The gonadal steroid estrogen exerts an important modulatory influence on the activity of multiple neuronal networks. In addition to classical genomic mechanisms of action, estrogen also exerts poorly understood rapid, nongenomic effects on neurons. To examine whether estrogen may exert rapid actions on intracellular signaling within gonadotropin-releasing hormone (GnRH) neurons in vivo, we examined the phosphorylation status of cAMP response element-binding protein (CREB) in these cells after the administration of 17-␤-estradiol to ovariectomized (OVX) mice. The percentage of GnRH neurons expressing phosphorylated CREB was increased more than sixfold ( p Ͻ 0.05) in a time-and dose-dependent manner by estrogen, with the increase first observed 15 min after estrogen administration. A series of in vitro studies demonstrated that estrogen acted directly on native GnRH neurons to phosphorylate CREB, but that estrogen conjugated to bovine serum albumin was without effect. The role of classical estrogen receptors (ERs) was evaluated using ER knock-out mice in vivo. The effect of estrogen on CREB phosphorylation in GnRH neurons was normal in ER␣ knock-out mice but completely absent in ER␤ knock-out mice. Finally, studies in intact female mice revealed levels of CREB phosphorylation within GnRH neurons that were equivalent to those of estrogen-treated OVX mice. These observations demonstrate that ER␤ mediates the rapid, direct effects of estrogen on the GnRH neuronal phenotype, and that these actions persist under physiological conditions. They also provide the first evidence for a role of ER␤ in nongenomic estrogen signaling within the brain in vivo.

Estrogen exerts classical genomic as well as rapid nongenomic actions on neurons. The mechanisms involved in rapid estrogen signaling are poorly defined, and the roles of the classical estrogen receptors (ERs alpha and beta) are unclear. We examined here the in vivo role of classical ERs in rapid estrogen actions by evaluating the estrogen-induced effects on two major signaling pathways within the brains of alphaER-, betaER-, and double alphabetaER-knockout (ERKO) ovariectomized female mice. Estrogen significantly (P < 0.05) increased the numbers of phospho-cAMP response element binding protein (phospho-CREB)-immunoreactive cells in specific brain regions of wild-type mice in a time-dependent manner beginning within 15 min. In brain areas that express predominantly ERbeta, this response was absent in betaERKO mice, whereas brain regions that express mostly ERalpha displayed no change in alphaERKO mice. In the medial preoptic nucleus (MPN), an area that expresses both ERs, the estrogen-induced phosphorylation of CREB was normal in both alphaERKO and betaERKO mice. However, estrogen had no effect on CREB phosphorylation in the MPN, or any other brain region, in double alphabetaERKO animals. Estrogen was also found to increase MAPK phosphorylation levels in a rapid (<15 min) manner within the MPN. In contrast to CREB signaling, this effect was lost in either alphaERKO or betaERKO mice. These data show that ERalpha and ERbeta play region- and pathway-specific roles in rapid estrogen actions throughout the brain. They further indicate an indispensable role for classical ERs in rapid estrogen actions in vivo and highlight the importance of ERs in coordinating both classical and rapid actions of estrogen.