Article Summary This week we’ll be reading this review article by Sudo et al. 2004. Please read the article closely, answer the following questions (save

Article Summary This week we’ll be reading this review article by Sudo et al. 2004.
Please read the article closely, answer the following questions (save your answers in a Word document and the copy and paste them here):

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Explain whether the results support or contradict the hypothesis (based on the discussion section).
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Don’t use quotes, you need to summarize the findings using your own words. J Physiol 558.1 (2004) pp 263–275 263

Postnatal microbial colonization programs
the hypothalamic–pituitary–adrenal system
for stress response in mice

Nobuyuki Sudo1,2, Yoichi Chida1, Yuji Aiba3,4, Junko Sonoda1, Naomi Oyama1, Xiao-Nian Yu1,
Chiharu Kubo1 and Yasuhiro Koga3

1 Department of Psychosomatic Medicine and 2 Department of Health Care Administration & Management, Graduate School of Medical Sciences,
Kyushu University, Fukuoka, Japan, 3 Department of Infectious Diseases, Tokai University School of Medicine, Isehara, Kanagawa, Japan and 4 Wakamoto
Pharmaceutical Co. Ltd, Ohi-machi, Kanagawa, Japan

Indigenous microbiota have several beneficial effects on host physiological functions; however,
little is known about whether or not postnatal microbial colonization can affect the
development of brain plasticity and a subsequent physiological system response. To test
the idea that such microbes may affect the development of neural systems that govern the
endocrine response to stress, we investigated hypothalamic–pituitary–adrenal (HPA) reaction
to stress by comparing germfree (GF), specific pathogen free (SPF) and gnotobiotic mice.
Plasma ACTH and corticosterone elevation in response to restraint stress was substantially
higher in GF mice than in SPF mice, but not in response to stimulation with ether.
Moreover, GF mice also exhibited reduced brain-derived neurotrophic factor expression
levels in the cortex and hippocampus relative to SPF mice. The exaggerated HPA stress
response by GF mice was reversed by reconstitution with Bifidobacterium infantis. In contrast,
monoassociation with enteropathogenic Escherichia coli, but not with its mutant strain devoid
of the translocated intimin receptor gene, enhanced the response to stress. Importantly, the
enhanced HPA response of GF mice was partly corrected by reconstitution with SPF faeces at
an early stage, but not by any reconstitution exerted at a later stage, which therefore indicates
that exposure to microbes at an early developmental stage is required for the HPA system to
become fully susceptible to inhibitory neural regulation. These results suggest that commensal
microbiota can affect the postnatal development of the HPA stress response in mice.

(Received 26 February 2004; accepted after revision 6 May 2004; first published online 7 May 2004)
Corresponding author N. Sudo: Department of Psychosomatic Medicine, Graduate School of Medical Sciences, Kyushu
University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. Email: sudo@hcam.med.kyushu-u.ac.jp

Early postnatal life represents a period of bacterial
colonization, a time when a previously sterile milieu is
inhabited by microorganisms that are likely to remain as
residents throughout the life of the animal. The human
intestine is more densely populated with microbes than
any other organ, and 1014 bacteria inhabit the gastro-
intestinal tract of adult humans, which exceeds the number
of eukaryotic cells (1013) of which the human body is
constituted (Tannock, 1999; Borrielo, 2002). Therefore,
it seems natural that such colonizing bacteria would
play a principal role in the postnatal maturation of the
mammalian immune system (Sudo et al. 1997; Hooper

Nobuyuki Sudo and Yoichi Chida contributed equally to this work.

& Gordon, 2001). In addition, these bacteria aid in the
digestion and absorption of macromolecules and act as
a barrier to gut pathogens by blocking attachment to
gut binding sites, which is the first step of bacterial
pathogenicity (Finlay & Falkow, 1990). Thus, there is no
doubt that most of our bacterial symbionts have several
beneficial effects on host physiological functions; however,
little is known about whether or not such microbes can
affect the development of brain plasticity and a subsequent
physiological system response.

The hypothalamic–pituitary–adrenal (HPA) axis is a
neuroendocrine system that is subjected to programming
by early life events. For example, as adults, neonatally
handled animals exhibit dampened HPA responses to
stress compared with non-handled animals (Meaney et al.

C© The Physiological Society 2004 DOI: 10.1113/jphysiol.2004.063388

264 N. Sudo and others J Physiol 558.1

1988). In contrast, adult animals exposed to repeated
periods of prolonged maternal deprivation as neonates
display increased HPA response to stress (Schmidt
et al. 2002). These effects persist throughout the life
of the animal and the resulting differences in HPA
activity are associated with the incidence of age-related
neuropathology (Meaney et al. 1988). Because of the close,
bidirectional communication between the neural and
immune systems (Turnbull & Rivier, 1999) early in life, a
time when the central nervous system (CNS) is especially
susceptible to environmental influences, we speculated
that such microbial colonization and subsequent immune
reaction during early life might alter the development of
HPA responsiveness.

To test this hypothesis, we investigated the HPA response
to stress by comparing genetically identical mice that
had no exposure to microorganisms (germfree; GF), mice
raised with a normal functional microbiota but not with
specific pathogens (specific pathogen free; SPF) and mice
raised with a selected group of organisms (gnotobiotic).

Methods

Animals

GF and SPF BALB/c mice (male, 9 weeks old)
were maintained in Trexler-type flexible-film plastic
isolators with sterile food and water (Sudo et al.
1997). Surveillance for bacterial contamination was
done by a periodic bacteriological examination of
faeces. To obtain Bifidobacterium infantis-, rabbit-derived
enteropathogenic Escherichia coli (EPEC)-, or EPEC
mutant strain (�Tir)-monoassociated mice whose flora
were composed of a single strain of bacterium, the parent
GF mice were administered a bacterium orally, and their
offspring thus became infected with this bacterium at the
neonate stage. These mice were used for the experiment at
9 weeks of age. To produce SPF flora-reconstituted mice,
the GF mice were inoculated with 0.5 ml of a 1 × 10−2
dilution of fresh SPF mouse faeces at either 1 or 3 weeks
before the commencement of the stress protocol. Such
reconstituted mice were subjected to the stress regimen
at 9 or 17 weeks of age. All experiments were approved by
the Ethics Committee for Animal Experiments of Kyushu
University.

Stress protocol

Acute restraint stress was applied by placing the animals
in a 50 ml conical tube (Nukina et al. 1998, 2001). Mice
were killed by cervical dislocation before (basal), and

immediately, 30, 60, or 120 min after being subjected
to 1 h of restraint stress. This procedure was performed
according to our Institutional Guidelines for Animal
Experiments. For ether stress (Diorio et al. 1993), animals
were maintained for 2.5 min in a glass container lined
with absorbent paper soaked with ether, then killed by
cervical dislocation before (basal), and immediately, 30,
60, or 120 min after ether exposure. Blood samples for
ACTH and corticosterone measurements were obtained
by cardiac puncture and stored at −80◦C before assay.
To avoid fluctuations in the biological measurements
resulting from differences in circadian rhythm, all samples
were collected at the same time of day (between 9.00 and
11.00 h).

Maternal behaviour

As described previously (Anisman et al. 1998; Francis et al.
1999), maternal behaviour was scored for three 60 min
observation periods daily (starting at 09.00, 13.00 and
17.00 h) on the second and third days postpartum. The
behaviour of each mother (n = 8) was scored every 4 min
(total of 360 samplings per group). The data were analysed
as the percentage of observations in which animals engaged
in the target behaviour. Individuals unaware of the origin
of the animals checked the following behaviours: mother
off pups, mother licking/grooming any pup, mother
nursing pups in an arched-back posture, nursing in a
‘blanket’ posture in which the mother lays over the pups,
or nursing in a passive posture in which the mother is
lying either on her back or side while the pups nurse.
Mean (± s.d.) frequency of the behaviours is expressed
as a percentage of the total observation.

Determination of ACTH and corticosterone levels

The plasma level of ACTH was measured by an immuno-
radiometric assay using the Allégro HS-ACTH kit (Nihon
Medi-Physics Co., Ltd, Nishinomiya Japan; Zahradnik
et al. 1989). In this assay, the concentration of ACTH is
linearly related to the amount of radioactivity bound to
the beads over a wide analytical range (1–1500 pg ml−1).
This assay system has been demonstrated to have a reliable
sensitivity of 1 pg ml−1.

The plasma level of corticosterone was measured using
a commercially available radioimmunoassay kit (ICN
Biomedicals, Costa Mesa, CA, USA). The concentration of
corticosterone in the serum samples was calculated from a
standard curve and expressed in nanograms per millilitre.
The detection limit of the assay was about 1 ng ml−1.

C© The Physiological Society 2004

J Physiol 558.1 Commensal microbiota and stress response 265

Determination of faecal bacterial flora

The faecal bacterial flora was determined according
to methods previously described (Mitsuoka et al. 1965;
Benno & Mitsuoka, 1992; Sudo et al. 2000). Briefly,
approximately 1 g (wet weight) was suspended in an
anaerobic diluent, and then serial tenfold dilutions from
10−1 to 10−8 were prepared. From the appropriate
dilution, a 0.05 ml aliquot was then spread on
three non-selective agar plates (trypticase soy blood
agar, glucose blood liver agar, Eggerth-Gagnon agar)
and selective agar plates to allow the incubation
of Streptococci, Enterobacteria, Staphylococci, yeasts,
Pseudomonas, Bacteroides, Bifidobacteria, Eubacteria,
Veillonella, Lactobacilli and Clostridia (Eiken, Tokyo,
Japan; OXOID, Basingstoke, UK). Staphylococcus medium
no. 110 supplemented with 2.5% egg yolk and 5 µg ml−1

methicillin (Nissui, Tokyo, Japan) was used for methicillin-
resistant Staphylococcus aureus and methicillin-sensitive
Staphylococcus aureus. After incubation for 2 days
(aerobes) or 3 days (anaerobes), 13 bacterial groups
and yeasts were identified by colonial and cellular
morphology, Gram staining, spore formation, and aerobic
and anaerobic growth.

Semi-quantitative RT-PCR methods

Gene expression levels of corticotropin-releasing factor
(CRH), glucocorticoid receptor (GR) and NMDA
receptor subunits (NR-1 and NR-2a) were analysed
by a semiquantitative RT-PCR method. The animals
were killed by cervical dislocation and each brain
was removed immediately. Total RNA was extracted
from the cortex, hippocampus and hypothalamus of
the GF and SPF mice using a commercially available
kit (Sepasol-RNA II, Nacalai Tesque, Kyoto, Japan),
according to the manufacturer’s instructions. Each brain
section was identified according to a stereotaxic atlas
(Paxinos & Franklin, 2001). To normalize the signals from
different RNA samples, glyceraldehydes-3-phosphate
dehydrogenase (GAPDH) mRNA was co-amplified as
an internal standard and then the relative values of
each transcript to GAPDH mRNA were calculated. In
preliminary experiments, optimal reaction parameters
were adjusted to obtain a linear relationship between
the number of PCR cycles and RT-PCR products and
between the initial amount of RNA and RT-PCR products.
Additionally, to verify the linearity of GAPDH signals
in each experiment, RT-PCR products were collected
after 26, 28, 30, 32 and 34 cycles in each sample and then
checked to see if the GAPDH signals would be within
the linear range of product accumulation. The following

primers were designed according to the method described
in the indicated literature, and their validity was checked
in our laboratory: GAPDH (Nukina et al. 2001), sense
primer 5′-TCCTGCACCACCAACTGCTTAG-3′, anti-
sense primer 5′-TCTTACTCCTTGGAGGCCATGT-3′;
c-Fos (Arrieta et al. 2000), sense primer
5′-CCCCTGTCAACACACAGGAC-3′, antisense
primer 5′-CCGATGCTCTGCGCTCTGC-3′;
CRH (Glasgow et al. 1999), sense primer
5′-AACTCAGAGCCCAAGTACGTTGAG-3′, antisense
primer 5′-TCACCCATGCGGATCAGAATC-3′;
GR (Kizaki et al. 1996), sense primer
5′-GCATGGAGAATTATGACCAC-3′, antisense primer 5′-
ATCAGATCAGGAGCAAAGCA-3′; NR-1 (Cai & Rhodes,
2001), sense primer 5′-CTCCCACCAGTCCAGCGTCT-
3′, antisense primer 5′-GTCATGTTCAGCATTGCGGC-
3′; NR-2a (Cai & Rhodes, 2001), sense primer 5′-
GGCTGTCAGCACTGAATCCAAAGG-3′, antisense
primer 5′-CGAAAGGCAGCTTCTGCAATGTGTG-3′.
PCR products were separated by electrophoresis on 1.5%
agarose gel and visualized by ethidium bromide staining
and UV light. Densitometric analysis was then done to
quantify the mRNA levels using public domain NIH
image software.

Measurement of neurotrophin and CRF protein levels

Sections of cortex, hippocampus and hypothalamus were
quickly removed after the animals were killed and samples
homogenized in a lysis buffer (137 mm NaCl, 20 mm Tris,
1% NP-40, 10% glycerol, 1 mm PMSF, 10 µg ml−1

aprotinin, 1 µg ml−1 leupeptin and 0.5 mm sodium
vanadate). Homogenates were centrifuged and the super-
natants used as enzyme-linked immunosorbent assay
(ELISA) samples. Neurotrophin protein concentrations
were determined by the method of Bradford, using bovine
serum albumin as a standard. ELISAs were performed
using the brain-derived neurotrophic factor (BDNF),
neurotrophin-3 (NT-3), or nerve growth factor (NGF)
Emax ImmunoAssay System kit (Promega, Madison, WI,
USA) according to the manufacturer’s instructions. CRF
protein levels in the hypothalamus were measured using
a commercially available radioimmunoassay kit (Phoenix
Pharmaceuticals Inc., Belmont, CA, USA).

Assays for cytokine levels in plasma

The plasma bioactivity of interleukin (IL)-6 was
determined by measuring the proliferation of the B9 cell
line, an IL-6-dependent B cell hybridoma, as previously
described (Nukina et al. 1998, 2001). The B9 cell line

C© The Physiological Society 2004

266 N. Sudo and others J Physiol 558.1

was kindly provided by Dr L. Aarden, the Netherlands
Red Cross Blood Transfusion Service, Amsterdam, the
Netherlands (Aarden, 1987). Briefly, B9 cells (5 × 103
per 100 µl) were cultured in 96-well microtiter plates
with serial dilutions of the plasma samples. After
72 h of incubation at 37◦C with 5% CO2, 20 µl of
MTT tetrazolium (5 mg ml−1; Research Organics Inc.,
Cleveland, OH, USA) was added to determine the
proliferation. After an additional 4 h of incubation the
supernatant was removed and 100 µl of 10% SDS with
0.01 n HCl was added to dissolve the crystals. The level of
cell proliferation was determined using a microplate ELISA
reader at 570 nm. The IL-6 activity in the plasma samples
was calculated based on a purified recombinant mouse
IL-6 standard (Boehringer Mannheim Corp., Mannheim,
Germany) run in the same assay. The sensitivity of the
assay was about 1 pg ml−1 and the specificity of the assay
was confirmed by using IL-6 neutralizing antibody, which
can antagonize 95% of the B9 cell proliferation induced by
the mouse plasma.

The IL-1β level of the plasma samples was assayed
using a commercially available ELISA kit (BioSource
International, Camarillo, CA, USA).

Anti-IL-6 treatment of mice

In some experiments, pre-treatment with anti-IL-6
antibody was done to neutralize the plasma IL-6 activity
upon exposure to Bifidobacterium infantis. Hybridoma
producing rat monoclonal antibody to mouse rIL-6,
MP5-20F3 clone (Starnes et al. 1990) was obtained
from the American Type Culture Collection (Rockville,
MD, USA) by courtesy of the DNAX Research
Institute of Molecular and Cellular Biology (Palo
Alto, CA, USA). Purified monoclonal antibodies from
the ascitic fluid from athymic nude mice were
then used in the experiment. The proper dosage of
anti-IL-6 antibody, that which sufficiently neutralizes
plasma IL-6 response, was determined according to
previous reports (Neta et al. 1992; Nukina et al. 1998).
Chromatographically purified rat IgG (Seikagaku Corp.,
Tokyo, Japan) was also used as a control antibody.

Statistical analysis

All data are expressed as the means ± s.d. The data were
analysed by Dunnett’s post hoc test after the factorial
analysis of variance. In some experiments, statistical
analysis was done using the Mann–Whitney U test. A
value of P < 0.05 was considered to indicate significant difference. Results Plasma ACTH and corticosterone responses of GF mice were more susceptible to restraint stress than those of SPF mice To investigate the difference in HPA response to stress stimuli between the GF and SPF mice, both groups of mice were subjected to either 1 h of restraint stress or ether exposure. Plasma ACTH and corticosterone elevation in response to restraint stress was substantially higher in GF mice than in SPF mice (Fig. 1A). When the mice were exposed to ether stimulus, no significant difference in plasma ACTH or corticosterone response was found between the groups of animals (Fig. 1B). GF mice revealed reduced expression levels of cortical GR transcript Morphological examination of the adrenal and pituitary glands by HE staining failed to reveal any obvious difference of structure or cell type between the GF and SPF mice. The average adrenal cortical thicknesses were 310 ± 35.4 µm in GF mice (n = 5) and 306.6 ± 28.8 µm in SPF mice (n = 6). Furthermore, when pituitary sections were stained with anti-ACTH antibodies, no quantitative difference was noted in the corticotrophs between the groups of animals. However, in the hypothalamus, the mRNA expression level of CRF transcript, a main stimulator of ACTH secretion from the pituitary gland, was significantly higher in GF mice than in SPF mice (relative values of CRF to GAPDH mRNA: GF 1.36 ± 0.10 versus SPF 0.85 ± 0.13, n = 5 per group, P < 0.05 by Mann–Whitney U test). Such elevated CRF mRNA levels in the hypothalamus of GF mice were also confirmed by CRF protein concentrations (GF 9.8 ± 3.4 ng mg−1 protein versus SPF 5.3 ± 2.7 ng mg−1 protein, n = 6 per group, P < 0.05). The mRNA expression level of GR, which negatively regulates HPA axis activation by inhibiting hypothalamic CRF gene expression, was significantly lower in the cortex, but not the hypothalamus or hippocampus, of GF mice than in SPF mice (relative values of GR to GAPDH mRNA: cortex, GF 0.94 ± 0.69 versus SPF 1.82 ± 0.62; hypothalamus, GF 0.49 ± 0.16 versus SPF 0.48 ± 0.34; hippocampus, GF 3.21 ± 0.72 versus SPF 3.92 ± 0.77; n = 4–7 per group, P < 0.05). The GF condition failed to affect maternal behaviour In view of previous data indicating that interaction of the dam with her litter can program HPA development (Liu et al. 1997, 2000), maternal behaviour was scored for three C© The Physiological Society 2004 J Physiol 558.1 Commensal microbiota and stress response 267 60 min observation periods daily on the second and third days postpartum. No difference in maternal behaviour was observed, with GF and SPF dams equally arch- backed-nursing or grooming/licking their pups (arch- backed nursing: GF 13.0 ± 2.9% versus SPF 11.8 ± 3.9%; grooming licking: GF 5.7 ± 1.3% versus SPF 5.2 ± 1.7%), suggesting that the enhanced HPA stress response of the GF mice was unlikely to have been due to reduced maternal contact. The GF mice showed reduced expression levels of cortical and hippocampal BDNF Previous reports demonstrated that early life events that take place during brain maturation can modulate Figure 1. Increased plasma ACTH and corticosterone response to restraint stress, but not to ether exposure in GF mice A, mice were subjected to a 1 h period of restraint stress (GF, n = 6–11 for each time point, total of 52 animals; SPF, n = 6–11 for each time point, total of 50 animals). The baseline data were obtained by cardiac puncture from mice that were killed by cervical dislocation before stress exposure. The baseline ACTH and corticosterone levels in the GF and SPF mice were 49 ± 12 pg ml−1 and 23 ± 4.2 ng ml−1, in the GF mice, 46 ± 13 pg ml−1 and 19 ± 5.6 ng ml−1, respectively. P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001 in Dunnett’s post hoc test between GF and SPF. B, GF and SPF mice failed to show any difference in HPA response to ether exposure (n = 6 for each time point, total of 30 animals per group). the expression of neurotrophins of cellular plasticity within selected brain regions (Liu et al. 2000; Roceri et al. 2002). We therefore compared the expression levels of neurotrophins and their related receptors in the various brain areas of the GF mice with those of the SPF mice. A semiquantitative RT-PCR analysis of NR subunits, neurotransmitters that regulate the expression of BDNF, showed decreased gene expression of cortical NR-1 and NR-2a and hippocampal NR-2a subunits in GF mice compared with SPF mice (Fig. 2A–C). Consistent with these results, the BDNF protein level in the cortex and hippocampus was significantly lower in GF mice than in SPF mice (Fig. 2D), whereas other neurotrophins, NT-3 and NGF, in these areas of GF mice were identical to those of SPF mice (NT-3: cortex, GF 66.8 ± 13.2 versus C© The Physiological Society 2004 268 N. Sudo and others J Physiol 558.1 SPF 65.8 ± 10.0; hippocampus, GF 126.5 ± 37.8 versus SPF 124.0 ± 14.1; NGF: cortex, GF 169.0 ± 87.0 versus SPF 188.7 ± 82.8; hippocampus, GF 136.4 ± 58.1 versus SPF 159.0 ± 87.5 pg (mg protein) −1). HPA response to stress in the gnotobiotic mice To further elucidate the involvement of gut microbiota in the HPA stress response, gnotobiotic mice whose flora were Figure 2. NR subunit gene expression and protein BDNF concentration A, a typical RT-PCR product with NR-1 specific primers revealed PCR products with the predicted 333 bp length of NR-1 mRNA. Total RNA was extracted from a single cerebral cortex of GF and SPF mice. NR-1 (B) and NR-2a transcripts (C) were detected by RT-PCR in the cortex, hypothalamus or hippocampus of GF and SPF mice (9 weeks old). Histograms show the relative band intensities on densitometric analysis as ratios of NR subunit and GAPDH mRNA after 30 cycles of amplification (n = 4–7 per group). D, BDNF protein concentration was measured by ELISA (n = 6–10 per group). ∗ P < 0.05, ∗∗ P < 0.01 by Mann–Whitney U test. composed of a single strain of bacterium at the neonate stage were tested for their susceptibility to restraint stress at 9 weeks of age. Monoassociation with Bifidobacterium infantis, which is a representative inhabitant of the neonate gut, dampened the HPA stress response to the SPF (Fig. 3). In accordance with previous reports that neonatal endotoxin treatment increases the HPA stress response later in life (Shanks et al. 1995, 2000), the hormonal stress response in rabbit-derived enteropathogenic Escherichia C© The Physiological Society 2004 J Physiol 558.1 Commensal microbiota and stress response 269 coli (EPEC)-monoassociated mice was substantially higher than that in GF mice, although no such exaggerated response was found in the mice reconstituted with an EPEC mutant strain, �Tir (Kenny et al. 1997), which is not internalized owing to defects in the translocated intimin receptor. Since there was no difference in the number of intestinal bacteria between the EPEC- and �Tir-associated mice, these results indicate that bacterial internalization to the intestinal epithelial layer is an indispensable condition that enables the EPEC strain to influence the regulatory system of the HPA response. Colonization by bacterium induced increases in the c-Fos mRNA levels in the paraventricular nucleus and in the corticosterone and cytokine concentrations in the plasma To further clarify the mechanism involved in the different sensitivity of each gnotobiotic mouse to stress, GF mice were orally inoculated with EPEC, �Tir or Bifidobacterium infantis. Then either the IL-1β and IL-6 levels in the plasma or the c-Fos expression level in the paraventricular nucleus, a marker for neuronal activity, was measured before and 6, 12 and 24 h after the inoculation with each bacterium. IL-1β and IL-6 levels in the plasma substantially increased and reached a peak at 12 h after the inoculation with EPEC (Fig. 4A and B). Administration of �Tir or Bifidobacterium infantis also triggered a small but significant increase in plasma IL-6 levels at 12 h after the inoculation without elevating the plasma IL-1β levels. Interestingly, the c-Fos mRNA level in the paraventricular nucleus was already elevated at 6 h after the inoculation regardless of which of the Figure 3. Effects of restraint stress on plasma ACTH and corticosterone levels in gnotobiotic mice Plasma ACTH and corticosterone levels were measured before or immediately after 1 h restraint in GF (n = 20), SPF (n = 18) and monoassociated mice (n = 18–24 per group) at 9 weeks of age. ∗ P < 0.05, ∗∗∗ P < 0.001 by Dunnett’s test. bacterial strains was used (Fig. 4C and D). This c-Fos response was accompanied by a concomitant increase in plasma corticosterone levels on exposure to microbes (control: basal 19 ± 10 ng ml−1, 6 h 25 ± 12 ng ml−1, 12 h 26 ± 16 ng ml−1, 24 h 17 ± 9 ng ml−1; Bifidobacterium: basal 20 ± 11 ng ml−1, 6 h 142 ± 36∗∗∗ ng ml−1, 12 h 35 ± 26 ng ml−1, 24 h 30 ± 19 ng ml−1; EPEC: basal 19 ± 8 ng ml−1, 6 h 175 ± 41∗∗∗ ng ml−1, 12 h 120 ± 29∗∗∗ ng ml−1, 24 h 86 ± 24∗∗ ng ml−1; �Tir: basal 22 ± 14 ng ml−1, 6 h 116 ± 32∗∗∗ ng ml−1, 12 h 60 ± 41 ng ml−1, 24 h 51 ± 32 ng ml−1; n = 5–7 for each time point; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 versus each corresponding basal value). Moreover, pretreatment with anti-IL-6 antibody failed to affect the elevated c-Fos response in the paraventricular nucleus (Fig. 5A and B) and corticosterone response in the plasma (Fig. 5C) on exposure to Bifidobacterium infantis. These results taken together indicate that visceral information derived from bacterial colonization can be transmitted to the brain at least partly through a humoral cytokine-independent pathway, probably via a neural route. A complete SPF flora partly reversed the HPA response to stress only when the flora was introduced at an early stage of development Finally, the effects of a complete SPF flora on the hormonal response to stress were examined. The enhanced HPA stress response of GF mice was partly corrected 3 weeks after reconstitution of SPF faeces at an early stage of development (Fig. 6A), while such correction was not found following any reconstitution exerted at a later stage (Fig. 6B). Bacterial examination of faecal samples revealed C© The Physiological Society 2004 270 N. Sudo and others J Physiol 558.1 Figure 4. Kinetics of cytokine concentration in the plasma and c-Fos gene expression in the paraventricular nucleus upon exposure to Bifidobacterium infantis, EPEC or ∆Tir GF mice at 5 weeks of age received a gavage of either 0.5 ml skimmed milk containing one of the bacterial strains (1 × 109 CFU; Bifidobacterium infantis, EPEC or �Tir) or skimmed milk alone (control), after which plasma and brain samples were collected before (basal) and 6, 12 or 24 h after inoculation with each bacterium. A, the plasma IL-1β level of the mice exposed to EPEC was measured by ELISA (n = 6–8 for each time point). No significant IL-1β elevation in the plasma was found after inoculation with Bifidobacterium infantis, �Tir or skimmed milk alone (control). B, plasma IL-6 levels were analysed by the B9 cell bioassay as described in the Methods (n = 6–8 for each time point). C, a typical RT-PCR product with c-Fos specific primers revealed PCR products with the predicted 247 bp length of c-Fos mRNA. D, histogram shows the relative band intensities on densitometric analysis as ratios of c-Fos and GAPDH mRNA after 30 cycles of amplification (n = 4 for each time point). ∗ P < 0.05, ∗∗ P < 0.01 and ∗∗∗ P < 0.001 were considered to be significantly different from the corresponding basal values. C© The Physiological Society 2004 J Physiol 558.1 Commensal microbiota and stress response 271 that colonization was almost completely accomplished 3 weeks after the administration of the SPF faeces, whereas aerobes, such as Escherichia coli, were more prominent than anaerobes 1 week after the administration of the SPF faeces. Collectively, these findings suggest that exposure to indigenous microbiota at an early developmental stage, when brain plasticity may still be preserved, is required for the HPA system to become fully susceptible to inhibitory neural regulation. Discussion Accumulating evidence has demonstrated a bidirectional communication between the brain and the gut. Researchers in this field preferentially call this cross-talk the ‘brain–gut …