Estrogen Induces Estrogen Receptor α-Dependent cAMP Response Element-Binding Protein Phosphorylation via Mitogen Activated Protein Kinase Pathway in Basal Forebrain Cholinergic Neurons In Vivo

Abstract

In addition to classical genomic mechanisms, estrogen also exerts nonclassical effects via a signal transduction system on neurons. To study whether estrogen has a nonclassical effect on basal forebrain cholinergic system, we measured the intensity of cAMP response element-binding protein (CREB) phosphorylation (pCREB) in cholinergic neurons after administration of 17β-estradiol to ovariectomized (OVX) mice. A significant time-dependent increase in the number of pCREB-positive cholinergic cells was detected after estrogen administration in the medial septum-diagonal band (MS-DB) and the substantia innominata (SI). The increase was first observed 15 min after estrogen administration. The role of classical estrogen receptors (ERs) was evaluated using ER knock-out mice in vivo. The estrogen-induced CREB phosphorylation in cholinergic neurons was present in ERβ knock-out mice but completely absent in ERα knock-out mice in MS-DB and SI. A series of in vitro studies demonstrated that estrogen acted directly on cholinergic neurons. Selective blockade of the mitogen activated protein kinase (MAPK) pathway in vivo completely prevented estrogen-induced CREB phosphorylation in cholinergic neurons in MS-DB and SI. In contrast, blockade of protein kinase A (PKA) was effective only in SI. Finally, studies in intact female mice revealed levels of CREB phosphorylation within cholinergic neurons that were similar to those of estrogen-treated OVX mice. These observations demonstrate an ERα-mediated nonclassical effect of estrogen on the cholinergic neurons and that these actions are present under physiological conditions. They also reveal the role of MAPK and PKA–MAPK pathway activation in nonclassical estrogen signaling in the basal forebrain cholinergic neurons in vivo.