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). 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.
Materials and Methods
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 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).
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, ± 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 μm
thick sections by light microscopy (Supplementary Fig. S1).