京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第21401号 / 生博第402号 / 新制||生||53(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 見学 美根子, 教授 上村 匡, 教授 渡邊 直樹 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

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Retinoic acid (RA) is critical for embryonic development and cell differentiation. Previous work in our laboratory has shown that blocking the RA-dependent increase in Pre-â cell leukemia transcription factors (PBX) mRNA and protein levels in P19 cells prevents them from differentiating to either endodermal or neuronal cells. This suggests that PBX is an important regulator of RA-induced differentiation of P19 cells. A microarray analysis was performed to identify PBX regulated genes, utilizing the empty vector P19 (TO3) and antisense to PBX (AS2) cell lines, during RA-induced differentiation of P19 cells into endodermal or neuronal cells. Among the genes identified by the microarray, Dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1 (DAX-1) and steroidogenic factor 1 (SF-1) were identified to be directly or indirectly regulated by PBX. Both DAX-1 and SF-1 proteins have only recently been reported to be present in preimplantation mouse embryos prior to the expression of steroidogenic enzymes, suggesting they may play a role in early mouse embryogenesis. To determine the roles of DAX-1 and SF-1 during RA-dependent differentiation, P19 cells that inducibly express either FLAG-DAX-1 or FLAG-SF-1 upon removal of doxicyclin were prepared. We found that overexpression of FLAG-DAX-1 had no effect on the RA-induced differentiation of P19 cells. However, FLAG-SF-1 overexpression prevented the RA-dependent loss of Oct-4, DAX-1 and the increase in COUP-TFI, COUP-TFII, and Ets-1 mRNA levels during the commitment stages of both endodermal and neuronal differentiation. Surprisingly, continued expression of SF-1 for seven days caused a RA-independent loss of Oct-4 protein. However, cells which continued to express SF-1 for seven days did not terminally differentiate into endodermal or neuronal cells in response to RA treatment. In addition, we found evidence for a feedback loop, where PBX reduces SF-1 mRNA expression and continued SF-1 expression blocks the RA-dependent increase in PBX protein levels. Our findings suggest that SF-1 plays a novel role in P19 cells where its level of expression is critical for the differentiation state of the cells. At basal levels SF-1 maintains the pluripotent state of the cells, while SF-1 levels must be dramatically reduced for cells to differentiate into both endodermal and neuronal cells upon RA treatment. However, at elevated levels above basal, SF-1 inhibits Oct-4 expression and leads to the induction of the expression of steroidogenic enzymes with a pattern consistent with adrenal cells in a RA-independent fashion. Taken together these data suggest that SF-1 plays a much more dynamic role in P19 cells than previously reported. / Biochemistry

This thesis is missing page 18, the other copies of the thesis are missing it as well. - Digitization Centre / The work reported in this thesis concerns a possible quantitative relationship between two bioelectric phenomena of the cat cerebral cortex: neuronal discharge and slow potentials. A review of literature dealing with research related to the present topic is given. This is followed by descriptions of the experimental procedure employed and the results obtained. A subsequent discussion in terms of current neurophysiological concepts relates present findings with those of other known research. The accompanying bibliography represents a fairly complete coverage of contemporary and recent experimental and theoretical work related to the present topic. / Thesis / Master of Arts (MA)

Although the processes used for temporarily storing and manipulating neural information have been extensively studied at the synaptic level far less attention has been given to the underlying cellular and molecular mechanisms that contribute to change in the intrinsic excitability of neurons. More importantly, how do these mechanisms of plasticity integrate with ongoing mechanisms of regulation of neural intrinsic excitability and, in turn, homeostasis of entire neural circuits? In this dissertation I describe the underlying mechanisms that contribute to persistent neural activity and, more globally, sensorimotor adaptation using weakly electric fish as my model system. Weakly electric fish have evolved a behavior adaptation known as the jamming avoidance response (JAR), and it is this adaptation that allows the organism to elevate its own electrical discharge in response to intraspecific interactions and subsequent distortions of the animal’s electric field. The elevation operates over a wide range and in vivo can last tens of hours upon cessation of a jamming stimulus. I demonstrate that the underlying mechanisms of the adaptation are mediated by calcium-dependent signaling in the pacemaker nucleus and that calcium-mediated phosphorylation plays an important role in the regulation of the long-term frequency elevation (LTFE). I demonstrate using an in vitro brain slice preparation from the weakly electric fish, Apteronotus leptorhynchus that the engram of memory formation depends on the cooperativity of calcium-dependent protein kinases and protein phosphatases. In addition, I show that the memory formation (in the form of LTFE) does not depend on the continued flux of calcium, but rather the phosphorylation events downstream of NMDA receptor activation. Moreover, I describe the differences in the expression of protein phosphatases and protein kinases as they relate to species-specific differences in sensorimotor adaptation. It is important to note that this is the first time that the cooperativity between different isoforms of protein kinase C (PKC) have been shown to play a role in graded long-term change in neuronal activity and, in turn, providing the neural basis of species-specific behavior. The neural adaptation of the electromotor system in weakly electric fish provides an excellent model system to study the underlying cellular and molecular events of vertebrate memory formation. / text

Os receptores canabinóides CB1 são abundantemente expressos nos núcleos da base (NB), sugerindo a participação do sistema canabinóide na Doença de Parkinson (DP). Os objetivos deste estudo foram investigar a localização do CB1 nos NB de ratos; avaliar o decurso temporal de sua expressão e de marcadores neuronais em modelo experimental da DP in vivo, e avaliar os efeitos do tratamento com compostos canabinóides em modelos experimentais da DP in vivo e in vitro. Nossos resultados mostraram que o CB1 é predominantemente expresso em neurônios GABAérgicos nos NB. A lesão dopaminérgica produziu mudanças temporais distintas da expressão do CB1 nas estruturas dos NB. O tratamento com o agonista canabinóide ACEA agravou à lesão dopaminérgica e o desempenho comportamental motor. Por outro lado, o tratamento com o antagonista AM 251, embora não tenha gerado diferenças neuroquímicas, gerou melhoras nos testes comportamentais. Por fim, em nosso modelo in vitro, o tratamento com inibidor de recaptação da anandamida AM 404 gerou uma discreta redução dos níveis de morte celular. / Cannabinoid receptors CB1 are abundantly expressed in the basal ganglia (BG), suggesting the involvement of the cannabinoid system in Parkinson\'s disease (PD). The objectives of this study were to investigate the location of CB1 in BG of rats; evaluate the time course expression of CB1 and neuronal markers in an experimental model of PD in vivo, and evaluate the effects of treatment with cannabinoids compounds in experimental models of PD in vivo and in vitro. Our results showed that CB1 is predominantly expressed in GABAergic neurons in BG. The dopamine lesion produced distinct temporal changes in the expression of CB1 in BG structures. Treatment with the cannabinoid agonist ACEA aggravated the dopaminergic lesion and the motor behavioral performance. Moreover, the treatment with the antagonist AM 251, although have not generated neurochemical changes,it promoted improvements in behavioral tests. Finally, in our in vitro model, the treatment with Anandamide transport inhibitor AM 404 led to a slight reduction in the levels of cell death.

A fisiopatologia da sepse se caracteriza por hipotensão acompanhada de aumento da secreção de vasopressina (AVP) na fase inicial e diminuição numa fase mais tardia. Essa hipotensão é em parte devido ao aumento da quantidade de óxido nítrico, que juntamente com outros mediadores tem sua produção aumentada durante a sepse. A óxido nítrico sintase (NOS) é responsável pela síntese deste mediador, e sua isoforma neuronal (nNOS) está presente no músculo esquelético, pulmões, testículos, próstata, pele e também nos neurônios vasopressinérgicos do hipotálamo. O presente trabalho avaliou a participação do óxido nítrico produzido pela isoforma neuronal de NOS sobre a secreção temporal de AVP durante a sepse experimental. Ratos Wistar receberam injeção i.p. de 7-nitroindazol (50mg/kg ou 80mg/kg), inibidor específico da NOS neuronal, ou DMSO 10% + óleo de gergelim na proporção 1:9 (veículo) e após 30 minutos foram submetidos ao estímulo séptico por ligadura e perfuração cecal (CLP) ou à operação fictícia (OF). Em um grupo de animais, a sobrevida foi avaliada durante 5 dias. Em outro grupo, os animais foram decapitados 0, 4, 6, 18 e 24 horas após a cirurgia e o sangue processado para determinação do hematócrito, sódio sérico, osmolalidade, proteínas, glicose, creatinina, nitrato sérico e AVP plasmática. A neurohipófise foi removida para a determinação do conteúdo de AVP, e o hipotálamo dissecado para a determinação da atividade da NOS total. A mortalidade observada após CLP não foi modificada com o pré-tratamento com 7-NI (50mg/kg), assim como os aumentos temporais de hematócrito, glicose e nitrato sérico observados. As proteínas plasmáticas e o sódio sérico apresentaram diminuição após CLP e o pré-tratamento com 7-NI antecipou a perda proteica e postergou a diminuição do sódio sérico. Os animais após CLP não apresentaram alterações de creatinina e osmolalidade, entretanto quando prétratados com 7-NI, apresentaram aumento em 6 e 18 horas e diminuição a partir de 4hs, respectivamente. A atividade da NOS total no hipotálamo aumentou nos tempos determinados de 4 e 24 horas após CLP e este aumento foi reduzido com o prétratamento com o 7-NI nas doses de 50 e 80mg/kg, respectivamente. O conteúdo neurohipofisário de AVP diminuiu em 4, 6 e 18 horas após CLP e o pré-tratamento com 7-NI reduziu os estoques apenas em 0 e 6 horas. As concentrações plasmáticas de vasopressina apresentaram-se aumentadas sómente 6 horas após CLP e o pré-tratamento não alterou essas concentrações. Esses resultados permitem concluir que o NO produzido pela NOS neuronal não teria uma tarefa substancial na secreção de vasopressina durante sepse experimental. / The pathophysiology of sepsis is caracterized by hypotension accompanied by increase of vasopressin secretion (AVP) in early phase and decrease during late phase. This hypotension is due, in part, to the increase of nitric oxide (NO) production, that, like other mediators, shows high production during sepsis. Nitric oxide synthase (NOS) is responsible by synthesis of NO. The neuronal isoform of NOS is present in skeletic muscle, testicles, prostate, skin and vasopressinergics neurons of hypothalamus. The present work evaluated the participation of nitric oxide produced by neuronal NOS in temporal vasopressin secretion during experimental sepsis. Rats Wistar received intraperitoneal injection of 7-nitroindazole (50 or 80 mg/kg), an inhibitor of neuronal nitric oxide synthase activity, or DMSO 10% + sesame oil in the proportion 1:9 (vehicle) and after 30 minutes, they were submited to septic stimulus by cecal ligation and puncture (CLP) or to sham operation. In one of the groups, the survival rate was evaluated during 5 days. In other group, the animals were decapited 0, 4, 6, 18 and 24 hours after CLP and the blood was processed to determinate haematocrit, serum sodium, osmolality, proteins, glucose, creatinine, serum nitrate, and plasma AVP. Neurohypophysis was removed to determination of vasopressin content, and hypotalamus was dissected to determinate total NOS activity. Mortality observed after CLP was not affected by periferal injection of 7-nitroindazole (50 mg/kg) as well as haematocrit, glucose and nitrate increase. Serum sodium and plasma protein decreased after CLP and the treatment antecipated the loss protein, and delayed serum sodium decrease. CLP animals didn\'t show creatinine and osmolality alterations, but when treated with 7-nitroindazole, showed increase 6 and 18 hours, and decrease 4 hours, respectively. NOS activity in hypothalamus increased 4 and 24 hours after CLP, and was reduced with 7-NI pretreatment (50 and 80 mg/kg respectively). AVP neurohypophysis content diminished 4, 6 and 18 hours after CLP and 7-NI reduced the content just at 0 and 6 hours. Vasopressin plasma concentration increased just 6 hours after CLP and 7-NI pretreatment didn\'t alter this parameter. We concluded that NO produced by neuronal NOS doesn\'t have a substantial role in vasopressin secretion during experimental sepsis.

Este trabalho teve como objetivo analisar os efeitos comportamentais e morfológicos da exposição ao campo magnético contínuo em um modelo experimental de doença de Huntington. Foram utilizados 68 ratos Wistar, machos, divididos em 6 grupos: GC (controle, n=12), GS (sham, n=12), GSM (sham magnético, n=8), GL (lesão, n=12), GEPS (polo sul, n=12) e GEPN (polo norte, n=12). O animais passaram por habituação ao Rota Rod durante 3 dias pré-cirúrgicos e por habituação ao monitor de atividade 24 horas antes da cirurgia. Após procedimentos cirúrgicos adequados, os GL, GEPS e GEPN receberam administração de ácido quinolínico (120nmol/2L) no núcleo estriado esquerdo. Os GS e GSM receberam administração de 2L de salina na mesma região. Ainda, nos GEPS e GEPN foi implantado no crânio de cada animal um magneto circular de neodímio (8x3mm) com potência de 3200 Gauss e no GSM foi realizado implante do mesmo material, sem estar magnetizado. No 7º dia pós-cirúrgico, os animais foram avaliados em relação à atividade motora espontânea no monitor de atividades, após 5 minutos da injeção subcutânea de apomorfina (2,5 mg/Kg), sendo que os animais do GC não receberam esta injeção, e atividade motora forçada no Rota Rod. Posteriormente aos experimentos os animais foram perfundidos e os encéfalos retirados para histologia. Os resultados da avaliação comportamental espontânea evidenciaram, para o comportamento de distância percorrida, um aumento significativo do GEPS em relação aos GC, GL e GEPN, e também do GSM e GS em relação ao GC, GL e GEPN. Observamos também uma diminuição significativa do GEPN em relação aos GS, GSM, GL e GEPS [F(5,62) = 3,19; p0,05]. Para o tempo de atividade, um aumento significativo do GEPS em relação aos GC e GEPN, e também do GSM e GS em relação ao GC, GL e GEPN. Observamos também uma diminuição significativa do GEPN em relação aos GS, GSM, GL e GEPS [F(5,62) = 5,46; p0,05]. Para o comportamento de cruzamentos, um aumento significativo do GEPS em relação aos GC e GEPN e também do GSM e GS em relação ao GC, GL e GEPN. Observamos também uma diminuição significativa do GEPN em relação aos GS, GSM, GL, e GEPS [F(5,62) = 3,31; p0,05]. E para o comportamento de giros anti-horários (ipsilaterais a lesão) um aumento significativo dos GL, GEPS e GEPN em relação aos GC, GS e GSM. Observamos também uma diminuição significativa dos GEPN e GEPS em relação ao GL e ainda uma diminuição do GEPN em relação ao GEPS [F(5,62) = 16,01; p0,05]. Os resultados referentes ao Rota Rod (atividade motora forçada) revelaram diminuição significativa do tempo de permanência no aparato do GL em relação aos demais GC, GS, GSM, GEPS e GEPN [(F(5,62) = 5,46; p0,05)]. A análise histológica revelou uma perda significativa de neurônios no núcleo estriado esquerdo do GL em relação aos demais GC, GS, GSM, GEPS e GEPN [(F(5,66) = 5,13; p0,05)]. Dessa forma, os resultados obtidos sugerem que a estimulação magnética exerce efeito neuroprotetor, com reversão das alterações comportamentais e morfológicas promovidas pelo ácido quinolínico. / The aim of this study was to analyze the behavioral and morphologic effects of the static magnetic field exposition in an animal model of Huntingtons disease. Sixty- eight male Wistar rats were used, placed in 6 groups: GC (control group, n=12), GS (sham group, n=12), GSM (sham magnetic group, n=8), GL (lesion group, n=12), GEPS (south pole stimulated group, n=12) and GEPN (north pole stimulated group, n=12). The animals passed through habituation to Rota Rod, during the 3 days pre-surgical, and to habituation to the activity monitor, 24 hours before surgery. After appropriate surgical procedures GL, GEPS and GEPN received administration of quinolinic acid (120nmol/ 2L) in the left striatum. The GS and GSM received administration of 2L of saline in the same region. Also, in GEPS and GEPN was implanted, on the skull of each animal, a circular neodymium magnet (8x3mm) with a power of 3200 gauss, the GSM was performed the implant of the same material, without being magnetized. On the seventh after surgery day, the animals were evaluated referring to spontaneous motor activity in the activity monitor, 5 minutes after subcutaneous injection of apomorphine (2.5 mg / kg), whereas the animals of the CG did not receive this injection, and forced motor activity in Rota Rod. Subsequently the experiments the animals were perfused and their brains removed for histology. The results showed to spontaneous behavioral assessment, related to the behavior of distance travelled, significant increase in GEPS compared to GC, GL e GEPN, and also in GSM and GS compared to GC, GL, and GEPN, and a decrease in GEPN compared to GC, GS, GSM, GL e GEPS [F (5,62) = 3.19, p 0.05]; for time of activity, a significant increase in GEPS compared to GC and GEPN, and also in GSM and GS compared to GC, GL and GEPN, and a decrease in GEPN compared to GS, GSM, GL e GEPS [F (5,62) = 5.46, p 0.05]; for the behavior of crossings, a significant increase in GEPS compared to GC and GEPN, and also in GSM and GS compared to GC, GL and GEPN, and a decrease in GEPN compared to GS, GSM, GL, e GEPS [F (5,62) = 3.31, p 0.05]; and to the behavior of anti hourly rotations, significant increase in GL, GEPS and GEPN compared to GC, GS, GSM, significant decrease in GEPN and GEPS compared to GL and also a decrease in GEPN compared to GEPS [F (5 , 62) = 16.01, p 0.05]. The results for the Rota Rod indicated a significant decrease in the permanency time on apparatus to GL compared to GC, GS, GSM, GEPS and GEPN [(F (5, 62) = 5.46, p 0.05)]. The histological analysis revealed a significant reduction in the number of neurons in the animals of GL compared to the others groups [F (5, 66) = 5, 13, p 0.05]. Therefore, the results suggest that magnetic stimulation exerts neuroprotective effect, with reversal of behavioral and morphological changes caused by quinolinic acid.

Os receptores para mineralocorticoides (MR) e glicocorticoides (GR) representam importantes sítios de ligação para os glicocorticoides. Enquanto a ativação crônica e excessiva de GR leva à atrofia do hipocampo, a ativação do MR é neuroprotetora. Considerando as alterações no giro denteado (GD), decorrentes do envelhecimento, e a possível participação do MR e GR neste processo, o objetivo deste trabalho foi correlacionar o desbalanço de tais receptores com a degeneração neuronal nesta região do hipocampo de cães idosos. Para isso, utilizaram-se cortes histológicos do hipocampo de 6 cadáveres caninos jovens/adultos (até 8 anos de idade) e 12 idosos (idade igual ou superior a 10 anos), de ambos os sexos e de qualquer raça, os quais foram submetidos à: coloração de Nissl, para a identificação de células nervosas; imuno-histoquímica, para o estudo da expressão do MR e GR; e marcação fluorescente (fluoro-jade B), para detecção de neurônios em degeneração. A camada polimórfica do GD de cães idosos apresentou redução (em 16%) na contagem de neurônios e maior número de neurônios em degeneração. Em conjunto com estas alterações celulares, a menor marcação/expressão do MR e a menor relação MR:GR foram correlacionadas com a degeneração neuronal na camada polimórfica do GD de cães idosos. Além disso, cadelas idosas apresentaram menor densidade celular na camada granular, quando comparadas aos machos idosos, sugerindo uma ação diferencial dos esteroides sexuais nas alterações do GD. Os resultados indicam que o desbalanço na relação MR:GR pode interferir na sobrevivência neuronal no GD de cães idosos. / The mineralocorticoid (MR) and glucocorticoid (GR) receptors bind the glucocorticoid hormones. The chronic and excessive GR activation leads to hippocampus atrophy. By contrast, MR activation is neuroprotective. Considering the aging changes in the dentate gyrus (DG) and the possible participation of MR and GR in this process, the objective of this study was to correlate the unbalance of these receptors with the neuronal degeneration in this hippocampal region of aged dogs. For that purpose, cadaveric histologic sections of hippocampus of 6 young/adult dogs (until 8 years old) and 12 aged dogs (more than 10 years old), of both sex and any breed, were included. The Nissl staining and imunochemistry were performed to identify nerve cells and to study the MR and GR expression, respectively. Moreover, fluorescent labeling (fluoro- Jade B) was used to detect degenerating neurons. The polimorfic layer of the DG of aged dogs showed reduction (of 16%) on the neurons counting and more degenerating neurons. Together with this cells changes, the less MR expression and MR:GR relation were correlated with the neuronal degeneration in the polimorfic layer of DG of aged dogs. Besides that, aged females presented lower cell density in the granular layer, when compared to aged males, suggesting a differential sex steroid action on changes in the GD. Our results indicate that unbalance on the MR:GR relation may interfere with neuronal survival in the DG of aged dogs.