Environmental Intervention as a Therapy for Adverse Programming by Ancestral Stress

Abstract

Ancestral stress can program stress sensitivity and health trajectories across multiple generations. While ancestral stress is uncontrollable to the filial generations, it is critical to identify therapies that overcome transgenerational programming. Here we report that prenatal stress in rats generates a transgenerationally heritable endocrine and epigenetic footprint and elevated stress sensitivity which can be alleviated by beneficial experiences in later life. Ancestral stress led to downregulated glucocorticoid receptor and prefrontal cortex neuronal densities along with precocious development of anxiety-like behaviours. Environmental enrichment (EE) during adolescence mitigated endocrine and neuronal markers of stress and improved miR-182 expression linked to brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) regulation in stressed lineages. Thus, EE may serve as a powerful intervention for adverse transgenerational programming through microRNA-mediated regulation of BDNF and NT-3 pathways. The identification of microRNAs that mediate the actions of EE highlights new therapeutic strategies for mental health conditions and psychiatric disease.

Figure 1. Enriched environment improves dysregulated hypothalamic-pituitary-adrenal axis activity.

(A) Illustration of critical components of the HPA axis assessed in this study. (B) Both transgenerational (TPS) and multigenerational (MPS) prenatal stress reduced glucocorticoid receptor markers in the hippocampus compared to the control group. Exposure to enriched environment increased glucocorticoid receptor count. (C) Circulating plasma corticosterone levels were reduced by enriched environment across all groups. Enrichment therapy thus improved HPA axis feedback regulation. Asterisks denote significances due to EE (*p < 0.05, **p < 0.01, ***p < 0.001); ^#denotes significant difference due to ancestral stress (^#p < 0.05).

Figure 2. Enriched environment improves stress-programmed glucocorticoid receptor cell count.

Stereological analysis of total GR-positive cells counted in hippocampal CA1-CA2, CA3 and dentate gyrus areas. Cell counts in control, transgenerationally (TPS) and multigenerationally (MPS) stressed rats revealed that ancestral stress reduced GR markers in the CA1-CA2 regions, increased markers in CA3 and decreased markers in the DG (TPS only). Enrichment increased total GR markers in all cases with the exception of the GR markers in the DG of the MPS and control animals. Asterisks denote significances due to EE (*p < 0.05, ***p < 0.001), ^#denotes significant difference due to stress (^#p < 0.05).

Figure 3. Ancestral stress reduced prefrontal cortex thickness.

(A) Image of a representative cresyl violet-stained coronal brain section corresponding to 3.70 mm relative to bregma illustrating the measurements of cortical thickness (DV: Dorsoventral, ML: Mediolateral, RF: Rhinal fissure, scale bar represents 2 mm). The right portion shows the gray scale 8-bit image used for mean gray value analysis. (B) DV thickness and (C) mean gray value were significantly decreased due to transgenerational and multigenerational stress, with no significant effect of enrichment therapy. PFC, prefrontal cortex. Asterisks denote significances (*p < 0.05, ***p < 0.001).

Figure 4. Enriched environment mitigates precocious anxiety-like behaviours induced by ancestral stress.

(A) Photograph of a rat in the automated open field and total distance travelled measured at P35, P60 and P100 showing an increase in movement time due to ancestral stress and a decrease due to enrichment. (B) Photograph of a rat in stretch-extend posture in the elevated plus maze at P35, P65 and P100 observed to measure the time spent risk assessing, showing an increase in the time spent risk assessing due to ancestral stress, and a decrease due to enrichment (at P65, TPS-EE and at P100, MPS-EE). Note that transgenerational and multigenerational stress generated precocious anxiety-like behavioural traits, and enrichment therapy normalized the developmental trajectory of these behaviours. Asterisks denote significances due to EE (*p < 0.05, **p < 0.01), ^#denotes significant difference due to stress (^#p < 0.05).

Figure 5. Trans- and multigenerational stress program miRNA expression profiles related to psychopathologies, which are reversed by enriched environment.

Fold changes of miRNAs in TPS and MPS rats in reference to controls. Note that ancestral stress led to up- or down-regulation of miRNA expression, respectively, and enrichment returned these changes to normal levels. Asterisks denote significances (FDR adjusted; *p < 0.05).

Figure 6. Animal model of transgenerational and multigenerational prenatal stress.

The animal model included a non-stress control lineage, a transgenerational stress lineage in which gestational stress only occurred in F0 dams, and a multigenerational stress lineage in which F0-F1-F2 dams were stressed during pregnancy. Males from four F3 litters per group were tested. Four male rats per litter were used; 2 animals from each litter were placed in a standard environment and 2 rats from each litter were placed in an enriched environment at the age of 35 days.