Infusions into the mPFC or DH were conducted as described previously (Kim et al., 2016 (link); Tuscher et al., 2016b (link)). Briefly, cyclodextrin-encapsulated 17β-E2 (Sigma-Aldrich) was dissolved in sterile 0.9% saline to a concentration of 10 µg/µl, and infused bilaterally into the DH or mPFC immediately after training. The vehicle, 2-hydroxypropyl-β-cyclodextrin (HBC; Sigma-Aldrich), was dissolved in saline to the same concentration of cyclodextrin present in the cyclodextrin-encapsulated E2 solution. 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)), resulting in a dose of 5-µg E2/hemisphere.
For DREADD experiments, stock solutions of clozapine-N-oxide (CNO, Cayman Chemical) were prepared by dissolving CNO in 100% dimethyl sulfoxide (DMSO) at a concentration of 100 mg/ml, and storing 10-µl aliquots at –20°C, as described previously (Tuscher et al., 2018 (link)). On the day of injection, CNO stock was thawed and diluted to 1 mg/ml in a solution of sterile 0.9% saline containing 2% DMSO. Intraperitoneal injections of 1-mg/kg CNO were administered immediately after training, followed directly by bilateral DH infusion of vehicle or E2.
For DREADD experiments, stock solutions of clozapine-N-oxide (CNO, Cayman Chemical) were prepared by dissolving CNO in 100% dimethyl sulfoxide (DMSO) at a concentration of 100 mg/ml, and storing 10-µl aliquots at –20°C, as described previously (Tuscher et al., 2018 (link)). On the day of injection, CNO stock was thawed and diluted to 1 mg/ml in a solution of sterile 0.9% saline containing 2% DMSO. Intraperitoneal injections of 1-mg/kg CNO were administered immediately after training, followed directly by bilateral DH infusion of vehicle or E2.
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