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).