina no MS/DBB sobre a via septo-hipocampal. Experimentos in vivo, comparando o efeito de microinfus?es no MS/DBB de solu??o salina (n = 5) ou nicotina (n = 8) em camundongos anestesiados por ketamina/xilazina mostraram um aumento na densidade de pot?ncia no espectro banda de gama (35 a 55 Hz) em ambas as estruturas (teste de Wilcoxon Rank-Sum, p = 0,038), mas sem alterar a coer?ncia entre as estruturas na mesma banda (teste de Wilcoxon Rank-Sum, p = 0,60). Houve tamb?m uma diminui??o na densidade pot?ncia na banda delta (1-3 Hz) ? oscila??o induzida pela ketamina. Realizamos tamb?m experimentos in vitro sobre o efeito da nicotina na voltagem de membrana e no potencial de a??o de neur?nios do MS/DBB. Registramos neur?nios (n=22) em current-clamp antes e depois da presen?a de nicotina no meio extracellular; 12 neur?nios responderam ? nicotina, metade aumentou a taxa de potenciais de a??o; outros seis diminu?ram, diferindo significativamente no limiar do potencial de a??o (- 47,3 ? 0,9 mV vs. -41 ? 1,9 mV, respectivamente, p = 0,007) e na largura do disparo (1,6 ? 0,08 vs. 2 ms ? 0,12 ms, respectivamente, p = 0,01). Al?m disso, realizamos outro conjunto de experimentos in vitro, relativo ? conectividade das tr?s grandes popula??es neuronais de MS / DBB que usam a acetilcolina, GABA ou glutamato como neurotransmissores. O registro pareado de patch-clamp mostrou que neur?nios glutamat?rgicos e GABA?rgicos realizam contatos intra-septais capazes de produzir correntes sin?pticas em neur?nios p?s-sin?pticos do MS/DBB. A probabilidade da conectividade entre diferentes popula??es neuronais foi implementada em um modelo realista que corrobora que a rede ? altamente sens?vel ? gera??o de ritmo gama. Juntamente com os dados dispon?veis, o conjunto completo de experi?ncias corrobora que a nicotina pode atuar como potenciador cognitivo, e um substrato eletrofisiol?gico prov?vel ? atrav?s da indu??o de oscila??o gama no circuito local do MS/DBB. / Nicotine administration in humans and rodents enhances memory and attention, and also has a positive effect in Alzheimer's Disease. The Medial Septum / Diagonal Band of Broca complex (MS/DBB) ? a main cholinergic system ? massively projects to the hippocampus through the fimbria-fornix, and this pathway is called the septohippocampal pathway. It has been demonstrated that the MS/DBB acts directly on the local field potential (LFP) rhythmic organization of the hippocampus, especially in the rhythmogenesis of Theta (4-8Hz) ? an oscillation intrinsically linked to hippocampus mnemonic function. In vitro experiments gave evidence that nicotine applied to the MS/DBB generates a local network Theta rhythm within the MS/DBB. Thus, the present study proposes to elucidate the function of nicotine in the MS/DBB on the septo-hippocampal pathway. In vivo experiments compared the effect of MS/DBB microinfusion of saline (n=5) and nicotine (n=8) on Ketamine/Xylazine anaesthetized mice. We observed power spectrum density in the Gamma range (35 to 55 Hz) increasing in both structures (Wilcoxon Rank-Sum test, p=0.038) but with no change in coherence between these structures in the same range (Wilcoxon Rank-Sum test, p=0.60). There was also a decrease in power of the ketamineinduced Delta oscillation (1 to 3 Hz). We also performed in vitro experiments on the effect of nicotine on membrane voltage and action potential. We patch-clamped 22 neurons in current-clamp mode; 12 neurons were responsive to nicotine, half of them increased firing rate and other 6 decreased, and they significantly differed in action potential threshold (-47.3?0.9 mV vs. -41?1.9 mV, respectively, p=0.007) and halfwidth time (1.6?0.08 ms vs. 2?0.12 ms, respectively, p=0.01). Furthermore, we performed another set of in vitro experiments concerning the connectivity of the three major neuronal populations of MS/DBB that use acetylcholine, GABA or glutamate as neurotransmitter. Paired patch-clamp recordings found that glutamatergic and GABAergic neurons realize intra-septal connections that produce sizable currents in MS/DBB postsynaptic neurons. The probability of connectivity between different neuronal populations gave rise to a MS/DBB topology that was implemented in a realistic model, which corroborates that the network is highly sensitive to the generation of Gamma rhythm. Together, the data available in the full set of experiments suggests that nicotine may act as a cognitive enhancer, by inducing gamma oscillation in the local circuitry of the MS/DBB.