Estrogen receptor beta mediates rapid estrogen actions on gonadotropin-releasing hormone neurons in vivo

Abstract

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-beta-estradiol to ovariectomized (OVX) mice. The percentage of GnRH neurons expressing phosphorylated CREB was increased more than sixfold (p < 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 ERalpha knock-out mice but completely absent in ERbeta 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 ERbeta 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 ERbeta in nongenomic estrogen signaling within the brain in vivo.

Figure 1.

Estrogen rapidly phosphorylates CREB within GnRH neurons in a time- and dose-dependent manner in vivo. Histograms show the percentage of GnRH neurons expressing CREB (A) and pCREB (B) immunoreactivity 15 min, 1 hr, and 4 hr after the administration of vehicle (V) (open bars) or 10μg of E2 (filled bars) to ovariectomized mice. C, The percentage increment in the numbers of GnRH neurons expressing pCREB immunoreactivity 1 hr after 1 or 10μg of E2 (filled bars) compared with vehicle-treated mice (V) (open bars). D, Photomicrograph shows nuclear pCREB immunoreactivity (black) and a double-labeled GnRH neuron (brown cytoplasm). Scale bar, 20 μm. ^*p < 0.05; n = 5–9 in all of the groups. Histograms show mean ± SEM.

Figure 2.

In vitro studies. Estrogen-dependent phosphorylation of CREB in GnRH neurons is direct and requires estrogen to pass into the cell. A, Histograms show the percentage of GnRH neurons expressing pCREB 1 hr after vehicle (V) (open bars) or 100 nm E2 (filled bars) in the presence of normal ACSF or with the addition of 0.5 μm TTX. B, Photomicrograph showing a single double-labeled GnRH plus pCREB neuron from the in vitro slice preparation. Scale bar, 20 μm. C, D, Histograms show the percentage of GnRH neurons expressing pCREB (C) or CREB (D) 1 hr after administration of vehicle (open bars), 100 nm E2–BSA (hatched bars), or 100 nm E2 (filled bars). ^*p < 0.05; n = 4–6 in all of the groups. Histograms show mean ± SEM.

Figure 3.

Estrogen requires ERβ to phosphorylate CREB in GnRH neurons in vivo. Histograms show the percentage of GnRH neurons expressing pCREB (A) and CREB (B) in wild-type littermates (WT), ERαKO mice, and ERβKO mice 1 hr after vehicle (V) (open bars) or 1μg of E2 (filled bars). ^*p < 0.05, n = 5–6 in all of the groups. Histograms show mean ± SEM.

Figure 4.

Differential dependence on ERβ for rapid CREB phosphorylation in brain. Histograms show the numbers (mean + SEM) of pCREB immunoreactive nuclei counted in the AVPV and CA1 hippocampus in wild-type littermates (WT), ERαKO mice, and ERβKO mice 1 hr after vehicle (V) (open bars) or 1 μg of E2 (filled bars). ^*p < 0.05; n = 5–6 in all of the groups. Histograms show mean ± SEM.

Figure 5.

CREB phosphorylation is elevated in intact female mice in vivo. Histograms show the percentage of GnRH neurons expressing pCREB (A) and CREB (B) and the numbers of GnRH-immunoreactive neurons (C) detected per section through the rPOA of OVX (open bars) and intact diestrous female mice (filled bars). ^*p < 0.05; n = 6 in all of the groups. Histograms show mean ± SEM.