The goal of this study was to elucidate the mechanisms of 17␤-estradiol (E 2 ) antioxidant and neuroprotective actions in stroke. The results reveal a novel extranuclear receptor-mediated antioxidant mechanism for E 2 during stroke, as well as a hypersensitivity of the CA3/CA4 region to ischemic injury after prolonged hypoestrogenicity. E 2 neuroprotection was shown to involve a profound attenuation of NADPH oxidase activation and superoxide production in hippocampal CA1 pyramidal neurons after stroke, an effect mediated by extranuclear estrogen receptor ␣ (ER␣)-mediated nongenomic signaling, involving Akt activation and subsequent phosphorylation/ inactivation of Rac1, a factor critical for activation of NOX2 NADPH oxidase. Intriguingly, E 2 nongenomic signaling, antioxidant action, and neuroprotection in the CA1 region were lost after long-term E 2 deprivation, and this loss was tissue specific because the uterus remained responsive to E 2 . Correspondingly, a remarkable loss of ER␣, but not ER␤, was observed in the CA1 after long-term E 2 deprivation, with no change observed in the uterus. As a whole, the study reveals a novel, membrane-mediated antioxidant mechanism in neurons by E 2 provides support and mechanistic insights for a "critical period" of E 2 replacement in the hippocampus and demonstrates a heretofore unknown hypersensitivity of the CA3/CA4 to ischemic injury after prolonged hypoestrogenicity.

Evidence from various sources suggested that the Gonadotropin-Releasing Hormone (GnRH) neuron does not contain glutamate receptors. Northern analysis of the hypothalamus showed the presence of NMDAR1, GluR1, GluR4 and GluR6 mRNA, while the pituitary showed the presence of NMDAR1, GluR1 and GluR6 mRNA. Western blot analysis also showed the presence of NMDAR1 and GluR1 protein. Since there are relatively few GnRH neurons in the hypothalamus, and GT1-7 cells have been considered to be a GnRH neuronal cell line, GT1-7 cells were studied in detail. GT1-7 cells contained NMDAR1 mRNA levels as shown by Northern analysis but did not contain GluR1, GluR4, or GluR6 mRNA. They did not show the presence of NMDAR1 and GluR1 protein by Western analysis. In addition, GT1-7 cells showed no NMDA receptor binding using the competitive inhibitor CGP-39563 and the noncompetitive inhibitor MK-801. Likewise, no binding was detected for kainate receptors. However, a small amount of binding for AMPA receptors was found in GT1-7 cells. GT1-7 cells did not exhibit glutamate toxicity and NMDA failed to elicit inward currents using patch-clamp techniques, although GABA did induce currents in the cells. As a whole, these studies suggest that GT1-7 cells lack or possess only low levels of ionotropic glutamate receptors.

Selective estrogen receptor modulators (SERMs) have been reported to enhance synaptic plasticity and improve cognitive performance in adult rats. SERMs have also been shown to induce neuroprotection against cerebral ischemia and other CNS insults. In this study, we sought to determine whether acute regulation of neurogenesis and spine remodeling could be a novel mechanism associated with neuroprotection induced by SERMs following cerebral ischemia. Toward this end, ovariectomized adult female rats were either implanted with pellets of 17β-estradiol (estrogen) or tamoxifen, or injected with raloxifene. After one week, cerebral ischemia was induced by the transient middle-cerebral artery occlusion (MCAO) method. Bromodeoxyuridine (BrdU) was injected to label dividing cells in brain. We analyzed neurogenesis and spine density at day-1 and day-5 post MCAO. In agreement with earlier findings, we observed a robust induction of neurogenesis in the ipsilateral subventricular zone (SVZ) of both the intact as well as ovariectomized female rats following MCAO. Interestingly, neurogenesis in the ipsilateral SVZ following ischemia was significantly higher in estrogen and raloxifene-treated animals compared to placebo-treated rats. In contrast, this enhancing effect on neurogenesis was not observed in tamoxifen-treated rats. Finally, both SERMs, as well as estrogen significantly reversed the spine density loss observed in the ischemic cortex at day-5 post ischemia. Taken, together these results reveal a profound structural remodeling potential of SERMs in the brain following cerebral ischemia.

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