Increased anxiety-like behavior during the post-stress period in mice exposed to repeated restraint stress

Abstract

Mice exposed to repeated restraint (RR: 2 h of restraint on each of 3 consecutive days) lose weight and do not return to the weight of non-stressed controls after restraint ends. These mice also exhibit an exaggerated endocrine response to mild stressors in the post-stress period. To determine if other aspects of the stress response are altered, NIH Swiss mice were repeatedly restrained then evaluated for anxiety-like behavior in various behavioral tests. Twelve days after the end of RR half of the control and RR mice were subjected to the mild stress of an intraperitoneal injection of saline before placement in an elevated plus maze. RR mice not subjected to mild stress showed the same level of anxiety as the control and RR mice exposed to mild stress. Placement in a light–dark box 20 days after restraint also indicated an increase in anxiety-like behavior in RR mice that had not been exposed to mild stress. In contrast, RR mice displayed no increase in anxiety-like behavior in the defensive withdrawal apparatus and the marble burying test 6 and 17 days, respectively, after restraint. RR mice released more corticosterone than non-restrained controls exposed to defensive withdrawal or EPM apparatus although baseline corticosterone remained at control levels. These results suggest that RR induces an exaggeration of both endocrine and behavioral responses to subsequent mild stressors. This post-stress hypersensitivity to mild stress may contribute to the sustained reduction in the body weight of RR animals.

Introduction

Mice and rats exposed to 2 h of restraint each morning for 3 consecutive days (repeated restraint, RR) lose weight during the period of restraint (Harris et al., 2001). It has been shown that this initial weight loss is due to an increase in energy expenditure and a decrease in food intake on the days of restraint (Harris et al., 2006). Following RR, food intake and energy expenditure return to pre-stress levels and the animals gain weight at the same rate as controls, however, they do not return to the weight of the controls (Harris et al., 2001, Harris et al., 2006). Although baseline serum corticosterone levels and the daily nadir of corticosterone release remain normal, RR animals exhibit an exaggerated corticosterone response to novel mild stressors presented 12 days after the end of repeated restraint (Harris et al., 2004). Although much of the work with this model has been carried out with rats, mice exposed to RR also show the same sustained down-regulation of body weight and hyperresponsiveness to subsequent mild stressors (Harris et al., 2001, Harris et al., 2004). Others have reported similar increased sensitivity to subsequent stressors in animals that have been acutely stressed. For example, rats exposed to one or three daily sessions of footshock show an exaggerated corticosterone response to a stress applied 10 days later (Caggiula et al., 1989, Gomez et al., 2002, Servatius et al., 1994). The increased sensitivity of the endocrine response to stressors in the post-stress period has been associated with the impaired ability of the glucocorticoids to suppress hypothalamic–pituitary–adrenal (HPA) axis activity and a lowering of the threshold for the initiation of endocrine responses to stress (Buwalda et al., 1997, Harris et al., 2004).

Exposure to stress results in a myriad of responses, of which activation of the HPA axis (Cook, 2004), an increase in energy expenditure (Harris et al., 2006), and a decrease in feeding and appetite (Harris et al., 2002b) are only a few. Exposure to stress also results in behavioral changes. For example, a period of acute restraint stress has been shown to increase anxiety-like behavior in three well-established behavior tests, the elevated plus maze (EPM), the defensive withdrawal paradigm, and the light–dark box measured immediately after the end of the restraint stress (Harris et al., 2001, Harris et al., 2002a, MacNeil et al., 1997, Morilak et al., 2003, Smagin et al., 1996). These behavioral tests rely on the conflict between the rodent's natural aversion to illuminated, open spaces and a tendency to explore novel areas. In addition, there is evidence that exposure to an acute stressor can result in sustained increases in anxiety-like behavior. In 1992, van Dijken et al. reported that rats exposed to a single session of inescapable foot shock demonstrated enhanced anxiety-like behavior in the open field apparatus 21 days later (Van Dijken et al., 1992). Adamec and Shallow (1993) found that a single, 5-min exposure to a predator caused exaggerated anxiety-like behavior in the EPM in rats 14 days later. In contrast to the EPM or light–dark box tests, the marble-burying test measures fear of a novel, invasive object (De Boer and Koolhaas, 2003, Njung'e and Handley, 1991). Therefore, this study was performed in order to determine whether RR induces long-lasting changes in anxiety-like behavior in various tests during the post-stress period in addition to causing the long-lasting hyperreactivity of the HPA axis. Six days after the end of repeated restraint, we measured behavior of controls and of mice that had been exposed to repeated restraint in a defensive withdrawal apparatus but found no difference in anxiety-type behaviors. Previously we had detected an exaggeration of the corticosterone response to mild stress in rats previously exposed to repeated restraint stress (Harris et al., 2004), therefore we subjected half of each of the control and restrained mice in this study to a mild stress before testing behavior in the elevated plus maze. The results of this test, performed 9–10 days after the end of repeated restraint, indicated that mice that had been exposed to repeated restraint but not mild stress were more anxious than mice that had never been exposed to any stress. Therefore, we did not use mild stress in any of the two final behavioral tests of marble burying 17 days after the end of restraint or activity in a light–dark box 19–20 days after the end of restraint.