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Comparative Study
. 2011 Feb 15;108(7):3047-52.
doi: 10.1073/pnas.1010529108. Epub 2011 Jan 31.

Normal gut microbiota modulates brain development and behavior

Rochellys Diaz Heijtz  1 , Shugui Wang, Farhana Anuar, Yu Qian, Britta Björkholm, Annika Samuelsson, Martin L Hibberd, Hans Forssberg, Sven Pettersson
Affiliations
Comparative Study

Normal gut microbiota modulates brain development and behavior

Rochellys Diaz Heijtz et al. Proc Natl Acad Sci U S A. .

Abstract

Microbial colonization of mammals is an evolution-driven process that modulate host physiology, many of which are associated with immunity and nutrient intake. Here, we report that colonization by gut microbiota impacts mammalian brain development and subsequent adult behavior. Using measures of motor activity and anxiety-like behavior, we demonstrate that germ free (GF) mice display increased motor activity and reduced anxiety, compared with specific pathogen free (SPF) mice with a normal gut microbiota. This behavioral phenotype is associated with altered expression of genes known to be involved in second messenger pathways and synaptic long-term potentiation in brain regions implicated in motor control and anxiety-like behavior. GF mice exposed to gut microbiota early in life display similar characteristics as SPF mice, including reduced expression of PSD-95 and synaptophysin in the striatum. Hence, our results suggest that the microbial colonization process initiates signaling mechanisms that affect neuronal circuits involved in motor control and anxiety behavior.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
GF mice display increased spontaneous motor activity. (A) Bars show cumulative distance traveled (meters) per zone and in the entire box (total) during the 60-min open field test session by SPF (open bars) and GF (filled bars) mice. (B) Average distance traveled (meters) measured in 10-min time bins across a 60-min session in an open field box. (Inset) Bars show cumulative distance traveled (meters) during the initial 10 min and the 20- to 60-min time interval of open field testing. (C) Representative tracks of movement patterns of SPF and GF mice at the 0–10, 30–40, and 50–60 min time intervals of the 60-min open field test session; distance traveled and rearing activity is shown in dark red and blue colors, respectively. (D) The time that SPF and GF mice spent in slow (>5 cm/s) or fast (>20 cm/s) locomotion during the initial 10 min of testing and the 20–60 min time interval. (E) Rearing activity of SPF (white), GF (black), and conventionalized (CON; light gray) mice. Circles show the average number of rears measured in 10-min time bins across a 60-min session in an open field box. (F) Rearing activity of SPF, GF, and adult CON mice (dark gray); lines connecting cumulative data in B, E, and F were drawn for clarity only. All data (A, B, and D–F) are presented as means (± SEM; n = 7–14 per group). *P < 0.05 compared with SPF mice.
Fig. 2.
Fig. 2.
GF mice display reduced anxiety-like behavior. (A) Bars show time (seconds) spent in the light and dark compartments during a 5-min light–dark box test by the SPF and GF mice. (B) Bars show time (seconds) spent in each area of the elevated plus maze by the SPF and GF mice during a 5-min test session. All data (A and B) are presented as means (±SEM; n = 7–9 per group). *P < 0.05 compared with SPF mice.
Fig. 3.
Fig. 3.
GF mice show elevated NA, DA, and 5-HT turnover in the striatum. The histograms depict the mean ratios (± SEM; n = 6 per group) for MHPG/NA (A), DOPAC/DA (B), and 5-HIAA/5-HT (C) in the striatum of male GF and SPF mice. Asterisks denote where GF mice differ significantly (P < 0.01) from SPF mice.
Fig. 4.
Fig. 4.
GF mice show altered expression of anxiety and synaptic plasticity-related genes. (A) Representative autoradiograms showing NGFI-A mRNA expression at the level of the frontal cortex of SPF and GF mice (OFC, orbital frontal cortex; AO, anterior olfactory region). (A') Bars show expression of NGFI-A mRNA (nCi/g) in the OFC and AO of SPF and GF mice. (B) Representative autoradiograms showing BDNF mRNA expression at the level of amygdala and dorsal hippocampus of SPF and GF mice (BLA, basolateral amygdala; CA1, CA1 region of the dorsal hippocampus). (B') Bars show expression of BDNF mRNA (nCi/g) in the BLA and CA1 region of SPF and GF mice. (C) Representative autoradiograms showing dopamine D1 receptor (Drd1a) mRNA expression at the level of the striatum and nucleus accumbens of SPF and GF mice (STR, striatum; Accb, nucleus accumbens, shell region). (C') Bars show expression of Drd1a mRNA (nCi/g) in the STR and Accb of SPF and GF mice. (D) Representative autoradiograms showing Drd1a mRNA expression at the level of the dorsal hippocampus of SPF and GF mice (DG, dentate gyrus; PtCx, parietal cortex, somatosensory area). (D') Bars show expression of Drd1a mRNA (nCi/g) in the DG and PtCx of SPF and GF mice. All data (A'–D') are expressed as means ± SEM, n = 8 per group. Filled bars represent GF mice. Open bars represent SPF mice. *P < 0.05, *P < 0.001 compared with SPF mice.
Fig. 5.
Fig. 5.
Expression profiling of GF mice and SPF mice brains. A heatmap of genes showing statistically significant (q < 5%) and fold change (>2) differences, between SPF (n = 6) and GF (n = 5) mice in the hippocampus (A), frontal cortex (B), and striatum (C). Each row represents the relative levels of expression of a single gene across all mice; each column represents the levels of expression for a single mouse. The colors red and green denote high and low expression, respectively. Differentially expressed genes were investigated for functional clustering by using Ingenuity Pathway Analysis software for canonical pathways (D), as described in Experimental Procedures.
Fig. 6.
Fig. 6.
GF mice show higher expression of synaptic-related proteins in the striatum compared with SPF mice. Representative Western blot films for synaptophysin (A) and PSD-95 (B) protein expression in the frontal cortex, striatum, and hippocampus of two male GF, SPF, and CON mice (for further details, see Table 1).
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