Cyclodextrin encapsulated e2

The aromatase inhibitor letrozole (Selleckchem, Houston, TX) was dissolved in sterile 0.9% saline and 2% dimethyl sulfoxide (DMSO) to concentrations of 0.01, 0.05, and 0.1 μg/μl. A volume of 0.5 μl was infused bilaterally into each side of the DH immediately after training. Vehicle-infused controls received infusions of sterile 0.9% saline and 2% DMSO at the same rate and total volume. Hippocampal infusions were conducted at a rate of 0.5 μl/min for 1 min per hemisphere as described previously (Fernandez et al., 2008 (link); Fortress et al., 2013 (link); Zhao et al., 2012 (link); Zhao et al., 2010b (link)), resulting in letrozole doses of 0.005, 0.025, and 0.05 μg/hemisphere. For experiments also involving ICV infusion of E₂, cyclodextrin-encapsulated E₂ (Sigma-Aldrich, St. Louis, MO) was dissolved in sterile 0.9% saline to a concentration of 10 μg/μl. The vehicle consisted of 2-hydroxypropyl-β-cyclodextrin (HBC; Sigma-Aldrich, St. Louis, MO) dissolved in saline to the same concentration of cyclodextrin present in the cyclodextrin-encapsulated E₂ solution. ICV infusions were conducted at the same rate as DH infusions (0.5 μl/min) for 2 min total, to allow for infusion of the same total volume at the same rate as DH infusions.

The Wnt/β-catenin inhibitor Dickkopf-1 (Dkk-1) was dissolved in sterile 0.9% saline to a concentration of 5 ng/μl. A volume of 0.5 μl Dkk-1 or saline vehicle was infused bilaterally into each side of the DH at a rate of 0.5 μl/min for 1 min per hemisphere immediately following training in OR and OP. cyclodextrin-encapsulated E₂ (Sigma-Aldrich, St. Louis, MO) was dissolved in sterile 0.9% saline to a concentration of 10 μg/μl. The vehicle consisted of 2-hydoxypropyl-β-cyclodextrin (HBC; Sigma-Aldrich, St. Louis, MO) dissolved in saline to the same concentration of cyclodextrin present in the cyclodextrin-encapsulated E₂ solution. A total volume of 1 μl E₂ or HBC was infused into the dorsal third ventricle at a rate of 0.5 μl/min for 2 min to allow for the same total volume as DH infusions at the same rate. This triple infusion protocol is routinely used by our laboratory to prevent potential damage to the DH from two infusions into the DH in rapid succession, and precludes possible interactions between the drugs within the DH and reduced drug efficacy that could result from the dilution of each drug in the larger ICV infusion volume (Fernandez et al., 2008 (link); Fan et al., 2010 (link); Zhao et al., 2010 (link), 2012 (link); Boulware et al., 2013 (link); Fortress et al., 2013 (link)).