A Comparison of Two Repeated Restraint Stress Paradigms on Hypothalamic-Pituitary-Adrenal Axis Habituation, Gonadal Status and Central Neuropeptide Expression in Adult Male Rats

The available evidence continues to illustrate an inhibitory influence of male gonadal activity on the hypothalamic-pituitary-adrenal (HPA) axis under acute stress. However, far less is known about how these systems interact during repeated stress. Because HPA output consistently declines across studies examining repeated restraint, the potential mechanisms mediating this habituation are often inferred as being equivalent, even though these studies use a spectrum of restraint durations and exposures. To test this generalisation, as well as to emphasise a potential influence of the male gonadal axis on the process of HPA habituation, we compared the effects of two commonly used paradigms of repeated restraint in the rodent: ten daily episodes of 0.5 h of restraint and five daily episodes of 3 h of restraint. Both paradigms produced comparable declines in adrenocorticotrophic hormone and corticosterone between the first and last day of testing. However, marked differences in testosterone levels, as well as corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP) expression, occurred between the two stress groups. Plasma testosterone levels remained relatively higher in animals exposed to 0.5 h of restraint compared to 3 h of restraint, whereas forebrain gonadotrophin-releasing hormone (GnRH) cell counts increased in both groups. AVP mRNA was increased after 3 h, but not after 0.5 h of repeated restraint, in the medial parvicellular paraventricular nucleus and in the posterior bed nucleus of the stria terminalis (BST), and increased with 0.5 h of repeated restraint in the medial amygdala. CRH mRNA was increased after 3 h, but not after 0.5 h of repeated restraint, in the central amygdala and anterior BST. The data obtained illustrate that, despite comparable declines in HPA responses, the pathways recruited for stress adaptation appear to be distinct between restraint groups. Given the extreme sensitivity of limbic AVP to testosterone, and conversely CRH to circulating glucocorticoids, whether differences in endocrine profiles might explain these neuropeptide differences remains to be seen. Nonetheless, the present study provides several new entry points for testing gonadal influences on stress-specific HPA habituation.