*2.1. Neuroendocrine Regulation*

The HPA axis is necessary for adaptation to stress. Physiological stimuli and stress activate the HPA axis (a three-organ hormonal cascade and feedback cycle) which leads to GC (cortisol in humans) secretion and release from the adrenal cortex (Box 1). Indeed, cortisol is both the primary GC which signals the stress response and the primary inhibitor of continuing HPA axis activity and of the autonomic stress responses [7].

### **Box 1.** The hypothalamic–pituitary–adrenal (HPA) axis.

Both physiological stimuli and stress trigger the HPA axis leading to glucocorticoids (GCs, cortisol in humans) release from the adrenal cortex:


Cortisol exerts crucial feedback e ffects on binding to GRs in the hypothalamus and the pituitary, which reduces the release of both corticotrophin-releasing hormone (CRH) and adrenocorticotrophic hormone (ACTH) [7]. GC-bound mGR releases endocannabinoids, which, in turn, inhibit the release of glutamate, which reduces the stimulation of the hypothalamic paraventricular nucleus (PVN) CRH neurons. GABAergic and neuropeptidergic inputs also play important roles in inhibiting CRH release. Transsynaptic GR mediated HPA axis inhibition also occurs in the hippocampus, as well as the prefrontal cortex and the hindbrain. These feedback e ffects are very important to stop the exposure to the catabolic actions of GC and the sympathetic nervous system e ffects. Norepinephrine is an important mediator of the central nervous system (CNS) and autonomic stress responses. Norepinephrine and CRH interact in the hypothalamus, locus coeruleus, and amygdala circuit, integrating autonomic and HPA axis responses to stress.

The HPA axis regulation is highly complex. In addition to trans-synaptic regulation, the secretion of CRH by the PVN is also regulated by GCs, arginine vasopressin, and oxytocin [48]. The secretion of ACTH is regulated by GCs. The sensitivity of the adrenal cortex to ACTH is another possible limiting step in HPA axis activity. For in the serum, cortisol is bound to proteins—mainly corticosteroid-binding globulin (CBG)—where only 5% of systemic cortisol is free and bioactive (Figure 1) [49]. The availability of CBG, which is negatively regulated by GC, is another influence on GC actions. At the cellular level, cortisol availability is regulated by tissue-specific enzymes, as well as the 11β-hydroxysteroid dehydrogenases (11β-HSDs). 11βHSD-1 is widely expressed in all the GC target tissues (e.g., neurons and glial cells), where it converts cortisone to active cortisol, whereas 11βHSD-2 has the opposite e ffect, particularly in the kidney (Figure 1) [50]. At the cellular level, cortisol actions are also regulated by the GR and its respective *NR3C1* gene, as well as co-chaperones of the GR, such as FKBP5, among others.

**Figure 1.** Modulators of the cellular action of glucocorticoids. These modulators include glucocorticoid availability, glucocorticoid receptor number and a ffinity, GRβ isoform expression, glucocorticoid receptor signalling, mineralocorticoid receptor signalling, glucocorticoid receptor–gene interaction, and glucocorticoid receptor single nucleotide polymorphisms. Abbreviations: 11β-HSD: 11-β-hydroxysteroid dehydrogenase; CBG: Corticosteroid-binding globulin; GR: glucocorticoid receptor; HSP: heat shock protein; MR: mineralocorticoid receptor; NF-κB: nuclear factor k B; SNPs: single nucleotide polymorphisms; HDAC2: histone deacetylase 2; AP-1: activator protein 1. Adapted by permission from [Nature] [51].

Cortisol alone, therefore, cannot be considered to reflect the HPA axis regulation of the stress response. Furthermore, cortisol response to stressful events is moderated by sex—with women showing greater variability in the HPA axis response than men [52,53]. Testosterone has been shown to inhibit the stress response, whereas in women, estradiol has an important moderating role in the activation of the HPA axis in response to stress [7]. HPA axis stress response is also moderated by the stage of development and the age of the subject, as well as the type, intensity, and duration of the stressful event. Additionally, also important to mention is the fact that cortisol is difficult to measure accurately to represent the reflection of HPA axis activity, as most methods show considerable intraindividual variability and, as described above, HPA axis function depends on a grea<sup>t</sup> variety of factors [54].
