Chronic subordination stress phase advances adrenal and anterior pituitary clock gene rhythms

Abstract

Circadian rhythms in glucocorticoids are the product of interactions between the hypothalamic-pituitary-adrenal (HPA) axis and the mammalian clock gene system. The adrenal clock can generate the glucocorticoid rhythm that in turn synchronizes other peripheral clocks to maintain homeostasis. Stress acutely activates and chronically upregulates the HPA axis, suggesting that the adrenal clock could be modulated by stress. However, there is no direct evidence that stress affects the adrenal clock rhythm. We tested the hypothesis that a model of chronic subordination stress (CSS) that has a major impact on HPA axis regulation, metabolism, and emotional behavior alters adrenal and pituitary clock gene rhythms. Clock gene rhythms were assessed using mPER2::Luciferase (PER2Luc) knockin mice in which in vitro bioluminescence rhythms reflect the Per2 clock gene expression. PER2Luc mice that experienced CSS for 2 wk showed positive energy balance reflected by increased body weight and food intake. Additionally, CSS phase advanced the adrenal (∼2 h) and the pituitary (∼1 h) PER2Luc rhythm compared with control mice. The activity rhythm was not affected. The adrenal clock phase shift was associated with increased feed conversion efficiency, suggesting that the metabolic phenotype in CSS mice may be related to altered adrenal clock rhythmicity. Interestingly, a single subordination experience followed by 8 h sensory housing also phase advanced the adrenal, but not the pituitary, PER2Luc rhythm. Overall, these data demonstrate a stress-induced phase shift in a peripheral clock gene rhythm and differential stress sensitivity of two peripheral clocks within the HPA axis, suggesting a link between clock desynchrony and individual vulnerability to stress.

Keywords: adrenal clock; circadian rhythm; corticosterone; subordination stress.

Fig. 1.

Chronic subordination stress (CSS) altered metabolic function and activated the hypothalamus-pituitary-adrenocortical (HPA) axis. A: schematic of the experimental procedure. Over a 14-day period, subordinate mice (black) undergo social interaction with dominant mice (white) for 10 min daily and then are housed together separated by a partition for the remaining 24 h. CSS increased body weight (B), food intake (C), feed conversion efficiency (D), adrenal weight (E), and thymus involution (F) (control, n = 19; CSS, n = 19). G: CSS increased plasma corticosterone [Zeitgeber time (ZT)2: control, n = 14, CSS, n = 19; ZT11: control, n = 24, CSS, n = 18; ZT14: control, n = 6, CSS, n = 6]. *P < 0.05, **P < 0.01, ***P < 0.0001.

Fig. 2.

CSS did not alter the mouse activity rhythms. A: average actograms of control (n = 8) and subordinate (n = 8) animals during the baseline and the stress phase. Animals were exposed to a 12:12 h light-dark cycle with lights on at 0500 h (ZT0). The experimental intervention (indicated by the black vertical arrow), either handling or social defeat encounter, occurred daily between ZT3 (0800 h) and ZT3.5 (0830 h). *Increased activity due to weekly cage change. B: period of the home-cage activity rhythm calculated throughout the stress phase is not different between control and subordinate mice.

Fig. 3.

CSS phase advanced the mPER2::Luciferase (PER2Luc) rhythm in adrenal and anterior pituitary explants. Representative experiment showing the PER2Luc rhythm in adrenal (A) and anterior pituitary (C) explants collected from a control and a CSS mouse. Adrenal (B) and pituitary (D) PER2Luc peak phase is advanced in subordinate mice by 2 and 1 h, respectively. The adrenal PER2Luc phase advance was negatively correlated with feed conversion efficiency in subordinate but not in control mice (E). No association was found for the pituitary phase advance and feed efficiency (F). Adrenal explants: control, n = 19, CSS, n = 19; pituitary explants: control, n = 17, CSS, n = 17. *P < 0.05, **P < 0.01.

Fig. 4.

A single subordination stress (SSS) episode acutely elevates corticosterone and phase advances the adrenal but not the anterior pituitary PER2Luc rhythm. A: schematic of the experimental procedures. Subordinate mouse (black) undergoes social interaction with dominant mouse (white) for 10 min and then mice are housed together separated by a partition for 8 h. B: corticosterone is elevated 30 min after the single defeat exposure (control, n = 8; SSS, n = 6). C: corticosterone remains elevated after a social defeat followed by 8 h sensory contact (control, n = 4; SSS, n = 5). D: PMT tracing of adrenal samples from representative control and subordinate mice. E: adrenal PER2Luc peak phase is advanced in subordinate mice (control, n = 5; SSS, n = 5). F: PMT tracing of pituitary samples from representative control and subordinate mice. G: pituitary PER2LUC peak phase is not altered in subordinate mice (control, n = 5; SSS, n = 5). *P < 0.05, **P < 0.01, ***P < 0.001.