Maternal Separation Combined With Limited Bedding Increases Anxiety-Like Behavior and Alters Hypothalamic-Pituitary-Adrenal Axis Function of Male BALB/cJ Mice.

Rodrigo Orso,Kerstin Camile Creutzberg,Erika Kestering-Ferreira,Saulo Gantes Tractenberg,Luis Eduardo Wearick-Silva,Rodrigo Grassi-Oliveira

doi:10.3389/fnbeh.2020.600766

Abstract

Early life stress (ELS) is considered a risk factor for the development of psychiatric conditions, including depression and anxiety disorder. Individuals that live in adverse environments are usually exposed to multiple stressors simultaneously, such as maternal neglect, maltreatment, and limited resources. Nevertheless, most pre-clinical ELS models are designed to explore the impact of these events separately. For this reason, this study aims to investigate the effects of a combined model of ELS on anxiety-like behavior and hypothalamic-pituitary-adrenal (HPA) axis related targets. From PND 2 to PND 15 BALB/cJ mice were exposed simultaneously to maternal separation (MS; 3 h per day) and limited bedding (LB; ELS group) or left undisturbed (CT group). Maternal behavior was recorded in intercalated days, from PND 1 to PND 9. Male offspring were tested for anxiety-like behavior from PND 53 to PND 55 in the open field test (OF), elevated plus-maze (EPM), and light/dark test (LD). After behavioral testing, animals were euthanized, and glucocorticoid receptor (Nr3c1), corticotrophin-releasing hormone (Crh), and its receptor type 1 (Crhr1) gene expression in the hypothalamus were measured. Moreover, plasma corticosterone levels were analyzed. We observed that ELS dams presented altered quality of maternal care, characterized by a decrease in arched-back nursing, and an increase in passive nursing. Stressed dams also showed an increase in the number of exits from the nest when compared to CT dams. Furthermore, ELS animals showed increased anxiety-like behavior in the OF, EPM, and LD. Regarding gene expression, we identified an increase in hypothalamus Crh levels of ELS group when compared to CT animals, while no differences in Nr3c1 and Crhr1 expression were observed. Finally, stressed animals showed decreased levels of plasma corticosterone when compared to the CT group. In conclusion, we observed an alteration in maternal behavior in ELS dams. Later in life, animals exposed to the combined model of ELS showed increased levels of anxiety-like behavior. Moreover, the central and peripheral HPA measures observed could indicate a dysregulation in HPA function provoked by ELS exposure.

Highlights

Over the years, animal studies have been trying to replicate human early life adverse conditions to model the impact of chronic stress exposure during sensitive periods of development
At postnatal day (PND) 2 no significant weight difference was observed between groups (Figure 2A), but shortly after the end of the stress protocol (PND 16), animals exposed to early life stress (ELS) present a significant reduction in body weight when compared to control group (CT) animals (t(12) = 4.933, p < 0.001; Figure 2B)
Post hoc analysis showed that at 1 PM this increase is only present at PND 5 (p = 0.016; interaction effect: F(12) = 5.586, p = 0.036; Supplementary Figure 1A), and it is not persistent when maternal behavior was recorded during late afternoon (5 PM)

Summary

Introduction

Animal studies have been trying to replicate human early life adverse conditions to model the impact of chronic stress exposure during sensitive periods of development. The meta-analysis of Wang et al (2020), reported that mice exposed to maternal separation (MS) showed no alteration in anxiety-like behavior, highlighting that MS studies face some uncertainties regarding publication bias (e.g., methodological and experimental procedures inconsistencies) that might affect replication and translational potential of studies. These inconsistencies in experimental ELS protocols may induce distinct degrees of stress exposure, attenuating or exacerbating the effects on both offspring and dams (Lehmann and Feldon, 2000; Tractenberg et al, 2016). How a translational approach using the combination of classical ELS models can affect stress-related parameters, such as anxiety-like behavior and hypothalamic-pituitary-adrenal (HPA) axis functioning is yet to be characterized

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Conclusion