Altered gut microbiome and autism like behavior are associated with parental high salt diet in male mice

Abstract

Neurodevelopmental disorders are conditions caused by the abnormal development of the central nervous system. Autism spectrum disorder (ASD) is currently the most common form of such disorders, affecting 1% of the population worldwide. Despite its prevalence, the mechanisms underlying ASD are not fully known. Recent studies have suggested that the maternal gut microbiome can have profound effects on neurodevelopment. Considering that the gut microbial composition is modulated by diet, we tested the hypothesis that ASD-like behavior could be linked to maternal diet and its associated gut dysbiosis. Therefore, we used a mouse model of parental high salt diet (HSD), and specifically evaluated social and exploratory behaviors in their control-fed offspring. Using 16S genome sequencing of fecal samples, we first show that (1) as expected, HSD changed the maternal gut microbiome, and (2) this altered gut microbiome was shared with the offspring. More importantly, behavioral analysis of the offspring showed hyperactivity, increased repetitive behaviors, and impaired sociability in adult male mice from HSD-fed parents. Taken together, our data suggests that parental HSD consumption is strongly associated with offspring ASD-like behavioral abnormalities via changes in gut microbiome.

Figure 1

HSD negatively regulates weight in male mice and does not result in reproductive changes in the parental generation. Body weight was measured weekly during the 8-week feeding protocol. (a) Each group shows a steady growth although HSD males were significantly smaller (n = 22 control male group, 21 HSD male group, 21 control female group, 22 HSD female group). (b) Average body weight at the end of feeding protocol per group. Reproductive health was measured by calculating pregnancy rate (c) and quantifying the number of pups per litter (d) in each pair (n = 14 control pairs, 15 HSD pairs). Both parameters show that HSD does not alter reproduction ability. Data are described as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, unpaired t-test (d), one-way ANOVA and Tukey’s test (b).

Figure 2

HSD does not alter exploratory behavior or memory tasks performance in the parental generation. The behavioral tests used were open field test (OFT), elevated plus maze (EPM), T-maze spontaneous alternation test and novel object recognition (NOR) test (n = 9 control males, 8 HSD males, 8 control females, 8 HSD females). Overall, no difference was observed in exploratory behavior in HSD-fed groups: (a) total distance traveled in the OFT, (b) time spent in the center area of the OFT, or open arm exploration in EPM (c). In addition, HSD-fed mice do not show memory impairment in the T-maze spontaneous alternation test (d), or in the NOR test after 1 h (e, although there is a clear trend toward reduced memory in HSD-fed male mice but it did not reach statistical significance). NOR test after 24 h shows similar results, no difference in memory performance (f). Data are described as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA and Tukey’s test.

Figure 3

Altered gut microbiome found in HSD-fed parental female mice is shared with their offspring. Gut microbiome data was obtained from fecal samples and analyzed from 16 s rRNA sequencing. (a) No difference was found in observed OTUs between control and HSD-fed dams as well as between their respective offspring (n = 5 control-fed dam, 5 HSD-fed dam, 10 mice from control-fed parent, 10 from HSD-fed parent). (b) Principal coordinate analysis (PCoA) plots of Bray–Curtis distance metrics show separate clustering of microbiome from parental and offspring generation. Black arrows indicate the direction of significant shift in microbiota composition between groups. (c) Summary of relative abundance of the top 14 most abundant microbial genera in the community sampled. Topmost abundant genera, Lactobacillus spp was significantly reduced, Akkermansia spp was increased and Bacteroides spp was unchanged in HSD-fed dams as well as in their offspring (d, e, f). Data are described as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA and Tukey’s test.

Figure 4

HSD does not change serum concentration of proinflammatory cytokines in female parental mice. Serum concentration on proinflammatory cytokines were measured using flow cytometry-based assay after offspring weaning. (a, b) Samples from HSD-fed female mice showed no change in the concentration of proinflammatory cytokines IL-17 (n = 9 in control and HSD-fed group) and TNF-α (n = 9 in control and n = 8 in HSD-fed group), or the (c) anti-inflammatory cytokine IL-10 (n = 6 control, n = 8 HSD-fed). (d, e) Proinflammatory cytokines IL-1β (d, n = 9 in control and HSD-fed group) and IL-23 (e, n = 8 in control and n = 9 in HSD-fed group) were not significantly reduced in HSD-fed females (n = 5 and n = 4 control, 5 HSD). Data are described as mean ± SEM.

Figure 5

Male offspring from HSD-fed mice are heavier and show ASD-like behavioral phenotype. Body weight of the offspring generation was measured from weaning to adulthood. (a) Male and female offspring from HSD-fed parents weigh significantly higher at the age of weaning and only male mice continued to weigh more until their 8th week (n = 30 in all groups). (b, c) Male offspring from HSD-fed parents show higher distance traveled and moving speed in the OFT (n = 13 male offspring from control-fed parents, 14 male offspring from HSD-fed parents, 12 female offspring from control-fed parents, 13 female offspring from HSD-fed parents). In the marble burying test, male offspring from HSD-fed parents show higher repetitive behavior (d). Offspring from HSD-fed parents show lower preference for the Social (S) chamber in the 3-chamber test compared to offspring from control-fed parents, (e) but no difference was observed in the urinary pheromone test (f). Data are described as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA and Tukey’s test.