Projeções do núcleo pré-óptico medial (MPN) para a porção ventromedial do núcleo ventromedial do hipotálamo (VMHvl) compõe um circuito de controle do comportamento sexual feminino (CSF) influenciado por pistas endógenas e exógenas. Este CSF no adulto só ocorre se o sistema nervoso se desenvolveu corretamente, por meio da ação de hormônios esteroidais. Por isso, objetivamos estudar as projeções do MPN para o VMHvl em fêmeas controle (GC) e ooforectomizados (GO). O GC aos 90 dias de idade foi submetido à cirurgia estereotáxica para injeção iontoforética do traçador anterógrado Phaseolus vulgaris no MPN. Já o GO aos 30 dias de idade, foi submetido à ooforectomia bilateral e aos 90 dias de idade submetido à mesma cirurgia que o GC. Como resultado, nos dois grupos observamos marcação anterógrada no VMHvl, assim como para outras regiões que integram o circuito do CSF; porém observamos uma possível redução das projeções no GO. Sendo assim, estabelecemos a conectividade do MPN com o VMHvl, contudo observamos que no GO há uma alteração na densidade e extensão dessas projeções. / Medial preoptic nucleus (MPN) outputs to the ventromedial portion of the ventromedial hypothalamic nucleus (VMHvl) participates in the female sexual behavior (FSB) control circuit influenced by endogenous and environmental cues. This FSB in adults only occurs if the nervous system has developed correctly, through the action of steroid hormones. Thus, our goal is to study the MPN outputs to the VMHvl in control (CG) and ovariectomized (OG) female animals. For the control group (CG), at 90 days of age, these females underwent stereotactic surgery for iontophoretic injection of the anterograde tracer Phaseolus vulgaris into the MPN. The oophorectomized group (OG), at 30 days of age, underwent oophorectomy and when they reach 90 days of age they underwent the same surgery as CG. As a results, in both experimental groups, we observed anterograde labeling in VMHvl, as well as for other regions that are part of the FBS circuit; but in these nuclei, we observed a possible reduction in the density of the projections on the OG. Thus, we establish the connectivity of the MPN with the VMHvl, however we observe that in the GO there is a change in the density and extension of these projections.

Orientador: Arielle Cristina Arena / Resumo: Os anti-inflamatórios não esteroidais (AINEs), entre eles o Ibuprofeno, são amplamente utilizados para o tratamento da dor e de processos inflamatórios, e estão entre as classes de medicamentos mais utilizadas por gestantes. Através da inibição da enzima ciclo-oxigenase, os AINEs inibem a síntese de prostaglandinas, compostos eicosanoides que atuam não somente como mediadores e moduladores inflamatórios, mas também em diversos processos fisiológicos do organismo, como no mecanismo de diferenciação sexual hipotalâmica. O processo de masculinização do hipotálamo é dependente de testosterona, que por ação da enzima citocromo P450 aromatase, é convertida em estradiol. Este hormônio regula positivamente a expressão da enzima ciclo-oxigenase no hipotálamo, aumentando a produção de prostaglandina do subtipo E2 (PGE2), que atua aumentando a formação de espinhas dendríticas no núcleo sexualmente dimórfico da área pré-optica (SDN-POA). Em virtude da importância da PGE2 no processo de diferenciação sexual hipotalâmica, torna-se preocupante o uso de anti-inflamatórios durante a gestação. Desta forma, o objetivo desse estudo foi avaliar os possíveis efeitos resultantes da exposição in utero e lactacional ao ibuprofeno e suas repercussões tardias sobre parâmetros reprodutivos masculinos em ratos machos. Para tanto, ratas prenhes foram expostas a três doses de ibuprofeno (10; 30; 60 mg/kg) entre a última semana de prenhez (Dias gestacionais 15-21) até o final da lactação (Dias pós-natal 21)... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Non-steroidal anti-inflammatory drugs (NSAID), including Ibuprofen, are widely used in the treatment of pain and inflammatory processes, and are of the most commonly classes of drugs used by pregnant women. By inhibiting the cyclooxygenase enzyme (COX), NSAID inhibit the synthesis of prostaglandins, eicosanoids compounds that act not only as mediators and inflammatory modulators, but also in various physiological processes of the organism, such as in the mechanism of sexual hypothalamic differentiation. The hypothalamus masculinization process is testosterone dependent, which by action of the aromatase cytochrome P450 enzyme is metabolized to estradiol. This hormone upregulates the expression of COX enzyme in the hypothalamus, increasing the production of prostaglandin E2, which acts by increasing the formation of dendritic spines in the neurons of the sexually dimorphic nucleus of the preoptic area in males (SDN-POA). Due to the importance of prostaglandin E2 in the process of hypothalamic sexual differentiation, the use of anti-inflammatory drugs during pregnancy is of concern. Thus, the aim of this study was to evaluate the possible effects resulting from in utero and lactation exposure to non-steroidal anti-inflammatory ibuprofen and its late repercussions on male reproductive parameters in male rats. For this, pregnant rats were exposed to three doses of ibuprofen (10; 30; 60 mg/kg) between the last week of pregnancy (Gestational Days 15-21) until the end of lactation (P... (Complete abstract click electronic access below) / Mestre

Les mâles sexuellement expérimentés présentent des modifications à long terme de l'arborisation dendritique, de modifications épigénétiques ainsi qu'une augmentation des niveaux d'expression de protéines associées à la neurotransmission glutamatergique et à la microglie dans l'aire pré-optique médiane (mPOA). En revanche, les concentrations plasmatiques et hypothalamiques en hormones stéroïdes ainsi que les propriétés du système nitrergique, connues pour être modulés par l'expérience sexuelle chez le rat, ne sont pas modifiées. Dans un second temps, l'implication de la voie de signalisation ERK1/2 dans la réponse comportementale associée à l'expérience sexuelle a été étudiée. Nous avons montré que cette voie de signalisation, activée dans la mPOA lors de l'accouplement, est potentialisée par l'expérience sexuelle. Nous avons ensuite démontré que son activation est possible par une action rapide (30 minutes) des stéroïdes. Enfin, nous avons montré que l'inhibition de la voie ERK1/2 avant un premier accouplement n'altère pas la mise en place de l'expérience sexuelle mais diminue de façon réversible la motivation sexuelle des mâles. Ainsi, l'expérience sexuelle est à l'origine de modifications structurales et biochimiques à long terme de la mPOA. Ces modifications, différentes de celles connues chez le rat, sont associées à une potentialisation de la voie de signalisation ERK1/2 activée de façon transitoires durant l'accouplement. Ces résultats mettent en évidence la nécessité d'élaborer un nouveau modèle, différent de celui établi chez le rat, permettant d'expliquer l'amélioration comportementale associée à l'expérience sexuelle chez la souris mâle. / Sexually experimented males exhibit long term modifications of the dendritic arborization, epigenetic modifications and increased levels of microglia and glutamate associated protein within the hypothalamic medial preoptic area (mPOA). However, hypothalamic and plasmatic concentration of steroid hormones and the nitrergic system are not impacted, contrary to data obtained in rat.The involvement of the ERK1/2 signaling pathway in the induction of sexual experience has also been studied. We showed that ERK1/2 pathway was activated within the mPOA during mating. This activation was increased in sexually experienced males. Furthermore, we showed ex vivo on hypothalamic slices that sex steroids were capable of rapidly (30 min) activate this pathway. Finally, the inhibition of ERK1/2 phosphorylation before the first mating did not disrupt the induction of sexual experience but decreased sexual motivation in a reversible manner.Taken together, these results indicate that long lasting and transitory plasticity mechanisms leading to sexual experience are different between rat and mouse. This indicate the necessity to elaborate a new molecular model associated with the behavioral improvement induced by sexual experience in male mouse.

The transmission of sex pheromone-mediated signals is essential for goldfish reproduction. However, the neural pathways underlying this reproductive signalling pathway in the goldfish brain is not well described. Lesioning experiments have shown previously that two brain areas, the preoptic area (POA) and the ventral telencephali pars ventralis (Vv) in particular, are important for reproduction. We used patch clamp electrophysiology to study the electrical activities of POA and Vv neurons. Based on the intrinsic properties of these neurons, we suggest there are five different functional classes of POA neurons and a single class of Vv neurons. In addition, by electrically stimulating the olfactory bulb (OB), we were able to show that this primary sensory structure makes monosynaptic glutamatergic connections with both POA and Vv neurons. While electrophysiology measures signalling events occurring at short time scales on the order of milliseconds to minutes, we were also interested in studying sex pheromone signalling in the goldfish brain over a long time scale. Thus, we describe changes in gene expression in male goldfish exposed to waterborne sex pheromones (17alpha,20beta dihydroxy-4-pregene-3-one and Prostaglandin-F2alpha) over 6 hours. We perform cDNA microarrays on Prostaglandin-F2alpha-treated fish to study the rapid modulation of transcription and define the signalling pathways affected. Our microarrays showed that 71 genes were differentially regulated (67 up and 4 down). Through gene ontology enrichment analysis, we found that these genes were involved in various biological processes such as RNA processing, neurotransmission, neuronal development, apoptosis, cellular metabolism and sexual reproduction. RT-PCRs were performed to validate our microarrays and to facilitate direct comparisons of the effects of the two sex pheromones, 17alpha,20beta dihydroxy-4-pregene-3-one and Prostaglandin-F2alpha. By combining electrophysiology and gene expression analyses, we were able to study sex-pheromone signalling on two different time scales. One short, occurring on the order of milliseconds to minutes, that involves electrical activities in the brain through the glutamatergic amino-3-hydroxy-5-methylisoxazole-4-propionate and N-methyl-D-aspartate receptors; and the other long occurring several hours later that involves changes in the gene expression levels of calmodulin and ependymin among other genes underlying neuroplasticity. Reproductive neuroplasticity in the goldfish may therefore require the activation of glutamatergic receptors which then activate downstream signals like calmodulin and ependymin to transform the sex pheromones-mediate signal into gene expression.

Numa primeira etapa, estudamos as conexões da VTA para o complexo VTA-substância negra (SN) utilizando a leucoaglutinina do Phaseolus vulgaris (PHA-L). Estas conexões são substanciais, topograficamente organizadas, com destaque para o pólo caudal da VTA que inerva bilateralmente toda a extensão deste complexo. Numa segunda etapa, estudamos as projeções da VTA para o hipotálamo. A VTA se projeta principalmente para a área pré-óptica lateral e área hipotalâmica lateral, a região subfornical posterior e o núcleo dorsomedial. Foram vistas poucas aposições entre varicosidades PHA-L+ e neurônios imunorreativos para orexina ou para hormônio concentrador de melanina. Por fim, estudamos a colocalização do substrato do receptor de insulina (IRS-1), IRS-1 fosforilado e fosfatidilinositol-3 quinase (PI3K) com tirosina hidroxilase (TH) ou com a subunidade B da toxina colérica (CTb) injetada no estriado. A maioria dos neurônios TH+ da VTA-SN expressa IRS-1; injeções de CTb no estriado resultaram em células duplamente marcadas para CTb/IRS-1, CTb/PI3K e CTb/IRS-1 fosforilado. / In a first step, we studied the connections of the VTA to the complex VTA-substantia nigra (SN) using the Phaseolus vulgaris leucoagglutinin (PHA-L). These connections are substantial, topographically organized, especially the caudal pole of the VTA, which innervates bilaterally throughout the length of this complex. In a second step, we studied the projections of the VTA to the hypothalamus. The VTA projected mainly to the lateral preoptic area, lateral hypothalamic area, posterior subfornical region and dorsomedial nucleus. Were observed few appositions between PHA-L+ varicosities and neurons immunoreactive for orexin or melanin-concentrating hormone. Finally, we studied the co-localization of the insulin receptor substrate-1 (IRS-1), IRS-1-phosphorylated and phosphatidylinositol-3 kinase (PI3K) with tyrosine hydroxylase (TH) or cholera toxin B subunit (CTb) injected into the striatum. Most TH+ neurons of the VTA-SN expressed IRS-1; CTb injections in the striatum resulted in cells double-labeled for CTb/IRS-1, CTb/PI3K and CTb/IRS-1 phosphorylated.

The transmission of sex pheromone-mediated signals is essential for goldfish reproduction. However, the neural pathways underlying this reproductive signalling pathway in the goldfish brain is not well described. Lesioning experiments have shown previously that two brain areas, the preoptic area (POA) and the ventral telencephali pars ventralis (Vv) in particular, are important for reproduction. We used patch clamp electrophysiology to study the electrical activities of POA and Vv neurons. Based on the intrinsic properties of these neurons, we suggest there are five different functional classes of POA neurons and a single class of Vv neurons. In addition, by electrically stimulating the olfactory bulb (OB), we were able to show that this primary sensory structure makes monosynaptic glutamatergic connections with both POA and Vv neurons. While electrophysiology measures signalling events occurring at short time scales on the order of milliseconds to minutes, we were also interested in studying sex pheromone signalling in the goldfish brain over a long time scale. Thus, we describe changes in gene expression in male goldfish exposed to waterborne sex pheromones (17alpha,20beta dihydroxy-4-pregene-3-one and Prostaglandin-F2alpha) over 6 hours. We perform cDNA microarrays on Prostaglandin-F2alpha-treated fish to study the rapid modulation of transcription and define the signalling pathways affected. Our microarrays showed that 71 genes were differentially regulated (67 up and 4 down). Through gene ontology enrichment analysis, we found that these genes were involved in various biological processes such as RNA processing, neurotransmission, neuronal development, apoptosis, cellular metabolism and sexual reproduction. RT-PCRs were performed to validate our microarrays and to facilitate direct comparisons of the effects of the two sex pheromones, 17alpha,20beta dihydroxy-4-pregene-3-one and Prostaglandin-F2alpha. By combining electrophysiology and gene expression analyses, we were able to study sex-pheromone signalling on two different time scales. One short, occurring on the order of milliseconds to minutes, that involves electrical activities in the brain through the glutamatergic amino-3-hydroxy-5-methylisoxazole-4-propionate and N-methyl-D-aspartate receptors; and the other long occurring several hours later that involves changes in the gene expression levels of calmodulin and ependymin among other genes underlying neuroplasticity. Reproductive neuroplasticity in the goldfish may therefore require the activation of glutamatergic receptors which then activate downstream signals like calmodulin and ependymin to transform the sex pheromones-mediate signal into gene expression.

Numa primeira etapa, estudamos as conexões da VTA para o complexo VTA-substância negra (SN) utilizando a leucoaglutinina do Phaseolus vulgaris (PHA-L). Estas conexões são substanciais, topograficamente organizadas, com destaque para o pólo caudal da VTA que inerva bilateralmente toda a extensão deste complexo. Numa segunda etapa, estudamos as projeções da VTA para o hipotálamo. A VTA se projeta principalmente para a área pré-óptica lateral e área hipotalâmica lateral, a região subfornical posterior e o núcleo dorsomedial. Foram vistas poucas aposições entre varicosidades PHA-L+ e neurônios imunorreativos para orexina ou para hormônio concentrador de melanina. Por fim, estudamos a colocalização do substrato do receptor de insulina (IRS-1), IRS-1 fosforilado e fosfatidilinositol-3 quinase (PI3K) com tirosina hidroxilase (TH) ou com a subunidade B da toxina colérica (CTb) injetada no estriado. A maioria dos neurônios TH+ da VTA-SN expressa IRS-1; injeções de CTb no estriado resultaram em células duplamente marcadas para CTb/IRS-1, CTb/PI3K e CTb/IRS-1 fosforilado. / In a first step, we studied the connections of the VTA to the complex VTA-substantia nigra (SN) using the Phaseolus vulgaris leucoagglutinin (PHA-L). These connections are substantial, topographically organized, especially the caudal pole of the VTA, which innervates bilaterally throughout the length of this complex. In a second step, we studied the projections of the VTA to the hypothalamus. The VTA projected mainly to the lateral preoptic area, lateral hypothalamic area, posterior subfornical region and dorsomedial nucleus. Were observed few appositions between PHA-L+ varicosities and neurons immunoreactive for orexin or melanin-concentrating hormone. Finally, we studied the co-localization of the insulin receptor substrate-1 (IRS-1), IRS-1-phosphorylated and phosphatidylinositol-3 kinase (PI3K) with tyrosine hydroxylase (TH) or cholera toxin B subunit (CTb) injected into the striatum. Most TH+ neurons of the VTA-SN expressed IRS-1; CTb injections in the striatum resulted in cells double-labeled for CTb/IRS-1, CTb/PI3K and CTb/IRS-1 phosphorylated.

The transmission of sex pheromone-mediated signals is essential for goldfish reproduction. However, the neural pathways underlying this reproductive signalling pathway in the goldfish brain is not well described. Lesioning experiments have shown previously that two brain areas, the preoptic area (POA) and the ventral telencephali pars ventralis (Vv) in particular, are important for reproduction. We used patch clamp electrophysiology to study the electrical activities of POA and Vv neurons. Based on the intrinsic properties of these neurons, we suggest there are five different functional classes of POA neurons and a single class of Vv neurons. In addition, by electrically stimulating the olfactory bulb (OB), we were able to show that this primary sensory structure makes monosynaptic glutamatergic connections with both POA and Vv neurons. While electrophysiology measures signalling events occurring at short time scales on the order of milliseconds to minutes, we were also interested in studying sex pheromone signalling in the goldfish brain over a long time scale. Thus, we describe changes in gene expression in male goldfish exposed to waterborne sex pheromones (17alpha,20beta dihydroxy-4-pregene-3-one and Prostaglandin-F2alpha) over 6 hours. We perform cDNA microarrays on Prostaglandin-F2alpha-treated fish to study the rapid modulation of transcription and define the signalling pathways affected. Our microarrays showed that 71 genes were differentially regulated (67 up and 4 down). Through gene ontology enrichment analysis, we found that these genes were involved in various biological processes such as RNA processing, neurotransmission, neuronal development, apoptosis, cellular metabolism and sexual reproduction. RT-PCRs were performed to validate our microarrays and to facilitate direct comparisons of the effects of the two sex pheromones, 17alpha,20beta dihydroxy-4-pregene-3-one and Prostaglandin-F2alpha. By combining electrophysiology and gene expression analyses, we were able to study sex-pheromone signalling on two different time scales. One short, occurring on the order of milliseconds to minutes, that involves electrical activities in the brain through the glutamatergic amino-3-hydroxy-5-methylisoxazole-4-propionate and N-methyl-D-aspartate receptors; and the other long occurring several hours later that involves changes in the gene expression levels of calmodulin and ependymin among other genes underlying neuroplasticity. Reproductive neuroplasticity in the goldfish may therefore require the activation of glutamatergic receptors which then activate downstream signals like calmodulin and ependymin to transform the sex pheromones-mediate signal into gene expression.