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.