Sex- and brain region-specific alterations in brain volume in germ-free mice

Abstract

Several lines of evidence demonstrate that microbiota influence brain development. Using high-resolution ex vivo magnetic resonance imaging (MRI), this study examined the impact of microbiota status on brain volume and revealed microbiota-related differences that were sex and brain region dependent. Cortical and hippocampal regions demonstrate increased sensitivity to microbiota status during the first 5 weeks of postnatal life, effects that were greater in male germ-free mice. Conventionalization of germ-free mice at puberty did not normalize brain volume changes. These data add to the existing literature and highlight the need to focus more attention on early-life microbiota-brain axis mechanisms in order to understand the regulatory role of the microbiome in brain development.

Keywords: Microbiome; Neuroscience.

Graphical abstract

Figure 1

Fly-through of coronal slices in the brain highlighting the voxel-wise significant absolute and relative volume (A) Absolute volume (left) and relative volume (right) differences between male and female germ-free (GF) and specific pathogen free (SPF) mice. For GF F, n = 13; GF M, n = 10; SPF F, n = 15; SPF M, n = 15. Orange/red highlighted regions were significantly larger in GF mice compared to SPF mice and blue highlighted regions were significantly smaller in GF mice. All changes highlighted were significant at an FDR value of < 0.05. Total brain volume in GF mice was significantly reduced compared to SPF mice (B). (C–F) Relative volume differences were observed in isocortex (C), cerebellum (D), fiber tracts (E), and hippocampus (F). ∗ shows FDR values < 0.05.

Figure 2

Fly-through of coronal slices in the brain highlighting the voxel-wise significant differences in absolute and relative volume between housing conditions Left: differences between altered Schaedler flora (ASF) and specific pathogen-free (SPF) mice, middle: differences between germ-free (GF) and SPF mice, right: differences between conventionalized GF (GFSPF) and SPF mice. Orange/red highlighted regions were significantly larger in SPF mice compared to GF mice and blue highlighted regions were significantly smaller in SPF mice. For GF F, n = 13; GF M, n = 10; SPF F, n = 15; SPF M, n = 15; GFSPF F, n = 11; GFSPF M, n = 12; ASF F, n = 6; ASF M, n = 6. All changes highlighted were significant at an FDR value of < 0.05.

Figure 3

Brain volume differences based on microbiota status Conventionalization of GF mice at birth with altered Schaedler flora (ASF) normalized total brain volume in female (A) and male (B) mice. Similarly, no relative brain volume differences were observed in isocortex or fiber tracts between SPF and ASF female (C, E) and male (D, F) mice. Total brain volume of female and male mice conventionalized at 5 weeks of age (GF/SPF) was reduced compared to SPF mice (A, B). In addition, GF/SPF showed reduced isocortex and fiber tract relative brain volume compared to SPF mice (C–F). For GF F, n = 13; GF M, n = 10; SPF F, n = 15; SPF M, n = 15; GFSPF F, n = 11; GFSPF M, n = 12; ASF F, n = 6; ASF M, n = 6. ∗ shows FDR values < 5% compared to sex-matched SPF mice; ∇ shows FDR values < 0.05 compared to sex-matched GF mice.

Figure 4

Hippocampal regional differences based on microbiota status (A) provides an anatomical view of the hippocampal subregions. Female mice showed no significant differences in relative volume of CA1 (B), CA2 (D), and CA3 (F) subregions. Smaller volume in the dentate gyrus was observed in GF and GF/SPF female mice compared to SPF female mice (H). Conventionalization of GF mice at birth with altered Schaedler flora (ASF) normalized hippocampal subregions volumes in male mice (C, E, G). Significant smaller volumes were observed in GF and GF/SPF male mice compared to SPF male mice in CA1 (C), CA2 (E), CA3 (G), and DG (I) subregions. No differences in hypothalamus were observed (J, K). For GF F, n = 13; GF M, n = 10; SPF F, n = 15; SPF M, n = 15; GFSPF F, n = 11; GFSPF M, n = 12; ASF F, n = 6; ASF M, n = 6. ∗ shows FDR values < 0.05 compared to sex-matched SPF mice; ∇ shows FDR values < 0.05 compared to sex-matched GF mice.