Os núcleos da rafe são agrupamentos celulares cujo principal tipo é o serotoninérgico. Em anuros adultos a estimulação elétrica e química da rafe bulbar não causa alteração na ventilação, enquanto que em mamíferos, dependendo da localização exata desta estimulação, a ventilação pode ser inibida ou estimulada. Ainda em mamíferos, os núcleos bulbares da rafe participam da resposta cardiorrespiratória à hipóxia (5% O2) e hipercarbia (5% CO2), enquanto que em anfíbios isto ainda não foi investigado. Portanto, o objetivo do presente trabalho foi investigar a participação dos núcleos da rafe nas respostas cardiorrespiratórias à hipóxia e hipercarbia em sapos (Rhinella schneideri). Para isso, primeiramente os núcleos da rafe foram localizados e identificados na espécie estudada. Imunorreatividade para proteínas Fosrelacionadas foi utilizada para verificar se estes núcleos são ativados ou inibidos durante o estímulo hipóxico e hipercárbico, e, adicionalmente, o papel dos núcleos magno e pontino da rafe nas respostas cardiorrespiratórias à hipóxia e hipercarbia foi investigado por meio da lesão nãoseletiva com ácido ibotênico. A ventilação pulmonar foi medida diretamente pelo método pneumotacográfico e a pressão arterial por canulação da artéria femoral. Os resultados demonstram que os núcleos bulbares da rafe são inibidos após o estímulo hipóxico e hipercárbico. A hipóxia causou aumento da ventilação pulmonar e da frequência cardíaca nos grupos veículo e lesado. A hipercarbia causou aumento da ventilação pulmonar em ambos os grupos. A lesão nãoseletiva dos núcleos magno e pontino da rafe não teve efeito na ventilação basal (durante normóxia normocárbica) e nas respostas cardiorrespiratórias à hipóxia e hipercarbia, sugerindo que estes núcleos não participam do controle da ventilação em condições basais ou durante a hipóxia e hipercarbia em sapos. / The raphe nuclei are brainstem cell groups and the main cell type is serotonergic. In adult anurans electrical or chemical stimulation of the medullary raphe has no effect on ventilation, whereas in mammals depending on the specific site of stimulation, an excitatory or inhibitory response is elicited. In mammals the medullary raphe participate in the ventilatory and cardiovascular response to hypoxia and hipercarbia while in amphibians there are no data available. Thus the present study was designed to investigate whether the raphe nuclei of toads (Rhinella schneideri) participate in the ventilatory and cardiovascular response to hypoxia (5% O2) and hipercarbia (5% CO2). First the raphe nuclei were located and identified. Fosrelated immunoreactivity was used to verify whether the raphe nuclei are activated or inhibited by hypoxia or hypercarbia. In addition, the role of raphe magnus and pontis in the respiratory and cardiovascular responses to hypoxia and hypercarbia were investigated by nonselective lesioning with ibotenic acid. Pulmonary ventilation was directly measured by pneumotachographic method and the arterial pressure by a femoral catheter. The results indicate that the medullary raphe nuclei are inhibited after hypoxic and hypercarbic stimulus (2,5h). Hypoxia caused hyperventilation and increase in the cardiac frequency in the vehicle and lesioned groups. Hypercarbia caused hyperventilation in both groups. Chemical lesions in the raphe magnus and pontis did not affect ventilatory or cardiovascular variables under resting conditions (normoxic normocárbica) or during the response to hypoxia or hipercarbia.

Chronic stress and dysfunction of the serotonergic system in the brain have been considered two of the major risks for development of depression. In this study, adult Fischer 344 rats were subjected to a regimen of chronic social defeat (CSD). To mimic stressful conditions, some rats were not exposed to CSD, but instead treated with corticosterone (CORT) in oral solution while maintained in their home cage. Protein levels of the serotonin transporter (SERT) in the dorsal raphe nucleus (DRN), hippocampus, frontal cortex, and amygdala were examined by Western blotting or immunofluorescence staining. The results showed that CSD up-regulated SERT protein levels in the DRN, hippocampus, frontal cortex, and amygdala regions. This up-regulation was abolished or prevented by adrenalectomy, or treatment with antagonists of corticosteroid receptors mifepristone and spironolactone, alone or in combination. Similarly, up-regulated SERT protein levels in these brain regions were also observed in rats treated with oral CORT ingestion, which was analogously prevented by treatment with mifepristone and spironolactone. Furthermore, both CSD- and CORT-induced up-regulation of SERT protein levels in the DRN and three brain regions were attenuated by simultaneous treatment with fluoxetine, an antidepressant that specifically inhibits serotonin reuptake. The results indicate that up-regulation in SERT protein levels in the DRN and forebrain limbic structures caused by CSD regimen was mainly motivated by CORT through corticosteroid receptors. The present findings demonstrate that chronic stress is closely correlated with the serotonergic system by acting on the regulation of the SERT expression in the DRN and its projection regions, which may contribute to the development of depression. Chronic stress and dysfunction of the serotonergic system are etiologically related to depression. In an attempt to explore their interaction, we found that chronic social defeat upregulated expression of serotonin transporter in the DRN and the projection regions, which may induce an alteration of serotonin transformation in the brain. This interaction may account for the development of this disease.

Insulin-like 6 immunoreactivity (irINSL6) was detected in Leydig cells of the mouse testis. In the brain, labeled somata were detected mainly in the caudal hypothalamus and midbrain. Double labeling the brainstem sections revealed that irINSL6 somata were 5-hydroxytryptamine (5-HT) positive. The presence of irINSL6 in discrete populations of hypothalamic and brainstem neurons and in Leydig cells of the testis suggests a diverse biological function of this novel peptide.

No description available.

Il y a longtemps qu’on a attribué à l’hippocampe un rôle central dans la mémoire, mais ce n’est pas son unique rôle. Un nombre grandissant d’études attestent que l’hippocampe peut être séparé en deux régions, dorsale et ventrale, qui sont fonctionnellement différentes. La partie dorsale de l’hippocampe est responsable du rôle classique dans la mémoire spatiale et contextuelle, alors que la région ventrale de l’hippocampe est importante dans l’expression de l’anxiété et de la motivation, entre autres. Les projections des noyaux du raphé, l’unique source d’afférences sérotoninergiques de l’hippocampe, auraient un rôle régulateur sur ses fonctions, dont le comportement anxieux. Toutefois, les fonctions de la projection sérotoninergique raphé-hippocampe ventral ne sont pas entièrement caractérisées et les différents rôles des sous-populations de neurones sérotoninergiques au sein même de la projection raphé-hippocampe ventral sont peu connus. Dans ce projet de recherche, nous avons utilisé des tests comportementaux et des outils optogénétiques, afin de déterminer le rôle de la projection sérotoninergique raphé-hippocampe ventral dans le comportement d’aversion. Notre hypothèse est que la sérotonine régule l’anxiété en agissant sur l’hippocampe ventral via cette projection. Nous démontrons entre autres que l’activation de la projection sérotoninergique raphé-hippocampe ventral induit une hausse de l’anxiété, mais spécifiquement chez les femelles. Nous démontrons aussi que l’activation de la projection réduit la locomotion. Nos données offrent un nouveau point de vue sur le rôle du raphé médian dans l’anxiété ainsi que sur l’importance du sexe dans l’expression du comportement anxieux. / It has been known for a long time that the hippocampus has a central role in memory, but it isn’t its sole function. A growing number of studies are showing that the hippocampus can be split in two regions, dorsal and ventral, that are functionally different. The dorsal part is responsible for the classic and well-known role of the hippocampus in spatial and contextual memory, while the ventral region is important for the expression of anxiety and motivation, among other roles. The only serotonergic input of the hippocampus are the raphe nuclei and it has been suggested that it has a regulatory effect over its functions, such as anxiety. Nonetheless, the functions of the raphe-ventral hippocampus serotonergic projection are not fully characterized and sub-populations of serotonergic neurons inside the projection itself aren’t known. In this research project, we used behavioral tests and optogenetic tools to determine whether the raphe to ventral hippocampus serotonergic projection is able to influence aversive behaviors. Our hypothesis is that serotonin regulates anxiety through its influence on the ventral hippocampus via the raphe-ventral hippocampus serotonergic projection. We found that optogenetic activation of the projection induces heightened anxiety, but only in female mice. Our data offer new insight as to how the median raphe regulates anxiety and the importance of sex in the expression of anxiety.

Estrogens greatly affect the activity and connectivity of serotonergic neural cell populations, which extend from clusters of nuclei in the brainstem, termed the raphe nuclei, where estrogen receptor β is the most abundantly expressed estrogen receptor subtype. Estrogenic effects on the raphe nuclei are primarily important for influencing various neuropsychological behaviors, including depression, mood swings and anxiety behaviors. Because of this connection, phases of intense hormone fluctuations for instance during menopause are often associated with several mood disturbances that often reduce the quality of life of menopausal women. Accordingly, long-term use of hormone replacement therapy appeared to be the method of choice for many menopausal women to help alleviate vasomotor symptoms, which may include neuropsychological changes such as depression. However, given the limitations and number of serious health risks attributed to hormone replacement therapy, natural compounds such as phytoestrogens are receiving widespread awareness due to their occurrence in medicinal plant extracts and a wide variety of food items including dietary supplements with respective health claims. Flavonoids, particularly the isoflavones and the naringenin-type flavanones, belong to a group of polyphenolic plant-derived secondary metabolites known to possess estrogen-like bioactivities. Nevertheless, little is known about their transactivational activity and their potential to regulate endogenous gene expression of estrogen responsive genes in the raphe nuclei due to the lack of suitable cellular models expressing sufficient amounts of functional estrogen receptor β. Hence, a raphe nuclei-derived cell line that expresses a functional estrogen receptor β was sought as a model to investigate effects of flavonoids in vitro. In this regard, RN46A-B14 cells derived from embryonic day 13 rat medullary raphe nuclei were primarily used in this study as the main cellular model. Nonetheless, expression of endogenous estrogen receptor β in these cells was not sufficient to pursue downstream investigations of estrogen-dependent activities. To overcome this deficit, a rat raphe nuclei-derived in vitro model that overexpresses a functional estrogen receptor β was initially established (herein termed RNDA cells) by stably transducing its parent cell line, RN46A-B14 cells, with a suitable lentiviral expression vector encoding a human estrogen receptor β gene. The stable expression and the functional characterization of the transgenic receptor was confirmed by Western blot analysis and luciferase reporter gene assays, respectively. The same reporter gene assay was used to scrutinize the transactivational activity of the flavonoids in RNDA cells. Key results revealed that Genistein, Daidzein, Equol, Naringenin and 8-Prenylnaringenin demonstrated high transactivational activity in a concentration-dependent manner by stimulating luciferase expression from an estrogen responsive element-regulated reporter gene construct transiently transfected in RNDA cells. Low transactivational activity was observed in RNDA cells in response to increasing concentrations of 7-(O-prenyl)naringenin-4'-acetate. However, no transactivational activity was noticed in response to 6-(1,1-Dimethylallyl)naringenin in the studied cell model. All effects elicited by the flavonoids were antagonized by the pure estrogen receptor antagonist, Fulvestrant, indicating that all substances act by binding to and activating the transgenic ERβ. Additional effects were observed in RNDA cells in response to a co-treatment of 1 µM of either Genistein or Daidzein, but not Equol, with 10 nM 17β-Estradiol. Slight antagonistic effects were observed in the same studied cell line when either 8-Prenylnaringenin or 7-(O-prenyl)naringenin-4'-acetate, but not Naringenin or 6-(1,1-Dimethylallyl)naringenin, were co-added with 17β-Estradiol. Results from the reporter gene assays were validated on the basis of regulation of mRNA expression of estrogen responsive genes following the global assessment of 17β-Estradiol-induced gene expression in this cell line using a DNA microarray technique. Out of 212 estrogen-regulated genes with at least two-fold change of expression, six were selected according to specific features of estrogenic regulation of expression. The expression of the six selected 17β-Estradiol-regulated genes was validated using quantitative real-time PCR analysis. The regulation of mRNA expression of the selected genes in response to the tested flavonoids was then investigated in RNDA cells. Additionally, because RNDA cells encode a temperature-sensitive mutant of the Simian Virus 40 large T-antigen, their neuronal differentiation is constitutive upon shifting them from conditions promoting proliferation (permissive temperature) to differentiation (non permissive temperature). Hence, the regulation of mRNA expression of the selected genes in response to the tested flavonoids was additionally investigated as RNDA cells differentiate. In RNDA cells grown under proliferative conditions, 17β-Estradiol up-regulated mRNA expression of camello-like 5, sex determining region Y-box 18 and keratin type I cytoskeletal 19. Similar effects were observed in response to 8-Prenylnaringenin, Genistein, Daidzein and Equol. In addition, 17β-Estradiol down-regulated mRNA expression of neurofilament medium polypeptide and zinc finger DHHC-type containing 2. Similar effects were observed in response to 8-Prenylnaringenin, Naringenin, Genistein, Daidzein and Equol. Yet, no effect was observed on the regulation of mRNA expression of solute carrier family 6 member 4 in response to 17β-Estradiol or the flavonoids in RNDA cells grown under proliferative conditions. When RNDA cells were shifted to conditions promoting differentiation, changes in cell morphology, in mRNA expression levels and in responsiveness towards 17β-Estradiol or the flavonoids were observed. These expression studies additionally highlighted some of the genes as indicator genes for RNDA cellular differentiation. The newly established RNDA cell line should prove useful to elucidate basic physiological properties of estrogen receptor β in the raphe nuclei. The present study should serve as the basis to help shed light on molecular and cellular mechanisms following the action of phytoestrogens, endocrine disruptors or other exogenous estrogen receptor ligands in neural cell populations, particularly the raphe nuclei, for further applications within the brain.

Made available in DSpace on 2014-12-17T15:37:01Z (GMT). No. of bitstreams: 1 JoacilGS_DISSERT.pdf: 4166780 bytes, checksum: d28a491081df0a86a997a1a2664b7535 (MD5) Previous issue date: 2010-12-28 / Serotonin or 5-hydroxytryptamine (5-HT) is a substance found in many tissues of the body, including as a neurotransmitter in the nervous system, in which may exert varied post-synaptic actions. Inside the neuro-axis, the location of 5-HT neurons is almost restricted to the raphe nuclei of the brainstem, such that 5-HT-immunoreactivity can be considered a marker of the raphe nuclei. The raphe nuclei are located in the brainstem, at or near the midline. The serotonergic groups were originally alphanumerically classified as B1 to B9 towards caudorrostral in rats and can be divided into upper and lower groups. In this study the distribution of serotonergic neurons was studied using immunohistochemistry in the brain of the rock cavy (Kerodon rupestris), a species of rodent endemic to Northeastern Brazil. The cytoarchitectonic location of serotonergic neurons was established in series of adjacent coronal and sagittal sections stained by the Nissl method and immunohistochemistry for 5-HT. Thus, we defined the raphe rostral linear, caudal linear, dorsal, median, and paramedian pontine raphe nuclei, and B9 cluster, constituting the rostral group, and the interpositus, magnus, obscure and palidus, constituting the caudal part of the group, comparable to which has been described for other mammalian species / A serotonina ou 5-hidroxitriptamina (5-HT) ? uma subst?ncia encontrada em muitos tecidos do organismo, inclusive no sistema nervoso como neurotransmissor, onde pode exercer a??es p?s-sin?pticas variadas. Dentro do neuro-eixo, a localiza??o dos neur?nios 5-HT ? quase absoluta nos n?cleos da rafe do tronco encef?lico, de tal maneira que 5-HT neuronal pode ser considerada um marcador dos n?cleos da rafe. Os n?cleos da rafe est?o localizados no tronco encef?lico, na linha m?dia ou suas proximidades. Os grupamentos serotonin?rgicos foram originalmente classificados alfanumericamente como B1 a B9 no sentido caudorrostral no rato e podem ser divididos em grupos superior e inferior. Neste trabalho a distribui??o dos neur?nios serotonin?rgicos foi estudada com imunoistoqu?mica no c?rebro do moc? (Kerodon rupestris), uma esp?cie de roedor end?mica da regi?o Nordeste do Brasil. A localiza??o citoarquitet?nica dos neur?nios serotonin?rgicos foi estabelecida em s?ries de sec??es coronais e sagitais adjacentes submetidas a colora??o pelo m?todo de Nissl e imunoistoqu?mica para 5-HT. Assim, foram delimitados os n?cleos da rafe linear rostral, linear caudal, dorsal, mediano, paramediano e pontino da rafe e grupamento B9, compondo o grupo rostral, e os n?cleos interp?sito, magno, obscuro e p?lido, compondo o grupo caudal, compar?vel ao que j? foi descrito para outras esp?cies de mam?feros

Estrogens greatly affect the activity and connectivity of serotonergic neural cell populations, which extend from clusters of nuclei in the brainstem, termed the raphe nuclei, where estrogen receptor β is the most abundantly expressed estrogen receptor subtype. Estrogenic effects on the raphe nuclei are primarily important for influencing various neuropsychological behaviors, including depression, mood swings and anxiety behaviors. Because of this connection, phases of intense hormone fluctuations for instance during menopause are often associated with several mood disturbances that often reduce the quality of life of menopausal women. Accordingly, long-term use of hormone replacement therapy appeared to be the method of choice for many menopausal women to help alleviate vasomotor symptoms, which may include neuropsychological changes such as depression. However, given the limitations and number of serious health risks attributed to hormone replacement therapy, natural compounds such as phytoestrogens are receiving widespread awareness due to their occurrence in medicinal plant extracts and a wide variety of food items including dietary supplements with respective health claims. Flavonoids, particularly the isoflavones and the naringenin-type flavanones, belong to a group of polyphenolic plant-derived secondary metabolites known to possess estrogen-like bioactivities. Nevertheless, little is known about their transactivational activity and their potential to regulate endogenous gene expression of estrogen responsive genes in the raphe nuclei due to the lack of suitable cellular models expressing sufficient amounts of functional estrogen receptor β. Hence, a raphe nuclei-derived cell line that expresses a functional estrogen receptor β was sought as a model to investigate effects of flavonoids in vitro. In this regard, RN46A-B14 cells derived from embryonic day 13 rat medullary raphe nuclei were primarily used in this study as the main cellular model. Nonetheless, expression of endogenous estrogen receptor β in these cells was not sufficient to pursue downstream investigations of estrogen-dependent activities. To overcome this deficit, a rat raphe nuclei-derived in vitro model that overexpresses a functional estrogen receptor β was initially established (herein termed RNDA cells) by stably transducing its parent cell line, RN46A-B14 cells, with a suitable lentiviral expression vector encoding a human estrogen receptor β gene. The stable expression and the functional characterization of the transgenic receptor was confirmed by Western blot analysis and luciferase reporter gene assays, respectively. The same reporter gene assay was used to scrutinize the transactivational activity of the flavonoids in RNDA cells. Key results revealed that Genistein, Daidzein, Equol, Naringenin and 8-Prenylnaringenin demonstrated high transactivational activity in a concentration-dependent manner by stimulating luciferase expression from an estrogen responsive element-regulated reporter gene construct transiently transfected in RNDA cells. Low transactivational activity was observed in RNDA cells in response to increasing concentrations of 7-(O-prenyl)naringenin-4'-acetate. However, no transactivational activity was noticed in response to 6-(1,1-Dimethylallyl)naringenin in the studied cell model. All effects elicited by the flavonoids were antagonized by the pure estrogen receptor antagonist, Fulvestrant, indicating that all substances act by binding to and activating the transgenic ERβ. Additional effects were observed in RNDA cells in response to a co-treatment of 1 µM of either Genistein or Daidzein, but not Equol, with 10 nM 17β-Estradiol. Slight antagonistic effects were observed in the same studied cell line when either 8-Prenylnaringenin or 7-(O-prenyl)naringenin-4'-acetate, but not Naringenin or 6-(1,1-Dimethylallyl)naringenin, were co-added with 17β-Estradiol. Results from the reporter gene assays were validated on the basis of regulation of mRNA expression of estrogen responsive genes following the global assessment of 17β-Estradiol-induced gene expression in this cell line using a DNA microarray technique. Out of 212 estrogen-regulated genes with at least two-fold change of expression, six were selected according to specific features of estrogenic regulation of expression. The expression of the six selected 17β-Estradiol-regulated genes was validated using quantitative real-time PCR analysis. The regulation of mRNA expression of the selected genes in response to the tested flavonoids was then investigated in RNDA cells. Additionally, because RNDA cells encode a temperature-sensitive mutant of the Simian Virus 40 large T-antigen, their neuronal differentiation is constitutive upon shifting them from conditions promoting proliferation (permissive temperature) to differentiation (non permissive temperature). Hence, the regulation of mRNA expression of the selected genes in response to the tested flavonoids was additionally investigated as RNDA cells differentiate. In RNDA cells grown under proliferative conditions, 17β-Estradiol up-regulated mRNA expression of camello-like 5, sex determining region Y-box 18 and keratin type I cytoskeletal 19. Similar effects were observed in response to 8-Prenylnaringenin, Genistein, Daidzein and Equol. In addition, 17β-Estradiol down-regulated mRNA expression of neurofilament medium polypeptide and zinc finger DHHC-type containing 2. Similar effects were observed in response to 8-Prenylnaringenin, Naringenin, Genistein, Daidzein and Equol. Yet, no effect was observed on the regulation of mRNA expression of solute carrier family 6 member 4 in response to 17β-Estradiol or the flavonoids in RNDA cells grown under proliferative conditions. When RNDA cells were shifted to conditions promoting differentiation, changes in cell morphology, in mRNA expression levels and in responsiveness towards 17β-Estradiol or the flavonoids were observed. These expression studies additionally highlighted some of the genes as indicator genes for RNDA cellular differentiation. The newly established RNDA cell line should prove useful to elucidate basic physiological properties of estrogen receptor β in the raphe nuclei. The present study should serve as the basis to help shed light on molecular and cellular mechanisms following the action of phytoestrogens, endocrine disruptors or other exogenous estrogen receptor ligands in neural cell populations, particularly the raphe nuclei, for further applications within the brain.