Article
https://doi.org/10.1038/s41467-023-40484-7
Asex-speci
fi
c thermogenic neurocircuit
induced by predator smell recruiting chole-
cystokinin neurons in the dorsomedial
hypothalamus
Predrag Jovanovic
1,2
, Allan-Hermann Pool
3
, Nancy Morones
1,2
, Yidan Wang
1,2
,
Edward Novinbakht
1,2
, Nareg Keshishian
1,2
, Kaitlyn Jang
1,2
,YukiOka
4
&
Celine E. Riera
1,2,5
Olfactory cues are vital for prey anim
als like rodents to perceive and evade
predators. Stress-induced hyperther
mia, via brown adipose tissue (BAT)
thermogenesis, boosts physical perfor
mance and facilitates escape. However,
many aspects of this response, includ
ing thermogenic control and sex-speci
fi
c
effects, remain enigmatic. Our study unveils that the predator odor tri-
methylthiazoline (TMT) elicits BAT th
ermogenesis, suppresses feeding, and
drives glucocorticoid release in female mice. Chemogenetic stimulation of
olfactory bulb (OB) mitral cells recapit
ulates the thermogenic output of this
response and associated stress hormon
e corticosterone release in female
mice. Neuronal projections from OB
to medial amygdala (MeA) and dor-
somedial hypothalamus (D
MH) exhibit female-speci
fi
c cFos activity toward
odors. Cell sorting and single-cell RNA-sequencing of DMH identify cholecys-
tokinin (CCK)-expressing
neurons as recipients of predator odor cues. Che-
mogenetic manipulation and neuronal silencing of DMH
CCK
neurons further
implicate these neurons in the propag
ation of predator odor-associated
thermogenesis and food intake suppression, highlighting their role in female
stress-induced hyperthermia.
The detection of stressful cues initiates a complex combination of
neuronal, behavioral and endocrine responses which ensure orga-
nismal survival. It is well established that neural circuits in the hypo-
thalamus are essential for coordinating mammalian responses to
perceived threats. A fundamental hypothalamic neuronal popula-
tion involved in promoting endocrine responses to stress is comprised
of corticotropin-releasing hormone (CRH) neurons found in the
paraventricular nucleus (PVH)
1
,
2
. Their stimulation controls the release
of cortisol in humans, and its rodent analog corticosterone (CORT)
through hypothalamic-pituitary-adrenal (HPA) axis activity, initiated
by release of CRH from the PVH into the pituitary portal
3
.CRHneurons
have been shown to be important in
mediating other stress-related
functions, including changes in behavior
2
and encoding of valence
4
.
A key role of the stress response is the mobilization of the body
’
s
energy stores to promote an adaptive response, such as
fl
eeing a
dangerous situation, however acute and chronic activation of stress
Received: 19 September 2022
Accepted: 31 July 2023
Check for updates
1
Center for Neural Science and Medicine, Department of Biomedical Sciences
, Cedars-Sinai Medical Center, 127 South San Vicente Boulevard, Los Angel
es,
CA 90048, USA.
2
Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, 127 South San Vicente Boulevard, Los Angeles, CA
90048, USA.
3
Department of Neuroscience, Department of Anesthesiology and Pain Management, Peter O
’
Donnell Jr. Brain Institute, University of Texas
Southwestern Medical Center, Dallas, TX, USA.
4
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA.
5
Department of Neurology, Cedars-Sinai Medical Center, 127 South San Vicente Boulevard, Los Angeles, CA 90048, USA.
e-mail:
Celine.riera@cshs.org
Nature Communications
| (2023) 14:4937
1
1234567890():,;
1234567890():,;
share different outcomes on energy metabolism
5
. Yet, the current
understanding on the neurocircuits associated with the different
metabolic consequences of acute and chronic stress is limited. Acute
CRH release mobilizes energy stores, including increasing brown adi-
pose tissue (BAT) thermogenesis, enhancing locomotion and sup-
pressing feeding to ensure a rapid response favoring animal survival, in
a process recognized as stress-induced hyperthermia
6
–
8
.Incontrast,
repeated stress is associated with HPA overactivity and chronic ele-
vation of CORT
5
. Prolonged elevation of CORT levels in rodents pro-
motes weight gain and higher adiposity, through the modulation of
feeding behavior leading to hyperphagia
9
, without impacting energy
expenditure
10
–
12
.
Although stress-induced hyperthermia is a fundamental auto-
nomic stress response observed in many mammalian species, the cen-
tral neuronal players required for this response have yet to be
characterized. Stress-induced hyperthermia has been observed in the
context of psychological stress, under the perception of danger, dis-
comfort or pain and upon central delivery of CRH
8
,
13
,
14
. These reports
have pointed at the dorsomedial hypothalamus (DMH) as a crucial
recipient and mediator of the stress hyperthermic response. In addition,
perception of the fear-in
ducing odor 2,4,5-trimethylthiazole (mT), a
synthetic compound related to the r
ed fox predator scent 2,4,5-tri-
methylthiazoline (TMT), is associated with cFos upregulation in the
DMH and an elevation of plasma CORT in mice, compared to control
scent
15
. Other predator odors commonly used in laboratory settings,
including cat and ferret odors, have been linked to the stimulation of
ventromedial hypothalamus (VMH) neurons for the propagation of fear
and aggressive responses
16
–
18
. The representation of diverse predator
odors is highly organized and spatially localized to subregions of the
medial amygdala (MeA) and VMH
17
,
18
. The MeA has been implicated in
fear and anxiety responses
19
, whereas the VMH has been associated with
defensive, escape, avoidance and panic-like behaviors
16
–
18
,
20
,
21
.Among
the various predator odors commonly used in lab settings driving
avoidance behavior in mice, TMT speci
fi
cally led to stimulus burying in
the cage, and did not induce VMH neurons stimulation
18
,suggesting
that this stressful odor i
s associated with a speci
fi
c avoidance behavior
possibly mediated by distinct neuronal targets.
It is well appreciated that the stress response is sexually
dimorphic, with women having a higher incidence of anxiety disorders
including panic disorders, and trauma-related disorders such as post-
traumatic stress disorders
22
. In rodents, presynaptic innervation using
synaptophysin staining density in
female rats has revealed higher
innervation preceding chronic variable stress in regions involved in
stress processing, including paraventricular nucleus of hypothalamus
(PVH), prefrontal cortex (PFC), bed nucleus of the stria terminalis
(BST) and basolateral amygdala
23
. However, after 14 days of chronic
variable stress, PVH, BST and amygdala had reduced synaptophysin
density in females but not in males. These data suggest that chronic
stress may be associated with increased susceptibility to stress-
associated disorders in females, yet the neurons involved in sexually
dimorphic regulation of stress are not known. In the present study, we
sought to identify neuronal populations involved in stress-induced
hyperthermia. Using a combination of predator odor presentation, as
well as chemogenetic manipulation of olfactory neurons, we found the
existence of a sexually dimorphic regulation of thermogenesis which
occurs speci
fi
cally in females. Combined anterograde tracing and
single cell RNA-sequencing identify DMH
CCK
neurons as a recipient of
olfactory signals and modulators of feeding and energy expenditure in
the female rodent brain.
Results
Fear odor detection promotes a sexually dimorphic regulation
of energy expenditure
Because of a pre-existing link be
tween the predator smell TMT and
weight loss in diet-induced obesity animals
15
, we explored the effects of
this aversive odor on energy homeos
tasis. Naïve and normal chow fed
C57BL/6 mice were exposed to TMT or control scent using a cotton swab
while subjected to indirect calorim
etry with the swab present through-
out the experiment (Fig.
1
a). Remarkably, CORT levels remained elevated
in TMT-exposed females compared to males after 90 min (Fig.
1
b). Swab
introduction with control odor led
to a transient increase in oxygen
consumption (VO
2
)inbothsexes,combinedwithhigheractivityfora
time period of 45
–
60 min (Fig.
1
c
–
f). In males, VO
2
and activity were
similar among control and TMT-treated mice for the
fi
rst 2 h after swab
introduction (Fig.
1
c, e). In females, subsequently upon predator odor
introduction, a signi
fi
cantly higher VO
2
response to TMT was observed,
together with elevated locomoto
r activity which persisted for
60
–
90 min (Fig.
1
d, f). Female
’
sgainofVO
2
was observed independently
of estrus cycle phase (Supplementary
Fig. 1a, b). Food intake was negli-
gible in the 3 h following odor detection in ad libitum-fed male and
female cohorts, with no signi
fi
cant differences between TMT and control
animals (Fig.
1
g, h). Using infrared imaging, we measured the tempera-
tureofthebrownadiposetissue(BAT)regionuponodorantpresenta-
tion,asBATplaysanessentialrolein
adaptive thermogenesis and energy
expenditure
24
,
25
. TMT odor was associated with a female-speci
fi
cBAT
temperature increase, peaking 15 min after odor introduction and
coinciding with the maximum VO
2
effect (Fig.
1
i, j). To further investigate
the impact of TMT on feeding, we assessed refeeding after an overnight
fast in the continuous presence of an
odorant stimulus (TMT or control
scent). Food intake was reduced in males and females exposed to TMT,
with a more pronounced effect in females, where exposure to predator
odor inhibited feeding (Fig.
1
k, l). These results indicate that TMT
exposure drives a sex-speci
fi
c stress response, characterized by
increased stress hormone production, higher movement and energy
expenditure in female mice upon predator odor detection.
We then probed the sexually dimorphic stimulation of odor pre-
sentation within the mediobasal hypothalamus (MBH) in light of the
recognized contribution of this re
gion to the integration of food and
predator cues
16
,
17
,
26
,
27
. Naïve mice of either sex were exposed to either a
food odor employing peanut butter oil scent, TMT as a predator odor,
and opposite sex urine scent as a pheromone scent (Fig.
1
m). We
validated these odors
’
ability to stimulate olfactory processing center
regions including the main olfactory bulb (MOB), accessory olfactory
bulb (AOB) and MeA nuclei (Supplementary Fig. 1c, d). As expected
3
,
4
,
volatile opposite sex urine exposure drove cFos neuronal activity in
AOB (Supp Fig. 1c). The posterior medial amygdala (pMeA), mainly
responsible for modulating responses related to reproduction
5
,
6
and
cues of predator presence
7
, was activated by opposite-sex odor and
TMT exposure in both sexes (Supplementary Fig. 1d). TMT odor ele-
vated neuronal activity within the ARC of both sexes (Fig.
1
n, o). In line
with previous observations
15
, TMT exposure led to a high cFos induc-
tion in the DMH in males, but a more pronounced cFos response was
visible in the female DMH (Fig.
1
p, q). Exposure to different odors did
not change neural activity in VMH and lateral hypothalamus (LH)
(Supplementary Fig. 1e, f). Interestingly, while all animals were calorie
replete during the experiments, peanut butter smell increased ARC
cFos activity selectively in females (Fig.
1
n, o).
Chemogenetic stimulation of mitral and tufted olfactory cells
increases energy expenditure in females
Our results indicate that a select aversive odor can drive changes in
female energy expenditure, feeding and glucocorticoid release. The
detection of odors in the nasal cavity is mediated by olfactory sensory
neurons (OSNs) which send axons to a speci
fi
cprojectionsiteinthe
olfactory bulb (OB). These axons form a glomerular structure where
each glomerulus integrates afferent signals from thousands of OSNs.
The output is then relayed via several dozen mitral/tufted cells
28
.
Mitral/tufted projections are distributed via the lateral olfactory tract
to a heterogeneous assemblage of secondary structures collectively
labeled as the olfactory cortex. The piriform cortex and the cortical
Article
https://doi.org/10.1038/s41467-023-40484-7
Nature Communications
| (2023) 14:4937
2
amygdala are the main recipients of inputs from the main OB (MOB),
and transmit olfactory information to the orbitofrontal cortex, the
insular cortex and hypothalamus. In order to gain a deeper under-
standing of the link between olfactory circuits and energy balance, we
assessed the output of MOB mitral/tufted cell stimulation in the
sexually dimorphic regulation of stress and energy balance. Given the
large size of the MOB, we employed a genetic approach to express an
activatory designer receptor exclusively activated by designer drug
(DREADD)
29
within all mitral/tufted cells of the MOB (Fig.
2
a). We
generated Tbx21-Cre;hM3D
loxP/loxP
transgenic mice expressing the
activatory Gq DREADD human M3 muscarinic acetylcholine receptors
(hM3D) in mitral and tufted cells of the MOB to test behavioral and
metabolic phenotypes resulting from OB
Tbx21
stimulation. We veri
fi
ed
the restriction of Cre expression within the MOB
30
(Supplementary
Fig. 2a). Injection of the DREADD agonist clozapine-N-oxide (CNO)
promoted cFos expression in mitral cells labelled by mCitrine, which
marks hM3D
+
neurons (Fig.
2
b, c). Female Cre
+
mice showed increased
ability to locate a buried food pellet, indicating higher motivation,
which was not observed in males (Fig.
2
d). Anxiety-like behavior
measured using the percent time spent in the center of an open
fi
eld
was also highly increased in female Cre
+
mice over female Cre
−
con-
trols, as observed by reduced time spent in the central area, whereas
abcd
efgh
ijkl
m
o
Males
Females
0
5000
10000
15000
20000
A
R
C
cFos
+
nuclei/mm
3
✱
✱✱✱
✱✱
Males
Females
0
5000
10000
15000
DMH
c
Fos
+
nucle
i/
m
m
3
✱
✱
Peanut Butter
TMT
Social cue
q
04590135180
2500
3000
3500
4000
4500
5000
5500
Time (min)
VO
2
(ml/h/kg)
Control males
TMT males
*
Swab
04590135180
0
10
20
30
Time (min)
Foo
d
intake (kcal/k
g
)
Swab
04590135180
3000
3500
4000
4500
5000
5500
Time (min)
VO
2
(ml/h/kg)
**
**
Swab
Control females
TMT Females
*
*
04590135180
0
10
20
30
Time (min)
F
o
od
i
ntake (kcal/kg)
Swab
04590135180
0
5000
10000
15000
20000
Time (min)
Activity (Counts)
*
Swab
04590135180
0
2000
4000
6000
8000
10000
Time (min)
Activity (Counts)
*
Swab
012346
0
100
200
300
400
Refeeding post fast (h)
F
o
od
i
n
tak
e(kca
l/k
g)
**
*
*
Swab
012346
0
100
200
300
400
500
Refeeding post fast (h)
Food intake (kcal/kg)
***
***
**
**
***
Swab
0
15 30 60 90 120 180
31.0
31.5
32.0
32.5
33.0
33.5
Time (min)
Swab
0 15306090120180
31.5
32.0
32.5
33.0
Time (min)
**
Swab
0
1000
2000
3000
4000
CORT (pg/ml)
✱✱✱✱
✱
Males Females
Control
TMT
cFos
n
Mineral oil
ARC
p
Fig. 1 | Effect of predator smell on energy metabolism and mediobasal hypo-
thalamus activity. a
Experimental design. Mice given TMT odor or control swab
were assessed by indirect calorimetry.
b
CORT levels measured 90 min following
TMT or control odor stimulus, males (light blue/blue) and females (pink/red),-
males,
N
= 12/control and
N
= 14/TMT, females,
N
=12pergroup,Two-wayAnova
with Tukey
’
s post hoc comparison.
c
,
d
VO
2
post-exposure to TMT,
N
=16per
group, Two-way Anova with Sidak
’
s post hoc comparison.
e
,
f
Activity 3 h post-
exposure to TMT, males,
N
= 16 per group, Two-way Anova with Sidak
’
sposthoc
comparison.
g
,
h
Food intake 3 h post-exposure to TMT, males,
N
=11pergroup,
females,
N
=12/controland
N
= 10/TMT.
I
,
j
Brown adipose tissue temperature 3 h
post-exposure to TMT, males,
N
= 11 per group, females,
N
= 11 per group, Two-way
Anova with Sidak
’
s post hoc comparison.
k
,
l
Food intake after an overnight fast in
male and female mice exposed to TMT or control scent, males,
N
= 9 control group
and
N
= 10 TMT group females,
N
= 10 per group, Two-way Anova with Sidak
’
s post
hoc comparison.
m
Schematic of the experiment. Mice given an odor were mon-
itored for changes in brain cFos expression.
n
,
o
cFos expression after odor
exposure in ARC and quanti
fi
cation,
N
= 5 per group, Two-way Anova with Dun-
nett
’
s post hoc comparison.
p
,
q
cFos expression after odor exposure in DMH and
quanti
fi
cation,
N
= 5 per group, Two-way Anova with Dunnett
’
s post hoc compar-
ison. All bar graphs are presented as mean values ± SEM. Table S1 contains the
detailed results of the statistical analysis. *
p
<0.05,**
p
< 0.01, ***
p
< 0.001. Source
data are provided as a Source Data
fi
le. corticosterone (CORT), arcuate nucleus of
the hypothalamus (ARC), dorsomedial hypothalamus (DMH), 2,4,5-trimethylthia-
zoline (TMT). Scale bar 100
μ
m.
Article
https://doi.org/10.1038/s41467-023-40484-7
Nature Communications
| (2023) 14:4937
3
b
a
f
e
d
c
g
p
j
l
k
cFos
Citrine
n
m
r
q
o
i
h
300um
Cre
+
Fig. 2 | Chemogenetic stimulation of olfactory mitral and tufted cells mod-
ulates energy expenditure in a sex-speci
fi
c manner. a
Schematic of the
experiment. Mice were either treated with CNO prior to behavior testing or
received saline prior to CNO in indirect calorimetry assays.
b
Immunostaining of
MOB from Tbx21-Cre
+
-hM3Dq
+
animal (Cre
+
), citrine (green) and cFos (red), scale
300
μ
m, Two independent experiments,
N
=6.
c
Analysis of cFos positive cells in
MOB, males (light blue/blue) and females (pink/red), males,
N
= 7 per group,
females,
N
= 8/Cre
−
and
N
= 6/Cre
+
, Two-way Anova with Tukey
’
s post hoc com-
parison.
d
Buried pellet test 20 min following CNO injection, males,
N
= 11/Cre-
and
N
= 6/Cre + , females,
N
= 9 per group, Two-way Anova with Tukey
’
s post hoc
comparison.
e
Open
fi
eld test 20 min following CNO injection,
N
= 11 per group,
Two-way Anova with Tukey
’
s post hoc comparison.
f
Levels of corticosterone
measured 90 min after CNO injection, males,
N
= 8 per group, females,
N
= 7 per
group.
g
,
h
VO
2
for 3 h post-CNO Injection, males,
N
= 14 per group, females,
N
= 13 per group, Two-way Anova with Sidak
’
s post hoc comparison.
i
,
j
Food
intake for 3 h post-CNO Injection, males,
N
= 14 per group, females,
N
= 13 per
group.
k
,
l
Total activity for 3 h post-CNO Injection, males,
N
= 14/per group,
females,
N
=13p.
m
,
n
Refeeding after overnight fasting post-CNO Injection,
males,
N
= 15/Cre
−
and
N
= 17/Cre + , females,
N
= 12/Cre
−
and
N
= 10/Cre + .
o
–
q
Representative infrared images of ROI selected to measure BAT temperature
and analysis of BAT temperature upon CNO injection, males,
N
= 8/Cre
−
and
N
= 11/Cre + , females,
N
= 9/Cre
−
and
N
= 12/Cre + , Three-way Anova.
r
Plasma
free fatty acid levels in mice collected 90 min post CNO delivery, males,
N
= 8 per
group, females,
N
= 7 per group, Two-way Anova with Tukey
’
s post hoc compar-
ison. All bar graphs are presented as mean values ± SEM. Table S2 contains the
detailed results of the statistical analysis. *
p
< 0.05, **
p
< 0.01, ***
p
< 0.001. Source
data are provided as a Source Data
fi
le. corticosterone (CORT), Clozapine-N-
Oxide (CNO), Brown adipose tissue (BAT).
Article
https://doi.org/10.1038/s41467-023-40484-7
Nature Communications
| (2023) 14:4937
4