A robust and reliable non-invasive test for stress responsivity in mice.

Annemarie Zimprich,Valerie Gailus-Durner,Sabine M Hã¶Lter,Helmut Fuchs,Carsten T Wotjak,Jan M Deussing,Lillian Garrett,Martin Hrabä› De Angelis,Wolfgang Wurst

doi:10.3389/fnbeh.2014.00125

Abstract

Stress and an altered stress response have been associated with many multifactorial diseases, such as psychiatric disorders or neurodegenerative diseases. As currently mouse mutants for each single gene are generated and phenotyped in a large-scale manner, it seems advisable also to test these mutants for alterations in their stress responses. Here we present the determinants of a robust and reliable non-invasive test for stress-responsivity in mice. Stress is applied through restraining the mice in tubes and recording behavior in the Open Field 20 min after cessation of the stress. Two hours, but not 15 or 50 min of restraint lead to a robust and reproducible increase in distance traveled and number of rearings during the first 5 min in the Open Field in C57BL/6 mice. This behavioral response is blocked by the corticosterone synthesis inhibitor metyrapone, but not by RU486 treatment, indicating that it depends on corticosteroid secretion, but is not mediated via the glucocorticoid receptor type II. We assumed that with a stress duration of 15 min one could detect hyper-responsivity, and with a stress duration of 2 h hypo-responsivity in mutant mouse lines. This was validated with two mutant lines known to show opposing effects on corticosterone secretion after stress exposure, corticotropin-releasing hormone (CRH) over-expressing mice and CRH receptor 1 knockout (KO) mice. Both lines showed the expected phenotype, i.e., increased stress responsivity in the CRH over-expressing mouse line (after 15 min restraint stress) and decreased stress responsivity in the CRHR1-KO mouse line (after 2 h of restraint stress). It is possible to repeat the acute stress test several times without the stressed animal adapting to it, and the behavioral response can be robustly evoked at different ages, in both sexes and in different mouse strains. Thus, locomotor and rearing behavior in the Open Field after an acute stress challenge can be used as reliable, non-invasive indicators of stress responsivity and corticosterone secretion in mice.

Highlights

Stress is a major risk-factor in many multifactorial diseases, such as cardiovascular diseases, psychiatric disorders like anxiety and depression, as well as neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease (Lupien et al, 1994; Black and Garbutt, 2002; Esch et al, 2002; Bunker et al, 2003; De Kloet et al, 2005; Sotiropoulos et al, 2008; Catania et al, 2009)
But not 15 or 50 min of restraint lead to a robust and reproducible increase in distance traveled and number of rearings during the first 5 min in the Open Field in C57BL/6 mice. This behavioral response is blocked by the corticosterone synthesis inhibitor metyrapone, but not by RU486 treatment, indicating that it depends on corticosteroid secretion, but is not mediated via the glucocorticoid receptor type II
The protocol we developed is easy and inexpensive to apply and provides first and foremost reproducible results, which makes it suitable for large-scale screening. It was established for C57BL/6 mice, as mouse mutants generated by the IKMC are on this genetic background, but as we show it works in BALB/cAnNCrl and C3H/HeNCrl mice, but not in 129S2/SvPasNCrl mice

Summary

Introduction

Stress is a major risk-factor in many multifactorial diseases, such as cardiovascular diseases, psychiatric disorders like anxiety and depression, as well as neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease (Lupien et al, 1994; Black and Garbutt, 2002; Esch et al, 2002; Bunker et al, 2003; De Kloet et al, 2005; Sotiropoulos et al, 2008; Catania et al, 2009). Exposure to a stressor activates the hypothalamic-pituitaryadrenal (HPA)-axis, by secretion of corticotropin-releasing hormone (CRH, aka CRF) and vasopressin from the paraventricular nucleus of the hypothalamus at the level of the median eminence (for review see Stratakis and Chrousos, 1995; Tsigos and Chrousos, 2002; De Kloet et al, 2005). Both neuropeptides in concert lead to the release of adrenocorticotropic hormone (ACTH) from the anterior pituitary into the circulation.

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Conclusion