Ru 28318

Nifedipine (NIF) and caffeine (caf) were obtained from Sigma-Aldrich. The RU-28318, thapsigargin (Tg) and the KB-R7943 were obtained from TOCRIS Bioscience (Bristol, UK). Fluo-4/AM and Fura-2/AM were obtained from Thermo Fisher Scientific (Waltham, MA, USA).

To determine the role of the corticosteroid receptors in the social approach behavior, we treated control male mice with either a MR antagonist or a GR antagonist. Mice (n = 8) received the GR antagonist RU38486 (Mifepristone, also known as a progesterone antagonist; Sigma; 10 mg/kg of body weight; Zhou et al., 2010 (link)) intraperitoneally 45 min before the start of the social approach test. RU38486 was dissolved in 0.9% NaCl containing 0.25% carboxymethylcellulose and 0.2% Tween-20. Solutions were prepared on the day before the injection and vortexed overnight.
A second group of mice (n = 8) received the MR antagonist RU28318 (Tocris Bioscience; 50 mg/kg body weight; dissolved in physiological saline (Schwabe et al., 2010 (link))) subcutaneously 45 min before the start of the social approach test. Solutions were prepared on the day before the injection, frozen, and defrosted 30 min before injection.
After the injection, mice returned to their home cage and the social approach test started 45 min later. These mice were tested in the habituation and sociability phase only. One week later the mice which previously had received RU28318 were tested again with new strangers in the sociability phase.

Injections were made using 10 μL-Hamilton syringes connected by PE-10 polyethylene tubing to a needle that was introduced into the brain through the guide cannula, as previously described (Sato et al., 1996 (link)). At the time of testing, metal obturators were removed, and the injection cannula (2 mm longer than the guide cannula) was inserted into the guide cannula. Injection volumes into the LV ranged from 1 to 2 μl, as described below. The metal obturators were replaced after injections.
Losartan potassium, AT1 receptor antagonist (Sigma-Aldrich, St Louis, MO, United States), was administered at the dose of 66 µg/1 μl, as described previously (Sato et al., 1996 (link)), RU28318, mineralocorticoid receptor antagonist (Tocris Bioscience, Ellisville, MO, United States) was used at the dose of 100 ng/2 µl, as described previously (Kim et al., 1998 (link)), dissolved in 0.15 M NaCl (saline) and 1% ethanol in 0.15 M NaCl (vehicle).