Metyrapone

d-(+)-Camphor was purchased from Aldrich and recrystallized from hot C₂H₅OH (mp 179-179.5 °C, uncorr, lit 179.8 °C⁷³ .) Metyrapone (Aldrich) was recrystallized from (C₂H₅)₂O/petroleum ether (l:l, v/v)⁷⁴ (mp 50-51 °C, lit 50-51 °C⁷³ ). Both compounds were dissolved in H₂O or buffer, except when camphor stocks were prepared at > 8 mM (solubility limit⁷³ ) in which case C₂H₅OH was used. 2-Adamantanone and 3,3,5,5-tetramethylcyclohexanone (both SigmaAldrich) were dissolved in H₂O at concentrations up to 2 mM, and norcamphor was soluble in H₂O up to at least 50 mM. (+)-α-Pinene is reported not to be soluble in H₂O at concentrations ≥18 μM (PubChem) and was dissolved in C₂H₅OH and diluted into aqueous solutions (≤1% C₂H₅OH, v/v) except in cases in which low concentrations were used without C₂H₅OH (<18 μM). In the substrate binding titrations (Figure 7C), the C₂H₅OH concentration reached 2% (v/v) and C₂H₅OH aliquots were also used in the reference cuvette.

Metyrapone (2-Methyl-1,2-di-3-pyridyl-1-propanone; 50mg/kg, Mety) (Sigma Cat. 856525) was dissolved in 10% ethanol vehicle and injected intraperitoneally (ip.),15 min prior to each stressor. The dose of Mety used has been shown to block stress induced elevations in plasma corticosterone (CORT) (Roozendaal et al., 1996 (link); Johnson & Yamamoto, 2010 (link)). The control group received 10% ethanol vehicle (ml/kg) injections ip.

Ethanol solutions (10% v/v) were prepared by diluting 95% alcohol (F.L. Carsetti s.n.c., Camerino, Italy) in tap water. The selective CRF1 receptor antagonist, antalarmin was obtained from National Institute on Alcohol Abuse and Alcoholism (NIAAA/NIH). As previously described (Cippitelli et al., 2012 (link)), antalarmin was suspended in a vehicle composed of 10% Tween 80 and distilled water. It was administered intraperitoneally (i.p.) at the doses of 10 mg/kg and 20 mg/kg in a volume of 1 ml/kg. This dosage was based on our previous work on the effect of antalarmin on alcohol consumption in Wistar rats (Cippitelli et al., 2012 (link)). The CORT synthesis inhibitor metyrapone (Sigma-Aldrich) was dissolved in 40% propylene glycol and then diluted in physiological saline. It was administered i.p. at doses of 50 mg/kg and 100 mg/kg in a volume of 2 ml/kg. The doses of metyrapone were based on a previous work that investigated the effect of metyrapone on relapse to cocaine seeking (Piazza et al., 1994 (link)). The glucocorticoid, CORT (Sigma-Aldrich) was dissolved in 1% Tween 80, then diluted in distilled water and sonicated. It was administered i.p. at doses of 2.5 mg/kg and 5 mg/kg in a volume of 1 ml/kg. Doses were chosen on the basis of previous studies (Brooks et al., 2004 (link); Graf et al., 2013 (link)).

To systemically inhibit corticosterone synthesis, mice were intraperitoneally injected with 50 mg/kg metyrapone (3292; R&D) and returned to their home cage. To block glucocorticoid receptors, mice were intraperitoneally injected with 40 mg/kg RU-486 (also known as mifepristone; M8046; Sigma-Aldrich) and returned to their home cage. metyrapone and RU-486 were freshly dissolved in 0.5% carboxymethylcellulose sodium (CMC; C9481; Sigma) on the day of the reciprocal social interaction test. Injection with 0.5% CMC was used as a baseline control. Behaviour testing was performed 40 min after injection.
For acute corticosterone exposure, mice were intraperitoneally injected on two consecutive days with 10 mg/kg corticosterone 2-hydroxypropyl-β-cyclodextrin complex (C174; Sigma-Aldrich) and returned to their home cage. The corticosterone 2-hydroxypropyl-β-cyclodextrin complex was used to facilitate solubility of the steroid. The reciprocal social interaction test was performed 40 min after the second corticosterone injection.
For DREADD-based chemogenetic activation or inactivation, mice expressing hM3Dq, hM3Di or mCherry were intraperitoneally injected with 2–3 mg/kg CNO (Enzo Life Sciences) and returned to their home cage. The reciprocal social interaction test was performed 32–40 min after the second CNO injection.