C2505

Total plasma CORT concentrations were assayed using CORT Enzyme Immunoassay (EIA) kits (Cat. No. ADI-900-097, Enzo Life Sciences, Plymouth Meeting, PA, USA) according to kit instructions, except for the concentration of steroid displacement reagent, which was increased twenty-fold. These conditions were chosen based on optimizations that were performed with samples from ADX rats that were spiked with known concentrations of exogenous CORT (Cat. No. C2505, Sigma-Aldrich). Samples were run in duplicate or triplicate and plates were read with a spectrophotometer set at 405 nm (Multiskan EX, VWR, West Chester, PA, USA). The dilution coefficient was applied during the analysis of the hormone concentrations to correct for the dilution of the samples. The inter- and intra-assay coefficients of variation were 10.1% and 8.4%, respectively.

Adult male Ankrd26−/− and WT mice were used for this experiment (n = 7 in each group). For bilateral adrenalectomy, mice were anesthetized with isoflurane and 0.5 mg/kg buprenorphine administered subcutaneously and the adrenals were removed in a sterile environment.
Ankrd26−/− and WT mice (n = 14) were adrenalectomized, and half of the mice (n = 7) were given corticosterone replacement (25 mg/L) in the drinking water available ad libitum immediately after the operation. corticosterone was purchased from Sigma (C2505), dissolved in ethanol and diluted in 0.9 % saline to reach a 0.6 % final concentration of ethanol.
A third group of Ankrd26−/− and WT mice (n = 7) were sham operated, and underwent the same procedure as the adrenalectomized mice, when adrenal glands were exposed but not removed. The non-steroid-supplemented groups (adrenalectomized and sham-operated) were given 0.9 % saline and 0.6 % ethanol instead of regular drinking water. The water consumption was recorded every 5 days. To confirm the successful adrenalectomy and the effectiveness of the corticosterone replacement blood samples were obtained from mandibular veins from each mouse after 2 h of the onset of the light cycle and serum corticosterone levels were determined by ELISA (Enzo Life Sciences) following the manufacturer’s instructions.

35 μg mL⁻¹ corticosterone (C2505, Sigma; St. Louis, MO) was dissolved in 0.45% β-cyclodextrin (C4767, Sigma; St. Louis, MO) in water. Fluoxetine (BG0197, Biotrend; Destin, FL) was dissolved at 160 μg mL⁻¹. corticosterone (5 mg kg⁻¹ day⁻¹) was delivered in the drinking water either alone or in the presence of Fluoxetine (18 mg kg⁻¹ day⁻¹).