Effects of early-life penicillin exposure on the gut microbiome and frontal cortex and amygdala gene expression

Abstract

We have established experimental systems to assess the effects of early-life exposures to antibiotics on the intestinal microbiota and gene expression in the brain. This model system is highly relevant to human exposure and may be developed into a preclinical model of neurodevelopmental disorders in which the gut-brain axis is perturbed, leading to organizational effects that permanently alter the structure and function of the brain. Exposing newborn mice to low-dose penicillin led to substantial changes in intestinal microbiota population structure and composition. Transcriptomic alterations implicate pathways perturbed in neurodevelopmental and neuropsychiatric disorders. There also were substantial effects on frontal cortex and amygdala gene expression by bioinformatic interrogation, affecting multiple pathways underlying neurodevelopment. Informatic analyses established linkages between specific intestinal microbial populations and the early-life expression of particular affected genes. These studies provide translational models to explore intestinal microbiome roles in the normal and abnormal maturation of the vulnerable central nervous system.

Keywords: developmental neuroscience; microbiome.

Graphical abstract

Figure 1

Study design: assessing effects of early-life antibiotics on gene expression in the brain Pregnant C57BL/6 mice were either exposed to low-dose penicillin G, as described [Cox], during the last week of pregnancy and until sacrifice of their pups on PND10 (STAT group; n = 8 pups), or exposed starting on the day of birth (STAT-Birth; n = 7 pups), or were unexposed (Control; n = 8 pups). The pups were sacrificed at PND10 for examination of microbiome and CNS transcription. Pups were born on P0, and all dams were sacrificed by P22.

Figure 2

Beta-diversity of pup intestinal contents, based on a Bray–Curtis distance matrix The significance of differences was tested by Permanova-pairwise test with 999 permutations (∗p < 0.05; ∗∗p < 0.01).

Figure 3

Heatmap representing taxa (at genus level) in the pup samples that are significantly different between STAT vs. Control and STAT-Birth vs. Control Each row represents a different taxon and each column a different treatment. Log₁₀ taxon relative abundance is shown in the scales for each intestinal sampling site at right. Significantly different taxa were inferred by MaAsLin2 analysis. (A) Cecal contents. (B) Colonic contents. (C) Small intestinal contents.

Figure 4

Gene expression in the frontal cortex and amygdala in the three study groups In the groups (STAT, STAT-Birth, and Control), there are three samples per pup. Global expression data for each sample in each site are represented by PCA. The significance of differences was tested by pairwise Mann–Whitney tests on PC1 (∗p < 0.05; ∗∗p < 0.01; not significant: p > 0.05). See also Figure S3.

Figure 5

Volcano plots of differentially expressed genes in the frontal cortex (left) and amygdala (right) between antibiotic-exposed and control pups Genes differentially expressed with p < 0.01 are represented by colored points (e.g., no change in gray, upregulation in red, and downregulation in blue). (A) Comparison of STAT vs. Control. (B) Comparison of STAT-Birth vs. Control.

Figure 6

Heatmap of genes in the frontal cortex and amygdala that are differentially expressed in the Control vs. STAT and/or the Control vs. STAT-Birth mice comparisons Genes that are significantly significant (p < 0.01 with log fold change > 1.5) are depicted. See also Figure S4.

Figure 7

Comparison of significantly expressed genes in the frontal cortex and amygdala from both STAT and STAT-Birth conditions with microbiota identified from MaAsLin2 analysis Normalized mean expression for each differentially expressed gene (determined by DEseq analysis) was divided into tertiles, which were used to group the relative abundance of each taxon in each sample. A pairwise Kruskal–Wallis rank-sum test was performed to compare the gene tertiles with relative abundance of each taxon identified by the MaAsLin2 analysis. Taxa with p value <0.05 were selected for the post-hoc Mann–Whitney test between the tertiles with Benjamini–Hochberg p value adjustment. Each of the taxa shown had at least one comparison with p value <0.05. (A) Frontal cortex vs colon. (B) Frontal cortex vs. small intestine (No significant differences were found in the comparison between frontal cortex vs. ceca). (C) Amygdala vs. ceca. (D) Amygdala vs. colon. (E) Amygdala vs. small intestine.