Corticosterone hbc complex

Animals underwent surgeries according to the different experimental designs under anesthesia with intramuscular ketamine (40–80 mg/kg; Anesket, Pisa Agropecuaria S.A. de C.V.) and intraperitoneal xylazine (5–8 mg/kg; Procin, Pisa Agropecuaria S.A. de C.V.). The adrenal glands were removed bilaterally by a cut on the back of the animal, and silicon catheters (SILASTIC) were inserted into the right jugular vein. After 1 week of recovery, the animals were given intravenous sterile saline (100 µl), dexamethasone (0.05 mg/kg; Sigma-Aldrich), or corticosterone–HBC complex (0.5 mg/kg Cort; Sigma-Aldrich). The animals were killed 7 min after the injection, as described previously.

To evaluate corticosterone levels after injection with corticosterone or vehicle, naïve P12 mice (n = 4–6 per time point per treatment group) were injected intraperitoneally with corticosterone (corticosterone-HBC complex 3 mg/kg dissolved in saline, total injection volume of 10 μl/g body weight) or vehicle (both obtained from Sigma-Aldrich, the Netherlands) between 8.30 and 9.00 a.m. Immediately before, or at 15, 30, 60, 120, 180, or 240 min after injection, mice were decapitated and trunk blood was collected. Between injection and decapitation, pups were returned to their home cage and left undisturbed. A separate group of non-injected mice was decapitated at the same time points to control for diurnal corticosterone variability.

After powdered food habituation and 24 h of fasting, grouped of mice were challenged for 1 h with 500 μl of conditioning stimuli, +Water, +SBT (1:500) or +Mt.Lion (pure Mt.Lion urine). Each stimulus was delivered on a cotton ball containing unreachable powdered food enclosed in a wire mesh. The quantification of the freezing time and the risk assessment behavior displayed by mice^{15 (link)} was performed for the first 15 min and respectively expressed as a percentage of time to freeze and as a mean score obtained per minute in a repeated measure design. Directly after euthanasia, blood samples were obtained by cardiocentesi, collected in EDTA-coated microtubes (Micro tube 1.3 ml K3E; Sarstedt), centrifuged at room temperature for 5 min at 10,000 × g and plasmas were kept at −80 °C until Elisa analysis^{15 (link)} (EIA Kit; Enzo Life Sciences). According to the kit manufacturer, corticosterone levels were measured in duplicate and a minimum of four mice were used to evaluate each situation. To mimic the observed increase of stress-related hormonal levels, corticosterone (Corticosterone: HBC complex; Sigma) in 0.9% saline vehicle were intraperitoneally injected (Cort. i.p., 10.0, 5.0, 2.5, 0.5, 0.0 mg kg⁻¹) with 100 μl per 10 g of mice bodyweight 30 min before behavioral assays^{64 (link)}.