sion of hERb2 inhibits ERa-mediated gene transcription and cell proliferation in vitro in MCF cells [19]. Hence it is considered a dominant negative isoform. Swaab’s group has demonstrated the expression of the alternatively spliced ERa mRNAs in post- mortem brain of Alzheimer’s Disease and that the dominant negative deletion (del.) 7 isoform appeared to be the major splice variant [20]. Several ERb splice variants have been identified in the rat hippocampus. One of these, rERb2, has an additional 54 bp nucleotides coding for extra 18 amino acids within the ligand binding domain that changes the conformation and reduces the binding affinity of rERb2 to estradiol by up to 30-fold [21,22]. In addition, rERb2 also exhibits weaker interactions with TIF2 and RAP250, two transcription coactivators [21,22]. The low binding affinity to estradiol and the ability to interact with coactivators make rERb2 a dominant negative receptor in the rat [23,24]. Therefore, although the rERb2 and hERb2 do not share the same spliced aa residues, both differ from ERb in the C-terminal ligand binding domain, both result in diminished ligand binding and both preferentially dimerize with ERa, decreasing normal ER-mediated functions [19]. It has been reported that ERb2 levels increase in the hippocampus [25,26], and pituitary [27] of OVX rats. This shift to prominent expression of the dominant negative receptor is reversible while there are still enough functional ERb, but at some yet to be defined point, the level of ERb2 expression disrupts normal ERb activity. We hypothesize that ERb2 expression increases with the duration of gonadal hormone deprivation and that the point at which ERb2 levels disrupt ER-mediated functions is the molecular mechanism underlying the closing of the therapeutic window for estrogen therapy. To test this hypothesis, rats were ovariectomized at 9 months of age and studied after Early ET (E2 treatment starting 6 days after OVX, equivalent to human early post-menopause [28,29]) and Late ET (E2 treatment starting 180 days after OVX, equivalent to starting 10–20 years after menopause, as was done in the WHIMS trial). ERb2 levels in peripheral blood cells were compared to those in the brain to determine whether these could be a valid marker for ERb2 expression in the brain. Materials and Methods Animal and treatments The animal protocol (#1168) was approved by the University of Mississippi Medical Center Animal Care and Use Committee and conformed to National Institutes of Health guidelines. UMMC is an AAALAC accredited facility. Six month old female or retired breeder (9 month-old) Sprague-Dawley rats were purchased from Charles River (Harlan Laboratories, Indianapolis, IN) and were housed and maintained on a 12/12-hour light/dark cycle (lights on 7:00 AM) and provided with unlimited access to food and water in the Laboratory Animal Facilities at the University Mississippi Medical Center. Ovariectomy The rats were anaesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg). The incision sites were shaved and cleansed with povidone iodine solution. Standard aseptic procedures were observed. Dorsal incisions were made in the lumbar region to reveal the dorsal fat pads covering the ovaries. Ovaries were removed after compression of the ovarian pedicle. After ovariectomy, the incisions were sutured. Rats were given Carprofen (subcutaneous, 5 mg/kg, once/day for 2 days), and ERb2 Determines the Therapeutic Window of ET returned to home cages for the number of days required by the experiment at which time they received E2 or vehicle injections. Groups The effects of removing ovarian hormones on the expression of ERb and ERb2 were first evaluated in adult reproductive (6 month-old) na¨ıve, sham OVX and OVX rats 14 days after surgery. Rats begin to show irregular estrous cycles at 9–12 months, as defined by prolonged cycles, interspersed with the normal 4–5 days rhythm [28,29]. As aging occurs, these animals transition into an acyclic, persistent anestrus status, in which ovulation has ceased [28,29] at 15 to 23 months of age. Therefore, in order to further study the role of ERb2 in Early and Late ET in post-menopausal females, female Sprague-Dawley rats, at 9 months old (compatible to human perimenopause), were ovariec- tomized (OVX, a rodent menopausal model) or sham-OVX. Estradiol treatments were initiated on day 7 (Early ET) or day 181 (Late ET) after OVX (Figure 1). In both Early and Late ET cohorts, 8 rats were sham-OVX, and 17 were OVX. Of the 17 OVX rats, 9 were treated with estradiol, while the other 8 were received vehicle. Treatment Rats were subcutaneously injected with estradiol, 30 mg/kg body weight or vehicle for 5 days (once per day). Although it has been reported by Tanapat that 10 mg/kg, but not 50 mg/kg, was the best concentration for promoting the proliferation of neural progenitor cells in the SGZ in OVX female rat hippocampus [30], the effects of estradiol at the doses between 10 and 50 mg/kg were not reported. In our previous dose response studies, we found that the concentration of 30 mg/kg estradiol showed the highest effects on neuroprotection and neurogenesis [16,31]. One day after the last estradiol injection, rats were assigned for a forced swim test (5 minutes) in random order. Animals were humanely sacrificed 7 days after the first estradiol injection and leukocytes, plasma, uterus, and brain were collected. One hemisphere of the brain was immediately dissected and several cortical regions and the hippocampus were frozen in liquid nitrogen and stored at 280uC for mRNA and protein assay. The other hemisphere was fixed in 4% paraformaldehyde (PFA). FACS assay for Ki-67 positive cells The assay was performed as previously described [32]. Briefly, the hippocampus was extracted from the PFA fixed brain hemispheres using consistent anatomical landmarks as criteria for dissection as described by Bilsland [33]. The white membranes were removed to avoid the contribution from subventricular zone and rostral migratory stream proliferative pools. Nuclei were extracted from the homogenate from individual hippocampus. Figure 1. Experimental flow chart for determining the impact of Early and Late estrogen therapy. Nine-month-old Sprague Dawley rats were ovariectomized and estradiol treatment was initiated 6 days (Early ET) or 180 days (Late ET) after OVX. After 6 days of treatment including two days behavioral tests, the rats were humanely sacrificed and brain and blood samples collected. doi:10.1371/journal.pone.0033493.g001 PLoS ONE | www.plosone.org 2 March 2012 | Volume 7 | Issue 3 | e33493