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).