Time-of-day-dependent adaptation of the HPA axis to predictable social defeat stress

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   Figure 1

   Bodyweight regulation and daily food intake across 19 days of day- vs nighttime social defeat stress. (A) Mice exposed to daily social defeat stress during the night at ZT13-15 (SDD) gain bodyweight similar to single-housed control (SHC) mice. In contrast, mice exposed to social defeat stress during the day at ZT1-3 (SDL) show significantly lower bodyweight on stress days than controls. On stress-free days, SDL mice regain weight, catching up with controls. (B) Cumulative food intake over the course of the experiment is comparable between SHC and SDL and significantly elevated when compared with SDD mice. (C) Comparison of anabolic indices (total bodyweight gain (g)/daily food intake (g)) under pre-stressed conditions (day -7 to 0) and over the whole course of the experiment (day 0 to 19). Energy-to-body mass conversion is significantly increased in SDD mice, while anabolic indices of mice exposed to chronic daytime stress (SDL) and controls (SHC) are unchanged. (D) 24-h food intake after the first SD exposure was comparable in all three experimental groups. On stress-free days, SDL mice have a slightly increased food intake, while food intake of SDD mice is reduced. SHC: single-housed controls (●); SDL: social defeat light (□); SDD: social defeat dark (■); data are presented as means ± s.e.m.; n = 15/group; (A and B): repeated measurement two-way ANOVA; (C and D): one-way ANOVA with Bonferroni post-hoc test; *P < 0.05; **P < 0.01; ***P < 0.001.
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   Figure 2

   Adaptation of CRH signalling following repeated social defeat stress. (A, B, D and E) Crh and Avp mRNA expression levels in the PVN (A and D) and the SON (B and E), as assessed by mRNA in situ hybridization on day 20 at the predicted time of stress without previous stressor exposure, were not affected by the stress paradigm. (C and F) Immunoblots for pituitary CRH-R1 and AVP-R1b protein expression reveal a downregulation after repeated social defeat during the night (SDD) compared with controls (SHC) at ZT14 (C and F). Predicted daytime stress does not influence CRH-R1 protein expression at ZT2, but also reduces AVP-R1b protein expression (F). Lower panel: representative blots showing either CRH-R1 or AVP-R1b immunoreactivity together with ß-TUBULIN as loading control. (G) Circulating ACTH concentrations at the time of expected SD are significantly reduced in SDD, but not in SDL compared with SHC mice. CRH: corticotrophin-releasing hormone; AVP: arginine vasopressin; PVN: paraventricular nucleus of the hypothalamus; SON: supraoptic nucleus of the hypothalamus; ACTH: adrenocorticotropic hormone; SHC: single-housed controls (●); SDL: social defeat light (□); SDD: social defeat dark (■); data are presented as mean ± s.e.m.; n = 6–9/group; unpaired t-tests were used for statistical comparison of SHC and SD mice at specific time points; *P < 0.05; **P < 0.01; ***P < 0.001.
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   Figure 3

   Adaptation of adrenal GC regulation following repeated social defeat stress. (A) MC2R protein levels show a robust diurnal rhythm with elevated expression during the night (ZT14) compared with the day (ZT2) in SHC mice investigated by immunoblots. Preceding daytime defeat stress has no impact on MC2R protein expression. Lower panel: representative blots showing MC2R immunoreactivity and β-TUBULIN as loading control. (B) 19 days of social defeat stress at night (SDD) led to reduced expression of SR-B1 at ZT14 compared with controls while daytime stress (SDL) does not influence SR-B1 protein expression at the time of expected stress exposure (ZT2). Lower panel: representative immunoblots for SR-B1 and β-TUBULIN as loading control. (C and D) Oil-Red O staining of adrenal gland sections does not reveal overt differences in lipid content among stress groups. (E) Adrenal gland weights are increased in response to SD independent of the time of stressor exposure. (F) In control animals (SHC), circulating corticosterone concentrations are elevated at ZT14 compared with ZT2. Nighttime levels (ZT14) of circulating corticosterone are decreased after 19 days of chronic nighttime social defeat stress (SDD). Chronic daytime stress (SDL) has no impact on circulating corticosterone concentrations at ZT2, the predicted time of stress. MC2R: melanocortin 2 receptor; SR-B1: scavenger receptor class B, member 1; SHC: single-housed controls (●); SDL: social defeat light (□); SDD: social defeat dark (■); data are presented as mean ± s.e.m.; n = 8–9/group; unpaired t-tests were used for statistical comparison of SHC and SD mice at specific time points; *P < 0.05; **P < 0.01; ***P < 0.001.
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   Figure 4

   Effects of repeated social defeat stress on diurnal corticoid excretion rhythms and acute stress responses. (A) Trailing circulating corticosterone levels, excreted corticoids in SHC mice are elevated during the night with a maximum at ZT13-17. This diurnal rhythm is altered in mice exposed to chronic social defeat stress. After SD exposure corticoid levels of SDD and SDL are elevated towards the end of the day and reduced during the night. No significant adaptations are observed for total 24-h excretion (A.U.C.; B) or rhythm amplitude (C). Hippocampal free GC content rises significantly after an acute 5-min swim test in all three groups, independent of prior social stressor exposure. (D) Baseline (ZT2-4), swim stress-induced increase in hippocampal free corticosterone (ZT6-8) and post-stress content (ZT8-10) as well as (E) calculated A.U.C.s were comparable in all three groups. Data are normalized to baseline values. SHC: single-housed controls (●); SDL: social defeat light (□); SDD: social defeat dark (■); data are presented as mean ± s.e.m.; A–C: n = 3–4/group, D, E: n = 7; A, D: repeated measurement two-way ANOVA; B, C, E: one-way ANOVA; ***P < 0.001 (relative to ZT2-4).

• © 2016 Society for Endocrinology