Normal gut microbiota modulates brain development and behavior Rochellys Diaz Heijtza,b,1, Shugui Wangc, Farhana Anuard, Yu Qiana,b, Britta Bj��rkholmd, Annika Samuelssond, Martin L. Hibberdc, Hans Forssbergb,e, and Sven Petterssonc,d,1 Departments of aNeuroscience, and dMicrobiology, Cell and Tumor Biology, Karolinska Institutet, 171 77 Stockholm, Sweden bStockholm Brain Institute, 171 77 Stockholm, Sweden cGenome Institute of Singapore, 02-01 Genome 138672, Singapore and eDepartment of Women���s and Children���s Health, Karolinska Institutet, 171 76 Stockholm, Sweden Edited by Arturo Zychlinsky, Max Planck Institute for Infection Biology, Berlin, Germany, and accepted by the Editorial Board January 4, 2011 (received for review August 11, 2010) 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 micro- biota. This behavioral phenotype is associated with altered expres- sion 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. developmental programming | microbiome | basal ganglia | cognitive behavior | synapse Eorganism���s arly lifeenvironmentalinfluenceshaveaprofoundimpactonthe later development, structure, and function. This phenomenon is called ���developmental programming,��� a process whereby an environmental factor acting during a sensitive or vul- nerabledevelopmentalperiodexertseffectsthatimpactonstructure and function of organs that, in some cases, will persist throughout life (1). One such environmental factor is the gut microbiota that, because of an evolutionary process, has adapted to coexist in com- mensal or symbiotic relationship with mammals (2). Immediately after birth, the newborn organism is rapidly and densely populated with complex forms of indigenous microbes. This process has been shown to contribute to developmental programming of epithelial barrier function, gut homeostasis, and angiogenesis, as well as the innate and host adaptive immune function (3, 4). Recent data indicate that gut microbiota have systemic effects on liver function (5���7), thus raising the possibility that gut microbiota can have de- velopmental effects in other organs elsewhere in the body. The functional development of the mammalian brain is of par- ticular interest because it has been shown to be susceptible to both internal and external environmental cues during perinatal life. Epidemiological studies have indicated an association between common neurodevelopmental disorders, such as autism and schizophrenia, and microbial pathogen infections during the peri- natal period (8, 9). These findings are supported by experimental studies in rodents, demonstrating that exposure to microbial pathogens during similar developmental periods result in behav- ioral abnormalities, including anxiety-like behavior and impaired cognitive function (10���12). In a recent study, it was shown that the commensal bacteria, Bifidobacteria infantis, could modulate tryp- tophan metabolism, suggesting that the normal gut microbiota can influence the precursor pool for serotonin (5-HT) (13). Here, we tested the hypothesis that the ���normal��� gut micro- biota is an integral part of the external environmental signals that modulate brain development and function. Results Germ Free (GF) Mice Display Increased Motor Activity and Reduced Anxiety-Like Behavior. In the first set of experiments, we subjected adult GF and specific pathogen free (SPF) mice with a normal gut microbiota to a battery of tests for exploratory activity and anxiety. GF and SPF mice were placed in a novel, open-field activity box. Their spontaneous motor activity, including loco- motor and rearing activities, were measured for 60 min. GF mice showed greater total distance traveled and more exploration of the center of the open field (P 0.05 Fig. 1A). There was also a trend for GF to display higher levels of rearing activity com- pared with SPF (GF vs. SPF, 489 �� 43 vs. 369 �� 50, P = 0.088). Both GF and SPF mice displayed similar locomotor activity (Fig. 1B) during the initial open field exposure, indicating that the increased locomotor activity in GF mice was not triggered by novelty. Instead, significant differences between groups were detected in habituation over time (repeated measures ANOVA, main effect F(1, 70) = 6.28, P 0.05). Thus, GF mice traveled a significantly longer distance (Fig. 1 B and C) and spent sig- nificantly (P 0.05) more time in both slow and fast locomotion (Fig. 1D) during the 20- to 60-min interval of testing. Given that certain microbial pathogens have been reported to induce anxiety-like behavior in animal models (10���12), we assessed whether the nonpathogenic gut microbiota could also affect anxiety-like behavior. For this purpose, we used two rodent tests of anxiety: the light���dark box test and the elevated plus maze (14). In the light���dark box test, GF mice spent significantly (P 0.05) more time in the light compartment of the box than control SPF mice (Fig. 2A). In the elevated plus maze test, GF mice spent significantly (P 0.05) more time in the open arm than SPF mice (Fig. 2B and Movies S1 and S2). The GF mice also engaged in riskier behavior than SPF as indicated by the great number of visits (GF vs. SPF: 3.9 �� 0.6 vs. 1.4 �� 0.69, P 0.05) to the ends of the open arms. There were no significant differences in the number of entries (GF vs. SPF: 13.14 �� 0.9 vs. 14.4 �� 1.5, P 0.1) and time spent (Fig. 2B) in the closed arm between GF and SPF mice. To test whether conventionalization in early life of GF mice could ���normalize��� the increased motor activity and alter the anxiety behavior, we conventionalized a new set of GF mice with microbiota obtained from SPF mice 30 d before mating and allowed the progeny to mature in an isolator with bacteria. Adult conventionalized offspring (CON) were behaviorally tested as described above. The rearing activity of the CON mice was sig- Author contributions: R.D.H., F.A., B.B., H.F., and S.P. designed research R.D.H., S.W., F.A., Y.Q., and A.S. performed research R.D.H., S.W., F.A., Y.Q., B.B., M.L.H., H.F., and S.P. analyzed data and R.D.H., F.A., H.F., and S.P. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. A.Z. is a guest editor invited by the Editorial Board. Freely available online through the PNAS open access option. 1 To whom correspondence may be addressed. E-mail: rochellys.heijtz@ki.se or sven. pettersson@ki.se. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1010529108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1010529108 PNAS | February 15, 2011 | vol. 108 | no. 7 | 3047���3052 NEUROSCIENCE

nificantly different from that of GF mice (P 0.05) and did not differ from that of SPF mice (Fig. 1E). CON mice normalized their locomotor activity to that of SPF mice during the later time interval of testing (P 0.05 Fig. S1), whereas their initial locomotor activity was similar to that of GF mice. In the light���box test, the behavioral pattern of CON mice was similar to that of GF mice and significantly different from that of SPF mice (P 0.05 Fig. S2). In the elevated plus maze test, the GF mice spent significantly (P 0.05) more time exploring the open arms compared with SPF mice. Introducing microbiota early in life to GF mice showed that CON mice altered their behavior and spent less time exploring the open arms (Fig. S3). EncouragedbytheobservationthatearlycolonizationofGFmice could normalize several behavioral patterns of GF mice, we ex- plored whether there is a sensitive/critical period for the effects of the normal gut microbiota on behavior. We therefore con- ventionalizedadultGFmiceandstudiedtheirbehaviorinopen field test as described above. Notably, conventionalization of adult mice failed to normalize the behavior of GF mice (Fig. 1F and Fig. S4). GF Mice Show Elevated Noradrenaline (NA), Dopamine (DA), and 5-HT Turnover in the Striatum. Anxiety-like behavior has been associ- ated with alterations in monoamine neurotransmission. There- fore, we investigated potential changes in the neurochemistry of GF mice in a new set of animals. The concentration of nor- adrenaline (NA), dopamine (DA), and 5-HT and their major metabolites, 3-methoxy-4-hydroxyphenylglycol (MHPG), dihy- droxyphenylacetic acid (DOPAC), and homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA), were measured in frontal cortex, striatum, and hippocampus of GF and SPF mice. The turnover rate of NA, DA, and 5-HT was significantly higher in the striatum of GF mice compared with SPF mice (Fig. 3). In contrast, no significant differences were found in the frontal cortex or hippocampus (Table S1). GF Mice Show Altered Expression of Synaptic Plasticity-Related Genes. Previous molecular and behavioral studies have impli- cated the immediate-early gene, nerve growth factor-inducible clone A (NGFI-A), and the synaptic plasticity-related gene, 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) Represen- tative 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, re- spectively. (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 con- ventionalized (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. 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. 3048 | www.pnas.org/cgi/doi/10.1073/pnas.1010529108 Diaz Heijtz et al.