Adaptation to single housing is dynamic: Changes in hormone levels, gene expression, signaling in the brain, and anxiety-like behavior in adult male C57B1/6J mice

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 C57B1/6J 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.