Behavioral tests
The experimental design is presented in Fig. 1. Behavioral testing was conducted on day 22 and 23. Novelty-suppressed feeding test was performed at 09:00 on day 22. Social interaction test was performed at 20:00 on day 22. Forced swim test was performed at 21:00 on day 22. Sucrose preference test was performed from 22:00 on day 22 to 10:00 on day 23. The rats were decapitated after the behavioral tests on day 23.
The novelty-suppressed feeding test (NSFT) was performed in an open field arena that contained five food pellets that were placed in the middle of the arena. The food deprivation started at 17:00 on day 21 and lasted for 16 hours. At 09:00 on day 22, individual rats were placed in the corner of the arena and allowed to freely explore it for 5 min. A food pellet was then placed in the center of the arena. The latency for the rat to leave the corner of the box and reach the food was recorded. The increase in latency to feed reflects anxiety-like phenotype. After the NSFT, the rats were returned to their home cages and allowed to eat food. There was no difference in food consumption within 60 min across the groups.
To evaluate sociability, the social interaction test (SIT) was performed as previously described (File & Seth, 2003). Social interaction test was performed at 20:00 on day 22. Each rat was placed for 5 min in an open field, facing the opposite corner. A novel unfamiliar rat of the same sex and similar weight was also in the arena. The time spent engaged in social interaction (e.g., sniffing, following, and grooming the partner) and total time spent moving around the arena (i.e., exploration) were recorded. The decrease in social interaction reflects anxiety-like phenotype.
Depressive-like behavior was assessed in the forced swim test (FST) as previously described (Porsolt et al,1977; Wang et al., 2015). For adaptation to swimming, on day 21 of CORT exposure, the rats were individually placed for 15 min in a 25 cm diameter × 60 cm height Plexiglas cylinder that was filled with 25°C ± 1°C water to a depth of 40 cm. At 21:00 on day 22, each rat was placed in the cylinder for 5 min, and behavior was recorded with video cameras that were oriented in different directions (top and side). Immobility time was assessed from the videotapes by a researcher who was blind to each rat’s treatment condition. Immobility was defined as the minimum movement that was necessary to keep the rat’s head above water. After the experiment, the rat was removed from the cylinder, dried with a towel, returned to its home cage, and further warmed and dried under a heat lamp. The water in the swim tank was changed for each rat.
Sucrose preference is considered an index of anhedonia (Snyder et al., 2011). Sucrose preference was performed before (baseline) and after chronic CORT exposure. Sucrose preference test (SPT) was conducted in a 5-day sucrose preference protocol. Briefly, rats were individually housed and habituated to two identical bottles filled with water on days 1 and 2, then replaced to two identical bottles filled with 1% sucrose solution on days 3 and 4. On day 5, rats were given a free choice between two bottles for 12 h, one filled with 1% sucrose solution and the other filled with water. The position of the bottles was switched in the middle of the test (6 h). The sucrose training and baseline test was performed 5 days before the CORT exposure. There was no difference in the sucrose preference across the groups in the baseline test. After 21 days of vehicle or CORT exposure, the SPT was performed for 12 h beginning at 22:00 on day 22 to 10:00 on day 23. Sucrose preference was calculated according to the following formula: Sucrose preference (%) = sucrose intake (g)/(sucrose intake [g] + water intake [g]) × 100%.

Western Blot

Rats were decapitated after the behavioral tests. Immediately after decapitation, the brains were quickly removed to a pre-chilled brain matrix. Bilateral punches (2 mm diameter) of the PVN (from bregma, -1.8 mm to -1.9 mm) and surrounding tissue were made with a hypodermic needle (12 gauge), guided by the Paxinos and Watson rat brain atlas (Paxinos & Watson, 1998). The process was performed on ice. The samples were stored in pre-chilled microcentrifuge tubes at -80 °C until the assay. The PVN tissues were sonicated in 0.5 ml of buffer/100 mg tissue. PVN protein was extracted and boiled in 1% sodium dodecyl sulfate (SDS) solution and quantified using a BCA assay kit (Pierce, Rockford, IL, USA) with bovine serum albumin as the standard. Equal amounts of protein (25 μg) were separated by SDS-polyacrylamide gel electrophoresis on an 8-12% gradient polyacrylamide gel and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% skim milk for 1 h at room temperature and incubated with primary antibodies, including anti-β-actin (1:1000; Cell Signaling Technology, Danvers, MA, USA), anti-GR (1:500; sc-1004, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-MR (1:1000; sc-71554, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-CRF (1:800; ab8901, Abcam, Shanghai, China) in TBS-T buffer (Tris-buffered saline + 0.1% Tween-20) at 4 °C overnight. After 3 × 10 min TBS-T washes, the blots were incubated with horseradish peroxidase-conjugated secondary antibodies (1:2000; Cell Signaling Technology, Danvers, MA, USA) for 2 h at room temperature. After 3 × 10 min TBS-T washes, the blots were developed with a chemiluminescence detection kit (Millipore, Billerica, MA, USA). Western blot bands were scanned with the GelDoc XR System (Bio-Rad, Hercules, CA, USA) and subsequently analyzed densitometrically using Image Lab software. The results were normalized to the protein expression level of β-actin.

The experimental design and statistical analysis

Data and statistical analysis comply with the recommendations of the British Journal of Pharmacology on experimental design and analysis in pharmacology (Curtis et al., 2018). In behavioral test, rats were equally assigned to two or four groups with simple randomization in each experiment. In intra-PVN administration experiments, the injection sites were verified by visual inspection or Nissl staining. The data from the animals whose injection site without in the PVN was excluded. The data was analyzed by a person who was blind to the treatments. The data are expressed as the mean ± SEM and analyzed by GraphPad Prism 8.0 (GraphPad Software Inc., San Diego, CA). The data presented in Fig. 2 was analyzed by unpaired Student’s t -test. The data presented in Fig. 3-5 was analyzed by one-way analysis of variance (ANOVA) followed by the Tukey’s post hoc test. Post hoc tests were conducted only if F in ANOVA achieved p < 0.05 and there was no significant variance inhomogeneity. The level of statistical significance was set at p< 0.05.

Results

Effects of chronic CORT administrationvia drinking water on behavior and GR, MR, and CRF levels in rats
We previously reported that rats that were exposed to CORT via drinking water for 21 days exhibited despair, anhedonia, anxiety, and sleep impairments (Ding et al., 2018). However, unknown was whether GRs, MRs, and CRF (i.e., the central component of the stress response) in the PVN are involved in its etiological mechanisms. We reconfirmed that the rats that received CORT via drinking water for 21 days exhibited a significant increase in immobility time in the FST (t 16 = 5.562, p < 0.01, Fig. 2A) and a significant decrease in sucrose preference in the SPT (t 16 = 8.441,p < 0.01, Fig. 2B). As shown in Fig. 2C, rats that received CORT exhibited less social interaction in the SIT (t 16 = 7.621, p < 0.01). In the NSFT, chronic CORT consumption significantly prolonged the latency to feed, reflecting anxiety-like behavior (t 16 = 6.305, p < 0.01, Fig. 2D). These data indicate that rats that received chronic CORT via drinking water exhibited both depressive- and anxiety-like behaviors. The Western blot analysis revealed that after chronic CORT treatment, the expression of CRF in the PVN significantly increased (t 8 = 11.98, p < 0.01, Fig. 2H), and both MR (t 8= 20.07, p < 0.01, Fig. 2E) and GR (t 8 = 9.315, p < 0.01, Fig. 2F) levels simultaneously decreased in the PVN, but the MR/GR ratio was unchanged (Fig. 2G).