Estradiol increases glutamate and gaba neurotransmission into gnrh neurons via retrograde no-signaling in proestrous mice during the positive estradiol feedback period

Abstract Surge release of gonadotropin releasing hormone (GnRH) is essential in the activation of pituitary gonadal unit at proestrus afternoon preceded by the rise of serum 17β-estradiol level during positive feedback period. Here we describe a mechanism of positive estradiol feedback regulation acting directly on GnRH-GFP neurons of mice. Whole cell clamp and loose patch recordings revealed that a high physiological dose of estradiol (200 pM), significantly increased firing rate at proestrus afternoon. The mPSC frequency at proestrus afternoon also increased, whereas decreased it at metestrus afternoon, and had no effect at proestrus morning. Inhibition of the estrogen receptor beta (ERβ), intracellular blockade of the Src kinase and PI3K and scavenge of nitric oxide inside GnRH neurons prevented the facilitatory estradiol effect indicating involvement of the ERβ/Src/PI3K/Akt/nNOS pathway in this fast, direct stimulatory effect. Immunohistochemistry localized soluble guanylate cyclase, the main nitric oxide receptor, in both glutamatergic and GABAergic terminals innervating GnRH neurons. Accordingly, estradiol facilitated neurotransmissions to GnRH neurons via both GABA A -R and glutamate/AMPA/kainate-R. These results indicate that estradiol acts directly on GnRH neurons via the ERβ/Akt/nNOS pathway at proestrus afternoon generating nitric oxide that retrogradely accelerates GABA and glutamate release from the presynaptic terminals contacting GnRH neurons. The newly explored mechanism might contribute to the regulation of the GnRH surge, a fundamental prerequisite of the ovulation. Significance Statement One of the major reasons of infertility is the failure in ovulation. Therefore, understanding of the neuronal processes resulting in proper ovulation is indispensable. Although hypothalamic GnRH neurons are regarded the master-neurons orchestrating reproduction, most of the relevant papers have suggested so far, that the estradiol positive feedback (indispensable for ovulation) acts in these neurons mostly indirectly, via hypothalamic nuclei free of GnRH neurons. Now we have presented strong evidence that estradiol exerts fast direct stimulatory action in GnRH neurons during the positive feedback, by activating retrograde nitric-oxide signaling to accelerate excitatory synaptic inputs to GnRH neurons. In addition, we suggest a putative mechanism, whereby indirect and direct actions of estradiol can work in concert regulating GnRH neurons.