Additional file 1 of Microbiota-indole 3-propionic acid-brain axis mediates abnormal synaptic pruning of hippocampal microglia and susceptibility to ASD in IUGR offspring

Additional file 1: Fig. S1. PCE-induced IUGR female rats did not show typical ASD-like manifestations. (A, C) Fetal body weight on the first day after birth. (B, D) IUGR rate. (E) An illustrative example of travel pathways of female rats in social choice test, open field test, and Y maze test. (F) Time of female rats spent in each zone in social choice test. (G) Marble burying index in marble burying test. (H) Time spent in the center, distance traveled in the center, and total distance traveled in open field test. (I) Spontaneous alteration rate in Y maze test. Dots in panels represent individual samples. Data are presented as mean ± SEM. *P < 0.05, ***P< 0.01. Fig. S2. AHR/NF-κB signaling is involved in ASD. (A) The top enriched pathways of differentially expressed genes by Clusterprofiler. (B) Gene set enrichment analysis (GSEA) plots shows NF-κB signaling pathway. Fig. S3. AHR intervention induces abnormalities of neurons and activation of microglia in the co-cultured primary hippocampal microglia and neurons in vitro. (A) Representative reconstruction of hippocampal neurons. Scale bar = 50 μm. (B) Dendritic length of hippocampal neurons. (C) Number of branch points in hippocampal neurons. (D) Dose-effect: cell viability after treated with different concentrations of CH-223191 (0, 5, 10, 20, 40 or 80 μM) for 2 h. (E) Time-effect: cell viability after treated with 10 μM CH-223191 at different time (0, 0.5, 1, 2, 4, and 8 h). (F) Protein levels of AHR and P-NF-κB. (G) mRNA levels of Ahr. (H) Morphology of primary microglia under the light microscope. Scale bar = 50 μm. (I) Morphology of primary microglia under the fluorescence microscope. Iba1 staining (green) and nuclear staining (DIPA, blue). The arrows point at activated microglia. Scale bar = 50 μm. (J) Microglia activation rate. Dots in panels represent individual samples. Data are presented as mean ± SEM. *P < 0.05, **P< 0.01, ***P < 0.001. Fig. S4. Correlation analysis and gut microbiota signatures of donor rats. (A) Spearman correlation between the IPA level and the P-NF-κB level in hippocampal tissue. (B) Correlations between the time spent in target zone and the Clostridium abundance. The correlations were evaluated by the Spearman correlation coefficient. (C) Alpha diversity represented by the Simpson index and the Shannon index. (D) Beta diversity as shown by the PCoA plot. An ellipse represents the 68% confidence interval of microbial distribution in each group. (E) LEfSe of differentiating genera or species in gut microbiota between groups (LDA > 2). Dots in panels represent individual samples. Fig. S5. Transplantation of microbiota from the IUGR rats causes dysregulation of AHR/NF-κB signaling and activation of hippocampal microglia. (A) Protein levels of AHR and P-NF-κB. (B) mRNA levels of Ahr. (C) Images under fluorescence microscopy showing different regions in hippocampus. Iba1 staining (green) and nuclear staining (DIPA, blue). Scale bar = 50 μm. (D-F) Number of Iba1+cells, number of endpoints per microglia., and process length. Dots in panels represent individual samples. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. Fig. S6. Transplantation of microbiota from the IUGR rats causes abnormalities of hippocampal neurons. (A) Representative reconstruction of hippocampal neurons. Scale bar = 50 μm. (B) Dendritic length of hippocampal neurons. (C) Number of branch points in hippocampal neurons. (D) Representative images of dendritic segments. Scale bar =10 μm. (E) Total spine density in the hippocampal neurons. (F) Mushroom spine density in hippocampal neurons. (J) Ultrastructure of neuronal synapses. Scale bar = 500 nm. (H) Synaptic vesicle numbers, synaptic cleft, postsynaptic density (PSD) thickness, and length of the synaptic active zone. (I) mRNA levels of Syn and Psd95. Data are presented as mean ± SEM. *P< 0.05, **P < 0.01, ***P < 0.001. Fig. S7. Postnatal IPA supplementation reverses changes in hippocampal neurons of IUGR rats. (A) Representative reconstruction of the hippocampal neurons. Scale bar = 50 μm. (B) Dendritic length of hippocampal neurons. (C) Number of branch points in hippocampal neurons. (D) Representative images of dendritic segments. Scale bar =10 μm. (E) Total spine density in hippocampal neurons. (F) Mushroom spine density in hippocampal neurons. (G) Ultrastructure of neuronal synapses. Scale bar = 500 nm. (H) Synaptic vesicle numbers, synaptic cleft, postsynaptic density (PSD) thickness, and length of the synaptic active zone. (I) mRNA levels of Syn and Psd95. Dots in panels represent individual samples. Data are presented as mean ± SEM. *P< 0.05, **P < 0.01, ***P < 0.001. Fig. S8. The methods section is supplemented. (A) The average daily food intake of offspring rats. Dots in panels represent individual samples. (B) Bacterial colonies formed under an anaerobic condition in the feces of SPF rats and pseudo-sterile rats.