Transgenerational inheritance of chronic adolescent stress: Effects of stress response and the amygdala transcriptome

Abstract

Adolescent stress can impact health and well-being not only during adulthood of the exposed individual but even in future generations. To investigate the molecular mechanisms underlying these long-term effects, we exposed adolescent males to stress and measured anxiety behaviors and gene expression in the amygdala-a critical region in the control of emotional states-in their progeny for two generations, offspring and grandoffspring. Male C57BL/6 mice underwent chronic unpredictable stress (CUS) for 2 weeks during adolescence and were used to produce two generations of offspring. Male and female offspring and grandoffspring were tested in behavioral assays to measure affective behavior and stress reactivity. Remarkably, transgenerational inheritance of paternal stress exposure produced a protective phenotype in the male, but not the female lineage. RNA-seq analysis of the amygdala from male offspring and grandoffspring identified differentially expressed genes (DEGs) in mice derived from fathers exposed to CUS. The DEGSs clustered into numerous pathways, and the "notch signaling" pathway was the most significantly altered in male grandoffspring. Therefore, we show that paternal stress exposure impacts future generations which manifest in behavioral changes and molecular adaptations.

Keywords: RNA-sequencing; adolescent stress; amygdala; chronic unpredictable stress; inheritance; multi-generational; notch signaling; stress; transcriptome; transgenerational.

Figure 1. Study design and timeline

Schematic representation of the experimental design. C57/B6 male mice were exposed to adolescent stress (F0) and then used for mating to generate the offspring. Both male and female offspring were tested for affective behaviors, and only males were used tissue collection for RNA-sequencing and mating to generate the grandoffspring (a). Diagram representation of the mice used for behavioral testing, mating, or RNA-sequencing in the stress-exposed mice, offspring, and grandoffspring (b). All mice were used only for one behavioral test, and mice used for mating or for RNA-sequencing were not evaluated in behavioral tests. Figure 1a describes the exact number of mice that were used in each behavioral test.

Figure 2. Paternal adolescent CUS exposure reduces male grandoffspring affective behavior and startle response

Male and female offspring were evaluated in the MB (a) and ASR (b) tests, and no behavioral changes between the mice derived from paternal stress or no stress controls were observed. Male and female grandoffspring were evaluated in the MB and ASR tests, male grandoffspring derived from the stress lineage (n=8) buried fewer marbles than those derived from the non-stress lineage (n=8) (c), and also had reduced startle amplitude (n=9) compared to controls (n=11) (d). This effect was not observed in female mice. MB no stress (n=9) stress (n=6), and ASR no stress (n= 9) and stress (n=9). **p = 0.0023, * p = 0.0398.

Figure 3. DEGs identified from RNA-sequencing of the amygdala from male offspring and grandoffspring

Volcano plots of the DEGs identified in the offspring and grandoffspring (a). Venn Diagram of the DEGs identified individually in the offspring or the grandoffspring and common between the two generations. Of the 174 common genes, a majority of the genes that are upregulated in offspring are downregulated in grandoffspring, and vice versa (b). Heat maps of DEGs identified in the 5 samples identified in each experimental group: stress derived male offspring vs. no stress derived male offspring, and stress derived male grandoffspring vs no stress derived male offspring (c). Network analysis of notch signaling pathway (d). DEGs identified in RNA-seq only are blue, genes with over 1.5fold differential expression in qPCR are yellow, and overlapping genes are green.