Repeated exposure to two stressors in sequence demonstrates that corticosterone and paraventricular nucleus of the hypothalamus interleukin-1β responses habituate independently

Abstract

A wide range of stress-related pathologies such as post-traumatic stress disorder are considered to arise from aberrant or maladaptive forms of stress adaptation. The hypothalamic-pituitary-adrenal (HPA) axis readily adapts to repeated stressor exposure, yet little is known about adaptation in neuroimmune responses to repeated or sequential stress challenges. In Experiment 1, rats were exposed to 10 days of restraint alone (60 minutes daily), forced swim alone (30 minutes daily) or daily sequential exposure to restraint (60 minutes) followed immediately by forced swim (30 minutes), termed sequential stress exposure. Habituation of the corticosterone (CORT) response occurred to restraint by 5 days and swim at 10 days, whereas rats exposed to sequential stress exposure failed to display habituation to the combined challenge. Experiment 2 compared 1 or 5 days of forced swim with sequential stress exposure and examined how each affected expression of several neuroimmune and cellular activation genes in the paraventricular nucleus of the hypothalamus (PVN), prefrontal cortex (PFC) and hippocampus (HPC). Sequential exposure to restraint and swim increased interleukin (IL)-1β in the PVN, an effect that was attenuated after 5 days. Sequential stress exposure also elicited IL-6 and tumour necrosis factor-α responses in the HPC and PFC, respectively, which did not habituate after 5 days. Experiment 3 tested whether prior habituation to restraint (5 days) would alter the IL-1β response evoked by swim exposure imposed immediately after the sixth day of restraint. Surprisingly, a history of repeated exposure to restraint attenuated the PVN IL-1β response after swim in comparison to acutely-exposed subjects despite an equivalent CORT response. Overall, these findings suggest that habituation of neuroimmune responses to stress proceeds: (i) independent of HPA axis habituation; (ii) likely requires more daily sessions of stress to develop; and (iii) IL-1β displays a greater tendency to habituate after repeated stress challenges compared to other stress-reactive cytokines.

Keywords: corticosterone; habituation; interleukin-1; multimodal stress; sequential stress.

Figure 1

Locations of brain punches for target structures, Experiment 1 design, and CORT adaptation results. A. Cingulate portion of the prefrontal cortex (Cing/PFC) - four punches, each 1.2 mm punch diameter, 1 mm punch deep. B. Paraventricular nucleus of the hypothalamus (PVN)-enriched punches – four punches, each 1.2 mm punch diameter, 1 mm punch depth. C. Hippocampus (HPC) – dorsal and ventral portions were combined and processed together. Dorsal – four punches, each 2 mm diameter, 1.5 mm depth. Ventral - four punches, each 1.2 mm diameter, 1 mm deep. D. In experiment 1, Rats were exposed to restraint alone, forced swim alone, or restraint immediately followed by forced swim for 10 days. Blood collection timepoints on days 1, 5, and 10 are indicated with red dots, body weight measurements are indicated with blue triangles, and organs were weighed at the point signified by the green square. Results from the corticosterone ELISA (n=8/group) are depicted for (E) the restraint alone group, (F) the forced swim alone group, and (G) the restraint + forced swim group. The * denotes where D1 differs from D10, # where D5 differs from D10, $ where D1 differs from D5, and ^ where D5 differs from D10.

Figure 2

Inflammatory cytokine and c-Fos real-time RTPCR results from Experiment 2. Rats were exposed to 1 or 5 days of forced swim or restraint immediately followed by forced swim. Immediately after stress on the last day of exposure, rats were rapidly decapitated and brains were collected. All data is expressed relative to the control group (homecage) and normalized to β-actin. Each group consisted of 6–8 subjects. Different letters across groups denote significant differences.

Figure 3

Design and results from Experiment 3. A. Rats were exposed to 5 days of repeated daily exposure to 60 minutes of restraint with a subsequent change in stressor (30 minutes of forced swim or an additional 30 minutes of restraint) on day 6, acutely exposed to 60 minutes of restraint followed immediately by 30 minutes of forced swim, received 5 days of restraint with no stress on day 6, or were non-stressed controls. B. Plasma corticosterone levels were assessed in each of the above groups (n=8). Different letters across groups denote significant differences. Real-time RTPCR data from the PVN (C), HPC (D), and PFC (E) expressed relative to the control group (homecage) and normalized to β-actin. Different letters across groups denote significant differences.