Microbiota regulate social behaviour via
stress response neurons in the brain
In the format provided by the
authors and unedited
Nature | www.nature.com/nature
Supplementary information
https://doi.org/10.1038/s41586-021-03669-y
Supplementary information for
Microbiota regulate social behavior via
stress response neurons in the brain
Wei
-
Li Wu
1,2,3*
, Mark D. Adame
1
, Chia
-
Wei Liou
2,3
, Jacob T. Barlow
1
,
Tzu
-
Ting Lai
2
,
Gil Sharon
1
, Catherine E. Schretter
1
, Brittany D.
Needham
1
, Ma
delyn I. Wang
1
, Weiyi
Tang
1
, James Ousey
4
, Yuan
-
Yuan Lin
2
, Tzu
-
Hsuan Yao
2
, Reem Abdel
-
Haq
1
, Keith
Beadle
1
, Viviana Gradinaru
1
, Rustem F. Ismagilov
1,4
& Sarkis K. Mazmanian
1*
1
Division of Biology and Biological Engineering, California Institute
of Technolo
gy, 1200 E.
California Boulevard, Pasadena, CA, 91125, USA
2
Department of Physiology, College of Medicine, National Cheng Kung University (NCKU),
1 University Rd., Tainan 70101, Taiwan
3
Institute of Basic Medical Sciences, College of Medicine
, National C
heng Kung University
(NCKU), 1 University Rd., Tainan 70101, Taiwan
4
Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200
E. California Boulevard, Pasadena, CA, 91125, USA
*
Correspondence:
wlwu@ncku.edu.tw
(W.
-
L.W.)
This PDF file includes:
Supplementary information
1
Supplementary information: Statistical tests and exact
P
values for each figure.
Supplementary Video 1: Social interaction test of CNO
-
injected
Crh
PVN
expressing
hM3Dq
. AAV
-
hSyn
-
DIO
-
hM3Dq
-
mCherry (hM3D
q) viruses were injected into the PVN
of
Crh
-
ires
-
Cre
mice to deliver hM3Dq. Shortly after CNO injection, the mouse exhibited
the reduction of social behavior toward novel mouse and the increase of
non
-
social
behavior
.
Supplementary Video 2: Social intera
ction test of
saline
-
injected
Crh
PVN
expressing
hM3Dq
. AAV
-
hSyn
-
DIO
-
hM3Dq
-
mCherry (hM3Dq) viruses were injected into the PVN
of
Crh
-
ires
-
Cre
mice to deliver hM3Dq. The mouse exhibited normal social behavior
toward novel mouse after
saline
(
vehicle
) injecti
on (same mouse with Supplementary
Video 1)
.
Supplementary information
1
Vasopressin and oxytocin in the PVN have been implicated i
n social
behavior
1
. While
SPF
and GF mice
contain
similar numbers of vasopressin expressing neurons,
GF mice have
fewer
oxytocin
-
expressing neurons in the PVN (Extended Data Fig. 5a
-
d). Moreover, levels
of gene expression for oxytocin, vasopressin, and the
ir receptors in the hypothalamus were
similar reg
ardless of microbiome status (Extended Data Fig. 5e), suggesting gut bacterial
effect
s
on social behavior are likely not through vasopressin while an oxytocin
-
dependent
pathway
cannot be completely ruled out
1
.
We
labeled neurons
in
the
PVN
to visualize the architecture of this brain region linked to the
HPA axis.
There were no overt changes in
the numbers of neurons in the PVN and their
proj
ections to the median eminence (ME) in GF mice (Extended Data Fig. 5f
-
i). Further,
we
observed no changes in
neuronal counts in the PVN
following ABX treatment in
a reporter
mouse (
Crh
-
ires
-
Cre;Ai14D
)
that e
nables visualization of
corticotrophin
-
releasing
hormone
(
CRH
)
-
expressing
neuron
, critical in the stress response of
mice (Extended Data Fig. 5j, k).
Thus, increased corticosterone levels in GF and ABX mice following social interaction are
not due to compe
nsatory effects of PVN neuronal density.
1
Donaldson, Z. R. & Young, L. J. Oxytocin, vasopressin, and the neurogenetics of
sociality.
Science
322
, 900
-
904 (2008).
Supplementary information: Statistical tests and e
xact
P
values for each figure.
Fig. 1
1b. Social activity statistics:
two
-
tailed unpaired
t
-
test
t=7.893 df=37
,
P<
0.0001****
1c. Social activity statistics:
two
-
tailed unpaired
t
-
test
t=6.917 df=14
,
P<
0.
0001****
1d. Social activity statistics:
two
-
tail
ed unpaired
t
-
test
t=4.777 df=50
,
P<
0.0001****
1f. c
-
Fos staining statistics:
two
-
tailed unpaired
t
-
test
PVN
:
t=2.724 df=10
,
P=
0.0214*
1g. c
-
Fos staining statistics:
two
-
tailed unpaired
t
-
test
adBNST
:
t=2.949 df=10
,
P=
0.0146*
1h. c
-
Fos staining statisti
cs:
two
-
tailed unpaired
t
-
test
DG
:
t=3.112 df=10
,
P=
0.0110*
1j. c
-
Fos staining statistics:
two
-
tailed unpaired
t
-
test
PVN
:
t=2.466 df=8
,
P=
0.0390*
1k. c
-
Fos staining
statistics:
two
-
tailed unpaired
t
-
test
adBNST
:
t=3.972 df=8
,
P=
0.0041**
1l. c
-
Fos stain
ing statistics:
two
-
tailed unpaired
t
-
test
DG
:
t=2.718 df=8
,
P=
0.0263*
1m. Corticosterone statistics:
two
-
tailed unpaired
t
-
test
t=3.558 df=29
,
P=
0.0013**
1n.
Corticosterone statistics:
two
-
tailed unpaired
t
-
test
t=2.557 df=14
,
P=
0.0228*
1o. Corticoster
one statistics:
two
-
tailed unpaired
t
-
test
t=3.526 df=58
,
P=
0.0008***
Fig. 2
2b. Corticosterone statistics:
two
-
way ANOVA with
Bonferroni
multiple comparison
post
-
hoc
test
SPF vs. GF: F (1, 17)=29.46,
P<
0.0001****
CMC vs. MET: F (1, 17)=114.4,
P<
0.0001****
Interaction: F (1, 17)=4.551,
P=
0.0478*
CMC vs. MET
SPF,
P<
0.0001****
GF,
P<
0.0001****
SPF vs. GF
CMC,
P=
0.0001***
MET,
P=
0.0584
2c.
Social activity statistics:
two
-
way ANOVA with
Bonferroni
multiple comparison
post
-
hoc
test
SPF vs. GF: F (
1, 17)=5.625,
P=
0.0298*
CMC vs. MET: F (1, 17)=4.699,
P=
0.0447*
Interaction: F (1, 17)=3.913,
P=
0.0643
CMC vs. MET
SPF
:
P>
0.9999
GF
:
P=
0.0212*
SPF vs. GF
CMC
:
P=
0.0157*
MET
:
P>
0.9999
2d. Corticosterone statistics:
one
-
way ANOVA with
Bonferroni
multiple
comparison
post
-
hoc
test
Main effect:
F (2, 37)=21.1
,
P<
0.0001****
Control
-
Sham vs. ABX
-
Sham:
P=
0.0001***
Control
-
Sham vs. ABX
-
ADX:
P=
0.
7113
ABX
-
Sham vs. ABX
-
ADX:
P<
0.0001****
2e. Social activity statistics:
two
-
way repeated measures ANOVA
with mixed ef
fects with
Bonferroni
multiple comparison
post
-
hoc
test
Control
-
Sham vs. ABX
-
Sham vs. ABX
-
ADX: F (2, 37)=9.105,
P=
0.0006***
Drugs: F (2.253, 79.61)=9.104,
P=
0.0002***
Interaction: F (6, 106)=6.785,
P<
0.0001****
1
st
CMC
Control
-
Sham vs. ABX
-
Sham:
P=
0.0032*
*
Control
-
Sham vs. ABX
-
ADX:
P>
0.9999
ABX
-
Sham vs. ABX
-
A
DX:
P=
0.001**
RU
-
486
Control
-
Sham vs. ABX
-
Sham:
P=
0.823
Control
-
Sham vs. ABX
-
ADX:
P=
0.0136*
ABX
-
Sham vs. ABX
-
ADX:
P=
0.0814
Metyrapone
Control
-
Sham vs. ABX
-
Sham:
P=
0.0242*
Control
-
Sham vs. ABX
-
ADX:
P=
0.0808
ABX
-
Sham vs. ABX
-
ADX:
P>
0.9999
2
nd
CMC
Control
-
Sham vs. ABX
-
Sham:
P<
0.0001****
Control
-
Sham vs. ABX
-
ADX:
P=
0.0917
ABX
-
Sham vs. ABX
-
ADX:
P=
0.0001***
Control
-
Sham
1
st
CMC vs. RU
-
486:
P=
0.175
1
st
CMC vs. Metyrapone:
P=
0.402
1
st
CMC vs. 2
nd
CMC:
P
=
0.6066
RU
-
486 vs. Metyrapone:
P>
0.9999
RU
-
486 vs.
2
nd
CMC:
P>
0.9999
Metyrapone vs. 2
nd
CMC:
P>
0.9999
ABX
-
Sham
1
st
CMC vs. RU
-
486:
P=
0.0012**
1
st
CMC vs. Metyrapone:
P<
0.0001****
1
st
CMC vs. 2
nd
CMC:
P=
0.5602
RU
-
486 vs. Metyrapone:
P=
0.0083**
RU
-
486 vs. 2
nd
CMC:
P=
0.0016**
Metyrapone vs. 2
nd
CMC:
P=
0.0029**
ABX
-
ADX
1
st
CMC vs. RU
-
486:
P=
0.2671
1
st
CMC vs. Metyrapone:
P>
0.9999
1
st
CMC vs. 2
nd
CMC:
P>
0.9999
RU
-
486 vs. Metyrapone:
P>
0.9999
RU
-
486 vs. 2
nd
CMC:
P=
0.3438
Metyrapone vs. 2
nd
CMC:
P>
0.9999
2f. So
cial activity statistics:
one
-
way ANOVA with
Bonferroni
multiple comparison
post
-
hoc
test
Main effect:
F (2, 19)=9.649
,
P=
0.0013**
Control
-
Sham vs. ABX
-
Sham:
P=
0.0050**
Control
-
Sham vs. ABX
-
SDV:
P=
0.0025**
ABX
-
Sham vs. ABX
-
SDV:
P>
0.9999
2h. Social activ
ity statistics:
two
-
way repeated measures ANOVA
with
Bonferroni
multiple
comparison
post
-
hoc
test
Subject: F (12, 24) = 2.112,
P=
0.05
75
Control vs.
Nr3c1
ΔDG
:
F (1, 12) = 4.677
,
P=
0.0515
Drugs:
F (
1.654
,
19.85
) = 10.94
,
P=
0.00
11
**
Interaction:
F (2, 24) = 4
.281
,
P=
0.0257*
Control
1
st
CMC vs. RU486:
P=
0.0
139
*
1
st
CMC vs. 2
nd
CMC:
P=
0.
1229
RU486 vs. 2
nd
CMC:
P
=
0.00
90
**
Nr3c1
ΔDG
1
st
CMC vs. RU486:
P>
0.9999
1
st
CMC vs. 2
nd
CMC:
P=
0.
5722
RU486 vs. 2
nd
CMC:
P=
0.
9103
Control vs.
Nr3c1
ΔDG
1
st
CMC:
P=
0.0
063
*
*
RU48
6:
P>
0.9999
2
nd
CMC:
P=
0.
1650
2i. Corticosterone statistics:
two
-
tailed unpaired
t
-
test
t=2.982 df=12
,
P=
0.0114*
2j. Social activity statistics:
two
-
way repeated measures ANOVA
with
Bonferroni
multiple
comparison
post
-
hoc
test
Subject:
F (12, 24) = 4.700
,
P=
0.
0006***
Control vs.
Nr3c1
ΔBNST
:
F (1, 12) = 9.394
,
P=
0.0098
**
Drugs:
F (
1.895
,
22.74
) = 9.236
,
P=
0.001
3
**
Interaction:
F (2, 24) = 7.213
,
P=
0.0035**
Control
1
st
CMC vs. RU486:
P
=
0.
0039
**
1
st
CMC vs. 2
nd
CMC:
P=
0.1223
RU486 vs. 2
nd
CMC:
P
=
0.000
5
***
Nr3c1
ΔBNST
1
st
CMC vs. RU486:
P
>
0.9999
1
st
CMC vs. 2
nd
CMC:
P>
0.9999
RU486 vs. 2
nd
CMC:
P>
0.9999
Control vs.
Nr3c1
ΔBNST
1
st
CMC:
P=
0.
0648
RU486:
P>
0.9999
2
nd
CMC:
P=
0.00
12
**
2k.
Corticosterone statistics:
two
-
tailed unpaired
t
-
test
t=2.168 df=12
,
P=
0.0510
2l. Social activity statisti
cs:
two
-
way repeated measures ANOVA
with mixed effects with
Bonferroni
multiple comparison
post
-
hoc
test
Control vs.
Nr3c1
ΔHYPO
:
F (1, 29) =
70.36
,
P<
0.0001****
Drugs:
F (
1.495
, 2
1.67
) = 5.905
,
P=
0.01
43
*
Interaction:
F (2, 29) = 4.033
,
P=
0.0285*
Control
1
st
CMC vs. RU486:
P=
0.0
170
*
1
st
CMC vs. 2
nd
CMC:
P>
0.9999
RU486 vs. 2
nd
CMC:
P=
0.0
627
Nr3c1
ΔHYPO
1
st
CMC vs. RU486:
P>
0.9999
1
st
CMC vs. 2
nd
CMC:
P
=
0.
1690
RU486 vs. 2
nd
CMC:
P
=0.3629
Control vs.
Nr3c1
ΔHYPO
1
st
CMC:
P=
0.0
012
**
RU486:
P<
0.0001****
2
nd
CMC:
P=
0.0
486
*
2m. Corticosterone statistics:
two
-
tailed unpaired
t
-
test
t=5.607 df=9
,
P=
0.0003***
Fig. 3
3a. Hypothalamus
Crh
gene expression statistics:
two
-
way ANOVA with
Bonferroni
multiple comparison
post
-
hoc
test
VEH vs.
ABX: F (1, 19)=8.675,
P=
0.0083**
Social stimulation vs. No social stimulation: F (1, 19)=13.67,
P=
0.0015**
Interaction: F (1, 19)=4.744,
P=
0.0422*
VEH vs. ABX:
No social stimulation,
P>
0.9999
Social stimulation,
P=
0.0044**
Social stimulation vs. No socia
l stimulation:
VEH,
P=
0.614
ABX,
P=
0.0009***
c. Social activity statistics:
two
-
way repeated measures ANOVA
with
Bonferroni
multiple
comparison
post
-
hoc
test
Subject:
F (19, 38) = 1.070
,
P=
0.
4151
mCherry vs. hM4Di:
F (1, 19) = 4.839
,
P=
0.0404*
Saline vs.
CNO:
F (1.654, 31.43) =
2.866
,
P=
0.0808
Interaction:
F (2, 38) = 9.463
,
P=
0.0005***
mCherry vs. hM4Di
1
st
VEH:
P>
0.9999
CNO:
P=
0.0075**
2
nd
VEH:
P>
0.9999
ABX mCherry
1
st
VEH vs. CNO:
P=
0.476
1
st
VEH vs. 2
nd
VEH:
P=
0.3592
CNO vs. 2
nd
VEH:
P=
0.0254*
ABX h
M4Di
1
st
VEH vs. CNO:
P=
0.0246*
1
st
VEH vs. 2
nd
VEH:
P=
0.3455
CNO vs. 2
nd
VEH:
P=
0.174
3d. Corticosterone statistics:
two
-
tailed paired
t
-
test
mCherry
:
t=0.1883 df=8
,
P=
0.8553
hM4Di
:
t=2.525 df=9
,
P=
0.0325*
3g. c
-
Fos staining statistics:
two
-
tailed unpai
red
t
-
test
PVN
:
t=2.274 df=19
,
P=
0.0347*
3i, Corticosterone statistics:
two
-
tailed paired
t
-
test
SPF mCherry
:
t=1.012 df=9
,
P=
0.3379
SPF hM3Dq
:
t=5.015 df=10
,
P=
0.0005***
3j. Social activity statistics:
two
-
way repeated measures ANOVA
with
Bonferroni
mul
tiple
comparison
post
-
hoc
test
Subject:
F (19, 38) = 4.145
,
P<
0.0001****
mCherry vs. hM3Dq: F (1, 19) = 13.66,
P=
0.0015**
Saline vs. CNO:
F (1.790, 34.00) = 10.82
,
P=
0.0004*
**
Interaction: F (2, 38) = 26.42,
P<
0.0001****
mCherry vs. hM3Dq
1
st
VEH:
P=
0.760
3
CNO:
P=
0.0001***
2
nd
VEH:
P>
0.9999
SPF mCherry
1
st
VEH vs. CNO:
P=
0.0827
1
st
VEH vs. 2
nd
VEH:
P=
0.8448
CNO vs. 2
nd
VEH:
P=
0.7816
SPF hM3Dq
1
st
VEH vs. CNO:
P=
0.0005***
1
st
VEH vs. 2
nd
VEH:
P=
0.366
CNO vs. 2
nd
VEH:
P<
0.0001****
3l. CRH (high) infusion
social activity statistics:
two
-
tailed paired
t
-
test
t=8.175 df=8
,
P<
0.0001****
3m. CRH (low) infusion social activity statistics:
two
-
tailed paired
t
-
test
t=3.953 df=6
,
P=
0.0075**
3o. DG glucocorticoid signaling agonism social activity statistics:
one
-
w
ay ANOVA
repeated measures with
Bonferroni
multiple comparison
post
-
hoc
test
Treatment effect: F (1.532, 9.194) = 70.73,
P<
0.0001**
**
VEH vs. CORT:
P=
0.0002***
VEH vs. DEX:
P=
0.0002***
3p. BNST glucocorticoid signaling agonism social activity statistics:
one
-
way ANOVA
repeated measures with mixed effects with
Bonferroni
multiple comparison
post
-
hoc
test
Treatment effect: F (0.5980,
3.588) = 16.57,
P=
0.0221*
VEH vs. CORT:
P=
0.0072**
VEH vs. DEX:
P=
0.0076**
Fig. 4
4b. Social activity statistics:
one
-
way ANOVA with
Bonferroni
multiple comparison
post
-
hoc
test
Main effect:
F (4, 35)=10.07
,
P<
0.0001****
AVNM vs. VNM:
P>
0.9999
AVNM vs. ANM:
P>
0.9999
AVNM vs. AVM:
P<
0.0001****
AVNM vs. AVN:
P>
0.9999
4c. Corticosterone statistics:
two
-
tailed unpaired
t
-
test
AVNM vs. AVM
: t
=2.162 df=14
,
P=
0.0484*
4d. Social activity statistics:
two
-
tailed unpaired
t
-
test
t=
4.988 df=24
,
P<
0.0001****
4e. Corticosterone statistics:
two
-
tailed unpaired
t
-
test
t=2.482 df=24
,
P=
0.0205*
4i. Social activity statistics:
two
-
w
ay repeated measures ANOVA
with
Bonferroni
multiple
comparison
post
-
hoc
test
Subject:
F (20, 20) =
2.795
,
P
=
0.
0131
*
VEH+Ctrl vs. ABX+Ctrl vs. ABX+E.f.:
F (2, 20) = 3.526,
P=
0.0488*
1
st
trial vs. 2
nd
trial: F (1, 20) = 5.237,
P=
0.0331
Interaction: F (2, 20
) = 4.315,
P=
0.0277*
1
st
vs. 2
nd
VEH+Ctrl:
P>
0.9999
ABX+Ctrl:
P>
0.9999
ABX+E.f.:
P=
0.0056**
1
st
trial
VEH+Ctrl vs. ABX+Ctrl:
P=
0.2886
VEH+Ctrl vs. ABX+E.f.:
P=
0.832
ABX+Ctrl vs. ABX+E.f.:
P>
0.999
2
nd
trial
VEH+Ctrl vs. ABX+Ctrl:
P=
0.1004
VEH+Ctrl vs. A
BX+E.f.:
P=
0.6402
ABX+Ctrl vs. ABX+E.f.:
P=
0.0047**
4j. Corticosterone statistics:
o
ne
-
way ANOVA with
Bonferroni
multiple comparison
post
-
hoc
test
Main effect: F (2, 19)=13.21,
P=
0.0003***
VEH+Ctrl vs. ABX+Ctrl:
P=
0.0015**
VEH vs. ABX+E.f.:
P>
0.9999
ABX+
Ctrl vs. ABX+E.f.:
P=
0.0006***
Extended data Fig. 1
1a. Detailed social activity statistics:
two
-
tailed unpaired
t
-
test
Anogenital sniff
:
t=5.552 df=37
,
P<
0.0001****
Nose
-
nose sniff
:
t=4.773 df=37
,
P<
0.0001****
Active approach
:
t=3.978 df=37
,
P=
0.0003***
Push
-
Crawl
:
t=2.947 df=37
,
P=
0.0055**
1b. Detailed s
ocial activity statistics:
two
-
tailed unpaired
t
-
test
Anogenital sniff
:
t=6.452 df=14
,
P<
0.0001****
Nose
-
nose sniff
:
t=3.585 df=14
,
P=
0.0030**
Active approach
:
t=3.714 df=14
,
P=
0.0023**
Push
-
Crawl
:
t=1.
158 df=14
,
P=
0.2662
1c. Social activity
statistics:
two
-
way ANOVA with
Bonferroni
multiple comparison
post
-
hoc
test
Subject microbiota effect: F (1, 51) = 72.70,
P<
0.0001****
Novel mouse microbiota effect:
F (1, 51) = 2.596,
P=
0.1133
Interaction: F (1, 51
) = 0.7374,
P=
0.3945
SPF novel mouse:
P<
0.0001****
GF novel mouse:
P<
0.0001****
1d. Detailed social activity statistics:
two
-
tailed unpaired
t
-
test
Anogenital sniff
:
t=3.703 df=50
,
P=
0.0005***
Nose
-
nose sniff
:
t=3.144 df=50
,
P=
0.0028**
Active approach
:
t
=3.792 df=50
,
P=
0.0004***
Push
-
Crawl
:
t=0.7444 df=50
,
P=
0.4601
1e. Social activity statistics:
two
-
tailed unpaired
t
-
test
t=0.3865 df=17
,
P=
0.7039
1f. Non
-
social activity statistics:
two
-
tailed unpaired
t
-
test
SPF
vs.
GF subjects
(
SPF novel mouse
):
t=0.24
4 df=37
,
P=
0.8086
SPF
vs.
GF subjects
(
GF novel mouse
):
t=1.653 df=14
,
P=
0.1205
VEH
vs.
ABX subjects
(
SPF novel mouse
):
t=0.6725 df=50
,
P=
0.5044
1g. Non
-
social activity statistics:
two
-
tailed unpaired
t
-
test
Digging
:
t=0.1678 df=16
,
P=
0.8689
Grooming
:
t=0
.7198 df=16
,
P=
0.4820
Rearing
:
t=0.7558 df=16
,
P=
0.4608
Total
:
t=1.422 df=16
,
P=
0.1743
1h left. Social activity statistics:
one
-
way
repeated measures
ANOVA
Main effect: F (1.722, 18.94) = 0.6116,
P=
0.5294
1h middle. Nose
-
to
-
nose sniff statistics:
one
-
wa
y
repeated measures
ANOVA
Main effect: F (1.941, 21.35) = 1.876,
P=
0.1784
1h right. Nose
-
to
-
tail sniff statistics:
one
-
way
repeated measures
ANOVA
Main effect:
F (1.675, 18.42) = 0.7903,
P=
0.4477
1i. Socia
l activity statistics:
one
-
way ANOVA
Main effect
:
F (2, 13) = 0.6098,
P=
0.5583
1j. Distance traveled statistics:
one
-
way ANOVA
Main effect:
F (2, 13) = 1.073,
P=
0.3705
1k. Female social activity statistics:
two
-
tailed unpaired
t
-
test
SPF vs. GF
:
t=3.193 df=17
,
P=
0.0053**
VEH vs. ABX
:
t=2.927 df=8
,
P=
0
.0191*
1l. Social activity statistics:
t
wo
-
way ANOVA with
Bonferroni
multiple comparison
post
-
hoc
test
SPF vs. GF: F (1, 40) = 96.47,
P<
0.0001****
Hours post
-
isolation: F (4, 40) = 0.5102,
P=
0.7285
I
nteraction: F (4, 40) = 0.6018,
P=
0.6635
SPF vs. GF
4
-
5
hr:
P=
0.0024**
5
-
6 hr:
P<
0.0001****
6
-
7 hr:
P<
0.0001****
7
-
8 hr:
P=
0.0028**
8
-
9 hr:
P
=0.0003***
1m. Social activity statistics:
o
ne
-
way ANOVA
Main effect:
F (2, 16) = 1.031,
P=
0.379
1n. 3
-
chamber social test:
Distance traveled
:
two
-
tailed unpaired
t
-
t
est
SPF vs. GF
:
t=3.249 df=17
,
P=
0.0047**
1o. 3
-
chamber social test:
Chamber duration: one
-
tailed paired
t
-
test
SPF: Mouse vs. Object
:
t=7.706 df=8
,
P<
0.0001****
GF: Mouse vs. Object
:
t=
4.787 df=9
,
P=
0.0005***
1o. 3
-
chamber social test:
Chamber entries:
one
-
tailed paired
t
-
test
SPF: Mouse vs. Object
:
t=1.148 df=8
,
P=
0.1420
GF: Mouse vs. Object
:
t=1.475 df=9
,
P=
0.0872
1p. 3
-
chamber social test:
Distance traveled: two
-
tailed unpaired
t
-
test
VEH vs. ABX
:
t=1.611 df=18
,
P=
0.1246
1q. 3
-
chamber social test:
Chamber duration: one
-
tailed paired
t
-
test
VEH: Mouse vs. Object
:
t=1.247 df=9
,
P=
0.1220
ABX: Mouse vs. Object
:
t=3.110 df=9
,
P=
0.0063**
1q. 3
-
chamber social test:
Chamber entries: one
-
tailed paired
t
-
test
VEH: Mouse vs. Object
:
t=0.8102 df=9
,
P=
0.2194
ABX: Mouse vs. Object
:
t=4.477 df=9
,
P=
0.0008***
1s. c
-
Fos staining statistics:
two
-
tailed unpaired
t
-
test
SPF vs. GF: t=1.804, df=10
,
P=
0.1014
VEH vs. ABX: t=1.800, df=8
,
P=
0.1095
1t. c
-
Fos staining statistics:
two
-
tailed unpaired
t
-
test
PVN
:
t=0.8376 d
f=9
,
P=
0.4239
adBNST
:
t=1.21 df=9
,
P=
0.2571
DG
:
t=1.958 df=9
,
P=
0.0818
BLA
:
t=1.215 df=9
,
P=
0.2552
1u. Corticosterone statistics:
two
-
way
repeated measures
ANOVA with
Bonferroni
multiple comparison
post
-
hoc
test
Subject: F (8, 16) = 1.739,
P=
0.1648
VEH vs
. ABX: F (1, 8) = 10.42,
P=
0.0121*
Time: F (
1.724
,
1
3.79
) = 129.2,
P<
0.0001****
Interaction: F (2, 16) = 2.006,
P=
0.1670
VEH vs. ABX
Iso:
P
=
0.
5775
0 hr,
P=
0.
0916
1 hr,
P=
0.
1751
Time: VEH
Iso vs. 0 hr:
P=
0.
0129*
Iso vs. 1 hr:
P
=
0.0
027
**
0 hr vs. 1 hr:
P
=
0
.00
23
**
Time: ABX
Iso vs. 0 hr:
P=
0.
0624
Iso vs. 1 hr:
P
=
0.00
16
**
0 hr vs. 1 hr:
P
=
0.00
62
**
1v. Corticosterone statistics:
two
-
way ANOVA with
Bonferroni
multiple comparison
post
-
hoc
test
ABX vs. GF: F (1, 27) = 6.006,
P=
0.0210*
Intact microbiota vs. No m
icrobiota: F (1, 27) = 8.229,
P=
0.0079**
Interaction: F (1, 27) = 4
.570,
P=
0.0417*
ABX vs. GF
Intact microbiota:
P>
0.9999
No microbiota:
P=
0.0085**
Intact microbiota vs. No microbiota
ABX:
P>
0.9999
GF:
P=
0.0125*
1w. Corticosterone statistics:
two
-
way A
NOVA
SPF vs. GF: F (1, 34) = 14.65,
P=
0.0005***
Hours post
-
isolation: F (4, 34) = 0.6051,
P=
0.6616
Interaction: F (4, 34) = 0.009598,
P=
0.9998
1x. Corticosterone statistics:
two
-
way ANOVA
VEH vs. ABX: F (1, 48) = 12.07,
P=
0.0011**
Hours post
-
isolation: F
(1, 48) = 1.873,
P=
0.1775
Interaction: F (1, 48)
= 0.1878,
P=
0.6667
1y. Social activity statistics:
one
-
way ANOVA with
Bonferroni
multiple comparison
post
-
hoc
test
Main effect:
F (2, 44) = 49.73,
P<
0.0001****
SPF vs. GF:
P<
0.0001****
SPF vs. exGF:
P=
0.00
01***
GF vs. exGF:
P<
0.0001****
1z. Corticosterone statistics:
one
-
way ANOVA with
Bonferroni
multiple comparison
post
-
hoc
test
Main effect:
F (2, 36) = 8.334,
P=
0.0011**
SPF vs. GF:
P=
0.0012**
SPF vs. exGF:
P>
0.9999
GF vs. exGF:
P=
0.0467*
Extended data
Fig. 2
2
a. Bacterial DNA statistics:
two
-
tailed unpaired
t
-
test
SPF vs. GF
:
t=4.583 df=6
,
P=
0.0038**
VEH vs. ABX
:
t=3.863 df=6
,
P=
0.0083**
2
b. Bacterial DNA statistics:
two
-
way
repeated measures
ANOVA with
Bonferroni
multiple
comparison
post
-
hoc
test
Subject: F (6, 12) = 0.5051,
P=
0.7933
VEH
vs. ABX: F (1, 6) = 3452,
P<
0.0001****
Time:
F (1.029, 6.177) = 3.720
,
P=
0.1002
Interaction: F (2, 12) = 3.72,
P=
0.0553
VEH
vs. ABX
1w:
VEH
vs. ABX
:
P=
0.001**
2w:
VEH
vs. ABX
:
P=
0.0001***
3w:
VEH
vs. ABX
:
P<
0.0001****
2
c. Bacterial DNA statistics:
two
-
way ANOVA with
Bonferroni
multiple comparison
post
-
hoc
test
Subject: F (6, 12) = 1.672,
P=
0.2112
VEH
vs. ABX: F (1, 6) = 106.3,
P<
0.0001****
Time:
F (1.630, 9.779) = 35.21
,
P<
0.0001****
Inter
action: F (2, 12) = 18.71,
P=
0.0002
VEH
vs. ABX
1w:
VEH
vs. ABX
:
P=
0.2237
2w:
VEH
vs. ABX
:
P=
0.0179*
3w:
VEH
vs. ABX
:
P<
0.0001****
Time_Vehicle
1w vs. 2w
:
P=
0.6526
1w vs. 3w
:
P=
0.002**
2w vs. 3w:
P=
0.0636
Time_ABX
1w vs. 2w
:
P=
0.0398
*
1w vs. 3w
:
P=
0.00
7
8
**
2w vs. 3w:
P>
0.9999
2
g. Observed species:
Kruskal
-
Wallis
AVNM vs Negative control, H=0.16819572,
P=
0.68172033, q=0.68172033
AVNM vs Vehicle, H=10.9600516,
P=
0.00093097***, q=0.00279292
Negative control vs Vehicle, H=4.6147260
3,
P=
0.03169856*, q=0.0475
4784
2
h. Faith’s phylogenetic diversity:
Kruskal
-
Wallis
AVNM vs Negative control, H=0.375,
P=
0.54029138, q=0.54029138
AVNM vs Vehicle, H=10.5992647,
P=
0.00113133**, q=0.00339398
Negative control vs Vehicle, H=6,
P=
0.01430588*, q=
0.02145882
2
i. Unweighted
UniFrac:
PERMANOVA
AVNM vs Negative control, Pseudo
-
F=1.19256034,
P=
0.2637, q=0.2637
AVNM vs Vehicle, Pseudo
-
F=11.5191588,
P=
0.0002***, q=0.0006
Negative control vs Vehicle, Pseudo
-
F=11.2825915,
P=
0.0071 **, q=0.01065
2
j. Weighted UniFrac:
PERMANOVA
AVNM
vs Negative control, Pseudo
-
F=1.01514874,
P=
0.3274, q=0.3274
AVNM vs Vehicle, Pseudo
-
F=15.3627766,
P=
0.0005***, q=0.0015
Negative control vs Vehi
cle, Pseudo
-
F=18.4648706,
P=
0.0055 **, q=0.00825
Extended data Fig. 3
3
a. Open
-
field (distance moved in 15 min) statistics:
two
-
tailed unpaired
t
-
test
t=0.2091 df=15
,
P=
0.8371
3
b. Open
-
field (center time in 15
min) statistics:
two
-
tailed unpaired
t
-
test
t=2.907 df=15
,
P=
0.0108*
3
c. Light
-
Dark box (light time in 10 min) s
tatistics:
two
-
tailed unpaired
t
-
test
t=2.184 df=16
,
P=
0.0442*
3
d. Elevated plus maze (open arm time in 5 min) statistics:
two
-
tailed unpaired
t
-
test
t=2.451 df=16
,
P=
0.0261*
3
e. Open
-
field (distance moved in 15 min) statistics:
two
-
tailed unpaired
t
-
tes
t
t=0.9382 df=18
,
P=
0.3606
3
f. Open
-
field (center time in 15 min) statistics:
two
-
tailed unpaired
t
-
test
t=0.9860 df=18
,
P
=
0.3372
3
g. Light
-
Dark box (light time in 10 min) statistics:
two
-
tailed unpaired
t
-
test
t=0.07870 df=18
,
P=
0.9381
3
h. Elevated plu
s maze (open arm time in 5 min) statistics:
two
-
tailed unpaired
t
-
test
t=0.1605 df=18
,
P=
0.8743
3
i. open
-
field
(distance moved in 60 min) statistics:
two
-
way repeated measures ANOVA
Subject: F (28, 140) = 2.010,
P=
0.0044**
VEH
vs. ABX: F (1, 28) = 0.2481
,
P=
0.6223
Time:
F (1.234, 34.55) = 3.521
,
P=
0.0
609
Interaction: F (5, 140) = 0.3179,
P=
0.9015
3
j. water consum
ption statistics:
two
-
way
repeated measures
ANOVA
Subject: F (8, 32) = 10.49,
P<
0.0001****
SPF vs. GF:
F (1, 8) = 0.1007
,
P=
0.
7591
Time:
F (1.91
5, 15.32) = 634.1
,
P<
0.0001****
Interaction:
F (4, 32) = 0.9299
,
P=
0.
4590
3
k. olfactory
habituation/dishabituation statistics:
two
-
way
repeated measures
ANOVA
with
Bonferroni
multiple comparison
post
-
hoc
test
Subject: F (19, 266) = 3.863,
P<
0.0001****
SP
F vs. GF:
F (1, 19) = 13.13
,
P=
0.0018**
Odors:
F (
4.431
,
84.18
) = 36.67
,
P<
0.0001****
Interaction:
F (14, 266) = 5.652
,
P<
0.0001****
SPF vs. GF
Water 1:
P>
0.9999
Water 2:
P>
0.9999
Water 3:
P>
0.9999
Almond 1:
P>
0.9999
Almond 2:
P=
0.
3966
Almond 3:
P>
0.9999
Banana 1:
P=
0.5
646
Banana 2:
P>
0.9999
Banana 3:
P>
0.9999
B6 Cage 1
-
1:
P
=
0.01
72
*
B6 Cage 1
-
2:
P
=
0.0
402
*
B6 Cage 1
-
3:
P
=
0.2028
BTBR Cage 1
-
1:
P>
0.9999
BTBR Cage 1
-
2:
P>
0.9999
BTBR Cage 1
-
3:
P=
0.
8881
3
l. olfactory habituation/dishabituation (soiled cage) st
atistics:
two
-
way
repeated
measures
ANOVA with
Bonferroni
multiple comparison
pos
t
-
hoc
test
Subject: F (19, 95) = 3.975,
P<
0.0001****
SPF vs. GF:
F (1, 19) = 0.4843
,
P=
0.4949
Odors:
F (4.010, 76.19) = 34.26
,
P<
0.0001****
Interaction:
F (5, 95) = 5.166
,
P=
0
.0003
***
SPF
B6 Cage 1
-
1 vs. B6 Cage 1
-
2:
P
=
0.
0020
**
B6 Cage 1
-
1 vs. B6 Cage 1
-
3:
P
=
0.000
2
***
B6 Cage 1
-
1 vs. BTBR Cage 1
-
1:
P>
0.9999
BTBR Cage 1
-
1 vs. BTBR Cage 1
-
2:
P
=
0.000
2
***
BTBR Cage 1
-
1 vs. BTBR Cage 1
-
3:
P<
0.0001****
GF
B6 Cage 1
-
1 vs. B6 Cage 1
-
2:
P=
0.
0120
*
B6 Cage 1
-
1 vs. B6 Cage 1
-
3:
P=
0.0
397
*
B6 Cage 1
-
1 vs. BTBR Cage 1
-
1:
P=
0.0
143
*
BTBR Cage 1
-
1 vs. BTBR Cage 1
-
2:
P<
0.0001****
BTBR Cage 1
-
1 vs. BTBR Cage 1
-
3:
P<
0.0001****
SPF vs. GF
B6 Cage 1
-
1:
P>
0.9999
B6 Cage 1
-
2:
P=
0.2822
B6 Cage 1
-
3:
P=
0.2699
BTBR Cage 1
-
1:
P=
0.3781
BTBR Cage 1
-
2:
P>
0.9999
BTBR Cage 1
-
3:
P>
0.9999
Extended data Fig. 4
4
a. qPCR statistics:
two
-
tailed unpaired
t
-
test
Arc
:
t=2.658 df=10
,
P=
0.0240*
Fos
:
t=3.785 df=10
,
P=
0.0036**
cJun
:
t=2.922 df=10
,
P=
0.0152*
JunB
:
t=3.32 df=10
,
P=
0.0077**
Egr
-
1
:
t=2.192 df=10
,
P=
0.0532
Egr
-
2
:
t=2.475 df=10
,
P=
0.0328*
Gadd45b
:
t=5.072 df=10
,
P=
0.0005***
Gadd45g
:
t=2.309 df=10
,
P=
0.0435*
Bdnf
:
t=2.966 df=10
,
P=
0.0142*
Map2
:
t=0.9036 df=10
,
P=
0.3874
4
b. qPCR statistics:
two
-
tailed un
paired
t
-
test
Arc
:
t=3.126 df=10
,
P=
0.0108*
Fos
:
t=5.916 df=10
,
P=
0.0001***
cJun
:
t=0.9066 df=10
,
P=
0.3859
JunB
:
t=1.777 df=10
,
P=
0.1060
Egr
-
1
:
t=4.096 df=10
,
P=
0.0022**
Egr
-
2
:
t=1.901 df=10
,
P=
0.0864
Gadd45b
:
t=0.1883 df=10
,
P=
0.8544
Gadd45g
:
t=0.8045 df=
10
,
P=
0.4398
Bdnf
:
t=1.552 df=10
,
P=
0.1517
Map2
:
t=0.2263 df=10
,
P=
0.8255
4
c. qPCR statistics:
two
-
tailed unpaired
t
-
test
Arc
:
t=0.7908 df=4
,
P=
0.4733
Fos
:
t=0.8264 df=4
,
P=
0.4550
cJun
:
t=0.544 df=4
,
P=
0.6153
JunB
:
t=0.544 df=4
,
P=
0.6154
Egr
-
1
:
t=0.9188 d
f=4
,
P=
0.4102
Egr
-
2
:
t=1.239 df=4
,
P=
0.2830
Gadd45b
:
t=0.8896 df=4
,
P=
0.4240
Gadd45g
:
t=2.276 df=4
,
P=
0.0852
Bdnf
:
t=0.02943 df=4
,
P=
0.9779
Map2
:
t=0.937 df=4
,
P=
0.4018
4
d. qPCR statistics:
two
-
tailed unpaired
t
-
test
Arc
:
t=0.2208 df=10
,
P=
0.8297
Fos
:
t=0
.6654 df=10
,
P=
0.5208
cJun
:
t=2.254 df=10
,
P=
0.0479*
JunB
:
t=2.649 df=10
,
P=
0.0244*
Egr
-
1
:
t=2.225 df=10
,
P=
0.0502
Egr
-
2
:
t=0.6359 df=10
,
P=
0.5391
Gadd45b
:
t=4.087 df=10
,
P=
0.0022**
Gadd45g
:
t=3.34 df=10
,
P=
0.0075**
Bdnf
:
t=
3.028 df=10
,
P=
0.0127*
Map2
:
t=1.
667 df=10
,
P=
0.1266
4
f. qPCR statistics:
two
-
tailed unpaired
t
-
test
Mrg1b
:
t=17.68 df=46
,
P<
0.0001****
Dsp
:
t=19.93 df=46
,
P<
0.0001****
Tdo2
:
t=20.25 df=46
,
P<
0.0001****
4
g. qPCR statistics:
two
-
tailed unpaired
t
-
test
Nr3c1
:
t=1.803 df=10
,
P=
0.1016
Nr3c2
:
t=0.1357 df=10
,
P=
0.8947
4
h. qPCR statistics:
two
-
tailed unpaired
t
-
test
Nr3c1
:
t=1.612 df=10
,
P=
0.1379
Nr3c2
:
t=0.0303
6
df=10
,
P=
0.9764
4
i. qPCR statistics:
two
-
tailed unpaired
t
-
test
Crh1
:
t=0.8771 df=9
,
P=
0.4032
Crh2
:
t=0.4293 df=9
,
P=
0.6778
Ucn
:
t=
1.236 df=9
,
P=
0.2477
Ucn2
:
t=2.114 df=9
,
P=
0.0636
Ucn3
:
t=1.975 df=9
,
P=
0.0797
Avp
:
t=0.01786 df=9
,
P=
0.9861
Oxt
:
t=1.598 df=9
,
P=
0.1445
4
j. qPCR
statistics:
two
-
tailed unpaired
t
-
test
Nr3c1
:
t=0.5107 df=10
,
P=
0.6206
Nr3c2
:
t=0.6873 df=10
,
P=
0.5075
4
k. q
PCR statistics:
two
-
tailed unpaired
t
-
test
Nr3c1
:
t=0.7211 df=10
,
P=
0.4873
Nr3c2
:
t=1.856 df=10
,
P=
0.0931
4
l. qPCR statistics:
two
-
tailed unpaired
t
-
test
Crh1
:
t=0.4691 df=10
,
P=
0.6490
Crh2
:
t=0.9035 df=10
,
P=
0.3875
Ucn
:
t=3.015 df=10
,
P=
0.0130*
Ucn2
:
t=1
.599 df=10
,
P=
0.1409
Ucn3
:
t=0.6816 df=10
,
P=
0.5110
Avp
:
t=0.5706 df=10
,
P=
0.5809
Oxt
:
t=0.2315 df=10
,
P=
0.8216
Extended data Fig. 5
5
c. AVP staining statistics:
two
-
tailed unpaired
t
-
test
t=0.8979 df=12
,
P=
0.3869
5
d. OXT staining statistics:
two
-
tailed unpaired
t
-
test
t=3.442 df=12
,
P=
0.0049**
5
e. qPCR statistics:
two
-
tailed unpaired
t
-
test
Avp
:
t=0.9073
df=6
,
P=
0.3992
Avpr1a
:
t=0.4982 df=6
,
P=
0.6361
Avpr1b
:
t=0.6378 df=6
,
P=
0.5471
Avpr2
:
t=1.38 df=6
,
P=
0.2168
Oxt
:
t=0.09407 df=6
,
P=
0.9281
O
xtr
:
t=1.071 df=6
,
P=
0.3252
5
g.
F
luorogold statistics:
two
-
way repeated measures ANOVA
Subject: F (6, 6) = 0.4886,
P=
0.7977
SPF vs. GF:
F (1, 6) = 1.732
,
P=
0.2362
Bregma:
F (1, 6) = 0.01217
,
P=
0.9158
Interaction:
F (1, 6) = 0.05600
,
P=
0.8208
5
i.
F
luorogo
ld statistics:
two
-
tailed unpaired
t
-
test
t=1.004 df=4
,
P=
0.3723
5
k.
F
luorogold statistics:
two
-
tailed unpaired
t
-
test
t=0.7995 df=6
,
P=
0.4545