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

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.

Keywords: Anxiety; Fibroblast growth factor 2; Glucocorticoid receptor; HPA axis; Mineralocorticoid receptor; Ribosomal protein S6.

Fig. 1.

Experimental design and timeline. PBS = phosphate-buffered saline, EPM = elevated plus maze.

Fig. 2.

Prolonged single housing modifies basal stress hormones and blunts the peripheral stress response. All mice show a rise in ACTH (A) and CORT (B) from AM to PM, as expected for the normal daily rhythms of these circulating hormones (p < 0.01). However, SH14 mice exhibit significantly higher basal ACTH and CORT levels than SH7 mice in the PM (A) and AM (B), respectively. All mice show a decrease in ACTH (C) and CORT (D) levels from 1 to 6 h post injection and EPM testing (p < 0.001). However, SH14 mice exhibit a significantly smaller rise in CORT than SH7 mice 1 h following injection stress and EPM testing (D). Data represent mean ± SEM. *p < 0.05.

Fig. 3.

Prolonged single housing blunts diurnal GR gene regulation. (A) SH7 mice show basal upregulation of GR mRNA expression in all areas of the dorsal HPC from AM to PM, while SH14 mice do not. (B) Representative GR in situ hybridization autoradiographs of the dorsal HPC from SH7 and SH14 mice sacrificed in the AM and PM. (C) SH14 mice show higher AM GR mRNA levels in all areas of the dorsal HPC, compared to SH7 mice. (D) GR mRNA levels are also significantly higher in the PVN of SH14 mice compared to SH7 mice. Data in (A) represent percent change from AM to PM ± the standard error of the percent change. Data in (C) and (D) represent mean ± SEM. *p < 0.05; **p < 0.01.

Fig. 4.

Prolonged single housing alters MR gene regulation and the MR/GR ratio in the CA2 subregion of the HPC. (A) Both SH7 and SH14 mice show a downregulation in MR gene expression in the dorsal HPC from AM to PM; the CA2 region is shown. However, SH14 mice sacrificed in the PM exhibit lower MR mRNA levels than SH7 mice sacrificed in the AM. (B) SH7 mice exhibit a significant decrease from AM to PM in the MR/GR mRNA ratio in the CA2 subregion of the dorsal HPC, in contrast to the SH14 mice that show little change from AM to PM. SH14 mice exhibit significantly lower and higher MR/GR ratios in the AM and PM, respectively, compared to SH7 mice. Data represent mean ± SEM. **p < 0.01; ***p < 0.001.

Fig. 5.

Prolonged single housing blunts FGF2 gene regulation in the HPC. (A) SH7 mice exhibit an increase in FGF2 gene expression from AM to PM in all areas of the dorsal HPC, while SH14 mice do not, except for the CA2 subregion. The FGF2 gene is highly expressed in the CA2, including its associated areas that are more medial: mCA2 that is more rostral to and continuous with the FC. (B) Representative FGF2 in situ hybridization autoradiographs of the dorsal HPC from SH7 and SH14 mice sacrificed in the AM and PM. (C) SH14 mice also show lower PM FGF2 levels compared to that of SH7 mice in several of the HPC subregions. Data in (A) represent percent change from AM to PM ± the standard error in the percent change. Data in (C) represent mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.

Fig. 6.

Prolonged single housing abrogates mTOR pathway activation in the HPC following acute stress. (A) Basal levels of S6 phosphorylation in the dorsal HPC are similar in SH7 and SH14 mice, but 1 h following acute injection stress and EPM testing, SH7 mice exhibit a significantly higher PO₄-S6/total S6 ratio than SH14 mice. This increased S6 phosphorylation in SH7 mice returns to basal levels by 6 h post-injection and behavior. (B) Representative immunoblots for PO₄- and total S6 corresponding to collated data for each experimental condition as shown in (A). Data represent mean ± SEM. ***p < 0.001.

Fig. 7.

Prolonged single-housing promotes anxiety-like behavior in the EPM. One hour following acute injection stress, SH14 mice made fewer entries (A), spent less time (B), and took longer to first enter (C) the open arms (OA) of the EPM than SH7 mice. Data represent mean ± SEM. **p < 0.01; ***p < 0.001.