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Estudo da interação entre ATP e glutamato em neurônios do núcleo paraventricular do hipotálamo e sua relação com a resposta simpatoexcitatória induzida por alterações na osmolaridade. / Study of the interaction between ATP and glutamate in neurons of the paraventricular nucleus of the hypothalamus and its relationship with the sympathoexcitatory response induced by changes in osmolarity.Hildebrando Candido Ferreira Neto 28 November 2014 (has links)
Neste trabalho investigamos a interação entre ATP-glutamato na modulação de potenciais de ação e atividade sináptica de neurônios PVN-RVLM, além de avaliar se esta interação induziria mudanças na atividade simpática lombar (ANSL) por estímulo osmótico. Utilizamos de técnicas de imunohistoquímica, whole-cell patch clamp e registro eletroneurográfico. Observou-se que o ATP aumenta a frequência de potenciais de ação em neurônios PVN-RVLM, efeito bloqueado por acido quinurênico (KYN) e PPADS. A injeção de ATP no PVN aumenta a ANSL (25 nmol: 72%), um efeito atenuado por PPADS e/ou KYN, e também por CNQX. O ATP não afeta a função sináptica, mas aumenta correntes glutamatérgicas induzidas por aplicação AMPA em 52%, a qual foi bloqueada por PPADS ou por quelação de Ca2+ intracelular. Além disso, o estímulo osmótico ativa neurônios do PVN que expressam receptores P2X2 e potencia as correntes mediadas por AMPA (53%), um efeito bloqueado por PPADS. Finalmente, demonstrou-se que receptores P2 no PVN são importantes na simpatoexcitação induzida por estímulo osmótico agudo. / In the present study we investigate the interaction of ATP-glutamate on the firing activity and synaptic function in PVN-RVLM neurons, besides whether that interaction would be translated in changes on sympathetic nerve activity (SNA) induced by osmotic stimulus. Immunohistochemistry, whole-cell patch clamp and electroneurography technical approaches were used. Our data have shown that ATP increases firing rate of PVN-RVLM neurons, an effect blocked by kynurenic acid (KYN) or PPADS. ATP injection into the PVN enhanced SNA (72%), which was attenuated by PPADS and/or KYN, or CNQX. ATP did not affect synaptic function but, glutamatergic currents evoked by AMPA application were augmented with ATP (AMPA area: 52%), blocked by PPADS and chelation of intracellular Ca2+. In addition, we observed that acute osmotic stimulus activates P2X2 expressing neurons in the PVN. Moreover, an osmotic challenge potentiated AMPA responses (53%), an effect blocked by PPADS. Finally, we demonstrated that P2 receptors in the PVN are important for osmotically-driven sympathoexcitation.
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Treinamento aeróbio x disfunção autonômica na hipertensão espontânea: uma abordagem molecular em núcleos centrais de regulação. / Aerobic training vs autoniomic dysfunction in spontaneous hypertension: a molecular approach in the autonomic control areas.Masson, Gustavo Santos 28 July 2014 (has links)
Disfunção autonômica, inflamação e estresse oxidativo são características da hipertensão. Investigamos a cronologia das adaptações fisiológicas e celulares induzidas pelo treinamento aeróbio em ratos espontaneamente hipertensos (SHR). SHR exibiam disfunção autonômica e, no núcleo Paraventricular no hipotálamo (PVN), estresse oxidativo e inflamação. Duas semanas de treinamento aeróbio normalizaram a função autonômica, estresse oxidativo, inflamação, ativação de microglia e conteúdo de HMGB no PVN. Após 8 semanas, SHR treinados apresentaram menor pressão arterial e resistência vascular periférica. Redução do conteúdo de HMGB1 consiste num mecanismo para explicar os benefícios do treinamento, já que infusão aguda intracerebroventricular de HMGB1 produziu disfunção autonômica e ativação de microglia pela sinalização do CxCr4. Assim, redução do estresse oxidativo e da inflamação induzida pelo treinamento contribui para a reversão da disfunção autonômica na hipertensão e a redução da liberação de HMGB1 explica estes benefícios. / Autonomic dysfunction, inflammation and oxidative stress are hallmarks in hypertension. We evaluated time-course of physiologic and cellular adaptations induced by aerobic training in spontaneous hypertensive rat (SHR). SHR showed autonomic dysfunction and, in the hypothalamic paraventricular nucleus (PVN), oxidative stress and inflammation. 2-weeks of aerobic training normalized autonomic function, oxidative stress, inflammation, microglia activation and HMGB1 content into the PVN. After 8-weeks, trained SHR exhibited lower arterial pressure and peripheral vascular resistance. Decrease of HMGB1 content is a mechanism to explain these training benefits, since HMGB1 intracerebroventricular acute infusion induced autonomic dysfunction, microglia activation through CxCr4 signaling. So, decrease of oxidative stress and inflammation induced by aerobic training contributes to reverse autonomic dysfunction in hypertension and decrease of HMGB1 content explains these benefits.
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Caracterización de los cambios celulares y moleculares en el sistema cerebral del estrés durante la dependencia de morfina / Cellular and molecular changes in the stress-responsive system during morphine dependenceNúñez Parra, María Cristina 11 November 2008 (has links)
Morphine withdrawal increases the HPA axis activity, which is dependent on a hyperactivity of noradrenergic pathways (NTS-A2) innervating the PVN. In this Thesis we found that morphine withdrawal resulted in an increase in ACTH and corticosterone secretion and a neuronal activation in the PVN. Additionally, we found robust increases in CRF and AVP hnRNAs in the PVN, concomitantly with an increase in c-Fos expression. Morphine withdrawal activated ERK1/2 in PVN and NTS, which could be involved in the c-Fos expression. Morphine withdrawal induced an increase in TH mRNA levels in the NTS-A2, total TH protein in the NTS and TH phosphorylation at Ser31 in PVN and NTS-A2, which resulted in an augmentation of TH activity in the PVN. The enhancement in TH phosphorylated at Ser31 was blocked by SL327 in both nuclei. Finally, we have found that adrenalectomy eliminated the hyperactivity of noradrenergic pathways innervating the PVN during morphine withdrawal. / La abstinencia a morfina aumenta la actividad del eje HHA, que depende de la hiperactivación de las vías noradrenérgicas (NTS-A2) que inervan al PVN. En esta Tesis encontramos que la abstinencia a morfina induce la liberación de ACTH y corticosterona y la activación neuronal del PVN. Además, observamos un aumento en los niveles de hnRNA para CRF y AVP en el PVN, concomitantemente con un incremento en la expresión de c-Fos. El síndrome de abstinencia a morfina activó a ERK1/2 en PVN y NTS. También indujo un incremento en el mRNA para TH en el NTS-A2, proteína TH total y fosforilación de TH en su Ser31 en PVN y NTS-A2, que se tradujo en un aumento en su actividad enzimática. El aumento de TH fosforilada en Ser31 fue bloqueado por SL327. Finalmente, la adrenalectomía eliminó la hiperactividad de las vías noradrenérgicas que inervan al PVN durante la abstinencia a morfina.
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Serotonergic Responsiveness in Hypothalamic NeuronsTung, Stephanie S. Y. 04 December 2012 (has links)
Serotonin (5-HT) has been implicated in energy homeostasis. There is growing evidence
that 5-HT, acting through the 5-HT1BR in the paraventricular nucleus of the hypothalamus
(PVN), is important to this regulation. To investigate the cellular events underlying 5-HT1BR action, a PVN neuronal cell model was established. The mHypoA-2/30 cell line expresses a complement of markers and neuropeptides specifically localized to the PVN. 5-HT induces neuronal activation in a dose-dependent manner as determined by an elevation in cFos mRNA levels. As 5-HT exerted limited transcriptional control, the integrity of 5-HT signaling machinery was assessed. 5-HT signals through cAMP and calcium secondary messenger systems by
suppressing cAMP and elevating intracellular calcium, effects that are mimicked by activating the 5-HT1BR and that are attenuated in the presence of inhibitors. These findings support the use of this novel PVN cell model for delineating components involved in direct 5-HT action in PVN neurons.
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Serotonergic Responsiveness in Hypothalamic NeuronsTung, Stephanie S. Y. 04 December 2012 (has links)
Serotonin (5-HT) has been implicated in energy homeostasis. There is growing evidence
that 5-HT, acting through the 5-HT1BR in the paraventricular nucleus of the hypothalamus
(PVN), is important to this regulation. To investigate the cellular events underlying 5-HT1BR action, a PVN neuronal cell model was established. The mHypoA-2/30 cell line expresses a complement of markers and neuropeptides specifically localized to the PVN. 5-HT induces neuronal activation in a dose-dependent manner as determined by an elevation in cFos mRNA levels. As 5-HT exerted limited transcriptional control, the integrity of 5-HT signaling machinery was assessed. 5-HT signals through cAMP and calcium secondary messenger systems by
suppressing cAMP and elevating intracellular calcium, effects that are mimicked by activating the 5-HT1BR and that are attenuated in the presence of inhibitors. These findings support the use of this novel PVN cell model for delineating components involved in direct 5-HT action in PVN neurons.
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Dissection du programme développemental du noyau paraventriculaire de l'hypothalamus.Caqueret, Aurore 03 1900 (has links)
Une cascade de facteurs de transcription composée de SIM1, ARNT2, OTP, BRN2 et SIM2 est requise pour la différenciation des cinq types cellulaires qui peuplent le noyau paraventriculaire (PVN) de l’hypothalamus, un régulateur critique de plusieurs processus physiologiques essentiels à la survie. De plus, l’haploinsuffisance de Sim1 est aussi une cause d’hyperphagie isolée chez la souris et chez l’homme. Nous désirons disséquer le programme développemental du PVN, via une approche intégrative, afin d’identifier de nouveaux gènes qui ont le potentiel de réguler l’homéostasie chez l’individu adulte.
Premièrement, nous avons utilisé une approche incluant l’analyse du transcriptome du PVN à différents stades du développement de la souris pour identifier de tels gènes. Nous avons comparé les transcriptomes de l’hypothalamus antérieur chez des embryons de souris Sim1+/+ et Sim1-/- à E12.5 issus de la même portée. De cette manière, nous avons identifié 56 gènes agissant en aval de Sim1 dont 5 facteurs de transcription - Irx3, Sax1, Rxrg, Ror et Neurod6. Nous avons également proposé un modèle de développement à deux couches de l’hypothalamus antérieur. Selon ce modèle, les gènes qui occupent un domaine médial dans la zone du manteau caractérisent des cellules qui peupleront le PVN alors que les gènes qui ont une expression latérale identifient des cellules qui donneront plus tard naissance aux structures ventrolatérales de l’hypothalamus. Nous avons aussi démontré que Sim1 est impliqué à la fois dans la différenciation, la migration et la prolifération des neurones qui peuplent le PVN tout comme Otp. Nous avons également isolé par microdissection au laser le PVN et l’hypothalamus médiobasal chez des souris de type sauvage à E14.5 pour en comparer les transcriptomes. Ceci nous a permis d’identifier 34 facteurs de transcription spécifiques au PVN et 76 facteurs spécifiques à l’hypothalamus médiobasal. Ces gènes représentent des régulateurs potentiels du développement hypothalamique.
Deuxièmement, nous avons identifié 3 blocs de séquences au sein de la région 5’ d’Otp qui sont conservés chez l’homme, la souris et le poisson. Nous avons construit un transgène qui est composé d’un fragment de 7 kb contenant ces blocs de séquences et d’un gène rapporteur. L’analyse de 4 lignées de souris a montré que ce transgène est uniquement exprimé dans le PVN en développement. Nous avons généré un deuxième transgène dans lequel le fragment de 7 kb est inséré en amont de l’ADNc de Brn2 ou Sim1 et de Gfp. Nous avons obtenu quatre lignées de souris dans lesquels le profil d’expression de Brn2 et de Gfp reproduit celui d’Otp. Nous étudierons le développement du PVN et la prise alimentaire chez ces souris. En parallèle, nous croisons ces lignées avec les souris déficientes en Sim1 pour déterminer si l’expression de Brn2 permet le développement des cellules du PVN en absence de Sim1. En résumé, nous avons généré le premier transgène qui est exprimé spécifiquement dans le PVN. Ce transgène constitue un outil critique pour la dissection du programme développemental de l’hypothalamus.
Troisièmement, nous avons caractérisé le développement de l’hypothalamus antérieur chez l’embryon de poulet qui représente un modèle intéressant pour réaliser des études de perte et de gain de fonction au cours du développement de cette structure. Il faut souligner que le modèle de développement à deux couches de l’hypothalamus antérieur semble être conservé chez l’embryon de poulet où il est aussi possible de classer les gènes selon leur profil d’expression médio-latéral et le devenir des régions qu’ils définissent.
Finalement, nous croyons que cette approche intégrative nous permettra d’identifier et de caractériser des régulateurs du développement du PVN qui pourront potentiellement être associés à des pathologies chez l’adulte telles que l’obésité ou l’hypertension. / A cascade of transcription factors composed of SIM1, ARNT2, OTP, BRN2 and SIM2 is required for the differentiation of the five major cell types populating the paraventricular nucleus (PVN) of the hypothalamus, a critical integrator of several homeostatic processes that are required for the survival of vertebrates. Haploinsufficency of Sim1 also causes isolated hyperphagia in mice and humans. The goal of our study is to dissect the developmental program of the PVN using an integrative approach in order to identify new genes that could potentially be implicated in the regulation of homeostasis in adults.
First, we used a comparative approach to analyse the PVN transcriptome at different developmental stages in mice embryos in order to identity new genes implicated in PVN development. We compared gene expression in the anterior hypothalamus of E12.5 Sim1-/- and Sim1+/+ littermate embryos using a microarray approach. We identified 56 genes acting downstream of Sim1 including 5 transcription factors - Irx3, Sax1, Rxrg, Ror and Neurod6. We proposed a model for the development of the anterior hypothalamus. In this model, the genes expressed in the medial domain of the mantle layer characterise cells that will form the PVN and genes expressed in the lateral domain identify cells that will give rise to ventrolateral areas of the hypothalamus. We also showed that Sim1, like Otp, is implicated in the differentiation, migration and proliferation of the neurons populating the PVN. Furthermore, we have isolated by laser captured microdissection the PVN and ARC nucleus in wild type mice at E14.5 and compared their transcriptomes. This technique allowed us to identity 34 transcription factors specific to the PVN and 76 factors specific to the ARC. These genes represent potential regulators of hypothalamic development.
Second, we identified 3 blocks of sequence in the 5’ region of Otp that are conserved between human, mouse and fish. We constructed a transgene which included a 7 Kb fragment encompassing these sequences followed by a reporter gene. The analysis of 4 mice strains showed that this transgene is specifically expressed in the prospective PVN. We have generated a second transgene in which the 7 Kb fragment is located upstream of the cDNA encoding Brn2 or Sim1 and Gfp. We obtained 4 mice strains in which the Brn2 and Gfp expression pattern is similar to the Otp expression pattern. These mice will be used to study PVN development and food intake. Also, to determine if Brn2 expression only – without Sim1 gene expression - allows the development of PVN cells, we are presently crossing these mice with Sim1 deficient mice. In conclusion, we have generated the first transgene that is specifically expressed in the PVN. This transgene constitutes a critical tool for dissecting the developmental program of the hypothalamus.
Third, we have characterised the development of the anterior hypothalamus of chick embryos which represent an interesting model for loss and gain of function experiments during the development of this brain region. Interestingly, our proposed model for the development of the anterior hypothalamus seems to be conserved in chick embryos. As a matter of fact, it is possible to classify genes according to their medio-lateral expression patterns and the outcome of the regions that they are defining.
Finally, we believe that this integrative approach will allow us to identify and characterize factors implicated in PVN development. From a clinical point of view, these factors could potentially be associated with pathologies such as obesity or arterial hypertension.
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Dissection du programme développemental du noyau paraventriculaire de l'hypothalamusCaqueret, Aurore 03 1900 (has links)
No description available.
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Treinamento aeróbio x disfunção autonômica na hipertensão espontânea: uma abordagem molecular em núcleos centrais de regulação. / Aerobic training vs autoniomic dysfunction in spontaneous hypertension: a molecular approach in the autonomic control areas.Gustavo Santos Masson 28 July 2014 (has links)
Disfunção autonômica, inflamação e estresse oxidativo são características da hipertensão. Investigamos a cronologia das adaptações fisiológicas e celulares induzidas pelo treinamento aeróbio em ratos espontaneamente hipertensos (SHR). SHR exibiam disfunção autonômica e, no núcleo Paraventricular no hipotálamo (PVN), estresse oxidativo e inflamação. Duas semanas de treinamento aeróbio normalizaram a função autonômica, estresse oxidativo, inflamação, ativação de microglia e conteúdo de HMGB no PVN. Após 8 semanas, SHR treinados apresentaram menor pressão arterial e resistência vascular periférica. Redução do conteúdo de HMGB1 consiste num mecanismo para explicar os benefícios do treinamento, já que infusão aguda intracerebroventricular de HMGB1 produziu disfunção autonômica e ativação de microglia pela sinalização do CxCr4. Assim, redução do estresse oxidativo e da inflamação induzida pelo treinamento contribui para a reversão da disfunção autonômica na hipertensão e a redução da liberação de HMGB1 explica estes benefícios. / Autonomic dysfunction, inflammation and oxidative stress are hallmarks in hypertension. We evaluated time-course of physiologic and cellular adaptations induced by aerobic training in spontaneous hypertensive rat (SHR). SHR showed autonomic dysfunction and, in the hypothalamic paraventricular nucleus (PVN), oxidative stress and inflammation. 2-weeks of aerobic training normalized autonomic function, oxidative stress, inflammation, microglia activation and HMGB1 content into the PVN. After 8-weeks, trained SHR exhibited lower arterial pressure and peripheral vascular resistance. Decrease of HMGB1 content is a mechanism to explain these training benefits, since HMGB1 intracerebroventricular acute infusion induced autonomic dysfunction, microglia activation through CxCr4 signaling. So, decrease of oxidative stress and inflammation induced by aerobic training contributes to reverse autonomic dysfunction in hypertension and decrease of HMGB1 content explains these benefits.
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Plasticity in the intermediolateral cell column of the spinal cord following injury to sympathetic postganglionic axonsGannon, Sean Michael 11 August 2014 (has links)
No description available.
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Central Mechanisms Regulating Pituitary-Adrenal Activity in Infant Guinea Pigs (Cavia porcellus) during Exposure to Psychological Stressors: Independent and Combined Effects of Maternal Separation and NoveltyMaken, Deborah Suzanne 11 December 2009 (has links)
No description available.
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