Early, but not late treatment with exogenous estradiol decreases the OVX-evoked ERb2 expression in cortex and hippocampus in rats Figure 3 presents the results of the ERb2 expression in hippocampus of 9 month old OVX female rats (OV) which were received Early (A) or Late (B) ET (OE) and compared with that in age matched sham OVX rats (SOV). In Early ET group, OVX induced a significant increase (21%, * p,0.05) in ERb2 immunoreactivity in 9 month old rats within 15 days of OVX compared to sham OVX. Six days of estradiol treatment initiated on day 7 after OVX repressed expression of ERb2 by 45% compared to OVX 9 month old rats receiving vehicle (#, p,0.01) and which was 25% lower than that in the sham OVX rats. In Late ET group, ERb2 expression was consistently higher (47% increase) in hippocampus of OVX rats than the sham OVX rats (Figure 3B). Initiation of estradiol treatment 6 months after OVX had no effect on the repression of ERb2 which was consistently higher (42%) in hippocampus of Late ET OVX rats compared to sham OVX rats (Figure 3B). Duration of ovarian hormone loss and efficacy of estradiol to increase cell proliferation in hippocampus Ki-67 is an endogenous transient cell proliferation marker expressed in nuclei of actively cycling cells which is not expressed in differentiating quiescent cells. The number of Ki-67-expressing ERb2 Determines the Therapeutic Window of ET cells was measured using flow cytometry in hippocampus of rats that received Early and Late ET. The Ki-67 positive cells were 41% less (p,0.05) in 15 month old compared to 9.5 month old sham OVX rats (Figure 4A), suggesting an age dependent decrease of hippocampal cell proliferation in female rats. The number of Ki-67 positive cells in 9 and 15 month-old OVX rats were similar despite the different duration of ovary loss, 6 or 180 days, suggesting that ovarian hormones have a major role in hippocampal cell proliferation. The number of Ki-67 positive cells in the hippocampus of rats receiving Early ET, in which E2 treatment was initiated 6 days after OVX, was twice than that of OVX rats not treated with E2 (p,0.01) and not significantly different from that of sham OVX rats (p.0.26). Proliferation of hippocampal cells was not increased in rats receiving Late ET, in which E2 treatment was initiated 180 days after OVX (p.0.36), compared to OVX vehicle control. There was a significant difference in hippocampal cell proliferation between the OVX rats that received Early or Late ET (p,0.01). Figure 3. Persistent loss of ovarian hormones diminishes the effects of estradiol on the regulation of ERb2 expression. ERb2 protein expression was evaluated by Western blot in hippocampus of rats OVX at 9 months of age in which E2 treatment was started 6 days (A, Early ET) or 180 days (B, Late ET) after OVX. Data is presented as average 6 SEM of optical density normalized with optical density of b- actin (n = 8 in SOV, 8 in OV and 9 in OE. * = p,0.05 vs. sham OVX; # = p,0.01 vs. sham OVX. doi:10.1371/journal.pone.0033493.g003 Figure 4. Persistent loss of ovarian hormones diminishes the response to estradiol on hippocampal cell proliferation and duration of forced swim. The effects of estradiol on hippocampal cell proliferation was determined by counting the number of Ki-67 positive nuclei in hippocampus (A); the antidepressant effect of estradiol was evaluated by forced on swim test (B), in rats that received Early or Late ET. Data is presented as average 6 SEM (n = 8 in SOV, 8 in OV and 9 in OE). doi:10.1371/journal.pone.0033493.g004 PLoS ONE | www.plosone.org 4 March 2012 | Volume 7 | Issue 3 | e33493