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\begin{document}

\title{Anti-stress effects of combined glucocorticoid and mineralocorticoid
receptor blockade in the paraventricular nucleus of the hypothalamus}



\author[1]{Hui Ding}%
\author[1]{Xiang-Yu Cui}%
\author[1]{Yu-Tong Liu}%
\author[1]{Su-Ying Cui}%
\author[1]{Xiao Hu}%
\author[1]{Hui-Ling Zhao}%
\author[1]{Yu Qin}%
\author[1]{Nurhumar Kurban}%
\author[1]{Yong-He Zhnag}%
\affil[1]{Peking University Health Science Centre}%


\vspace{-1em}



  
  \date{March 14, 2025}


\begingroup
\let\center\flushleft
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\selectlanguage{english}
\begin{abstract}
Background and Purpose: Mineralocorticoid receptors (MRs),
glucocorticoid receptors (GRs) and corticotropin-releasing factor (CRF)
in the paraventricular nucleus of the hypothalamus (PVN) are implicated
in the stress response. The present study investigated the role of GRs
and MRs in the PVN in regulating depressive and anxiety-like behaviors.
Experimental Approach: To model chronic stress, rats were exposed to
chronic corticosterone treatment via drinking water for 21 days, and the
GR antagonist RU486 and MR antagonist spironolactone, alone and
combined, were directly injected in the PVN daily for 7 days before the
behavioral tests. Depressive- and anxiety-like behaviors were evaluated
in forced swim test, sucrose preference test, novelty-suppressed feeding
test and social interaction test. The expression of GRs, MRs and CRF
were detected by Western-Blot. Key Results: The rats exposed to
corticosterone exhibited depressive- and anxiety-like behaviors. The
expression of GRs and MRs decreased, and CRF levels increased in the
PVN. The intra-PVN administration of RU486 increased the levels of GRs
and CRF without influencing depressive- or anxiety-like behaviors. The
spironolactone-treated group exhibited an increase in MRs without
influencing GRs and CRF in the PVN, and improved anxiety-like behaviors.
Interestingly, the intra-PVN administration of RU486 and spironolactone
combined restored the expression of GRs, MRs, and CRF and improved
depressive- and anxiety-like behaviors. Conclusion and Implications:
These results suggest that the simultaneous restoration of GRs, MRs, and
CRF in the PVN in this rat model of stress might play an important role
in the treatment of depression and anxiety.%
\end{abstract}%



\sloppy


\textbf{Introduction}

The hypothalamic-pituitary-adrenal (HPA) axis is the driver of
neuroendocrine stress responses. In response to stress, neurons in the
parvocellular domain of the paraventricular nucleus of the hypothalamus
(PVN) are activated to release corticotropin-releasing factor (CRF) and
arginine vasopressin (Jiang et al., 2019). CRF coordinates with arginine
vasopressin to stimulate anterior pituitary corticotrophs to release
adrenocorticotropic hormone (ACTH) into the circulatory system, and ACTH
targets the zona fasciculata of the adrenal glands, resulting in the
synthesis and secretion of glucocorticoids (GCs; cortisol in humans,
corticosterone {[}CORT{]} in rodents) (Ulrich-Lai \& Herman,
2009)\emph{.} Glucocorticoids regulate HPA axis activity by limiting
their own secretion via a negative feedback mechanism by acting on
mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs)
(Binder \& Nemeroff, 2010). These receptors are expressed in neurons
that innervate the PVN trans-synaptically and are also found in the PVN
(Chen et al., 2014). In the rat PVN, the co-localization of GRs and MRs
was found in the parvocellular region, where CRF neurons mainly reside
(Han et al., 2005).

The PVN is one of the primary sites of GC negative feedback regulation
of the HPA axis. As the central driving force, CRF-producing neurons in
the PVN play a key role in determining the state of activation of the
HPA axis and are closely involved in stress responses and the
pathogenesis of depression (Lloyd \& Nemeroff, 2011). Although the exact
functional mechanism of GRs and MRs in the modulation of CRF expression
remains obscure, MRs and GRs may exert differential regulatory effects
on the expression of CRF and operate synergistically in feedback actions
of GCs. As the final common pathway for stress integration by the brain,
the PVN is ultimately responsible for both normal and pathological
features of HPA axis stress responses. Despite progress to date, still
unclear are the mechanisms that underlie chronic stress-related PVN
drive, the modification of PVN secretagogue signaling, and synaptic
plasticity.

Long periods of stress influence the function of GRs and MRs. However,
the precise functions of GRs and MRs in the PVN and possible changes
that occur in the pathogenesis of depression remain poorly understood
(Nguyen et al., 2017), thus hampering our ability to develop strategies
to mitigate stress and diseases that are related to stress adaptation.

The predominant hypothesis that CRF hypersecretion plays a leading role
in the pathogenesis of major depression has received support from
studies that showed that severely depressed patients exhibited high
cerebrospinal fluid (CSF) levels of CRF (Gold et al., 2002) and
postmortem studies of depressed suicide victims that found very high CSF
CRF levels (Pandey et al., 2019). Consistent with the finding that
patients with major depression have the highest incidence of HPA axis
hyperactivity, studies of depressed suicide victims have revealed high
CRF mRNA expression and CRF immunoreactivity in hypothalamic PVN neurons
(Deussing \& Chen, 2018).

Glucocorticoid receptors, MRs, and CRF are strongly implicated in the
stress response. In the present study, we hypothesized that the
regulation of GRs and MRs in the PVN may influence depressive-like
behaviors by modulating CRF in the PVN. We investigated the functions of
GRs and MRs and their actions on CRF production in the PVN during
chronic stress by exploring the behavioral effects of subcutaneous
injections or intra-PVN infusions of the GR antagonist RU486
(mifepristone) and MR antagonist spironolactone. We utilized a rat model
of depression that was induced by 21 days of CORT administration via
drinking water, which produces a series of depressive- and anxiety-like
behaviors. We first tested a series of depressive-like behaviors in the
forced swim test (FST), sucrose preference test (SPT),
novelty-suppressed feeding test (NSFT), and social interaction test
(SIT). We then measured the levels of GRs, MRs, and CRF in the PVN.
Finally, we co-administered spironolactone and RU486 both subcutaneously
and directly in the PVN to evaluate possible correlations between these
depressive- and anxiety-like behaviors and changes in GRs, MRs, and CRF
in the PVN.

\subsubsection*{Materials and Methods}

{\label{materials-and-methods}}

\subsubsection*{Animals}

{\label{animals}}

Male Wistar rats (Grade I) were purchased from the Animal Center of
Peking University (Beijing, China). The animals weighed 250-270 g at the
beginning of the experiments. The rats were maintained on a 12 h/12 h
light/dark cycle (lights on at 09:00) with \emph{ad libitum} access to
food and water. The rats were housed individually in plastic cages. The
animal protocols were approved by the Peking University Committee on
Animal Care and Use (permission no. LA 2015101).

\subsubsection*{Surgery for cannula
implantation}

{\label{surgery-for-cannula-implantation}}

Under xylazine (6 mg/kg, i.p.) and ketamine (80 mg/kg, i.p.) anesthesia,
the rats were implanted with a double-guide cannula (27-gauge; Plastics
One, Roanoke, VA, USA) for drug administration into the PVN. Briefly,
the cannula was implanted with the tip 0.5 mm above the PVN
(anterior/posterior, -1.8 mm; medial/lateral, \selectlanguage{ngerman}± 0.4 mm; dorsal/ventral,
-7.0 mm). The guide cannula was secured with acrylic cement and two
stainless steel screws that were anchored to the skull. The cannula
placements were histologically verified using Nissl staining of 30 \selectlanguage{greek}μ\selectlanguage{english}m
thick sections by light microscopy (Supplementary Fig. S1).

\textbf{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 \selectlanguage{greek}μ\selectlanguage{english}g/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 \emph{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 \selectlanguage{greek}μ\selectlanguage{english}g/\selectlanguage{greek}μ\selectlanguage{english}l. 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 \selectlanguage{greek}μ\selectlanguage{english}g/\selectlanguage{greek}μ\selectlanguage{english}l. From day 16 to day 22,
drug or vehicle was injected once daily into the PVN in a 0.25 \selectlanguage{greek}μ\selectlanguage{english}l volume
over 2 min at 08:00\textasciitilde{}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.

\textbf{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 \selectlanguage{ngerman}× 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\%.

\subsubsection*{}

{\label{section}}

\subsubsection*{Western Blot}

{\label{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 \selectlanguage{greek}μ\selectlanguage{english}g)
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-\selectlanguage{greek}β-\selectlanguage{english}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 \selectlanguage{ngerman}°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 \selectlanguage{greek}β-\selectlanguage{english}actin.

{\label{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-buffer100-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-ux3bcg-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-ux3b2-actin-11000-cell-signaling-technology-danvers-ma-usa-anti-gr-1500-sc-1004-santa-cruz-biotechnology-santa-cruz-ca-usa-anti-mr-11000-sc-71554-santa-cruz-biotechnology-santa-cruz-ca-usa-and-anti-crf-1800-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-12000-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-ux3b2-actin.}}

\subsubsection*{The experimental design and statistical
analysis}

{\label{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 \selectlanguage{ngerman}± 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 \emph{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 \emph{F} in ANOVA
achieved \emph{p} \textless{} 0.05 and there was no significant variance
inhomogeneity. The level of statistical significance was set at
\emph{p}\textless{} 0.05.

\subsubsection*{}

{\label{section-1}}

\subsubsection*{Results}

{\label{results}}

Effects of chronic CORT administration\emph{via} drinking water on
behavior and GR, MR, and CRF levels in rats

{\label{effects-of-chronic-cort-administration-via-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 (\emph{t}
\textsubscript{16} = 5.562, \emph{p} \textless{} 0.01, Fig. 2A) and a
significant decrease in sucrose preference in the SPT (\emph{t}
\textsubscript{16} = 8.441,\emph{p} \textless{} 0.01, Fig. 2B). As shown
in Fig. 2C, rats that received CORT exhibited less social interaction in
the SIT (\emph{t} \textsubscript{16} = 7.621, \emph{p} \textless{}
0.01). In the NSFT, chronic CORT consumption significantly prolonged the
latency to feed, reflecting anxiety-like behavior (\emph{t}
\textsubscript{16} = 6.305, \emph{p} \textless{} 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 (\emph{t} \textsubscript{8} =
11.98, \emph{p} \textless{} 0.01, Fig. 2H), and both MR (\emph{t}
\textsubscript{8}= 20.07, \emph{p} \textless{} 0.01, Fig. 2E) and GR
(\emph{t} \textsubscript{8} = 9.315, \emph{p} \textless{} 0.01, Fig. 2F)
levels simultaneously decreased in the PVN, but the MR/GR ratio was
unchanged (Fig. 2G).

\textbf{Effects of intra-PVN infusion of RU486 on behavior in rats that
were chronically treated with CORT}

We investigated the effects of RU486 on depressive- and anxiety-like
behaviors and the levels of GRs, MRs, and CRF in the PVN in rats that
were treated with CORT + vehicle. On day 16 during CORT consumption at
08:00, the rats received an intra-PVN infusion of RU486 for 7 days. As
shown in Fig. 3, rats that were treated with CORT + vehicle also
exhibited depressive-like behaviors in the FST (\emph{F}
\textsubscript{3, 23} = 42.47, \emph{p} \textless{} 0.01, Fig. 3A) and
SPT (\emph{F} \textsubscript{3, 23} = 7.599, \emph{p} \textless{} 0.01,
Fig. 3B) and anxiety-like behaviors in the SIT (\emph{F}
\textsubscript{3, 23} = 24.03, \emph{p} \textless{} 0.01, Fig. 3C) and
NSFT (\emph{F} \textsubscript{3, 23} = 6.225, \emph{p}\textless{} 0.01,
Fig. 3D). The intra-PVN infusion of RU486 did not influence CORT-induced
depressive- or anxiety-like behaviors (Fig. 3A-D). Western blot revealed
that the intra-PVN infusion of RU486 restored GR expression in the PVN
(\emph{F} \textsubscript{3, 16} = 15.88, \emph{p} \textless{} 0.01, Fig.
3F) without marked improvement in MR levels in the PVN (Fig. 3E) and
significantly decreased the MR/GR ratio (\emph{F} \textsubscript{3, 16}
= 8.628, \emph{p} \textless{} 0.01, Fig. 3G). Unexpectedly, intra-PVN
RU486 administration augmented the increase in CRF levels compared with
rats that were treated with CORT + vehicle (\emph{F} \textsubscript{3,
16} = 94.75, \emph{p} \textless{} 0.01, Fig. 3H). These data suggest
that although GR levels in the PVN in rats that were treated with CORT +
vehicle rats were restored to normal by the intra-PVN infusion of RU486
alone, the MR/GR ratio was still imbalanced, and CRF levels remained
high. Thus, depressive- and anxiety-like behaviors in rats that were
chronically treated with CORT were not inhibited.

Effects of intra-PVN infusion of spironolactone on behavior in rats that
were chronically treated with CORT

{\label{effects-of-intra-pvn-infusion-of-spironolactone-on-behavior-in-rats-that-were-chronically-treated-with-cort}}

To elucidate the involvement of MRs in the PVN in the behavioral
alterations that were induced by chronic CORT exposure, the MR
antagonist spironolactone was microinjected into the PVN on day 16 of
CORT consumption at 08:00 for 7 days. Depressive-like behavior,
including immobility time in the FST (Fig. 4A) and sucrose preference in
the SPT (Fig. 4B), were not reversed by intra-PVN spironolactone
administration. In contrast, the intra-PVN infusion of spironolactone in
CORT-exposed rats exerted anxiolytic-like effects, reflected by an
increase in social interaction in the SIT (\emph{F} \textsubscript{3,
22}= 18.76, \emph{p} \textless{} 0.01, Fig. 4C) and a shorter feeding
latency in the NSFT (\emph{F} \textsubscript{3, 22} = 19.07,
\emph{p}\textless{} 0.01, Fig. 4D). The intra-PVN injection of
spironolactone reversed the decrease in MR levels in the PVN that was
induced by chronic CORT exposure (\emph{F} \textsubscript{3, 16} =
26.15, \emph{p} \textless{} 0.01, Fig. 4E). Chronic CORT exposure
significantly altered GR and CRF expression, and these changes were
unaffected by intra-PVN spironolactone administration (Fig. 4F, H).
Because MR levels returned to normal but GRs levels failed to recover,
the MR/GR ratio was two-times higher than normal and notably out of
balance (\emph{F} \textsubscript{3, 16} = 7.344, \emph{p} \textless{}
0.01, Fig. 4G). Based on these results, the CORT-induced decrease in MR
levels in the PVN may be associated with anxiety-like behavior, and
intra-PVN spironolactone administration restored MR levels to normal,
which may be related to its anxiolytic effect. Meanwhile, the failure of
GR and CRF levels and the MR/GR ratio to return to normal in the PVN in
CORT-treated rats may explain why the intra-PVN infusion of
spironolactone did not produce an antidepressant effect.

Effects of co-administration of RU486 and spironolactone in the PVN on
behavior and GR, MR and CRF levels in rats that were chronically treated
with CORT

{\label{effects-of-co-administration-of-ru486-and-spironolactone-in-the-pvn-on-behavior-and-gr-mr-and-crf-levels-in-rats-that-were-chronically-treated-with-cort}}

Accumulating evidence indicates that MRs cooperate with GRs in the
stress response (Spencer et al., 1998; Mattsson et al., 2009; Jadavji et
al., 2011). In this experiment, we co-administered the GR antagonist
RU486 and MR antagonist spironolactone in the PVN. RU486 was
microinjected in the PVN once daily beginning on day 16 of CORT
consumption at 08:00 for 7 days. Spironolactone was administered on the
same days, 40 min after RU486 administration. Combined GR and MR
blockade in the PVN in rats that were chronically treated with CORT
restored behavior in the FST (\emph{F} \textsubscript{3,22} =
15.78,\emph{p} \textless{} 0.01, Fig. 5A), SPT (\emph{F}
\textsubscript{3,22} = 9.56, \emph{p} \textless{} 0.01, Fig. 5B), SIT
(\emph{F} \textsubscript{3,22} = 16.68, \emph{p} \textless{} 0.01, Fig.
5C) and NSFT (\emph{F} \textsubscript{3,22} = 18.99, \emph{p}
\textless{} 0.01; Fig. 5D) and the levels of MRs (\emph{F}
\textsubscript{3,16} = 14.64, \emph{p} \textless{} 0.01, Fig. 5E), GRs
(\emph{F} \textsubscript{3,16} = 33.93, \emph{p} \textless{} 0.01, Fig.
5F) and CRF (\emph{F} \textsubscript{3,16} = 22.60, \emph{p} \textless{}
0.01, Fig. 5H) in the PVN. Altogether, these data suggested
co-administration of the MR and GR antagonists in the PVN restored the
levels of MRs, GRs, and CRF and the MR/GR ratio in the PVN to normal in
CORT-exposed rats, thus inhibiting depressive- and anxiety-like
behaviors.

\textbf{Discussion}

In the present study, we hypothesized that the regulation of GRs and MRs
in the PVN may influence depressive-like behaviors by modulating CRF in
the PVN. After exogenous CORT exposure via drinking water for 21 days,
plasma corticosterone rhythms were clearly altered in the present rat
model (Supplementary Fig. S2), which resulted in the overproduction of
CRF and reductions of GR and MR expression in the PVN. Intra-PVN
administration of only the combination of the GR antagonist RU486 and MR
antagonist spironolactone inhibited depressive- or anxiety-like
behaviors and restored MR, GR, and CRF levels and the MR/GR ratio in the
PVN to normal. However, intra-PVN administration of either RU486 or
spironolactone alone did not inhibit depressive-like behaviors.
Intra-PVN administration of spironolactone alone inhibit anxiety-like
behaviors.

Glucocorticoid receptors are richly expressed in the medial
parvocellular PVN and co-localize with CRF, thus placing the PVN in a
prime position to control the output of neurons that activate the HPA
axis (Liposits et al., 1987; Uht et al., 1988). Although the PVN is well
known to be the major site of CRF synthesis in the brain (De Souza,
1995), with projections to many extrahypothalamic areas that are
involved in various behaviors, its role in stress-related behaviors has
not been studied extensively. Chronic variable stress reduces the
expression of GRs in the medial parvocellular PVN. CRF expression is
negatively regulated by GCs, and GRs may play a role in the observed
upregulation of CRF during chronic stress (Herman et al., 1995). This
negative correlation between GR and CRF levels (i.e., downregulation of
GRs and upregulation of CRF) was also observed in the PVN in the present
rat model. However, the intra-PVN infusion of RU486 returned GRs level
in the PVN to normal, had no effect on MR, decreased the MR/GR ratio,
and increased CRF levels, and depressive- and anxiety-like behaviors did
not improve. These results showed that simply increasing the expression
of GRs in the PVN without reversing CRF to normal levels could not
improve either depressive- or anxiety-like behavior. Therefore, one
unresolved issue is why the recovery of GRs did not return CRF levels to
normal but rather further increased the abnormally high levels of CRF.
One possibility is the recruitment of other mechanisms beyond the
negative regulation of CRF by GRs. Lamberts et al. reported that RU486
activated the HPA axis, resulting in a resetting of this system at a
higher level at which the diurnal rhythm and responsiveness to CRF
stimulation were maintained, whereas the sensitivity to dexamethasone
diminished, and these changes were caused by the induction of partial
cortisol receptor resistance during RU486 therapy (Lamberts et al.,
1991). Based on its mechanism of action and evidence that the
restoration of normal hypothalamic CRF activity by RU486 appears to be
the rate-limiting step in HPA axis recovery (Muglia et al., 2000),
central GR blockade may deprive the hypothalamus from GR activity,
thereby stimulating CRF-producing neurons in the hypothalamus and
resulting in the further enhancement of CRF.

The HPA axis has long been recognized for its involvement in depression,
with a focus on cortisol/CORT and less of an emphasis on aldosterone as
a stress hormone. A recent study found that aldosterone acted
selectively in mood-regulating brain areas, without competing with
cortisol/CORT (Murck et al., 2014). Although receptors for aldosterone
(i.e., MRs) are known to be expressed in the PVN, its role in the local
regulation of HPA axis function has been less explored. The MR
antagonist spironolactone inhibits the effects of mineralocorticoids by
displacing them from MRs in the PVN. Wu et al. (2013) found that chronic
subcutaneous CORT treatment triggered several depressive-like behaviors
and downregulated MR expression in parallel in the hippocampus and
hypothalamus. On the other hand, they also reported that intraperitoneal
spironolactone administration for 7 days in animals that were
subcutaneously treated with CORT reduced immobility time in the FST and
improved performance in a novel object recognition test (Wu et al.,
2013). In the present study, CORT-exposed rats via drinking water also
exhibited lower MR expression in the prefrontal cortex, hippocampus, and
amygdala (data not shown). Bitran et al. (1998) hypothesized that the
anxiolytic effects of an MR antagonist may be more pronounced in animals
whose hippocampal MRs are activated by CORT or the MR agonist
aldosterone. In the present study, the intra-PVN infusion of
spironolactone restored MR levels in the PVN to normal and improved
anxiety-like behaviors in the NSFT and SIT, without altering CRF levels
in the PVN, but it did not improve depressive-like behaviors in the FST
or SPT. However, still needing to be confirmed is whether the anxiolytic
effects were caused by the restoration of MRs in the PVN in the present
rat model. Thus, further studies should seek to determine the
pharmacological specificity of the effects of intra-PVN spironolactone
administration to exclude possible actions of spironolactone at sites
other than MRs.

The balance between MRs and GRs during chronic stress plays an important
role in the etiology of depression (De Kloet et al., 1998). The
pharmacological modulation of GRs and MRs also influences the endocrine
stress response and depressive-like behaviors in rodents. The ways in
which MRs and GRs interact to influence the endocrine system and related
behaviors are still being clarified. In the present study, RU486 alone
restored the downregulation of GR levels in the PVN to normal but did
not influence the downregulation of MRs, thus making the MR/GR ratio
significantly lower than normal and failing to suppress depressive- and
anxiety-like behaviors (Fig. 3). Spironolactone treatment alone restored
MR levels in the PVN to normal but did not influence the downregulation
of GRs, thus making the MR/GR ratio significantly higher than normal and
inhibiting anxiety-like behaviors in the NSFT and SIT (Fig. 4). When
RU486 and spironolactone were administered together, MR and GR levels
and the MR/GR ratio returned to normal, and depressive- and anxiety-like
behaviors significantly improved (Fig. 5). The present findings suggest
that the normalization of MR, GR, and CRF levels and the MR/GR ratio may
be necessary conditions to inhibit depressive- and anxiety-like
behaviors in the present rat model. Nonetheless, the restoration of MR
levels in the PVN to normal may be a sufficient condition to improve
anxiety-like behaviors.

CRF also acts within other regions of the brain where it directly
contributes to stress-responsive behavior. Indeed, some depressed
patients had high CSF levels of CRF, and these levels are normalized in
patients who respond to antidepressant treatment, suggesting that these
alterations of CRF levels contribute to the symptomology of depression
(Pandey et al., 2019). In the central nervous system, patients had high
levels of CRF in the PVN and in monoaminergic nuclei, including the
locus coeruleus and raphe (Austin et al., 2003; Bissette et al., 2003).
Elevations of plasma CRF levels in depressed individuals are ameliorated
by successful electroconvulsive shock therapy (Nemeroff et al., 1991)
and antidepressant treatment (Heuser et al., 1998). The causal
relationship between depression and CRF hyperactivity and receptor
expression is unclear. The present results suggest that a state of
equilibrium between GRs and MRs in the PVN may improve the function of
the HPA axis, which can improve depressive- and anxiety-like behaviors.
However, the specific ways in which GRs, MRs, and CRF in the PVN are
involved in the pathogenesis of depression and anxiety need to be
elucidated.

The present study has some limitations. We only examined male rats to
eliminate gender influences that are known to modulate experimentally
induced emotion and HPA activity. The significance of sex differences in
PVN function is virtually unexplored. Therefore, we do not know whether
our results are generalizable to female rats. Furthermore, we did not
detect the activity of HPA function. Therefore, unclear was the state of
the HPA axis when antidepressant effects were induced by GR and MR
blockade. One possibility is that the target sites of the antidepressant
effects of RU486 and spironolactone may not be in the PVN.

Overall, the present study found that the combination of pharmacological
GR and MR antagonism in the PVN prevented the pathogenesis of
depressive- and anxiety-like behaviors in rats that were chronically
treated with CORT, confirming that both MRs and GRs in the PVN play an
important role in CRF function. Such a combined pharmacological approach
may be a novel strategy to prevent hypercortisolism- or chronic
stress-induced depression and its metabolic complications. Much work
still needs to be done to achieve a more complete understanding of the
role of GRs, MRs, and CRF in the PVN in depressive- and anxiety-like
behaviors.

\textbf{Acknowledgements}

This work was supported by grants from the National Natural Science
Foundation of China (No. 81872851 and 81871038) and National Scientific
\& Technological major special project (No. 2019ZX09301-127). The
authors declare no competing interests.

\textbf{Author contributions}

H.D. and X.Y.C. designed, conducted, and analyzed the experiments with
oversight by Y.H.Z.. Y.T.L. and X.H. performed forced swim test. H.L.Z
and Y.Q. performed sucrose preference test. N.K. performed
novelty-suppressed feeding test. H.D., S.Y.C. and Y.H.Z. wrote the
paper. All authors reviewed and approved the final manuscript.

\textbf{Conflict of interest statement}

The authors declare no conflicts of interest.



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\textbf{Figure Legends}

\textbf{Fig. 1.} Experimental design. Rats received surgery of
implantation of cannula into PVN. The rats were exposed to the vehicle
or CORT via drinking water for 21 days from day 1 to day 22. The rats
received intra-PVN injections of RU486 and spironolactone, alone or
combined from day 16 to day 22. The rats underwent behavioral
experiments including novelty-suppressed feeding test, social
interaction test, forced swim test and sucrose preference test on day 22
and day 23. The rats were decapitated after the behavioral tests on day
23.

\textbf{Fig. 2.} Chronic CORT treatment for 21 days induced depressive-
and anxiety-like behaviors, decreased GR and MR protein levels, and
increased CRF protein levels in the PVN. Immobility time in the FST (A)
and sucrose preference in the SPT (B) were detected to evaluate
depressive-like behaviors. Social interaction in the SIT (C) and latency
to feed in the NSFT (D) were detected to evaluate anxiety-like behaviors
(\emph{n\textsubscript{1}} , \emph{n\textsubscript{2}} = 9). (E-I)
Quantification of MRs (E), GRs (F), the MR/GR ratio (G), and CRF (H) in
the PVN and representative Western blots (I) after 21 days of vehicle or
CORT administration. \selectlanguage{greek}β-\selectlanguage{english}actin is shown as a quantitative loading control
(\emph{n\textsubscript{1}} , \emph{n\textsubscript{2}} = 5). The data
are expressed as mean \selectlanguage{ngerman}± SEM. *\emph{p} \textless{} 0.05,
**\emph{p}\textless{} 0.01, \emph{vs} . vehicle group (unpaired
Student's\emph{t} -test).

\par\null

\textbf{Fig. 3.} Intra-PVN administration of RU486 for 7 days before the
test had no effect on depressive- or anxiety-like behaviors in rats that
were chronically exposed to CORT via drinking water for 21 days.
Immobility time in the FST (A) and sucrose preference in the SPT (B)
were detected to evaluate depressive-like behaviors. Social interaction
in the SIT (C) and latency to feed in the NSFT (D) were detected to
evaluate anxiety-like behaviors (\emph{n\textsubscript{1}}
,\emph{n\textsubscript{2}} , \emph{n\textsubscript{3}} =
6,\emph{n\textsubscript{4}} = 9). (E-I) Quantification of MRs (E), GRs
(F), the MR/GR ratio (G), and CRF (H) in the PVN and representative
Western blots (I). \selectlanguage{greek}β-\selectlanguage{english}actin is shown as a quantitative loading control
(\emph{n\textsubscript{1}} , \emph{n\textsubscript{2}}
,\emph{n\textsubscript{3}} , \emph{n\textsubscript{4}} = 5). The data
are expressed as mean \selectlanguage{ngerman}± SEM. *\emph{p} \textless{} 0.05,
**\emph{p}\textless{} 0.01, \emph{vs} . vehicle group;
\textsuperscript{\#}\emph{p}\textless{} 0.05,
\textsuperscript{\#\#}\emph{p} \textless{} 0.01,\emph{vs} . CORT alone
group (one-way ANOVA followed by the Tukey's post hoc test).

\par\null

\textbf{Fig. 4.} Intra-PVN infusion of spironolactone (SPL) for 7 days
before the test ameliorated chronic CORT-induced anxiety-like behaviors.
Immobility time in the FST (A) and sucrose preference in the SPT (B)
were detected to evaluate depressive-like behaviors. Social interaction
in the SIT (C) and latency to feed in the NSFT (D) were detected to
evaluate anxiety-like behaviors (\emph{n\textsubscript{1}}
,\emph{n\textsubscript{2}} , \emph{n\textsubscript{3}} =
6,\emph{n\textsubscript{4}} = 8). (E-I) Quantification of GRs (E), MRs
(F), the MR/GR ratio (G), and CRF (H) in the PVN and representative
Western blots (I). \selectlanguage{greek}β-\selectlanguage{english}actin is shown as a quantitative loading control
(\emph{n\textsubscript{1}} , \emph{n\textsubscript{2}}
,\emph{n\textsubscript{3}} , \emph{n\textsubscript{4}} = 5). The data
are expressed as mean \selectlanguage{ngerman}± SEM. *\emph{p} \textless{} 0.05,
**\emph{p}\textless{} 0.01, \emph{vs} . vehicle group;
\textsuperscript{\#}\emph{p}\textless{} 0.05,
\textsuperscript{\#\#}\emph{p} \textless{} 0.01,\emph{vs} . CORT alone
group (one-way ANOVA followed by the Tukey's post hoc test).

\textbf{Fig. 5.}Co-administration of RU486 and spironolactone in the PVN
produced anxiolytic- and antidepressant-like effects. Immobility time in
the FST (A) and sucrose preference in the SPT (B) were detected to
evaluate depressive-like behaviors. Social interaction in the SIT (C)
and latency to feed in the NSFT (D) were detected to evaluate
anxiety-like behaviors (\emph{n\textsubscript{1}} ,
\emph{n\textsubscript{2}} ,\emph{n\textsubscript{3}} = 6,
\emph{n\textsubscript{4}} = 8). (E-I) Quantification of MRs (E), GRs
(F), the MR/GR ratio (G), and CRF (H) in the PVN and representative
Western blots (I). \selectlanguage{greek}β-\selectlanguage{english}actin is shown as a quantitative loading control
(\emph{n\textsubscript{1}} ,\emph{n\textsubscript{2}} ,
\emph{n\textsubscript{3}} ,\emph{n\textsubscript{4}} = 5). The data are
expressed as mean \selectlanguage{ngerman}± SEM. *\emph{p} \textless{} 0.05, **\emph{p}
\textless{} 0.01, \emph{vs} . vehicle group;
\textsuperscript{\#}\emph{p} \textless{}
0.05,\textsuperscript{\#\#}\emph{p} \textless{} 0.01, \emph{vs} . CORT
alone group (one-way ANOVA followed by the Tukey's post hoc test).\selectlanguage{english}
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\includegraphics[width=0.70\columnwidth]{figures/Fih/Fih}

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