The type of stress matters: repeated injection and permanent social isolation stress in male mice have a differential effect on anxiety- and depressive-like behaviours, and associated biological alterations

Abstract

Chronic stress can alter the immune system, adult hippocampal neurogenesis and induce anxiety- and depressive-like behaviour in rodents. However, previous studies have not discriminated between the effect(s) of different types of stress on these behavioural and biological outcomes. We investigated the effect(s) of repeated injection vs. permanent social isolation on behaviour, stress responsivity, immune system functioning and hippocampal neurogenesis, in young adult male mice, and found that the type of stress exposure does indeed matter. Exposure to 6 weeks of repeated injection resulted in an anxiety-like phenotype, decreased systemic inflammation (i.e., reduced plasma levels of TNFα and IL4), increased corticosterone reactivity, increased microglial activation and decreased neuronal differentiation in the dentate gyrus (DG). In contrast, exposure to 6 weeks of permanent social isolation resulted in a depressive-like phenotype, increased plasma levels of TNFα, decreased plasma levels of IL10 and VEGF, decreased corticosterone reactivity, decreased microglial cell density and increased cell density for radial glia, s100β-positive cells and mature neuroblasts-all in the DG. Interestingly, combining the two distinct stress paradigms did not have an additive effect on behavioural and biological outcomes, but resulted in yet a different phenotype, characterized by increased anxiety-like behaviour, decreased plasma levels of IL1β, IL4 and VEGF, and decreased hippocampal neuronal differentiation, without altered neuroinflammation or corticosterone reactivity. These findings demonstrate that different forms of chronic stress can differentially alter both behavioural and biological outcomes in young adult male mice, and that combining multiple stressors may not necessarily cause more severe pathological outcomes.

Fig. 1. Schematic representation of the experimental procedure.

Mice were exposed to one of four experimental conditions after an initial 1-week habituation period: (i) stress-free control group (socially-housed in pairs and injection naive—Group 1); (ii) repeated injection stress (socially-housed in pairs and repeatedly injected—Group 2); (iii) social isolation stress (permanent social isolation and injection naive—Group 3); and (iv) combined stress (permanent isolation and repeatedly injected—Group 4). Behavioural testing began after 6 weeks of treatment and was carried out under stringent environmental control, using standardized test procedures, between 09:00 and 15:00, unless otherwise stated. Blood was collected 24 h before and 30 min after acute stress exposure, i.e., the Porsolt Swim Test, in the final week of behavioural testing. Animals were culled 24 h later and brain tissue extracted. a Housing for sibling pair-housed animals (Groups 1 and 2): large techniplast cages (45 × 32 × 20 cm) were set up with sawdust, a red house, white house, nesting material and a plastic enrichment tube. Larger cages with more nesting material, shelter and enrichment were deemed necessary to reduce aggression between sibling pair-housed animals. b Housing for singly-housed animals (Groups 3 and 4): small tecniplast cages (32 × 16 × 14 cm) were set up with sawdust, a white house and nesting material. Note: a larger sample size was used for control animals (n = 20) given the risk of group housing such an aggressive strain (Deacon, 2006; Charles Rivers Laboratories International, Inc., 2012; www.criver.com).

Fig. 2. Effect(s) of repeated injection, social isolation and combined stress on anxiety- and depressive-like behaviour.

a Mean (±SEM) centre duration in the OFT (F[3,46] = 8.9, p < 0.001). b Median (±IQR) latency to feed in the NSFT (K[4,50] = 21.6, p < 0.001). c Mean (±SEM) % sucrose preference consumption in the SPT (F[3,46] = 3.0, p = 0.04). d Mean (±SEM) immobility duration in the PST (F[3,46] = 2.9, p = 0.045). *p < 0.05; **p < 0.01; ***p < 0.001 relative to control (adjusted p-values). Analyses: one-way ANOVA or Kruskal-Wallis with Bonferroni or Dunn’s post hoc correction, respectively.

Fig. 3. Effect(s) of repeated injection, social isolation and combined stress on corticosterone responsivity and systemic inflammation.

a Mean (±SEM) plasma corticosterone levels before and after acute stress exposure. b Mean (±SEM) plasma TNF-α levels before and after acute stress exposure. c Mean (±SEM) plasma IL1β levels before and after acute stress exposure. d Mean (±SEM) plasma IL2 levels before and after acute stress exposure. e Mean (±SEM) plasma IL4 levels before and after acute stress exposure. f Mean (±SEM) plasma IL10 levels before and after acute stress exposure. g Mean (±SEM) plasma VEGF levels before and after acute stress exposure. *p < 0.05; **p < 0.01; ***p < 0.001 (adjusted p-values). Analyses: repeated-measures two-way ANOVA with Bonferroni post hoc comparison.

Fig. 4. Effect(s) of repeated injection, social isolation and combined stress on microglial biology.

a Mean (±SEM) Iba1-positive cell immunoreactivity in the dentate gyrus (Effect of exposure: F[3,138] = 5.7, p = 0.002; Effect of region: F[2,138] = 1.2, p = 0.29; Interaction: F[3,138] = 0.9, p = 0.45). b Representative photomicrographs of the ventral (i) and dorsal (ii) dentate gyrus stained for Iba1 for repeatedly injected and socially isolated mice, respectively, all relative to controls. Images captured at ×2 and ×20 magnification, scale bar = 50 μm. c mean (±SEM) Iba1-positive cell density in the ventral dentate gyrus for repeatedly injected mice (t [28] = 2.7, p = 0.02). d Mean (±SEM) Iba1-positive cell density in the dorsal dentate gyrus for socially isolated mice (t [28] = 4.2, p = 0.001). e Representative photomicrographs and associated sholl masks of Iba1 cells in the ventral dentate gyrus of repeatedly injected and control animals. f Representative photomicrographs and associated skeletons of Iba1 cells in the ventral dentate gyrus of repeatedly injected and control animals. g Representative photomicrographs and associated sholl masks of Iba1 cells in the dorsal dentate gyrus of socially isolated and control animals. Images e–g were taken at ×40 magnification, scale bar = 40 μm. In Sholl masks, red denotes a greater degree of branching complexity, whereas blue denotes the lowest degree of branching complexity. *p < 0.05; **p < 0.01; ***p < 0.001 (adjusted p-values). Analyses: two-way ANOVA with Bonferroni post hoc comparison or Independent samples t-test.

Fig. 5. Effect(s) of repeated injection, social isolation and combined stress on astrocyte biology.

a Mean (±SEM) GFAP-positive cell immunoreactivity in the dentate gyrus (Effect of exposure: F[3,138] = 15.7, p < 0.001; Effect of region: F[2,138] = 2.1, p = 0.15; Interaction: F[3,138] = 0.9, p = 0.45). b Representative photomicrograph of the dorsal dentate gyrus from a representative control stained for GFAP (×10 magnification; scale bar = 100 μm) and close-up representative photomicrographs for each experimental group (×20 magnification; scale bar = 50 μm). c mean (±SEM) GFAP-positive cell density in the dentate gyrus stratified by GFAP cell type and dentate gyrus region (Effect of exposure (a): F[1,168] = 6.9, p = 0.01; Effect of region (b): F[2,168] = 3.6, p = 0.06; Effect of cell type (c): F[1,168] = 350.8, p < 0.001. Interactions: (a × b) F[1,168] = 0.03, p = 0.87; (a × c) F[1,168] = 5.2, p = 0.03; (b × c) F[1,168] = 2.2, p = .15; (a × b × c) F[1,168] = 0.02, p = 0.88). d Representative photomicrographs of a radial glial cell (GFAP+/SOX2+) and astrocyte (GFAP+/SOX2−) taken from socially isolated animals (×40 magnification; scale bar = 40 μm) and representative maximum intensity 2D z-stack projections of ten confocal images taken from socially isolated and control animals (×20 magnification; scale bar = 50 μm). e Mean (±SEM) s100β-positive cell density in the dentate gyrus (Effect of exposure: F[1,84] = 24.0, p < 0.001; Effect of region: F[2,84] = 0.2, p = 0.64; Interaction: F[1,84] = 0.3, p = 0.61). f Representative photomicrographs of s100β-positive cells in the ventral dentate gyrus of controls and socially isolated mice (×40 magnification; scale bar = 50 μm). *p < 0.05; **p < 0.01; ***p < 0.001 (adjusted p-values). Analyses: two-way ANOVA with Bonferroni post hoc comparison or three-way factorial ANOVA with Bonferroni post hoc comparison.

Fig. 6. Effect(s) of repeated injection, social isolation and combined stress on neurogenesis.

a Mean (±SEM) DCX-positive cell density in the dentate gyrus (Effect of exposure: F[3,138] = 28.9; p < 0.001; Effect of region: F[2,138] = 2.0; p = 0.14; Interaction: F[6,138] = 0.3; p = 0.94). b Representative photomicrographs of the dorsal dentate gyrus for all experimental groups stained for DCX (×10 magnification; scale bar = 100 μm). c Mean (±SEM) DCX-positive cell morphology in the dentate gyrus (Effect of exposure (a): F[3,368] = 26.9, p < 0.001; Effect of region (b): F[1,368] = 2.2, p = 0.14; Effect of cell type (c): F[3,368] = 20.3, p < 0.001; Interactions: (a × b) F[3,368] = 1.1, p = 0.36; (a × c) F[9,368] = 2.2, p = 0.02; (b × c); F[3,368] = 1.5, p = 0.22; (a × b × c); F[9,368] = 0.8, p = 0.59). *p < 0.05; **p < 0.01; ***p < 0.001 (adjusted p-values). Analyses: two-way ANOVA with Bonferroni post hoc comparison or three-way factorial ANOVA with Bonferroni post hoc comparison.