Adaptation to single housing is dynamic: Changes in hormone levels, gene expression, signaling in the brain, and anxiety-like behavior in adult male C57Bl/6J mice

Highlights

• Extension of single housing from 7 to 14 days disrupts the HPA axis in C57Bl/6J mice.

• Expression of hippocampal GR, MR and FGF2 genes are altered from 7 to 14 days of single housing.

• The hippocampal mTOR pathway and anxiety-like behavior are changed by stress and single housing.

• Multiple biological markers must be used to completely assess dynamic effects of single housing.

Abstract

For basic research, rodents are often housed in individual cages prior to behavioral testing. However, aspects of the experimental design, such as duration of isolation and timing of animal manipulation, may unintentionally introduce variance into collected data. Thus, we examined temporal correlates of acclimation of C57Bl/6J mice to single housing in a novel environment following two commonly used experimental time periods (7 or 14 days, SH7 or SH14). We measured circulating stress hormones (adrenocorticotropic hormone and corticosterone), basally or after injection stress, hippocampal gene expression of transcripts implicated in stress and affect regulation: the glucocorticoid receptor (GR), the mineralocorticoid receptor (MR), including the MR/GR ratio, and fibroblast growth factor 2 (FGF2). We also measured signaling in the mammalian target of rapamycin (mTOR) pathway. The basal elevation of stress hormones in the SH14 group is accompanied by a blunting in the circadian rhythms of GR and FGF2 hippocampal gene expression, and the MR/GR ratio, that is observed in SH7 mice. Following mild stress, the endocrine response and hippocampal mTOR pathway signaling are decreased in the SH14 mice. These neural and endocrine changes at 14 days of single housing likely underlie increased anxiety-like behavior measured in an elevated plus maze test. We conclude that multiple measures of stress responsiveness change dynamically between one and two weeks of single housing. The ramifications of these alterations should be considered when designing animal experiments since such hidden sources of variance might cause lack of replicability and misinterpretation of data.

Introduction

Animals, like humans, are constantly interacting with the environment, including both its physical and social aspects, while simultaneously defining their own territory. A disruption of the balance between territoriality and social interactions, such as crowding or social isolation, triggers stress responses with neurobiological consequences. These responses, which can be adaptive or maladaptive depending on the time frame, manifest as changes in the hypothalamic-pituitary-adrenal (HPA) axis as well as in the neural circuitry that regulates stress responsivity and affect regulation. Replicability and consistency of experimental results are essential for rigorous scientific research. However, hidden sources of variability may exist in an experimental design. Rodents are usually acclimated to a new environment prior to experimental testing, often with changes in how they are housed. Yet, the temporal aspects of animals’ responses to this adaptation are often not considered.

Approximately seven days of acclimation has been typically considered to be sufficient time for stress hormones such as corticosterone (CORT) to return to basal levels in rodents, based on reports that concentrations of plasma CORT of single-housed animals are similar to those of group-housed animals at 7 days (Bartolomucci et al., 2003; Haller et al., 2000; Reis et al., 2012; Tuli et al., 1995), 14 days and up to 42 days (Arndt et al., 2009; Bartolomucci et al., 2003; Hunt and Hambly, 2006; Tuli et al., 1995). One study has shown resting CORT levels in isolated rodents to be even lower than group-housed animals with no differences in circadian rhythm at 18 days of housing (Nichols and Chevins, 1981).

Single housing is the standard condition for experiments in many laboratories. For example, researchers employing surgical procedures often single house animals at least following surgery so that there is no interference among animals while they heal. Other researchers house animals individually to eliminate order effects during behavioral testing (Arndt et al., 2009; Chesler et al., 2002; Lyte et al., 2005). When animals are group-housed, the experimenter needs to remove one animal at a time for testing. Once a cage is disturbed by removing an animal, the other animals exhibit stress responses that would likely confound their performance in a variety of behavioral tests (Arndt et al., 2009). For example, when testing anxiety-like behavior in the elevated plus maze (EPM) or the light/dark box, the goal is to measure spontaneous behavior in a novel environment that is not influenced by prior handling or cage disturbance. Thus, it is easier to test behavior in animals, with and without prior surgery, when they are single-housed.

Another rationale for single housing animals comes into play when determining the effects of genotype on phenotype. Genotype may have the predominant effect on phenotype in single-housed animals, as Nagy et al. (2002) report when examining body composition. In contrast, increased variance in group-housed animals, as compared to those single-housed, may be due to the effects of behavioral and social interactions on the phenotype. Thus, single housing animals may enhance the probability of detecting significant differences because of lower variances in the dependent variables among experimental groups, especially if the findings of Nagy et al. (2002) can be extrapolated to behavioral variables.

Group housing can modify behavior and hormonal levels in variable ways since co-housed mice form social hierarchies (Wang et al., 2014). For example, subordinate CD-1 male mice have higher plasma corticosterone when group-housed in the presence of an alpha male. Conversely, alpha males have higher levels of this stress hormone when mice are pair-housed (Williamson et al., 2017). When social isolation is used, the length can range from 24 h of single housing prior to a specific behavioral test (Parmigiani et al., 1999) to behavioral testing performed after extended periods of time such as 3–8 weeks (Simler et al., 1982; Kempf et al., 1984; van der Veen et al., 2007). Since the goal of our study was to determine how a set of inter-related variables changes dynamically in C57Bl/6J mice when they are single-housed for 7 or 14 days immediately after delivery, conditions often used by researchers, group housing was not included as an experimental variable.

Thus, we examined temporal aspects of rodents' acclimation to single housing in a new environment by assessing stress hormone levels and expression of stress- and affect-related genes in the brain. We determined how the response to single housing, in and of itself, might be a significant variable affecting stress hormone levels over this time period, and following an acute stress (PBS injection). We also measured gene expression of the glucocorticoid receptor (GR), the mineralocorticoid receptor (MR), and fibroblast growth factor 2 (FGF2), all key players in regulating stress and affect. We focused on the hippocampus (HPC) which is involved in controlling the basal tone and rhythmicity of the HPA axis (Akil et al., 1991), terminating the stress response (Herman et al., 2005; Cullinan et al., 1993), and regulating emotional responsiveness (Admon et al., 2009; Chaudhury et al., 2014; Eren-Kocak et al., 2011). We measured phosphorylation of ribosomal protein S6 (S6) in the HPC as a marker of mTOR pathway activation since mTOR signaling is linked in the brain to translational control, synaptic plasticity, developmental disorders, and psychiatric illness (Bockaert and Marin, 2015; Hoeffer and Klann, 2010; Huber et al., 2015). Finally, we tested PBS-injected mice in the EPM for anxiety-like behavior as an end point known to be sensitive to changes in the level of stress. This multivariate study reveals that acclimation to a novel environment, from gene expression to behavior, does not reach a stable endpoint at one week, but rather is dynamic. Therefore, this acclimation feature should be taken into consideration when designing animal research studies.