The effects of stress during early postnatal periods on behavior and hippocampal neuroplasticity markers in adult male mice
Introduction
Disrupted maternal care early in life interferes with the proper development of the infant and may result in the manifestation of neurobiological and behavioral abnormalities in adulthood. Several rodent models have been developed to study the effects of early-life stressors on brain functioning. Early-life exposure to maternal separation, maternal deprivation as well as neonatal handling and limited bedding/nesting were shown to have long-lasting neurobehavioral consequences (Levine, 1956, Rosenfeld et al., 1992, Plotsky and Meaney, 1993, Gilles et al., 1996, Brunson et al., 2005).
The limited bedding/nesting paradigm is increasingly used as a rodent model for early-life stress (ELS). Dams that are confronted with insufficient nesting material leave the nest more frequently. This results in chronically fragmented and unpredictable maternal care (Brunson et al., 2005). In turn, the inadequate care acts as a major stressor to the pups; for example, the frequency of nest leaving by the dam was associated with reduced body weight of the pups (Rice et al., 2008). Because the dam is present and instrumental to the stress induction, this model has been argued to be a better translation to the human condition than other models of ELS, such as the maternal separation model (Molet et al., 2014).
ELS induction by limited bedding/nesting in mice is typically initiated at postnatal day (PND) 2 and lasts until PND 9 (Molet et al., 2014). ELS induction from PND 2–9 by limited bedding/nesting reduces bodyweight, leads to elevated basal plasma corticosterone and increases adrenal weights early after the stressor but are absent in adulthood (Brunson et al., 2005, Rice et al., 2008, Raineki et al., 2010, Naninck et al., 2015). The results from other ELS paradigms demonstrate that the timing of the stressor is an important factor to consider (Molet et al., 2014). The onset of ELS induced by maternal deprivation may have dramatic effects on animal physiology (van Oers et al., 1997). Whether the timing of stress induction in the limited bedding/nesting paradigm may also determine impact of subsequent effects on brain and behavior has not yet been explored.
Maternal separation in rats has been shown to alter anxiety-like behavior and to affect the hippocampal GABAergic system (Barbosa Neto et al., 2012, Martisova et al., 2012). Importantly, developmental perturbations to the GABAergic system were found to affect adult anxiety-like behavior, but the timing in which the manipulation took place appears to dictate the ultimate effects. Specifically, the groups of mice treated with the GABAa agonist muscimol from PND 3–5 or from PND 14–16 both displayed enhanced anxiety-like behavior in adulthood but the effect was much stronger in mice treated from PND 3–5 compared to the group treated from PND 14–16 (Salari et al., 2015). The GABAa receptor is a heteropentameric structure containing two α-, two β- and often a γ-subunit. Because the γ2 subunit of the GABAa receptor is a pivotal element for the binding and functioning of the anxiolytic benzodiazepines (Pritchett et al., 1989), the abnormal cerebral expression of this subunit during development may thus be anticipated to interfere with anxiety-like behavior in adulthood. Concordantly, transient inactivation of the γ2 subunit of the GABAa receptor (Gabrg2) from PND 13–14 but not from PND 27–28 was found to increase anxiety-like behavior in mice (Shen et al., 2012). Hence, the GABA-ergic system is strongly implicated in ELS-induced anxiety-related alterations in this system occurring in the later period seem to be especially relevant. In accordance with this suggestion, studies employing maternal separation show that the late developmental stage (after PND 9) may be a particularly vulnerable period for the stress-induced programming of anxiety-like behavior (Huot et al., 2001, Benetti et al., 2009).
In adulthood, rats and mice subjected to ELS by restricted bedding/nesting from PND 2–9 exhibited defects in hippocampal synaptic plasticity and displayed associated cognitive impairments (Brunson et al., 2005, Wang et al., 2011, Kohl et al., 2015). Cognitive deficits in spatial and object memory were observed in the hippocampal-dependent Morris water maze and in the novel object recognition task (Brunson et al., 2005, Wang et al., 2011). In addition, social recognition was impaired in adult mice that were submitted to the limited bedding/nesting paradigm (Kohl et al., 2015), and social memory formation is known to depend on intact hippocampal functioning (Bannerman et al., 2001, Bannerman et al., 2002, Squires et al., 2006). Recent evidence also implicates the cell adhesion molecules, neuroligins (NLGNs) and nectins, in ELS-induced adjustments in hippocampal synaptic plasticity (Wang et al., 2013, Liao et al., 2014, Kohl et al., 2015). The importance of the timing of the limited bedding/nesting paradigm on cognition and hippocampal cell adhesion molecules has not been studied.
Here we compared the effects of ELS in the limited bedding/nesting paradigm when applied either, as in most previous studies, during the first postnatal days (PND 2–9; ELS-early) or during the immediately subsequent week (PND 10–17; ELS-late). We investigated the effects of ELS-early and ELS-late on animalś anxiety, social behavior and cognition in adulthood and on the hippocampal gene expression of the γ2 subunit of the GABAa receptor (Gabrg2), NLGNs (Nlgn-1, Nlgn-2 and Nlgn-3) and nectins (nectin-1 and nectin-3).
Section snippets
Animals
Upon arrival, BALB/cJ and C57Bl/6J mice (Charles River Laboratories, Bois des Oncins, France) were housed in the animal facility of the Ecole Polytèchnique Fédérale de Lausanne (EPFL, Lausanne, Vaud, Switzerland) in ventilated cages on a 12-h light/dark cycle (lights on at 7 am), controlled for temperature (23 ± 1 °C) and humidity (50 ± 15%) with food and water available ad libitum. Mice were habituated for 2 weeks before breeding. The breeding pairs consisted of male BALB/cJ × female C57bl/6J, and
Bodyweight
Body weight was measured both before weaning (postnatal days – PND 9, 17 and after (at PND 24 and at 4, 6, 7, 8, 10 and 12 weeks of age; Fig. 1B). The ELS-early group initially displayed a reduced body weight (Fig. 1A: PND 9: Mann–Whitney U = 32, p < 0.001; PND 17: F2.85 = 38.4, p < 0.001 post-test t = 8.7 p < 0.001 control vs. ELS-early; PND24: F2,68 = 14.0, p < 0.001, post-test control vs. ELS-early t = 5.1, p < 0.001) that persisted during the post-weaning period (Fig. 1B: F2,270 = 32.4, p < 0.001, post-tests control
Discussion
The impact of early-life stressors was previously shown to depend on the timing of disturbance induction (Molet et al., 2014). Here, we tested the hypothesis that variation in the onset of ELS during the early postnatal period, as induced by the limited bedding/nesting paradigm, would differentially impact anxiety, social behavior and cognitive processing in male mice at adulthood and would be accompanied by alterations in the hippocampal gene expression of the GABA-ergic system and/or the cell
Conclusion
We show here that the timing of stress induction in the limited bedding/nesting paradigm for mice differentially shapes adult anxiety-like behavior and body weight development, whereas the stress-induced effects on adult cognition and social behavior were nondiscriminatory. Importantly, our findings kept their validity when taking into account putative litter effects. The enhanced anxiety displayed by ELS-late mice was associated with hippocampal Gabrg2 expression, and although ELS-early and
Acknowledgments
Christine Fülling is thanked for assistance with the writing of this manuscript. This study was funded by grants from the Swiss National Science Foundation (31003A-152614; NCCR Synapsy), the EU FP7 project MATRICS (No 603016), Oak Foundation, and intra-mural funding from the EPFL to CS.
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These authors contributed equally to this study.