These results suggest that ET increases the proliferation of hippocampal cells, in rats when the estradiol was given soon after OVX, but not after prolonged loss of ovarian hormones. Antidepressant efficacy of estrogen therapy correlates with the duration of ovarian hormone loss before therapy is initiated The forced swim test was performed to evaluate the antidepressant effects of estradiol in Early and Late ET rats (Figure 4B). The swim time of 15 month-old sham OVX rats was 53615 sec, 57% less than the 9-month old sham-OVX rats (123627 sec, p,0.01). The swim time of 9 month-old rats OVX for 6 days was 44% less than that of age-matched sham-OVX rats (p,0.05). The swim time of rats receiving estradiol 6 days after OVX was 153652 sec, significantly higher than that in age matched OVX rats that received vehicle (p,0.01). OVX did not change the swim time of 15 month old female rats and there was no difference in swim time between 9 month-old rats OVX for 6 days and 15 month-old rats OVX for 6 months (OV). The swim time of rats that started to receive estradiol 180 days after OVX was 7168 sec (Figure 4B), no difference was observed with that in 15 month old sham-OVX and OVX rats. These findings suggest that exogenous estradiol may serve as an anti-depressant agent in rats when the estradiol is given soon after OVX, but not after prolonged loss of ovarian hormones. ERb2 expression in circulating white blood cells (WBC) reflects the expression of ERb2 in hippocampus If the ERb2 expression in circulating WBC correlates with that in the hippocampus, then it will be a peripheral, easily accessible, laboratory reference marker for ET prescription in clinic. Therefore, we measured and compared ERb2 expression protein extraction of the WBC from the whole blood and hippocampus by Western blot. The results of Western blot analysis demonstrated an increase in expression of ERb2, but not of ERb, in WBC of the rats OVX for 6 days. Similar to the expression pattern of ERb2 in hippocampus, Early ET also produced a reduction of expression ERb2 in WBC of female rats (Figure 5A). The association of the optical densities obtained from the WBC and hippocampus of each samples were analyzed by Pearson Correlation as plotted in Figure 5. The results indicated that the expression levels of ERb2 in WBC and hippocampus are highly associated (r = 0.93, p,0.05) in OVX rats that received vehicle (OV) or Early ET (OE) (Figure 5B). Discussion The causes for the discrepant results from basic science studies of the effects of estrogen therapy after ovarian hormone loss and the results of the WHIMS trial are no doubt complex. Reanalyzes of the WHIMS data, meta-analyses of early clinical trials, and meta-analysis of studies of ET in bilateral oophorectomy [5], as well as more recent prospective studies, support the theory of a critical period or therapeutic window soon after menopause during which time the initiation of estrogen therapy maintains normal neural and cognitive functions, while initiation of ET well after menopause is ineffective or harmful. The molecular mechanisms for this window are unknown. Recent studies suggest that the C terminus of Hsc70-interacting protein (CHIP)-mediated degradation of hippocampal estrogen receptor-a [37] may serve as a molecular mechanism for the critical therapeutic window for post-menopausal ET [37,38]. We present another potential mechanism, the increased expression of the dominant negative ERb isoform, ERb2, which appears to be a ERb2 Determines the Therapeutic Window of ET Figure 5. Expression of ERb2 in white blood cells (WBC) correlates with that in the Hippocampus. The expression of ERb2 in peripheral blood WBC and hippocampus in rats that received Early ET (OE) or vehicle (OV) was measured by Western blot. A. The representative images of ERb2 Western blot of hippocampus and WBC. B. Pearson correlation of the optical density of ERb2 protein expression in extracts of hippocampus and peripheral leukocytes. doi:10.1371/journal.pone.0033493.g005 crucial determinant of the neuroprotective and neurogenic efficacy of ET responsible, at least in part, for the loss of estrogen response after protracted absence of ovarian hormones in a rat model of menopause. The 18-amino acid insertion of ERb2 lies within helix 6, a structure immediately following the b-turn within the ligand binding domain and in the center of the ligand binding pocket [39]. Recombinant protein generated by in vitro transcription/ translation showed that ERb2 had markedly reduced ligand binding (K(D) = 17.7+/24.7 nM) compared with ERb bound 3H- estradiol (K(D) = 0.56+/20.19 nM) [40], demonstrating that the 18 amino acids insertion significantly alters the structure of the ERb ligand binding pocket and impedes binding to ligands. Because their DNA binding domains are the same, ERb2 and ERb have similar binding affinity for palindromic estrogen responsive elements (EREs) in vitro and in vivo [40]. ERb2 has little transcriptional activity except at high ligand concentrations in NIH 3T3 cells with transactivated ERE-tk-CAT reporter genes, due to low ligand binding affinity. Co-transfected ERb2 has been found to significantly inhibit the transcriptional activities of ERa and ERb in several studies [22,25,40–42]. The most plausible mechanism is that ERb2 competes as a homodimer or heterodimer with the homodimers of ERa or ERb in binding to ERE. Both human and rodent ERb2 forms heterodimers of ERa/ ERb2 or ERb/ERb2, as demonstrated by gel shift assay [17,22,41], and the ERa/ERb2 heterodimer shows significantly higher affinity to ERE than that of the homodimer of ERa [42]. Nuclear receptors form strong dimers that are essential for their function as transcription factors and ligand binding affects dimer stability. ER dimerization is ligand independent [43,44]. Un- liganded ER-LBDs exist as stable dimers with a slow dissociation rate (t1/2 = 3963 min at 28 C) which is further slowed (#7-fold) by the addition of various ligands [45]. Dimerization of ERa and ERb is E2- and antiestrogen (EM-652)-independent in human embryo kidney 293 cells using the fluorescence resonance energy transfer (FRET) technique [46] and bioluminescence resonance PLoS ONE | www.plosone.org 5 March 2012 | Volume 7 | Issue 3 | e33493