variation (Windle et al. 1998b), making it crucial that samples be collected at the same time each day (e.g., at the nadir of the rhythm). In contrast, urine and feces yield an index of stress reactivity over hours or possibly several days (steady-state samples) and are, therefore, less vulnerable to circadian variation. To date, only hair can provide a chronic index of stress covering a period of several months or more. Assessment of cortisol in hair is presumably unaffected by circadian variation and can be obtained at any time of day.
The above information is relevant for understanding and interpreting what might be revealed about stress and distress by examining the activation of the HPA axis. The two most likely ways to assess distress are to (1) examine differences in basal glucocorticoid levels and identify animals outside a “normal range” or (2) obtain glucocorticoid levels before and after the imposition of a stressor. The first approach is problematic because it assumes that a certain concentration of glucocorticoids indicates distress, although there is no scientific evidence to support this assumption. Moreover, as many sampling methods may themselves activate the stress response, there are no standardized ranges for basal glucocorticoid concentrations. The second approach is also problematic for two reasons: first, the putative relationship between the magnitude of change in glucocorticoid concentrations and distress has not been established; and second, both positive and aversive stimuli activate the HPA axis. Finally, the development of stress or distress is not necessarily associated with activation of the HPA axis, as hormonal changes are not necessarily present under all clearly stressful conditions. For example, animals that experience chronic neuropathic pain do not exhibit changes in circadian corticosteroid levels or oscillations in HPA responsivity to restraint, despite the presence of neuropathic pain markers (mechanical allodynia and hyperalgesia) and activation of central pain and stress circuits in the amygdala (Bomholt et al. 2005; Ulrich-Lai et al. 2006).
As is the case with the hormones of the HPA axis, stressors alter the secretion of other endocrine factors (e.g., prolactin, growth hormone, luteinizing hormone, α-melanocyte stimulating hormone [α-MSH], and oxytocin). Serum levels of these hormones can be effectively used to monitor the temporal dynamics of stress responses. While some (prolactin, α-MSH, oxytocin) increase during stress, others decrease (growth hormone, luteinizing hormone, prolactin), depending on the animal species and the physiological state in which stress occurs (Armario et al. 1984; for more references see Additional References). Due to the fact that these hormones are also released in response to other stimuli (e.g., suckling of young, ultra-