Chronic psychosocial stress affects corticotropin-releasing factor in the paraventricular nucleus and central extended amygdala as well as urocortin 1 in the non-preganglionic Edinger-Westphal nucleus of the tree shrew

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Summary

Stressful stimuli evoke neuronal and neuroendocrine responses helping an organism to adapt to changed environmental conditions. Chronic stressors may induce maladaptive responses leading to psychiatric diseases, such as anxiety and major depression. A suitable animal model to unravel mechanisms involved in the control of adaptation to chronic stress is the psychological subordination stress in the male tree shrew. Subordinate male tree shrews exhibit chronic hypothalamo–pituitary–adrenal (HPA) activation as reflected in continuously elevated cortisol secretion, and structural changes in the hippocampal formation. Corticotropin-releasing factor (CRF) is the major peptide released upon activation of the HPA axis in response to stress. Recent evidence suggests that besides CRF, urocortin 1 (Ucn1) also plays a role in stress adaptation. We have tested the significance of CRF and Ucn1 in adaptation to chronic psychosocial stress in male tree shrews exposed for 35 days to daily psychosocial conflict, by performing semi-quantitative immunocytochemistry for CRF in the parvocellular hypothalamic paraventricular nucleus (pPVN), extended amygdala, viz. central extended amygdala (CeA) and dorsolateral nucleus of the bed nucleus of the stria terminalis (BNSTdl) as well as that for Ucn1 in the non-preganglionic Edinger-Westphal nucleus (npEW). Compared to unstressed animals, psychosocial stress resulted in an immediate and sustained activation of the HPA axis and sympathetic tone as well as reduced testosterone concentration and decreased body and testis weights vs. non-stressed tree shrews. In the pPVN, the number of CRF-immunoreactive neurons and the specific signal density of CRF-immunoreactive fiber terminals in the CeA were strongly reduced (−300 and −40%, respectively; P<0.05), whereas no significant difference in CRF fiber density was found in BNSTdl. The npEW revealed 4 times less Ucn1-immunoreactive neurons (P<0.05). These clear effects on both Ucn1- and CRF-neuropeptide contents may reflect a crucial mechanism enabling the animal to adapt successfully to the stressors, and point to the significance of the pPVN, CeA and npEW in stress-induced brain diseases.

Introduction

Adaptation to a continuously changing environment is fundamental for maintaining physical and mental health. Failed adaptation may lead to stress-related psychiatric disorders, such as anxiety and major depression. These mental disorders impair the lives of more then 10–15% of the human population (Reul and Holsboer, 2002), explaining why animal models to unravel brain circuitries involved in stress adaptation are of crucial importance. A promising model is the chronic psychosocial stress paradigm in male tree shrews. Coexistence of two males in visual and olfactory contact leads to a stable dominant/subordinate relationship, with the subordinates showing obvious changes in behavioral, neuroendocrine and central nervous activity that are similar to the signs and symptoms observed during episodes of depression in human patients (Fuchs et al., 2004; Fuchs, 2005). More specifically, subordinates loose body weight, have reduced locomotor activity and their circadian rhythm is profoundly disturbed. They also exhibit increased concentration of cortisol, increased adrenal weight and diminished gonadal function as well as reduced hippocampal volume and proliferation rate of the granule precursor cells in the dentate gyrus (for review see Fuchs, 2005). Interestingly, all of these responses can be normalized by antidepressants, suggesting that this psychosocial stress paradigm is a valid model with high face and predictive validity for human depression (Fuchs and Flugge, 2002; van Kampen et al., 2002; Fuchs, 2005). Moreover, the tree shrew is a non-rodent species that is phylogenetically situated between insectivores and primates, with major similarities to the latter (Martin, 1990; Kriegs et al., 2007).

The hypothalamic–pituitary–adrenal (HPA) axis is an extremely sensitive physiological system, which is activated by a wide range of physiological and psychological stressors, leading to the release of corticotropin-releasing factor (CRF), in the parvocellular hypothalamic paraventricular nucleus (pPVN), adrenocorticotrope hormone (ACTH) in the pituitary gland and glucocorticoids in the adrenal cortex (e.g. Vale et al., 1981; Arborelius et al., 1999; de Kloet, 2000; Tsigos and Chrousos, 2002). In addition to the pPVN, CRF is also found in stress-related brain areas such as the central extended amygdala, viz. central nucleus of the amygdala (CeA) and dorsolateral nucleus of the bed nucleus of the stria terminalis (BNSTdl; Merchenthaler et al., 1982; Herman et al., 2005; McEwen, 2007).

The stress response with the activation of the HPA axis, however, is meant to be of a limited duration, thus chronic stress leads to a complete habituation of the HPA axis stress response, which is an adaptive phenomenon protecting the organism from the adverse effects of sustained elevated levels of circulating glucocorticoids (Viau and Sawchenko, 2002; Armario et al., 2004; de Kloet et al., 2005). However, in certain conditions, if there are high glucocorticoids together with a chronic stressor, the steroids act in brain in a feed-forward fashion to recruit a stress-response network and the HPA axis remains hyperactive (for review see Dallman et al., 2006). This maladaptive coping strategy may result in increased incidence of stress-induced brain diseases, such as major depression (Tsigos and Chrousos, 2002; Strohle and Holsboer, 2003; de Kloet et al., 2005; Bale, 2006; Muller and Holsboer, 2006). In line with this notion, accumulating evidence suggests that dysregulation of CRF may play a role in depression (Holsboer et al., 1984a, Holsboer et al., 1984b; Banki et al., 1987; Rubin et al., 1987; Nemeroff, 1988), and that this dysregulation may be corrected by antidepressant drug treatment (Nemeroff, 1992; De Bellis et al., 1993).

A neuropeptide related to CRF and participating in the physiology of the stress adaptation, is urocortin 1 (Ucn1; Vaughan et al., 1995). In mammals, Ucn1 is most abundantly expressed in the non-preganglionic Edinger-Westphal nucleus (npEW; Vaughan et al., 1995; Kozicz et al., 1998; Bittencourt et al., 1999; Ryabinin et al., 2005). The EW is classically known as a cholinergic, preganglionic parasympathetic midbrain nucleus projecting to the ciliary ganglion. However, evidence has accumulated that the structure and the function of the EW are more complex than originally thought, and the existence of anatomically and functionally distinct neuron populations within the EW has been postulated (Saper et al., 1976; Burde et al., 1982; Vasconcelos et al., 2003; Ryabinin et al., 2005; Laursen and Rekling, 2006; and unpublished observations of the authors). Therefore, to distinguish EW cells that are positive for Ucn1 but do not innervate the ciliary ganglion from those that exhibit choline acetyltransferase immunopositivity and do project to the ciliary ganglion, the terminology of npEW has been introduced (Weitemier et al., 2005; Ryabinin et al., 2005). Ucn1 neurons in the npEW are recruited by various kinds of stressors (Weninger et al., 2000; Kozicz et al., 2001; Gaszner et al., 2004; Korosi et al., 2005). It has been demonstrated that in the npEW Ucn1 mRNA levels are upregulated in CRF-deficient mice compared to the wild-type mice (Weninger et al., 2000). Therefore, an inverse relationship between CRF- and Ucn1-containing neuronal systems has been hypothesized and supported by the finding of down-regulated Ucn1 mRNA expression in the npEW of CRF over-expressing mice (Weninger et al., 2000; Kozicz et al., 2004). Furthermore, Ucn1 neurons in the npEW in the mouse show a non-habituating Fos response to a chronic homotypic ether challenge that also resulted in a marked down-regulation of npEW Ucn1 mRNA levels as compared to acutely stressed animals (Korosi et al., 2005). In addition, Ucn1-null mice exhibit increased anxiety-like behavior (Vetter et al., 2002), as well as impaired HPA axis adaptation to repeated restraint stress (Zalutskaya et al., 2007). From these results it was concluded that CRF and Ucn1 neuronal systems are closely (inter)related and play a crucial role in regulating adaptation to stress.

Based on the above and the fact that altered brain peptide contents are considered as good indicators of stress adaptation, here we tested the hypothesis that in the male tree shrew, chronic psychological stress would alter the secretory dynamics of Ucn1 in npEW and that of CRF in pPVN, CeA and BNSTdl.

Section snippets

Animal experimentation

Twelve experimentally naive adult male tree shrews (Tupaia belangeri) were obtained from the breeding colony at the German Primate Center (Göttingen, Germany). They were housed individually on a 12 h light 12 h dark cycle with ad libitum access to food and water. All experiments were in accordance with the European Communities Council Directive of November 24, 1986 (86/EEC) and with the US National Institutes of Health Guide for the Care and Use of Laboratory Animals, and had been approved by the

Body weight

Chronic psychosocial stress resulted in a significant and maintained reduction in body weight in subordinate animals (100%=217 g) as compared to non-stressed control animals (100%=224 g) starting with the onset of the conflict situation (Figure 1a).

Neuroendocrine parameters

Repeated stressful experience resulted in a sustained elevation of urinary-free cortisol (100%=265 pg/μMol creatinine; Figure 1b) vs. controls (100%=125 pg/μMol creatinine). Two-way ANOVA (stress×time) revealed a significant difference between groups (P

Discussion

In this study, we have tested our hypothesis that chronic psychosocial stress in the tree shrew affects the secretory dynamics of CRF and Ucn1 in three main centers involved in adaptation to various stressful conditions viz. the npEW, pPVN, CeA and BNSTdl. The major findings of this study are: (1) subordinate tree shrews exhibited chronic HPA axis activation; (2) subordinate tree shrews also showed a considerable change in the secretory activity of npEW Ucn1 and pPVN CRF neurons; (3)

Conclusions

In the present study, we have demonstrated the impact of a naturally occurring form of stress on the modulation of CRF in pPVN, CeA and BNSTdl as well as that of Ucn1 in npEW. The observed considerable alterations indicate that each of the three brain centers and both neuropeptides are involved in a substantially strong response by the brain to the chronic psychosocial stressor. However, to elucidate whether these responses are adaptive or maladaptive in nature, longitudinal studies of activity

Role of the funding source

Funding for this study was provided by The Nederlands Organization for Scientific Research (NWO); Grant no.: #864.05.008. NWO had no further role in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication.

Conflict of interest

The authors declare no conflict of interest.

Acknowledgement

The authors are grateful to Dr. M. Heistermann for the determination of serum testosterone.

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