Prenatal stress disrupts social behavior, cortical neurobiology and commensal microbes in adult male offspring

Abstract

In utero and early neonatal exposure to maternal stress is linked with psychiatric disorders, and the underlying mechanisms are currently being elucidated. We used a prenatal stressor in pregnant mice to examine novel relationships between prenatal stress exposure, changes in the gut microbiome, and social behavior. Here, we show that males exposed to prenatal stress had a significant reduction in social behavior in adulthood, with increased corticosterone release following social interaction. Male offspring exposed to prenatal stress also had neuroinflammation, decreased oxytocin receptor, and decreased serotonin metabolism in their cortex in adulthood, which are linked to decreased social behavior. Finally, we found a significant difference in commensal microbes, including decreases in Bacteroides and Parabacteroides, in adult male offspring exposed to prenatal stress when compared to non-stressed controls. Our findings indicate that gestation is a critical window where maternal stress contributes to the development of aberrant social behaviors and alterations in cortical neurobiology, and that prenatal stress is sufficient to disrupt the male gut-brain axis into adulthood.

Keywords: Microbiome; Neuroinflammation; Prenatal stress; Social behavior.

Fig. 1.

Prenatal stress leads to decreased social interaction in male offspring. Pregnant Female C57BL6 mice were exposed to restraint stress for 2h a day from E10–E16 or left as non-stressed control mice. Separate cohorts of male offspring at P60–70 were tested in a battery of behavioral tests. A) Social interaction index was determined in male offspring of PNS exposed mice. Social approach was reduced in males exposed to PNS when compared with control (t (16)=2.080, p≤0.05) (B) distance travelled (t (16)=0.913, p=0.38) and C) side preference (t (16)=1.042, p=0.32) in the social approach paradigm did not differ. D) Time spent in the open arm of the elevated plus maze did not change with PNS (t (19)=0.070, p=0.95). E) Cognition, as assessed by preference for a novel object was not influenced by PNS (t (9)=0.475, p=0.65). F) Depressive-like behavior was examined in the tail suspension test and did not change following PNS (t (8) 0.453, p=0.66). Bars represent the mean ± SEM. Means with (*) are significantly different from controls.

Fig. 2.

Prenatal stress decreases serotonergic metabolism in adult males. Cortex and plasma were collected from adult males exposed to prenatal stress (PNS) and control C57BL/6 mice and HPLC analysis of 5HT and its metabolite 5HIAA were performed. A. HPLC analysis shows a trend towards an increase in 5HT (Fig. 2A; t (20)=−1.358, p=0.1) in the cortex of males exposed to PNS when compared to non-exposed male controls. B. HPLC analysis demonstrates a significant decrease in 5HIAA (Fig. 2B; t(20)=2.910, p=0.008) in the cortex of males exposed to PNS when compared to non-exposed male controls. C. Significant decrease in the ratio of 5HIAA/5HT (t (20)=3.172, p=0.005)) in the cortex of males exposed to PNS when compared to non-exposed controls indicates an overall increase in serotonin. D. qPCR analysis shows significant decrease (t (22)=2.696, p=0.01) in MAO-A in the cortex of adult males exposed to PNS when compared to non-exposed males. (n=10–12/group) E. In a separate cohort, HPLC analysis of 5HT shows significant increase in the plasma of males exposed to PNS when compared to non-exposed controls (t (15)=3.095, p=0.01). Bars represent the mean ± SEM. Means with (*) are significantly different from controls.

Fig. 3.

Prenatal stress increases corticosterone response to social interaction and reduces oxytocin receptor levels in adult males. A cohort of male C57/BL6 offspring exposed to prenatal stress (PNS) and control offspring were tested in the social behavior paradigm. Blood was collected immediately after completion of social behavior. A. Corticosterone (CORT) significantly increased in males immediately after social interaction (t (17)=2.588, p=0.02) B. qPCR analysis shows a significant increase in CRH in the cortex of PNS exposed males when compared to non-exposed controls (t (22)=2.696, p=0.01) C. qPCR analysis shows a significant decrease in OXTR in the cortex of adult males exposed to PNS when compared to non-exposed males. (t (22)=2.782, p=0.01) Bars represent the mean ± SEM. Means with (*) are significantly different from controls.

Fig. 4.

Prenatal stress leads to cortical neuroinflammation in adult males. Prenatal Stress (PNS) increased cytokine production and microglial activation in brain tissue collected from adult males. A, B. qPCR analysis shows a significant increase in IL-6 (t (21)=2.487, p=0.02) and IL-1β (t (22)=2.770, p=0.01) in the cortex of PNS exposed males when compared to non-exposed controls. C. Representative images of IBA-1 labeled prefrontal cortex from PNS exposed adult males, and non-exposed controls. D. Exposure to PNS caused microglia activation with increased proportional area of Iba-1 immunofluorescence in the prefrontal cortex (t (14)=3.463, p=0.01) Bars represent the mean ± SEM. Means with (*) are significantly different from controls.

Fig. 5.

Prenatal stress causes microbial changes in male offspring. Sequences from stool samples from stressor-exposed male offspring in adulthood plottedseparately from samples from non-stressed control male offspring on a Principal Coordinate Analysis (PCoA) using weighted UniFrac distances. This difference was statistically significant using the Adonis statistic (p < 0.05). B. PD Whole Tree measure of alpha diversity was not significantly different in males exposed to PNS and unexposed males. C. Relative abundances of sequences classified at the family or genus taxonomic level in male offspring exposed to PNS compared to non-exposed males. D. The relative abundances of Bacteroides and Parabacteroides were significantly different in males exposed to PNS compared to non-exposed males. A total of 14 non-stress male offspring and 15 stressed male offspring from 6 non-stress and 9 stressed dams were examined for microbiome samples. *p < .001 after multiple test correction.