Ovariectomy and Estradiol Treatment

Adult mice were bilaterally ovariectomized under general anesthesia with isoflurane. For estradiol treatment, a 5-mm-long Silastic capsule (inner diameter: 1.57 mm; outer diameter: 2.41 mm) containing crystalline 17β-estradiol (E2758, Sigma-Aldrich, USA), diluted 1:1 with cholesterol (C8667, Sigma Aldrich), was inserted subcutaneously at the nape of the neck at the same time as ovariectomy, as previously described [44]. This is a routinely used method of exogenously elevating circulating E2 levels in mice [44-46]. Brain tissue from OVX mice was collected 3 weeks after ovariectomy and OVX + E2 mice 6 weeks after ovariectomy.