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Heinzmann, Jan-Michael (2012): Central and peripheral aspects of hypothalamic-pituitary-adrenal (HPA) axis dysfunction: insights from mice selectively bred for extremes in stress reactivity. Dissertation, LMU München: Faculty of Biology
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Abstract

An aberrant regulation of the hypothalamic-pituitary-adrenal (HPA) axis is closely associated with the pathophysiology of affective disorders such as major depression (MD). Accordingly, patients suffering from MD frequently show profound neuroendocrine alterations with hyper- or hypo-cortisolism as a result of a dysregulated stress hormone system. Focussing on this key endophenotype of MD, the ‘stress reactivity’ (SR) mouse model was recently established, consisting of three independent mouse lines, the high (HR), intermediate (IR) and low (LR) stress reactivity line, selectively bred for differences in their corticosterone (CORT) secretion in response to a psychological stressor. Previous studies revealed distinct differences between HR, IR and LR animals regarding sleep architecture, activity rhythms, emotional behaviour, cognition as well as neuroendocrine functions, resembling several endophenotypes observed in depressed patients. In the series of studies presented in this work, we aimed to investigate whether the differences between HR, IR and LR mice were restricted to the peripheral phenomenon of adrenal CORT secretion, or whether these endophenotypes were brought about by an aberrant regulation of upstream control centres of the HPA axis. To this end, we performed experiments investigating all functional levels of HPA axis control, i.e. the adrenals, the pituitary and brain centres known to be involved in the neuroendocrine stress response. Moreover, we assessed the expression of corticosteroid-binding globulin (CBG), which contributes to the transport and delivery of CORT to its target tissues. Finally, we studied HPA axis regulatory mechanisms by means of the combined dexamethasone/corticotropin-releasing hormone (Dex/CRH) test. At the level of the adrenal, we found that pharmacological inhibition of the biosynthesis and secretion of CORT using metyrapone had a significant impact on the stress-coping behaviour of HR, IR and LR animals as determined in the forced swim test (FST). As another peripheral factor influencing the secretion of CORT, we assessed the adrenal sensitivity of the animals to adrenocorticotropic hormone (ACTH) in vivo. After a Dex-mediated inhibition of endogenous ACTH release from the anterior pituitary, LR animals showed a markedly reduced CORT surge compared to HR mice in response to a stimulation of the adrenals with two doses of exogenous ACTH, indicating an enhanced adrenal sensitivity in HR mice and a blunted responsiveness to ACTH in LR mice. In addition, we found significant differences in plasma CBG levels between the three mouse lines (HR>IR>LR), concomitant with differences in free plasma CORT both, basal and in response to 15 min restraint stress (HR>IR>LR). Since only free CORT is biologically active, these results indicate that CBG might play a role in the endophenotypes of the SR mouse lines. At the pituitary level, we detected significantly altered ACTH protein levels (HR>IR≥LR) and proopiomelanocortin mRNA expression (HR>IR>LR), suggesting a differential activation of the anterior pituitary between the three lines, which is in line with the observed differences in stress reactivity. In the brain, we assessed the neuronal activation induced by an acute stressor in regions known to be involved in HPA axis function such as the prefrontal cortex, the basolateral amygdala, the hippocampus and the paraventricular nucleus of the hypothalamus (PVN) using c-fos in-situ hybridisation. Acute stress exposure markedly increased neuronal activation in all investigated brain areas. However, significant differences in the neuronal excitation between the three lines were only detected in the PVN (HR>IR>LR), indicating an altered activation of the animals’ HPA system orchestrated by this nucleus. Finally, we assessed HPA axis regulatory mechanisms by means of the Dex/CRH test. Our results revealed considerable similarities to clinical studies, with HR mice showing signs of Dex non-suppression in addition to an overshooting CORT surge after CRH stimulation, mirroring the HPA axis hyper-active state of patients suffering from the psychotic or melancholic subtype of MD. In contrast, LR animals presented a strong Dex-induced CORT suppression and a blunted response to the CRH stimulation, resembling the situation observed in atypically depressed patients. Strikingly, chronic fluoxetine (Flx) treatment enhanced the negative feedback regulation of the HPA axis in all three lines of the SR mouse model. In particular, the Dex-mediated CORT suppression in HR mice was restored by Flx. Furthermore, Flx affected the stress-coping behaviour of the animals in the FST. Flx induced a reduction in active coping, indicating an attenuation of the hyper-aroused state, particularly in HR mice. Taken together, the series of studies presented here demonstrated that the SR mouse model shows functional alterations on all levels of the HPA axis - peripheral, central and regarding the regulation – similar to the endophenotypes of MD patients, thus revealing a high level of face and construct validity of the model. Hence, the SR mouse model can serve as a valuable tool in the discovery and validation of new drug targets and improve already existing treatments of MD, particularly those targeting the HPA system.