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. 2020 May 8;10:7737. doi: 10.1038/s41598-020-64173-3

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Colonization with conventional microbiota and bifidobacteria facilitates early microglial reactivity and mid-late microglial ramification (a) Representative image of gating strategy for flow cytometric analysis. (b) Results plotted as percentage of CD11b⁺ CD45^low microglia/total microglia at each timepoint. Ramified microglia possess the phenotype CD11b⁺,CD45^low. At P4, GF mice have an increased % of microglia expressing markers of ramified microglia, relative to BIF mice. (One-Way ANOVA; *p<0.05, **p<0.01, *** p<0.001; n = 3 male animals/group/timepoint) (c) Microglia were labeled using an anti-IBA1 (Ionized calcium binding adaptor molecule 1) antibody. Scale = 50 μm. Representative images of staining in cerebellar white matter of P4 pups in each group, demonstrating fewer ameboid microglia in P4 germ-free mice (d) Morphological classification and quantification of ramified vs. ameboid microglia in P4 mice (**** p<0.0001, n = 3 mice/group, with 3 fields per mouse). (e) Expression of Tnf gene from whole tissue homogenate in each region (Cb = Cerebellum, Cort = frontal cortex, Hipp = Hippocampus) (data generated from PCR array data, therefore statistical significance and error bars are not shown for these pooled samples; n = 5 male mice/group/timepoint/brain region) (f) qPCR analysis of GF and BIF mouse cerebellum for markers of phagocytic microglia. (One-Way ANOVA; *p<0.05; n = 8 male animals/group/timepoint) (g) qPCR analysis of GF and BIF mouse cerebellum for scavenger receptors, which are required for proper phagocytosis. (One-Way ANOVA; *p<0.05; n = 8 male animals/group/timepoint).