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.