Membrane estrogen receptors mediate calcium signaling and MAP kinase activation in individual hippocampal neurons

Previously we demonstrated that 17β-Estradiol (E2) induced rapid Ca ²⁺ influx via L-type calcium channel activation, which was required for activation of Src/ERK/CREB/Bcl2 signaling cascade and subsequent induction of neuroprotective and neurotrophic responses in rat hippocampal and cortical neurons (Wu et al., 2005; Zhao et al., 2005). The current study determined the presence and specificity of membrane E2 binding sites and the functional consequence of E2 binding to membrane receptors in individual neurons. Using E2-BSA-FITC (fluorescein isothiocyanate) macromolecular complex, membrane E2 binding sites were observed in hippocampal neurons. Punctate FITC signal was observed on plasma membrane of soma and neuronal processes in E2-BSA-FITC binding neurons. No membrane binding was observed with BSA-FITC. Specificity of binding was demonstrated by competition with excess un-conjugated E2. An ERa specific agonist, PPT, and an ERb agonist, DPN, partially competed for E2-BSA-FITC binding. Imaging of intracellular Ca ²⁺ ([Ca ²⁺]i) in live neurons, revealed rapid Ca ²⁺ responses in E2-BSA-FITC binding neurons within minutes that culminated in a greater [Ca ²⁺]i rise and [Ca ²⁺]i spikes at > 20 min. The same neurons in which E2-BSA-FITC induced a [Ca ²⁺]i rise also exhibited activated pERK (extracellular signal-regulated kinase) that was translocated to the nucleus. Immunofluorescent analyses demonstrated that both excitatory and inhibitory neuronal markers labeled subpopulations of E2-BSA-FITC binding neurons. All E2-BSA-FITC binding neurons expressed L-type calcium channels. These results demonstrate, at a single cell level, that E2 membrane receptors mediate the rapid signaling cascades required for E2 neuroprotective and neurotrophic effects in hippocampal neurons. These results are discussed with respect to therapeutic targets of estrogen therapy in brain.