Cortisol

The brain tissues (n=42, 6 per treatment group) were dissected into three brain regions: Area 1, Area 2, and Area 3. Macro-dissected brain samples (Area 2 and Area 3) were washed into D-PBS (Nacalai Tesque) and treated by Trypsin-EDTA solutions (Gibco). The pellet was resuspended in Leibovitz L-15 medium (Gibco) supplemented with 10% Fetal Bovine Serum (FBS) and 0.1% Penicillin/Streptomycin antibiotics (Gibco) and cultured to a pre-coated 12-well plate (BD, New Jersey, USA). After 24 h of incubation, cortisol (100 nM and 1,000 nM, Nacalai Tesque), DEX phosphate (10 nM, and 100 nM, Sigma), and 5-HT (10 μM, and 100 μM, Sigma) were supplemented for 24 h. 5-HT receptor antagonist Mirtazapine (5-HT receptor 2 and 3 antagonists) (Tocris Bioscience) and Metergoline (5-HT receptor 1 antagonist) (Sigma) at 1 µM were added 30 min before the 5-HT treatment (10 μM) for 24 h. The treatment dose and duration were referred to recent studies (49 (link)–51 (link, link)). The cell viability was determined using the Muse Count & Viability kit (Luminex). The average cell viability was 80.5%.