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SUPPLEMENTARY FIGURES 1
-
8
Supplementary figure 1. Assessment of different estrus cycle phase effects on oxygen consumption
upon exposure to predator smell and effect of predator smell on neuronal activity in mediobasal
hypothalamus. a.
Oxygen consumption 3 hours post
-
exposure to TM
T in females divided into groups by their
estrus cycle phase,
No odor,
N=
10/pro
-
estrus and N=4/meta
-
diestrus, TMT,
N=
9
/pro
-
estrus and N=
5
/meta
-
diestrus
.
b.
O
xygen consumption
at first time point
post
-
exposure to TMT,
No odor, N=10/pro
-
estrus and
N=4/meta
-
d
iestrus, TMT, N=9/pro
-
estrus and N=5/meta
-
diestrus
,
Two
-
way Anova with Sidak’s post hoc
comparison
.
c,d,e, f.
cFos expression after odor exposure in AOB, pMeA, LH and VMH, respectively
,
N=
5 per
group, N=4 per group for AOB control females, LH and VMH
food odor males
,
Two
-
way Anova with Sidak’s
post hoc comparison
,
s
cale 100μm
.
All bar graphs are presented as mean values ± SEM.
Table S
8
contains
the detailed results of the statistical analysis
.
*p < 0.05, **p < 0.01, ***p < 0.001
,
*
*
**p < 0.0
0
01
. Source
data are
provided as a Source Data file.
Supplementary figure 2. Sexually dimorphic modulation of energy expenditure upon activation of
mitral and tufted olfactory cells. a.
Representative picture of MOE and MOB of Tbx21
-
Cre
+
/LSL
-
TdTomato
+
mouse, TdTomato (red)
,
s
cale 300μm
.
b.
Oxygen consumption
3 hours post
-
injection of saline or CNO,
males,
N=5
per group, females, N=5/Cre
+
+Saline and N=6/Cre
-
+CNO
.
c.
Analysis of covariance (ANCOVA) of energy
expenditure (EE) following CNO injection with body weight as covariance,
males,
N=1
4/Cre
-
and N=
15
/Cre
+
,
females,
N=1
3
/Cre
-
and N=1
4
/Cre
+
,
S
D
: males, Cre
-
0.0
47
, Cre
+
0.0
55
, females,
Cre
-
0.0
31
, Cre
+
0.0
37
.
d.
Oxygen consumption measured during day and night phase
s
fo
l
lowing CNO injection, blue (males), red
(females),
males,
N=14
per group
, females, N=13
per group
.
e.
Representative picture of mCherry expression
in mitral cell layer of MOB upon injection of AAV
-
DIO
-
mCherry in MOB of Tbx
-
Cre mice, mCherry (red)
and
schematic representation of virus delivery in MOB
, Scale bar 100μm
.
f
,g
.
Oxygen consumption and activity 3
hours post
-
injection of CNO,
males,
N=
6 per group, females, N=5/AAV
-
mCherry and N=7/AAV
-
hM3Dq
.
h.
Experimental design.
Tbx21
-
Cre
+
/LSL
-
TdTomato
+
or control mice were treated with saline prior to CNO that
was administ
e
red for
three
consecutive day
s
,
b
ig panel: Oxygen consumption over three days of consecutive
injections of CNO, small panel:
Average oxygen consumption measured for 3 hours post
-
injec
tion of CNO,
N=14 per group, females, N=13 per group
,
Two
-
way Anova with Tukey’s post hoc comparison
.
i
.
Average
oxygen consumption rate
(left panel) and oxygen consumption rate of brown adipose tissue measured post
-
injection of CNO
(right panel)
,
males,
N=
8 per group, females, N=10 per group,
Two
-
way Anova with Tukey’s
post hoc comparison
.
j
.
RTqPCR against
thermogenesis
genes in brown ad
ipose tissue of
mice
post
-
injection
of CNO
,
males, N=5/Cre
-
and N=7/Cre
+
, females,
N=
5
/Cre
-
and N=
7
/Cre
+
.
All bar graphs are presented as
mean values ± SEM.
Table S
9
contains the detailed results of the statistical analysis.
*p < 0.05, **p < 0.01,
***p < 0.001
,
*
*
**p < 0.0
0
01
. Source data are provided as a Source Data file.
Supplementary figure 3.
cFos expression in VMH post
-
injection of CNO. a.
Quantification of cFos
expression in VMH following CNO injection
,
males, N=7 per group, females, N=8/Cre
-
and N=6/Cre
+
,
Two
-
way
Anova with Tukey’s post hoc comparison
.
b.
Immunostaining of cFos
(green) in VMH
,
s
cale 50μm
.
All bar
graphs are presented as mean values ± SEM.
Table S
10
contains the detailed results of the statistical analysis.
*p < 0.05, **p < 0.01. Source data are provided as a Source Data file.
Supplementary figu
re
4
.
Recruitment of GABAergic and glutamatergic MBH neurons by chemogenetic
stimulation of MOB neurons
. a.
Representative images of immunohistochemical and in situ hybridization co
-
staini
n
g in female ARC, LH and DMH
for Vgat (green), Vglut2 (magenta) and
cFos protein (red), green arrows
point to colocalization of Vgat/cFos signal, pink
arrows point to colocalization of Vg
lut2
/cFos signal
, Scale bar
50μm and 100μm
.
b.
Quantification of
colocalization between Vgat/cFos and
Vglut2/cFos
signal in ARC, LH
and DMH of male and female mice,
N=5 per group, N=4/ARC males, Unpaired t
-
test, Two
-
tailed.
Source data
are provided as a Source Data file.
Supplementary figure 5.
Activation of
LepR
-
expressing neurons upon olfactory stimuli. a.
Left upper
panel:
Experimental design. Brain tissue of Cre
+
-
hM3Dq
+
and control mice were collected 90 minutes post
-
injection of CNO
, Left lower panel: Quantification of LepR/cFos signal in ARC, DMH and
LH, N=5
per group
,
Right panel:
Representative images of immunohistochemical and in situ hybridization co
-
staining in female
ARC,
DMH
and
LH
for
LepR
(green) and cFos protein (red),
yellow cells express LepR/cFos
,
s
cale 100μm
.
b.
Left
upper
panel: Experime
ntal design. Brain tissue of
WT mice exposed to cotton swab with or without TMT
for 30 min
w
as
collected
at
90 minutes
time point
,
Left lower panel: Quantification of LepR/cFos signal in ARC,
DMH and LH, N=
4
per group
,
Unpaired t
-
test, Two
-
tailed
,
Right panel: Representative images of
immunohistochemical and in situ hybridization co
-
staining in female ARC, DMH and LH for LepR (green) and
cFos protein (red), yellow cells express LepR/cFos
,
s
cale 100μm
.
All bar graphs are presented as mean
values ± S
EM.
Table S
11
contains the detailed results of the statistical analysis
.
*p < 0.05. Source data are
provided as a Source Data file.
Supplementary figure
6
.
Anterograde tracing from MOB revels neuronal TdTomato positive cells in
hypothalamus.
a,b
.
Immunostaining of
NeuN
(
blue
)
,
G
FAP
(green) and mCherry
(red)
in
mediobasal
hypothalamus
, Two independent experiment
s
(N=5)
, scale 100μm
.
Supplementary figure
7
.
Single
-
cell
analysis of dorsomedial hypothalamus.
a
.
Uniform Manifold
Approximation and Projection (UMAP) plot of
inhibitory
c
lusters (5664 cells)
.
b.
Expression of top genes
in
each inhibitory cluster. Dot size indicates
the
percent of nuclei expressing gene and col
or indicates intensity of
expression.
c.
Uniform Manifold Approximation and Projection (UMAP) plot of
excitatory
c
lusters (
3972
cells).
d.
Expression of top genes
in
each
excitatory
cluster. Dot size indicates
the
percent of nuclei expressing the
gene and
color indicates intensity of expression.
Source data are provided as a Source Data file.
Supplementary figure
8
.
Representation of cluster nomenclature genes across inhibitory and
excitatory clusters
. a.
Violin plot
representing the expression levels of genes used for cluster nomenclature in
inhibitory clusters (5664 cells).
b.
Violin
plot representing the expression levels of genes used for cluster
nomenclature in excitatory clusters (3972 cells
).
Source data are pro
vided as a Source Data file.
Supplementary Table S1
(related to Figure 1)
b
)
Two
-
way Anova with Tukey’s post hoc
comparison, N=12
-
14/group
F
Sex
(1, 46)=17.54,
P=0.0001
F
Sex x
Treatment
(1, 46)=11,32,
P=0.0016
c,d)
Three
-
way Anova, N=1
6
/group
F
Time
(5.816, 348.9) = 47.56, P<0.0001
F
Sex
(1, 60) = 47.75
,
P<0.0001
F
Time x
Treatment
(9, 540) = 3.280,
P=0.0007
F
Sex x
Treatment
(1, 60) = 7.280,
P=0.0090
c)
Two
-
way Anova with Sidak’s post hoc
comparison, N=16/group
F
Time
(5.095, 152.8) = 23.79
,
P<0.0001
F
Time x
Treatment
(9, 270) = 2.359
P
=
0
.0141
d)
Two
-
way Anova with Sidak’s post hoc
comparison, N=16/group
F
Time
(5.403, 162.1) = 24.27
,
P<0.0001
F
Treatment
(1, 30) = 8.073
,
P=0.0080
F
Time x
Treatment
(9, 270) = 1.929
,
P=0.0481
e,f)
Three
-
way Anova, N=16/group
F
Time
(5.010, 300.6) = 21.40, P<0.0001
F
Sex
(1, 60) = 12.44,
P=0.0008
F
Time x
Treatment
(9, 540) = 2.790,
P=0.0033
F
Time x Sex
(9, 540) = 3.166,
P=0.0010
e)
Two
-
way Anova with Sidak’s post hoc
comparison, N=16/group
F
Time
(4.120, 123.6) = 14.65, P<
0.0001
F
Time x
Treatment
(9, 270) = 2.264,
P=0.0186
f)
Two
-
way Anova with Sidak’s post hoc
comparison, N=16/group
F
Time
(5.261, 157.8) = 7.471, P<0.0001
F
Treatment
(1, 30) = 4.836,
P=0.0357
I,j
)
Three
-
way Anova, N=
1
1
/group
F
Time
(4.733, 189.3) = 6.226, P<0.0001
F
Time x
Sex x
Treatment
(6, 240) = 2.751,
P=0.0132
j)
Two
-
way Anova with Sidak’s post hoc
comparison, N=1
1
/group
F
Time x
Treatment
F (6, 120) = 2.680, P=0.0178
k,l)
Three
-
way Anova, N=
9
-
10
/group
F
Time
F (1.934, 67
.69) = 99.12, P<0.0001
F
Sex
(1, 35) = 4.478, P=0.0415
F
Treatment
(1, 35) = 51.28, P<0.0001
F
Time x
Treatment
(5, 175) = 29.21, P<0.0001
F
Sex x
Treatment
(1, 35) = 5.362,
P=0.0266
k)
Two
-
way Anova with Sidak’s post hoc
comparison, N=
9
-
10
/group
F
Time
(1.557, 26.46) = 49.13, P<0.0001
F
Treatment
F (1, 17) = 8.911, P=0.0083
F
Time x
Treatment
(5, 85) = 3.826
,
P=0.0036
l)
Two
-
way Anova with Sidak’s post hoc
comparison, N=10/group
F
Time
(2.088, 37.59) = 51.12, P<0.0001
F
Treatment
(1, 18) =
62.98, P<0.0001
F
Time x
Treatment
(5, 90) = 42.82, P<0.0001
o)
Two
-
way Anova with
Dunnett
’s post hoc
comparison, N=
5
/group
F
Sex
(1, 32) = 7.739,
P=0.0090
F
Treatment
(3, 32) = 8.972,
P=0.0002
q)
Two
-
way Anova with Dunnett’s post hoc
comparison, N=5/
group
F
Treatment
(3, 32) =
7.887
, P=0.000
4