energy transfer (BRET) assays [44]. The dimers of ERb2/ERb2, ERb2/ERb or ERb2/ERa are predicted to bind to E2 at least 30 times less [25,41,42], thus their transcriptional activity is similarly reduced. Hence the efficacy of estradiol is diminished when expression of ERb2 increases, as occurs after loss of ovarian estrogen. While the hypothesis is that in the absence of E2 in the rat and human the estrogen insensitive ERb2 accumulates and obstructs normal function of the remaining ER [22,41,42], the possibility of the accumulation of another ligand for ERb2 that causes it to act as a repressor of normal ER function should not be discounted [42]. The relevance of our rat model to the human after menopause is based on the following. First, the human ERb2 and rat ERb2 are functionally comparable. Both hERb2 and rERb2 differ from ERb in the ligand-binding domain, both exhibit similar dominant negative function, and both form dimers with ERa or ERb [18,25,26,47–50]. Second, estropause in rodent shares similarities with human menopause. The rodent estrous cycle is 4–5 days long, consisting of proestrus, estrus, diestrus I, and diestrus II. Rats begin to show irregular cycles at 9–12 months, defined by prolonged cycles, interspersed with the normal 4–5 days rhythm, followed by a persistent acyclic, anestrus state with no ovulation between 15 and 23 months of age [28,29]. Rat estropause differs from monkey and human menopause in that aged rats retain a much larger number of primary oocytes, resulting in higher estrogen levels at the onset of persistent diestrus than those seen in primates after menopause [28] [51,52]. To limit this difference, the ovaries were removed from rats entering estropause, thus providing a model that mimics both the age and ovarian hormone status of the post menopausal woman. The female rats receiving estradiol on day 7, or 181 after OVX at 9 months of age is analogous to estrogen therapy in a human early and decades later after menopause, respectively. The ages of the rats used, 6, 9 and 15 months, are thought to correspond with adult reproductive, peri-menopasue, and late post-menopause in women. In the current study ERb2 expression increased in the brains of intact rats with age and estropause, natural ovarian failure, as well as with ovariectomy. The progressive increase in ERb2 from 6, to 9 to 15 months of age in the sham control correlated with a significant decrease in NPC proliferation in the hippocampus, as well as swim time in a forced swim test. Similarly, Nerve growth factor and brain Insulin-like Growth Factor decrease in with age rats [53–56]. Loss of neurons and a decrease in NPC proliferation are associated with depression and dementia, and preservation of NPC proliferation is suggested as a mechanism for the antidepressant effect of estrogen treatment after menopause and ovariectomy [57–60]. Others and we have demonstrated a time dependent loss of the effect of estradiol on NPC proliferation in dentate gyrus SGZ in OVX rats [30,61]. Unbiased stereology in rats at 6 months of age demonstrated that compared to the vehicle treated OVX group, estradiol administration initiated 6 days after OVX increased SGZ NPC proliferation by 40%, while ET initiated 28 days post-OVX had no significant effect on OVX-induced decline of NPC proliferation [30,61]. Using a similar protocol, we used References 1. Brinton RD (2004) Impact of estrogen therapy on Alzheimer’s disease: a fork in the road? CNS Drugs 18: 405–422. 2. Brinton RD (2009) Estrogen-induced plasticity from cells to circuits: predictions for cognitive function. Trends Pharmacol Sci 30: 212–222. 3. Simpkins JW, Perez E, Wang X, Yang S, Wen Y, et al. (2009) The Potential for Estrogens in Preventing Alzheimer’s Disease. Ther Adv Neurol Disord 2: 31–49. 4. Singh M, Dykens JA, Simpkins JW (2006) Novel mechanisms for estrogen- induced neuroprotection. Exp Biol Med (Maywood) 231: 514–521. ERb2 Determines the Therapeutic Window of ET unbiased steriology to measure BrdU positive cells in hippocampal SGZ in rats in which ET was initiated 14 days after OVX and observed a 13.2% increase in proliferation (unpublished data). Ki-67 is an endogenous transient cell proliferating marker expressed in nuclei of actively cycling and does not express in the differentiating quiescent cells. In our previous study we observed a 43% increase in hippocampal cell proliferation upon estradiol treatment of OVX rats by counting Ki-67 positive cell numbers using flow cytometry [32]. While analysis by flow cytometry does not determine the type of proliferating cell, this relative increase is similar to the 40% increase of BrdU cells in SGZ in the estradiol treated group reported in Tanapat’s study [30], suggesting that the cell proliferation detected by Ki-67 positive cells in whole hippocampus reflects the dividing potential of NPC in SGZ, though proliferation of other cells may also be involved. In the current study hippocampal cell proliferation and swim time were not significantly different between intact 15-month-old rats and those ovariectomized 6 months before at 9 months of age, when ovarian function in rats normally declines. This suggests that this model is appropriate for comparison to the WHIMS study in which ET was initiated a decade or more after natural menopause in many of the women. ET initiated 6 days after OVX in rats reversed the decrease in hippocampal cell proliferation produced by OVX, however ET initiated 180 days after OVX, a situation analogous to initiating ET a decade or more after natural menopause, did not. In summary, our study confirmed and extended the evidence for a consistent progressive increase in ERb2 associated with the loss of ovarian hormones. The accumulation of the dominant negative ERb2 correlated with duration of ovarian hormone loss and the inability of ET to reverse the decrease in neuronal proliferation and function and performance in the forced swim test for depression in rats. Accelerated ERb2 expression and accumulation in women may be a mechanism for the loss of efficacy of estrogen therapy if treatment is delayed for too long after menopause. Significant risks of ET in women have been identified, particularly in those in which ET is initiated long after menopause. Expression of ERb2 in circulating leukocytes paralleled that in the brain, suggesting the potential for using WBC ERb2 levels as a clinically useful biomarker to identify the therapeutic window for effective ET. Measurement of ERb2 expression in peripheral WBC could provide a guide for when to initiate post-menopausal estrogen therapy and to avoid therapy when the risk/benefit ratio is unfavorable. Furthermore, determi- nation of the factors leading to increased ERb2 expression could lead to targeted non-estrogen therapies and/or expand the therapeutic window and delay the depression and cognitive loss that has been associated with menopause. Author Contributions Conceived and designed the experiments: JMW CS SB RDB EG. Performed the experiments: JMW SA XH RH SH RI XO IP. Analyzed the data: JMW SA XH XO EG CS RB IP. Wrote the paper: JMW EG XO RDB CS. 5. Rocca WA, Grossardt BR, Shuster LT (2011) Oophorectomy, menopause, estrogen treatment, and cognitive aging: clinical evidence for a window of opportunity. Brain Res 1379: 188–198. 6. Yaffe K, Sawaya G, Lieberburg I, Grady D (1998) Estrogen therapy in postmenopausal women: effects on cognitive function and dementia. Jama 279: 688–695. 7. Rossouw JE (2008) Postmenopausal hormone therapy for disease prevention: have we learned any lessons from the past? Clin Pharmacol Ther 83: 14–16. PLoS ONE | www.plosone.org 6 March 2012 | Volume 7 | Issue 3 | e33493