Drugs administration
CORT powder (corticosterone, TCI, Tokyo, Japan) was dissolved in 100% ethanol and then added to the drinking water to a final concentration of 200 μg/mL and 1% ethanol. Vehicle is 1% ethanol in the drinking water. Water bottles were wrapped in tinfoil to avoid light-induced degradation of the CORT. Freshly prepared vehicle or CORT was replaced every 2 days. From 21:00 on day 1 to 21:00 on day 22, the rats were exposed to vehicle or CORT for 21 days via drinking water. At an average water intake of 50 mL/rat/day, the corresponding daily dose of CORT was 10 mg. There was no difference in average daily water intake across the groups. The administration of CORT via drinking water significantly increased circulating CORT levels and induced the absence of circadian rhythm of CORT. This chronic elevation of CORT dysregulates the HPA axis, which is associated clinical depression and anxiety. Preliminary results showed that the activation of HPA axis and the behavioral parameters were not affected by the 21 days of 1% ethanol exposure.
The GR antagonist RU486 (M1732, Tokyo Chemical Industry, Tokyo, Japan) was dissolved in 1% ethanol, which 0.9% saline was added to a final concentration of 1 μg/μl. The MR antagonist spironolactone (ab141289, Abcam, Shanghai, China) was dissolved in 1% ethanol, which 0.9% saline was added to a final concentration of 0.2 μg/μl. From day 16 to day 22, drug or vehicle was injected once daily into the PVN in a 0.25 μl volume over 2 min at 08:00~09:00. The injection cannula was kept in place for an additional 2 min to allow the drug to completely diffuse from the tip. The rats received intra-PVN injections of RU486 and spironolactone, alone or combined.