Regulação dos neurônios KNDy pelo estradiol: papel da kisspeptina e dinorfina no controle da secreção de prolactina e hormônio luteinizante

Abstract

Kisspeptin, neurokinin and dynorphin (KNDy) neurons in the arcuate nucleus (ARC) are implicated in luteinizing hormone (LH) pulsatility. Previous evidence has also shown that KNDy neurons have extensive projections onto tuberoinfundibular dopaminergic (TIDA) neurons that are involved in prolactin (PRL) secretion. In rodents, estradiol (E2) induces concurrent preovulatory surges of LH and PRL. We aimed to test the hypothesis that KNDy neurons are major player in regulating this release of PRL. The activity of KNDy neurons was evaluated using double-label immunohistochemistry to c-Fos and kisspeptin in the ARC. The number of kisspeptin-immunoreactive (ir) neurons was lower on proestrus and estrus compared with diestrus. Nevertheless, the percentage of kisspeptin-ir neurons expressing c-Fos was increased on proestrous afternoon by the time of LH and PRL surges, confirmed using two anti-c-Fos antibodies. Male rats, in turn, displayed fewer kisspeptin-ir neurons in the ARC than diestrous females with unchanged coexpression of c-Fos during the day. The effects of E2 were evaluated in the positive-feedback model of ovariectomized (OVX) rats treated with E2 (OVX+E2). The number of kisspeptin-ir neurons in the ARC was reduced in OVX+E2 compared with OVX rats. Similarly to proestrus, however, the percentage of kisspeptin-ir neurons expressing c-Fos was increased in OVX+E2 rats at 18:00 h. This response, as well as the LH surge, was blocked by the anti-estrogen tamoxifen. Moreover, the activation of kisspeptin neurons in OVX+E2 rats was positively correlated with the activity of gonadotropin-releasing hormone (GnRH) neurons and inversely correlated with the expression of phosphorylated tyrosine hydroxylase (S40pTH) in the median eminence, used as an index of TIDA neuron activity. The effects of E2 on expression of KNDy genes in the ARC were determined using qPCR. E2 inhibited the expression of Kiss1 and had no effect on Nkb mRNA. In contrast, OVX+E2 rats displayed an increased expression of prodynorphin (Pdyn) mRNA at 18:00 h, coinciding with the E2-induced LH and PRL surges. We then used transgenic rats expressing Cre-recombinase under the TH promoter to fill the cell bodies and dendrites of TIDA neurons with GFP, allowing investigation of dynorphin and kisspeptin inputs onto these cells. OVX+E2 rats displayed more dynorphin-ir neurons in the ARC in opposition to less kisspeptin-ir cells. E2 also increased the number of dynorphin inputs on the soma and dendrites of TIDA neurons whereas decreased the kisspeptin ones. Thus, we provide evidence of a dual effect of E2 on the regulation of KNDy neurons. E2 reduces kisspeptin expression as part of the negative-feedback effect on LH. Concurrently, E2 induces an afternoon rise in the activity of KNDy neurons, higher expression of dynorphin and increased dynorphin inputs on TIDA neurons. These findings reveal a novel mechanism through which KNDy neurons may control the E2-induced PRL secretion.