Evidence for nongenomic actions of steroids is now coming from a variety of fields of steroid research. Mechanisms of steroid action are being studied with regard to the membrane receptors and the activation of second messengers. The present study investigated the mechanism for the rapid effect of estrogen on acutely dissociated hippocampal CA1 neurons by using the whole-cell, voltage-clamp recording. Under the perforated patch configuration, 17P-estradiol potentiated kainateinduced currents in 38% of tested neurons. The potentiation was stereospecific, rapid in onset, and reversible after the removal of the steroid. Dose-response curves show that the potentiation by l-/P-estradiol was evident at a concentration as low as 10 nM and saturated at IO PM. 17/3-Estradiol did not affect the kinetics (i.e., affinity and cooperativity) and reversal potential of kainate-induced currents. This suggests that the potentiation did not result from direct interaction with kainate receptors nor the activation of ion channels other than kainate receptor-channels.The potentiation by 17/3-estradiol was similar to the enhancement of kainate-induced currents evoked by 8-bromo-CAMP, and was modulated by an inhibitor of phosphodiesterase (IBMX). The estrogen potentiation was blocked by a specific blocker of PKA (&I-CAMPS). Under standard recording configuration, the effect was significantly affected by intracellular perfusing with GDP-p-S or GTP-y-S. The data suggest that the potentiation of kainate-induced currents by 17P-estradiol was likely a G-protein(s) coupled, CAMPdependent phosphorylation event. By involvement of this nongenomic mechanism, estrogen may play a role in the modulation of excitatory synaptic transmission in the hippocampus.

17Beta-estradiol can potentiate kainate-induced currents in isolated hippocampal CA1 neurons. The action of estrogen was rapid in onset, steroid and stereospecific, and reversible. The potentiation could be mimicked by 8-bromo-cAMP, an activator of protein kinase A. As the hippocampus expresses both isoforms of the intracellular estrogen receptor (ER alpha and ER beta), the role of ERs in the rapid action of 17beta-estradiol remains elusive. Here we report that the rapid action of 17beta-estradiol is independent from the classical ER activation in the modulation of membrane excitability. Under whole cell voltage clamp recording configuration, 17beta-estradiol-induced potentiation was observed in both wild-type and the ER alpha gene knockout mice. The perfusion or incubation of ICI 182,780, which blocks both ER alpha and ER beta, did not affect estrogen potentiation in either group. Further study showed that adenosine 3',5'-cyclic-monophosphothioate Rp-isomer, a specific inhibitor of protein kinase A, completely blocked the potentiation observed with the application of 17beta-estradiol in ER alpha gene knockout mice. Our results provide evidence that a distinct estrogen-binding site exists, which appears to be coupled to alpha-amino-3-hydroxyl-5-methyl-4-isoxazole proprionic acid/kainate receptors by a cAMP-dependent phosphorylation process.

Using whole‐cell voltage‐clamp recordings of dissociated hippocampal CA1 neurones, we demonstrated that 17β‐oestradiol rapidly potentiates kainate‐induced currents when applied either to the outside or the inside of the neurone. However, when the steroid was conjugated to bovine serum albumin (E2‐BSA), application to either the extracellular plasma membrane (E2‐BSAout) or the cytosolic side of the cell (E2‐BSAin) had no observable effect on kainate‐induced currents. However, when applied stimultaneously to both sides of the plasma membrane, E2‐BSA potentiated kainate‐induced currents. Application of E2‐BSAout and GTPγSin potentiated kainate‐induced currents. The potentiation of kainate‐induced currents by 17β‐oestradiol was occluded by cholera toxin pretreatment and appeared to be pertussis toxin insensitive. E2‐BSAin prolonged the effect of 8‐bromoadenosine 3′,5′ cyclic monophosphate (8‐bromo‐cAMP) on kainate‐induced currents. The recovery from the 8‐bromo‐cAMP response was found to be a function of the concentration of E2‐BSAin. The application of ATPγSin occluded the effect of 17β‐oestradiol. These results suggest that the non‐genomic action of 17β‐oestradiol in the potentiation of kainate‐induced currents is mediated via an action on Gs protein‐coupled receptors. This operates in concert with an internal action of 17β‐oestradiol on a cAMP‐dependent phosphorylation.

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