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Significance Thermal homeostasis is essential for survival in mammals. Although it is known that temperature-sensitive neurons in the hypothalamus can control body temperature, the precise neural types and dynamics of neurons responding to changes in environmental temperature are not well defined. In this study, we identified subsets of temperature-activated neurons in two hypothalamic nuclei, the preoptic area (POA) and the dorsomedial hypothalamus (DMH), and showed that modulating their activity can lead to alterations in core temperature. The data further suggest that heat-activated GABAergic neurons in the POA reduce the activity of cold-activated neurons in the DMH, which function to increase thermogenesis and physical activity. These data identify a neural circuit that controls core temperature and thermogenesis.
Requirement of preoptic GABAergic neurons in reducing T core. (A) Heat-induced (38 °C, 2 h) cFos colocalized with the GABAergic marker GAD67 in the vLPO (no. of cFos + and GAD67 + /no. of cFos + = 36.3 ± 2.4%, n = 3). The dashed white lines indicate boundaries between subregions. (B) Scheme of optogenetic modulation and viral expression of ChR2 (excitatory) or hGtACR1 (inhibitory) in vLPO Vgat neurons. The dashed yellow lines indicate the positions of optical inserts. (C) Slice recordings of neurons expressing ChR2 (Upper) or hGtACR1 (Lower). Blue light (blue, 6 mW, 40 Hz) faithfully elicited photocurrents in ChR2-expressing neurons in the vLPO. A blue light pulse (6 mW) completely silenced hGtACR1-expressing neurons in the vLPO. (D and E) T core (D) and activity (E) changes after optogenetic stimulation in mice expressing ChR2 (Upper, n = 4) or hGtACR1 (Lower, n = 3) in vLPO Vgat neurons. Stimulation protocol for ChR2: unilateral light pulses for 2 s (473 nm, 10 mW, 20 Hz, 40% on) followed by a 2-s break, with the sequence repeating for 30 min. For hGtACR1: bilateral light on for 30 s (473 nm, 6 mW) followed by a 90-s break, with the sequence repeating for 30 min. Bar graph of activity changes (average of 10-min interval) are shown in the right. Baselines (b.s.) represents the average counts between t =-30 and-20 min. (20-30 min) represents the average of counts between t = 20 and 30 min. (Scale bars: A, 100 μm; B; 200 μm.) All data are plotted as mean ± SEM. The P values compared with control group (eYFP) are calculated based on statistical tests listed in SI Appendix, Table S1. *P ≤ 0.05; **P ≤ 0.01. aca, anterior commissure, anterior part; B, bregma; dLPO and vLPO, dorsal and ventral part of lateral preoptic nucleus respectively; MPO, medial preoptic nucleus; 3V, third ventricle; VLPO, ventrolateral preoptic nucleus.
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Correction
NEUROSCIENCE
Correction for A hypothalamic circuit that controls body tem-
perature,by Zheng-Dong Zhao, Wen Z. Yang, Cuicui Gao, Xin
Fu, Wen Zhang, Qian Zhou, Wanpeng Chen, Xinyan Ni, Jun-Kai
Lin, Juan Yang, Xiao-Hong Xu, and Wei L. Shen (first published
January 4, 2017; 10.1073/pnas.1616255114).
The authors note that the grant number X-040214-002 should
instead appear as 31400955.
www.pnas.org/cgi/doi/10.1073/pnas.1701881114
www.pnas.org PNAS
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CORRECTION
A hypothalamic circuit that controls body temperature
Zheng-Dong Zhao ()
a,b,c,1
, Wen Z. Yang
a,b,c,1
, Cuicui Gao
a,b,c
, Xin Fu
a,b,c
, Wen Zhang
d
, Qian Zhou
a,b,c
,
Wanpeng Chen
a,b,c
, Xinyan Ni
a
, Jun-Kai Lin
d
, Juan Yang
a
, Xiao-Hong Xu
d
, and Wei L. Shen ()
a,2
a
School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China;
b
Institute of Neuroscience, Shanghai Institutes for Biological
Sciences, Chinese Academy of Sciences, Shanghai, 200031, China;
c
University of Chinese Academy of Sciences, Beijing, 100049, China; and
d
Institute of
Neuroscience, State Key Laboratory of Neuroscience, Chinese Academy of Sciences Center for Excellence in Brain Science and Intelligence Technology,
Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200031, China
Edited by David J. Mangelsdorf, The University of Texas Southwestern Medical Center, Dallas, TX, and approved December 9, 2016 (received for review
October 3, 2016)
The homeostatic control of body temperature is essential for survival
in mammals and is known to be regulated in part by temperature-
sensitive neurons in the hypothalamus. However, the specific neural
pathways and corresponding neural populations have not been fully
elucidated. To identify these pathways, we used cFos staining to
identify neurons that are activated by a thermal challenge and found
induced expression in subsets of neurons within the ventral part of the
lateral preoptic nucleus (vLPO) and the dorsal part of the dorsomedial
hypothalamus (DMD). Activation of GABAergic neurons in the vLPO
using optogenetics reduced body temperature, along with a decrease
in physical activity. Optogenetic inhibition of these neurons resulted in
fever-level hyperthermia. These GABAergic neurons project from the
vLPO to the DMD and optogenetic stimulation of the nerve terminals
in the DMD also reduced body temperature and activity. Electrophys-
iological recording revealed that the vLPO GABAergic neurons sup-
pressed neural activity in DMD neurons, and fiber photometry of
calcium transients revealed that DMD neurons were activated by cold.
Accordingly, activation of DMD neurons using designer receptors
exclusively activated by designer drugs (DREADDs) or optogenetics
increased body temperature with a strong increase in energy expen-
diture and activity. Finally, optogenetic inhibition of DMD neurons
triggered hypothermia, similar to stimulation of the GABAergic neu-
rons in the vLPO. Thus, vLPO GABAergic neurons suppressed the ther-
mogenic effect of DMD neurons. In aggregate, our data identify
vLPODMD neural pathways that reduce core temperature in re-
sponse to a thermal challenge, and we show that outputs from the
DMD can induce activity-induced thermogenesis.
thermoregulation
|
preoptic area
|
dorsomedial hypothalamus
|
fiber photometry
|
energy expenditure
Adult mammals, including humans, precisely maintain core
body temperature (T
core
) within a narrow range. This system
is essential for survival because a significant deviation of T
core
can adversely affect cellular metabolism. Changes in environ-
mental temperature activate homeostatic responses that, in turn,
regulate energy expenditure, adaptive thermogenesis, and phys-
ical activity in an attempt to maintain near-optimal T
core
. Pre-
vious studies have shown that the preoptic area (POA) of the
hypothalamus plays an important role in maintaining a stable
T
core
via afferent inputs from skin thermoreceptors. The direct
sensing of changes in skin temperature, in turn, activates POA
efferent signals that control thermal effector organs (1, 2). For
example, in response to a heat stress, warmth receptors within
the dorsal root ganglion provide excitatory input to POA neu-
rons via relay neurons in the dorsal lateral parabrachial nucleus
(3). In addition, recordings from in vivo models and in slice pre-
parations identify a group of intrinsic warm-sensitive neurons
(WSNs) (2040%) that may enable animals to directly detect brain
warmth and promote heat loss (4). Thermosensitive transient re-
ceptor potential channel M2 (TRPM2), which is expressed in the
POA, may be part of the heat sensor in WSNs that can limit fever
and induce hypothermia (5). Furthermore, activation of gluta-
matergic neurons in several POA subareas results in severe hypo-
thermia, whereas activation of subsets of POA GABAergic neurons
hasbeenfoundtohaveonlyaminimalornoeffectonT
core
(5, 6).
However, the precise nature of the POA projections that play a role
in suppressing thermogenesis have not been elucidated. Thus, al-
though several relevant brain regions have been suggested to act
in concert with the POA to regulate T
core
(1, 2), including the dor-
somedial hypothalamus (DMH), the periventricular nucleus, and
the raphe pallidus nucleus, functional connections between the
POA and other thermoregulatory regions are largely unknown.
In the present study, we began by defining sites in which cFos
staining was induced by changes in environmental temperature.
These studies identified temperature-activated neurons in the
ventral part of the lateral preoptic nucleus (vLPO) and the dorsal
part of the DMH (DMD). We confirmed that DMD neurons
responded to cold by using fiber photometry, and tested the ability
of these and POA neurons to affect T
core
, energy expenditure (EE),
and physical activity by using optogenetics (7) or designer receptors
exclusively activated by designer drugs (DREADDs) (8). These
functional studies identified neural pathways in which heat-activated
GABAergic neurons in the vLPO inhibit cold-activated neurons
in the DMD to suppress thermogenesis and lower T
core
.
Results
Activation of GABAergic POA Neurons Is Sufficient To Drive Hypothermia.
The POA is known to receive input from cold- and heat-sensitive
neurons (1). However, although recent data have identified some of
the neural substrates for temperature sensing (5, 6), components of
the neural circuit(s) that respond to thermal challenges have not been
Significance
Thermal homeostasis is essential for survival in mammals. Al-
though it is known that temperature-sensitive neurons in the
hypothalamus can control body temperature, the precise neu-
ral types and dynamics of neurons responding to changes in
environmental temperature are not well defined. In this study,
we identified subsets of temperature-activated neurons in two
hypothalamic nuclei, the preoptic area (POA) and the dorso-
medial hypothalamus (DMH), and showed that modulating
their activity can lead to alterations in core temperature. The
data further suggest that heat-activated GABAergic neurons in
the POA reduce the activity of cold-activated neurons in the
DMH, which function to increase thermogenesis and physical
activity. These data identify a neural circuit that controls core
temperature and thermogenesis.
Author contributions: Z.-D.Z., W.Z.Y., X.-H.X., and W.L.S. designed research; Z.-D.Z., W.Z.Y.,
C.G., X.F., W.Z., Q.Z., W.C., X.N., J.-K.L., and J.Y. performed research; Z.-D.Z., W.Z.Y., C.G.,
X.F., W.Z., Q.Z., and W.C. analyzed data; and Z.-D.Z., W.Z.Y., and W.L.S. wrote
the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
See Commentary on page 1765.
1
Z.-D.Z. and W.Z.Y. contributed equally to this work.
2
To whom correspondence should be addressed. Email: shenwei@shanghaitech.edu.cn.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1616255114/-/DCSupplemental.
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fully elucidated. For example, there are conflicting data concerning
the potential role of GABAergic neurons in the POA (2, 5, 6). To
identify these key neural populations, we characterized the pattern of
cFos activation following a thermal challenge (SI Appendix,Fig.S1).
We found heat-induced cFos expression in the medial preoptic area
(MPO) and the vLPO, and it colocalized with the GABAergic marker
GAD67 (Fig. 1Aand SI Appendix,Fig.S5A). Both the MPO and the
vLPO have been suggested to play important roles in thermoregula-
tion(1,2,911). To test the function of the vLPO neuronal pop-
ulation, we targeted expression of channelrhodopsin-2 (ChR2) fused
with eYFP to GABAergic neurons within the vLPO by injecting Cre-
dependent adeno-associated virus (AAV) 5 viruses into the vLPO of
Vgat-IRES-Cre driver mice (Vgat stands for vesicular GABA trans-
porter) (Fig. 1B). In slice recordings, we confirmed that the delivery of
blue light to ChR2-expressing vLPO
Vgat
neurons resulted in neural
excitation (Fig. 1C). In vivo, we found that light-induced activation of
vLPO
Vgat
neurons triggered a rapid reduction in T
core
with a decrease
in physical activity (ΔT=1.8 ±0.09 °C at t=30 min, mean ±SEM,
Fig. 1 Dand E) in freely behaving mice. This effect was specific to
ChR2, because injection of control AAV5 viruses (expressing eYFP)
did not significantly affect T
core
or activity.
Next, we asked whether inhibiting these neurons was sufficient
to drive hyperthermia. Using the Guillardia theta anion chan-
nelrhodopsin 1 (hGtACR1), which robustly silences neural ac-
tivity in response to blue-yellow light (12), we found that blue
light delivery was sufficient to silence neurons in slice recordings
(Fig. 1C). Remarkably, light stimulation of mice expressing
hGtACR1 in vLPO
Vgat
neurons caused severe hyperthermia with
elevated activity levels (maximal T
core
=40.6 °C, Fig. 1 Dand E).
Indeed, we needed to minimize the time in which animals re-
ceived light illumination to prevent hyperthermia-induced death.
We also tested the function of MPO GABAergic neurons by
targeted expressing of ChR2 to GABAergic neurons within the
MPO of Vgat-IRES-Cre driver mice (SI Appendix, Fig. S5B).
The GABAergic neurons in the MPO have been suggested to
play important roles in thermoregulation (1, 2). Surprisingly, we
found that optogenetic activation of these MPO
Vgat
neurons did
not significantly affect T
core
or activity (SI Appendix, Fig. S5 C
and D). Similar to our results, DREADD activation of these
neurons has a minimal effect on T
core
(5).
Thus, our results establish that activation of GABAergic neurons
within a preoptic subregion (vLPO) can inhibit thermogenesis,
whereas inhibition of these neurons dramatically raises core tem-
perature. We next explored the functional targets of these neurons.
Critical Role of the POADMDConnectioninReducingT
core
.We
thought that vLPO
Vgat
neurons might project to the DMH because
the DMH is known to participate in thermoregulation, and because
we observed that thermal stimuli induced strong cFos staining within
the DMD (SI Appendix,Fig.S1). To test whether vLPO
Vgat
neu-
rons directly innervate DMD neurons, we first performed antero-
grade tracing from these neurons by injecting ChR2 into vLPO
Vgat
neurons (Fig. 2A) and found staining of nerve terminals in the
DMD (Fig. 2B). We then performed retrograde labeling by injecting
the retrograde protein, cholera toxin B subunit (CTb; ref. 13) into
the DMD and found it labeled many neurons in the POA, in-
cluding heat-activated neurons (as indicated by induction of cFos)
in the vLPO (Fig. 2C).
We tested the function of this vLPODMD projection by
stimulating vLPO
Vgat
terminals in the DMD after viral injection of
ChR2 into the vLPO (Fig. 2D). We found that stimulation with blue
light triggered a significant reduction of T
core
(ΔT=2.3 ±0.6 °C at
t=60 min, mean ±SEM, Fig. 2E) along with a decrease in activity
(Fig. 2F). The magnitude of the effect was similar to that observed
after direct stimulation of Vgat neurons in the vLPO. Thus, stim-
ulation of vLPO
Vgat
nerve terminals in the DMD recapitulated the
phenotype observed when vLPO
Vgat
cell bodies were stimulated
(Fig. 1 Dand E,Upper).
These functional data suggest that vLPO
Vgat
neurons inhibit
DMD neurons and reduce core temperature and thermogenesis.
We next set out to confirm this inhibition directly by recording
inhibitory postsynaptic currents (IPSCs) in DMD neurons after
vLPO
Vgat
terminals were stimulated. This recording is important
because our data could also be explained by inhibition of axon
fibers that pass through the DMD without directly innervating
DMD neurons. In slice preparations, we confirmed that stimu-
lation of vLPO
Vgat
terminals expressing ChR2 by blue light-
induced IPSCs in the DMD neurons and that these currents were
blocked by a GABA
A
receptor antagonist, bicuculline (Fig. 2G).
The onset of these currents was <3 ms after light delivery, which
is within the time range of monosynaptic transmission (14). Fur-
thermore, to identify which types of DMD neurons are synaptically
connected to the vLPO
Vgat
neurons, we used the recording pipette
to isolate mRNA from individual, light-responsive DMD neurons
immediately after recordings and analyzed gene expression by
single-cell reverse transcription PCR (RT-PCR) (15). These single-
cell RT-PCR data showed that there were both Vgat
+
and Vglut2
+
postsynaptic neurons in the DMD, with the majority being Vgat
+
(Fig. 2Hand SI Appendix,Fig.S6).
Several reports (1621) have suggested that DMD neurons
can regulate thermogenesis, but the inputs to these neurons have
not been elucidated. Our studies in anterograde and retrograde
B = 0.14
Heat GAD67 / cFos (29 38oC)
A
MPO
dLPO
aca
3V
B
C
hGtACR1
ChR2
D
Time (min)
Tcore (°C)
-20 0 20 40 60
35
37
39
41 Light
**
hGtACR1
Control
Body Temperature Physical Activity
20 mV
5 s
Light on
E
vLPOVgat
Light
vLPO
vLPOVgat
3V
3V
aca
aca
Time (min)
-20 0 20 40 60
34
35
36
37
38 Light ChR2
Control
**
Act. (counts)
vLPO
vLPO
MPO
MPO
ChR2 / DAPI
hGtACR1 / DAPI
VLPO
VLPO
Fig. 1. Requirement of preoptic GABAergic neurons in reducing T
core
.
(A) Heat-induced (38 °C, 2 h) cFos colocalized with the GABAergic marker
GAD67 in the vLPO (no. of cFos
+
and GAD67
+
/no. of cFos
+
=36.3 ±2.4%,
n=3). The dashed white lines indicate boundaries between subregions.
(B) Scheme of optogenetic modulation and viral expression of ChR2 (excit-
atory) or hGtACR1 (inhibitory) in vLPO
Vgat
neurons. The dashed yellow lines
indicate the positions of optical inserts. (C) Slice recordings of neurons
expressing ChR2 (Upper) or hGtACR1 (Lower). Blue light (blue, 6 mW, 40 Hz)
faithfully elicited photocurrents in ChR2-expressing neurons in the vLPO. A
blue light pulse (6 mW) completely silenced hGtACR1-expressing neurons in
the vLPO. (Dand E)T
core
(D) and activity (E) changes after optogenetic
stimulation in mice expressing ChR2 (Upper,n=4) or hGtACR1 (Lower,n=3)
in vLPO
Vgat
neurons. Stimulation protocol for ChR2: unilateral light pulses for
2 s (473 nm, 10 mW, 20 Hz, 40% on) followed by a 2-s break, with the se-
quence repeating for 30 min. For hGtACR1: bilateral light on for 30 s (473 nm,
6 mW) followed by a 90-s break, with the sequence repeating for 30 min. Bar
graph of activity changes (average of 10-min interval) are shown in the right.
Baselines (b.s.) represents the average counts between t=30 and 20 min.
(2030 min) represents the average of counts between t=20 and 30 min.
(Scale bars: A,100μm; B;200μm.) All data are plotted as mean ±SEM. The
Pvalues compared with control group (eYFP) are calculated based on statistical
tests listed in SI Appendix, Table S1.*P0.05; **P0.01. aca, anterior com-
missure, anterior part; B, bregma; dLPO and vLPO, dorsal and ventral part of
lateral preoptic nucleus respectively;MPO,medialpreopticnucleus;3V,third
ventricle; VLPO, ventrolateral preoptic nucleus.
Zhao et al. PNAS
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2043
NEUROSCIENCE SEE COMMENTARY
tracing, optogenetic activation of terminals from vLPO
Vgat
neurons in the DMD, slice recordings, and single-cell RT-PCR
suggest the possibility that there are functional connections be-
tween the vLPO
Vgat
neurons and DMD
Vgat
or DMD
Vglut2
neurons
to regulate core temperature. We next tested these connections
directly by monitoring and modulating the activity of neurons in
the DMD.
Fiber Photometry Reveals That DMD Neurons Are Cold Activated. The
observation that inhibitory neurons that project to the DMD
cause hypothermia suggests that neurons within the DMD may
be cold sensitive (via either direct or indirect inputs) and act to
raise core temperature (which would explain why inhibiting their
inhibitors would raise T
core
; Fig. 1D). We first tested this hy-
pothesis by using the calcium reporter GCaMP6f (22) to directly
measure neural Ca
2+
signals by fiber photometry after changes
in environmental temperature. Similar experimental setups
have yielded stable recordings from the dorsal raphe (23) (Fig.
3A). We recorded Ca
2+
signals from both glutamatergic
(DMD
Vglut2
) and GABAergic (DMD
Vgat
) neurons separately by
injecting a Cre-dependent version of GCaMP6f into Vglut2-
IRES-Cre and Vgat-IRES-Cre mice, respectively (Fig. 3B). We
controlled floor temperature (T
floor
) of mice with a Peltier controller
(Fig. 3C). The DMD
Vglut2
neurons showed significant Ca
2+
signals
in response to cooling (2513 °C, maximal response, ΔF/F
0_max
=
16.5 ±2.6%, mean ±SEM; Fig. 3D,Left), but not to warming (25
3C,Fig.3D,Center). The responses occurred within seconds of the
temperature shift and diminished quickly in cold. Approximately 2 min
following the temperature shift (t=110120 s), mean neuronal activity
levels (ΔF/F
0(110120 s)
) were not higher than baseline (Fig. 3D,Right).
We also observed an even larger Ca
2+
increases in response to
cooling in DMD
Vgat
neurons (2513 °C) (ΔF/F
0_max
=22.7 ±2.8%,
mean ±SEM; Fig. 3E,Left). This Ca
2+
signal increased rapidly
(within seconds) after the temperature shift and diminished more
slowly than in DMD
Vglut2
neurons. ΔF/F
0(110120s)
was significantly
larger than baselines (Fig. 3E,Right). We fitted the response curve
by using a sigmoidal function and found the full width at half
maximum (FWFM or T
1/2 max
)oftheDMD
Vgat
neurons was longer
than that of DMD
Vglut2
neurons (T
1/2 max
=22.6 s and 16.5 s, re-
spectively; SI Appendix,Fig.S8Aand B).
Taken together, these results indicate that both GABAergic
and glutamatergic neurons in the DMD are activated by cooling.
The response of DMD
Vgat
neurons lasted longer than that
of DMD
Vglut2
neurons. All of our temperature stimuli-evoked
calcium responses appeared to result from periphery sensory input,
rather than from a slow change in body temperature, based on
D
H
bic.
wash
vLPOVgat DMD
Light
ChR2
IPSCs
aca
3V
AB
3V
DMD
MPO vLPO
CTb
Vgat
& ChR2
vLPO DMD
DMD
ChR2
terminals
C
G
Vglut2
Vgat
Undefined
Single-cell RT-PCR
Cell number
vLPO DMD
dLPO
DMV
3V
Vgat
& ChR2
Light
CTb /DAPI /cFos (29 38oC)
Injection site CTb in the POA
VMH
EF
DMD
B = -1.82
Tcore ( C)
Act. (counts)
VLPO
Fig. 2. Critical role of the vLPO
Vgat
DMD connection in reducing T
core
.
(A) Scheme for anterograde tracing using ChR2 and retrograde tracing using
CTb. (B) vLPO
Vgat & ChR2
terminals in the DMD. (C) vLPO neurons were ret-
rogradely labeled by CTb injected in the DMD, which colocalized with heat-
induced cFos. White arrows indicate the colocalization of cFos and CTb.
(Scale bars: 100 μm.) (D) Scheme for terminal optogenetic stimulation in the
DMD after ChR2 injection into vLPO
Vgat
neurons. Both injection and stimu-
lation were bilateral. (Eand F) Bilateral terminal optogenetic stimulation in
the DMD-reduced T
core
(E) and activity (F)(n=5). Illumination protocol: light
pulses for 2 s (473 nm, 10 mW, 20 Hz, 40%) followed by a 2-s break, with the
sequence repeating for 1 h. Bar graph of activity changes (average of 20-min
interval) are shown in F. Baselines (b.s.) represents the average of counts in
between t=30 and 10 min. (4060 min) represents the average of counts
between t=40 and 60 min. (G) Induction of inhibitory postsynaptic currents
(IPSCs) in DMD neurons by light stimulation (blue, 6 mW, 2 ms) of ChR2-
expressing terminals projected from vLPO
Vgat
neurons. IPSCs were blocked
by bicuculline (bic.), and partially recovered after wash. Thick lines indicate
the mean, whereas the shaded areas indicate SD. (H) Single-cell RT-PCR
analysis of recorded cells. All data are plotted as mean ±SEM (except in G);
Pvalues compared with control group (532 nm) are calculated based on
statistical tests listed in SI Appendix, Table S1.*P0.05; ***P0.001. DMD
and DMV, dorsal and ventral part of dorsomedial hypothalamic nucleus re-
spectively; VMH, ventromedial hypothalamic nucleus.
Detector
Laser
Dichroic
473nm
525nm
Thermal pad
GCaMP6f 3V
DMDVglut2 DMDVgat
3V
-120 -60 0 60 120
3
13
23
33
-120 -60 0 60 120
-10
0
10
20
30
Floor temp.
DMD
DMV
DMD
DMV
DMDVglut2
DMDVgat
Floor temp.
DMDVglut2
DMDVgat
DMDVglut2
DMDVgat
-120 -60 0 60 120
-10
0
10
20
30
Time (s)
AB
C
D
E
Fig. 3. DMD neural dynamics in response to thermal stimuli. (A) Scheme of
fiber photometry setup. (B) The expression of GCaMP6f in the DMD driven
by Vglut2-IRES-Cre or Vgat-IRES-Cre. The dashed yellow lines indicate the
positions of optical inserts. The dashed white lines indicate the boundary of
the DMH. (Scale bars: 200 μm.) (C) Controlled floor temperature (T
floor
) with
a Peltier controller. (Left) Cooling traces (2513 °C). (Right) Heating traces
(2538 °C). (D) Cooling, but not warming-activated DMD
Vglut2
neurons. Thick
lines indicate the mean, whereas the shaded areas indicate SEM. ΔF/F
o
represents change in GCaMP6f fluorescence from the mean level before the
floor temperature change. b.s. represents baseline, which is the averaged
ΔF/F
o
between t=20 and 120 s. (010) and (110120 s) represent the
averaged ΔF/F
o
between t=(010 s) and t=(110120 s), respectively (n=3).
(E) Cold, but not warmth, activated DMD
Vgat
neurons (n=4). All data are
plotted as mean ±SEM. The Pvalues, compared with baselines, are calcu-
lated based on statistical tests listed in SI Appendix, Table S1.*P0.05;
***P0.001; ns, not significant. DMD and DMV, dorsal and ventral part of
dorsomedial hypothalamic nucleus, respectively; 3V, third ventricle.
2044
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www.pnas.org/cgi/doi/10.1073/pnas.1616255114 Zhao et al.
the fast-rising kinetics. The calcium responses occurred within
seconds after the temperature shift (Fig. 3 Dand E), whereas the
changes in T
core
happened minutes after the temperature shift
(SI Appendix, Fig. S2).
DMD Neurons Promote Thermogenesis. The finding that cooling
leads to abrupt Ca
2+
transients in DMD
Vglut2
neurons suggests
that these neurons might play a functional role in cold-induced
thermogenesis. We next tested this by using DREADDs to
activate DMD
Vglut2
neurons remotely, allowing us to measure
energy expenditure (EE) without being impeded by optical
fibers required for optogenetics. As predicted, we found that
injection of the ligand clozapine N-oxide (CNO) into mice
expressing designed human M3 muscarinic receptor coupled to Gq
(hM3D) (8) in DMD
Vglut2
neurons strongly increased T
core
,along
with an increase in EE and physical activity (ΔT=1.4 ±0.3 °C,
ΔEE =47.3 ±5.1% at t=100 min, mean ±SEM; Fig. 4 AD). These
increases are direct evidence that activation of DMD
Vglut2
neurons
promotes thermogenesis, which is consistent with previous studies
showing that the DMD may send excitatory (or glutamatergic) pro-
jections to promote cold- or febrile-induced thermogenesis (16, 17,
24, 25).
The findings that cooling induced calcium transients in
DMD
Vgat
neurons (Fig. 3E) and that cold-induced cFos colo-
calized with GAD67 in the DMD (SI Appendix, Fig. S7A) suggest
that these neurons could also be important for regulating cold-
induced thermogenesis. We tested this by using both DREADDs
and optogenetics to activate DMD
Vgat
neurons. We found that
injection of CNO, but not saline, into mice expressing h3MD
in DMD
Vgat
neurons resulted in a slow, yet long-lasting increase
in T
core
, with an increase in EE and activity (ΔT=1.3 ±0.2 °C,
ΔEE =47.4 ±7.5% at t=100 min, mean ±SEM; Fig. 4 EH).
We fitted the response curve with a sigmoidal function and cal-
culated maximum change rate (k) and full width at half maximum
(FWFM) (SI Appendix,Fig.S8Cand D). For the same dose of
CNO, the kfor the T
core
, EE, and activity response curves were
smaller for DMD
Vgat
neural activation than was observed after
DMD
Vglut2
neural activation. However, the FWFWs were longer
after DMD
Vgat
neural activation versus activation of DMD
Vglut2
(SI
Appendix,Fig.S8Cand D). The kinetics of these biological re-
sponses matched that seen by recording from these neurons,
which indicates that cold-induced Ca
2+
transients of DMD
Vgat
neurons diminished more slowly than that of DMD
Vglut2
neurons
(Fig. 3 Dand Eand SI Appendix,Fig.S8Aand B). Also, we
observed an increase in T
core
and activity after optogenetic
stimulation of mice expressing ChR2 in DMD
Vgat
neurons (SI
Appendix, Fig. S8 BD). In aggregate, these data show that ac-
tivation of both glutamatergic and GABAergic neurons in the
DMD can increase T
core
, EE, and activity, although the kinetics
of the biological responses to activation of these neurons were
different.
Inhibition of DMD Neurons Is Sufficient To Drive Hypothermia. The
finding that vLPO
Vgat
inputs to the DMD lower core tempera-
ture led us to predict that optogenetic inhibition of DMD neu-
rons would have a similar effect. This prediction was tested by
injecting AAV9 viruses expressing hGtACR1 into the DMD of
Vglut2-IRES-Cre or Vgat-IRES-Cre driver mice (thereby driving
hGtACR1 expression in DMD
Vglut2
or DMD
Vgat
neurons, re-
spectively). We found that blue light stimulation of mice expressing
hGtACR1 in DMD
Vglut2
neurons resulted in a significant reduction
in T
core
, along with a decrease in activity (ΔT=1.3 ±0.2 °C at t=
60 min, mean ±SEM; Fig. 5 Aand B), similar to optogenetic
activation of the vLPO
Vgat
neurons (Fig. 1 Dand E). Similarly,
blue light stimulation of mice expressing hGtACR1 in DMD
Vgat
neurons resulted in significant reductions in T
core
, along with a
decrease in activity (ΔT=2.5 ±0.5 °C at t=60 min, mean ±SEM;
Fig. 5 Cand D). Taken together, these results define elements of a
POADMH neural circuit in the hypothalamus (including
vLPO
Vgat
DMD
Vglut2
and vLPO
Vgat
DMD
Vgat
connections) that
regulate thermogenesis.
Discussion
The maintenance of a stable core temperature is essential for
survival. Our study elucidates a central mechanism through
which changes in core temperature (in response to alternations
in ambient temperature or other stimuli) elicit a set of adaptive
thermogenic responses that defend T
core
. In aggregate, we find
that in response to a heat challenge, heat-activated GABAergic
neurons in the vLPO directly inhibit the activity of cold-activated
glutamatergic and GABAergic neurons in the DMD to lower
T
core
, and do so (in part) by suppressing EE and activity. Thus,
we have elucidated pathways for controlling body temperature
and activity-induced thermogenesis.
In connection with our study, previous studies have suggested
that the vLPO is an important site for thermoregulation. Local
warming of the vLPO causes paw vasodilation in rats (9), sug-
gesting the existence of WSNs in this area that can drive hypo-
thermia. Also, the vLPO is labeled when a retrograde tracer
(pseudo rabies virus) is injected into the interscapular brown
adipose tissue (BAT) of rats (26), suggesting its involvement in
controlling BAT thermogenesis. Interestingly, a recent study
discovered that a heat-sensitive channel, TRPM2, in the POA
(including the vLPO) may be part of the WSN heat sensor to
limit fever (5). Thus, it will be interesting to see whether TRPM2
is important for vLPO neurons to detect brain warmth and
lower T
core
.
We found that heat activated a subset of GABAergic neurons in
the vLPO, which then lowered T
core
. Although a similar (or
stronger) effect was observed after activation of glutamatergic
neurons in several preoptic subregions [MPA (6), and MPO (5)],
activation of GABAergic neurons in these areas has a small effect
on T
core
, and the role of GABAergic neurons in the vLPO was not
studied previously. We found that activation of GABAergic neu-
rons in the vLPO is sufficient to induce hypothermia (Fig. 1 Band
D). However, this activation does not include the ventrolateral
preoptic nucleus (VLPO), which is important for sleep regulation
(27, 28) but may be dispensable for thermoregulation, because its
lesion has a minimal effect on T
core
(27). The vLPO GABAergic
neurons comprise a functionally confirmed GABAergic popula-
tion in the POA. In addition, we found that inhibiting vLPO
GABAergic neurons induced fever-level hyperthermia, suggesting
that these neurons can control T
core
in either direction (Fig. 1D),
and that these neurons provide a key entry point for studying the
neural mechanisms controlling thermogenesis. Furthermore, we
found heat can also activate a subset of glutamatergic neurons in
thevLPO,whichcandriveseverehypothermia and hypoactivity (SI
DMD
DMV
BytivitcAlacisyhPerutarepmeTydoB
F
Time (min)
-60 0 60 120 180 240
0.15
0.20
0.25
0.30
0.35 **
Energy Expenditure
DMD
Vglut2 & hM3D
Vgat & hM3D
A
DMD
E
CD
HG
3V
3V
DMD
DMV
Fig. 4. Activation of DMD neurons increases body temperature. (A) Sche-
matic and representative images of viral expression of hM3D in DMD Vglut2-
IRES-Cre
+
neurons. (BD) Activation of DMD
Vgut2
neurons (n=5) by CNO
injection (i.p. at t=0, 1.5 mg/kg body weight) resulted in significant in-
creases in T
core
(B), EE (C), and activity (D). (E) Schematic and representative
images of viral expression of hM3D in DMD Vgat-IRES-Cre
+
neurons.
(FH) Activation of DMD
Vgat
neurons (n=8) by CNO injection (i.p. at t=0,
1.5 mg/kg body weight) resulted in significant increases in T
core
(F), EE (G),
and activity (H). (Scale bars: 100 μm.) All data are plotted as mean ±SEM. The
Pvalues, compared with the saline group, are calculated based on statistical
tests listed in SI Appendix, Table S1.**P0.01; ***P0.001.
Zhao et al. PNAS
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NEUROSCIENCE SEE COMMENTARY
Appendix,Fig.S3). Thus, vLPO GABAergic neurons might re-
ceive local inputs from vLPO glutamatergic neurons or other
preoptic glutamatergic neurons as proposed (1, 2) (such as the
MnPO, because the MnPO receives inputs from the periph-
ery (3) and sends glutamatergic outputs specifically to the
vLPO; SI Appendix,Fig.S4). These inputs must be functionally
confirmed.
In addition to the POA, the DMH is also an important site for
thermoregulation. Although a POADMH connection has been
proposed to play a role in thermoregulation (1, 2), direct func-
tional confirmation of this connection is lacking. For example,
pioneering studies suggest that there are anatomical connections
between DMH neurons and MnPO or MPO neurons, and DMH
thermogenic neurons are differentially regulated by GABAergic
and glutamatergic inputs (18, 24, 25). However, it is equally likely
for these inputs to arise from preoptic subregions (such as the
MnPO or MPO) vs. other brain regions. In addition, activation of
GABAergic neurons in subregions including the MnPO and MPO
have a small effect on T
core
(refs. 5 and 6 and SI Appendix,Fig.S5).
We thus wondered whether vLPO
Vgat
neurons innervate DMD
neurons and reduced T
core
in response to heat by inhibiting
DMD neurons. To test this hypothesis, we combined antero-
grade and retrograde tracing, terminal optogenetic stimulation,
IPSC recording, and single-cell RT-PCR (Fig. 2) to show that
vLPO
Vgat
DMD connections are critical for bidirectionally
regulating T
core
and activity. Interestingly, we found that the
vLPO
Vgat
neurons preferentially innervated DMD
Vgat
neurons,
compared with DMD
Vglut2
neurons (Fig. 2H).
Previous studies have shown that DMD neurons, especially
glutamatergic neurons, play an important role in promoting
thermogenesis (1618, 29, 30). However, the response of these
neurons to thermal stimuli was unknown. Here, we recorded
calcium dynamics in the DMD by using fiber photometry and
found, surprisingly, that both glutamatergic and GABAergic
neurons were activated by cooling (2513 °C; Fig. 3 Dand E), but
that these neurons responded with different dynamics. The glu-
tamatergic neurons responded more abruptly and their response
decayed more quickly compared with the GABAergic neurons
(SI Appendix, Fig. S5). Consistent with these kinetics, we found
that increases in T
core
, EE, and activity levels resulting from
CNO-mediated activation of DMD glutamatergic neurons arose
more rapidly and decayed more quickly than those elicited by
CNO-mediated activation of DMD GABAergic neurons (Fig. 4
and SI Appendix, Fig. S8).
The role played by GABAergic neurons in the DMD in
thermoregulation has not been studied previously, although the
DMD contains more GABAergic than glutamatergic neurons
(31). Here, we have shown unequivocally that these GABAergic
neurons are essential for controlling thermogenesis. Their acti-
vation strongly promoted increases in T
core
, EE, and physical
activity (Fig. 4 EHand SI Appendix, Fig. S5), whereas their
suppression reduced T
core
and activity (Fig. 5 Cand D). We
therefore have uncovered a type of thermogenic neuron.
Several reports have suggested that neurons expressing the leptin
receptor (LepR) in the DMD are important for thermogenesis
(1921). Cold-induced cFos expression colocalizes with LepR
(21), and activation of these LepR neurons increases T
core
and
EE (19). Our immunostaining results suggest that DMD
LepR
neurons contain both glutamatergic and GABAergic types, with the
majority being glutamatergic (SI Appendix,Fig.S9). Thus,
DMD
LepR
neurons might be a downstream target of the vLPO
Vgat
neurons.
It remains unclear how DMD neurons are connected with
premotor neurons to direct thermogenesis. Early observations
suggested that glutamatergic neurons may project directly to
premotor neurons within the rostral medullary region (rMR) to
promote thermogenesis (1618). It has also been reported that
rMR premotor neurons receive inhibitory inputs from other
medullary regions (32, 33). Thus, DMD
Vglut2
neurons may di-
rectly innervate rMR premotor neurons to promote thermo-
genesis, and DMD
Vgat
neurons might disinhibit rMR neurons by
suppressing their inhibitory inputs.
Delineating the specific neural cell types involved in thermo-
regulation is a key step toward understanding these critical
neural circuits. Using the PhosphoTRAP approach (34), which
enables the immunoprecipitation of translational ribosomes via
phosphorylated ribosomal protein S6 (a marker of neural activ-
ity), we (SI Appendix, Fig. S10) and Tan et al. (35) independently
discovered that brain-derived neurotrophic factor (BDNF), a
classic neurotrophic factor (36), is transcriptionally activated
following heat challenge and is a novel marker for heat-activated
neurons. It is interesting to see that heat affects the expression of
a neurotrophic factor, suggesting that long-term heat challenge
may affect nerve growth and cause remodeling of thermoregu-
latory networks. The exact role played by BDNF in this context
must be further tested. BDNF-expressing neurons in the ven-
tromedial preoptic nucleus (VMPO) can drive hypothermia
without affecting physical activity (35). Thus, the circuits in-
volving VMPO
BDNF
neurons and vLPO
Vgat
neurons may act
coordinately to regulate EE, BAT thermogenesis, activity, and
vasodilation to lower T
core
.
Our results establish a neural circuit (Fig. 5E) for regulating
heat loss behaviors, in which environmental heat indirectly ac-
tivates POA glutamatergic neurons (refs. 13, 5, and 6 and SI
Appendix, Fig. S3), which may, in turn, activate a population of
GABAergic neurons in the vLPO (Fig. 1 and SI Appendix, Fig.
S4). The vLPO GABAergic neurons inhibit thermogenic neu-
rons in the DMD to suppress EE and activity, thereby lowering
T
core
(Figs. 2, 4, and 5). The DMD neurons include both gluta-
matergic and GABAergic subtypes and are cold activated (Fig. 2
and 3). DMD glutamatergic neurons may send excitatory input
to premotor neurons in the rMR to stimulate thermogenesis (16
18). DMD GABAergic neurons might disinhibit rMR premotor
neurons by suppressing their inhibitory inputs (32, 33).
Glutamatergic GABAergic
Time (min)
Tcore (°C)
-30 0 30 60 90
33
34
35
36
37
Vglut2 &
hGtACR1
Light BA ytivitcAlacisyhPerutarepmeTydoB
DC
DMDVgat 0
50
100
150
200
250
b.s. 30- 60 min
*
Tcore (C)
Eundefined
DMDVglut2
Heat Thermogenesis
EE, activity
POA DMD
rMR
vLPO
Fig. 5. Optogenetic inhibition of DMD neurons induces hypothermia.
(Aand B) Bilateral inhibition of DMD
Vgut2
neurons via hGtACR1 resulted in
significant decreases in T
core
(A) and activity (B)(n=4). ΔT
core
represents the
T
core
changes from the mean level before light delivery (t=30 to 10 min).
The baseline (b.s.) (average of t=30 to 10 min) and t=60 min are in the
bar graph. The average of activity in 30-min intervals between t=40 and
10 min (baseline, b.s.) and between t=30 and 60 min are shown in the bar
graph. Stimulation protocol: light on for 30 s (473 nm, 10 mW) followed by a
90-s break, with the sequence repeating for 1 h. (Cand D) Bilateral inhibition
of DMD
Vgat
neurons (n=4) via hGtACR1 resulted in significant decreases in
T
core
(C) and activity (D). Stimulation protocol is the same as in A.(E) Model
for heat-induced suppression of thermogenesis. Sold line represents the
connection verified in the current study. Dash lines represents proposed
connections based our data and other reports. (+), activation. (), inhibition.
All data are plotted as mean ±SEM. The Pvalues, compared with baseline
(b.s.), are calculated based on statistical tests listed in SI Appendix, Table S1.
*P0.05; **P0.01. DMD, dorsal part of dorsomedial hypothalamic nu-
cleus; POA, preoptic area; rMR, rostral medullary region; vLPO, ventral part
of lateral preoptic nucleus.
2046
|
www.pnas.org/cgi/doi/10.1073/pnas.1616255114 Zhao et al.
Materials and Methods
Mice. Animal care and use conformed to institutional guidelines of Shang-
haiTech University, Shanghai Biomodel Organism Co., and governmental
regulations. All experiments were performed on adult mice (816 wk old).
Mice were housed under controlled temperature (2225 °C) in a 12-h reverse
light/dark cycle (light time, 8 PM to 8 AM) with a standard chow diet [4%
(wt/wt) fat SPF Rodent Feed] and ad libitum drinking water. The following
mice strains were from Jackson Laboratory (USA): C57BL/6J (000664); Vglut2-
IRES-Cre (016963); Vgat-IRES-Cre (028862); Ai14 (007914); LepR-Cre (008320);
Gad67-GFP (006340).
AAV Vectors. We used Cre-inducible AAV vectors (titer >10
12
) from the fol-
lowing sources: the Vector Core at the University of North Carolina at Chapel
Hill (AAV5-EF1a-DIO-hChR2(H134R)-eYFP, AAV5-EF1a-DIO-eYFP, AAV5-
hSyn-DIO-hM3D-mCherry, and AAV5-hSyn-DIO-hM4D-mCherry), the Vector
Core at the University of Pennsylvania (AAV5-Syn-Flex-GCaMP6f), and
Shanghai Taitool Bioscience Co. (AAV9-hSyn-GtACR1-P2A-EGFP). The latter
construct was a gift by Minmin Luo, National Institute of Biological Sciences,
Beijing, originally described in ref. 12.
Surgeries. A detailed description is provided in SI Appendix, SI Materials and
Methods. We delivered 0.2 μL (unless specified) of AAV virus through a
pulled-glass pipette and a pressure microinjector (Nanoject II, 3-000-205A,
Drummond). The fiber-optic inserts (200 μm i.d., AniLab Co.) were chroni-
cally implanted (200 μm above viral injection sites) and secured with dental
cement.
Behavioral Assays. A detailed description is provided in SI Appendix, SI
Materials and Methods.T
core
, EE, and activity were monitored by animal
monitoring system with temperature telemetry (Columbus, with G2 E-Mitter
transponders).
Immunohistochemistry. A detailed description is provided in SI Appendix, SI
Materials and Methods.
Fiber Photometry. A detailed description is provided in SI Appendix, SI Ma-
terials and Methods. The GCaMP6f fluorescence signals were acquired with a
fiber photometry system (Fscope, Biolinkoptics, China). The floor tempera-
ture was controlled by a Peltier controller by customized Labview code
(National Instrument) described in ref. 37.
Electrophysiological Recordings and Single-Cell RT-PCR. A detailed description
is provided in SI Appendix, SI Materials and Methods.
PhosphoTRAP and mRNA Sequencing. A detailed description is provided in SI
Appendix, SI Materials and Methods. The procedure of PhosphoTRAP was
similar as described in ref. 34 with some modification.
RNA Fluorescent In Situ Hybridization. A detailed description is provided in SI
Appendix, SI Materials and Methods. RNA probes against Vgat, GAD67,
Vglut2, and BDNF were constructed according to the description on Allen
Brain Atlas (www.brain-map.org).
ACKNOWLEDGMENTS. We thank Dr. Jeff Friedman for help with Phospho-
TRAP; Drs. Ji Hu, Minmin Luo, Cheng Zhan, Jun Liao, Yan Zou, Pengyu
Huang, and Winnie Shum for reagent share; Junjie Luo, Rongfeng Hu, and
Yi Li for help with statistics; and Shen Xian HuiWechat group for valuable
discussion. This study is funded by National Nature Science Foundation of
China Grants X-0402-14-002 (to W.L.S.) and 31471065 (to X.-H.X.), Ministry of
Science and Technology Office China 973 program (to X.-H.X.), the Thousand
Young Talents Program of China (to W.L.S. and X.-H.X.), and the Shanghai-
Tech University start-up fund.
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... A calcium imaging study demonstrated that GABA-and glutamatergic DMH neurons are equally activated by cooling and acute chemogenetic activation increased energy expenditure, locomotor activity and body temperature, however, body weight and other metabolic parameters were not considered [45]. We speculate that the strong induction of locomotor activity in free moving Vglut2 and Vgat animals will contribute to increased body temperature and is further supported by the fact that in anesthetized mice (without confounding locomotion) chemogenic activation of GABA-and glutamatergic neurons did not induce BAT temperature. ...
... Cold-activated dDMH/DHA Lepr and dDMH/DHA Glut neurons receive inhibitory inputs from warm-sensing POA neurons [3,12,54]. We and others suggested -against dogma -that these inputs originate from glutamatergic, but not GABAergic, POA neurons to mediate warminduced suppression of energy expenditure and body temperature [45,[55][56][57] and was confirmed by single cell profiling of the preoptic region [58]. Thus, warm-sensing, glutamatergic POA neurons that suppress energy expenditure and body temperature are inconsistent with a direct innervation of cold-activated dDMH/DHA Lepr neurons, and instead should require GABA interneurons [59,60]. ...
... Conversely, sleep inducing GABAergic galanin expressing neurons in the ventrolateral preoptic region (VLPO) also directly innervate the DMH, and decreased body temperature (and likely energy expenditure) with neuronal activation [45,61], and thus may directly inhibit dDMH/ DHA Lepr neurons. Our data indicates that Glp1r is enriched in all GABAergic DMH Lepr clusters, but it remains unclear if these clusters all represent projection neurons (to ARC) or if they may identify local interneurons no genetic markers are known for their distinction. ...
... Por ejemplo, los terneros Murrah machos criados en establos abiertos sin sombra y en un clima tropical húmedo alcanzaron temperaturas corporales entre 38.5 ± 0.37°C y 40.5 ± 0.10°C durante el mediodía (Das et al., 1999), mientras que hembras Murrah lactantes y no preñadas expuestas a potreros sin sombra alcanzaron una temperatura ambiente de hasta 39.0°C (Silva et al., 2011). El hipotálamo puede reconocer tales aumentos en la temperatura corporal y los receptores termosensibles cutáneos también pueden enviar señales excitadoras a las neuronas sensibles al calor (WSN) en el ganglio de la raíz dorsal (DRG) de la médula espinal (Zhao et al., 2017). Desde la médula espinal, la señal se transmite al POA utilizando la parte dorsal del núcleo parabraquial lateral (LPBd) donde participan las neuronas glutamatérgicas (Morrison y Nakamura, 2019). ...
... Heat defense activities, including induction of vasodilation, reduction of energy expenditure or interscapular brown adipose tissue (iBAT) thermogenesis, and suppression of muscle shivering activity, are highly involved with the mPOADMH, mPOAraphe pallidus nucleus (RPa) or DMH RPa circuits [19]. Of note, warm-sensitive preoptic neurons exhibit high heterogeneity, expressing specific or overlapped markers such as Bdnf, CCK, ERα, galanin, Pgds2, Qrfp, Nos1, Vglut2/Pacap/leptin receptor, Vgat, Trpm2 and Trpc4 [20][21][22][23][24][25][26][27]. Recent researches indicate that HA can enhance the acquired thermal adaptation ability by promoting the proliferation and differentiation of nascent neurons in the mPOA, while the neuronal identity and circuitry of HA-improved mPOA neurons are poorly understood [28]. ...
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Rationale: Record-breaking heatwaves caused by greenhouse effects lead to multiple hyperthermia disorders, the most serious of which is exertional heat stroke (EHS) with the mortality reaching 60 %. Repeat exercise with heat exposure, termed heat acclimation (HA), protects against EHS by fine-tuning feedback control of body temperature (Tb), the mechanism of which is opaque. This study aimed to explore the molecular and neural circuit mechanisms of the HA training against EHS. Methods: Male C57BL/6 mice (6-8 weeks) and male TRPV1-Cre mice (6-8 weeks) were used in our experiments. The EHS model with or without HA training were established for this study. RNA sequencing, qPCR, immunoblot, immunofluorescent assays, calcium imaging, optogenetic/ chemical genetic intervention, virus tracing, patch clamp, and other methods were employed to investigate the molecular mechanism and neural circuit by which HA training improves the function of the medial preoptic area (mPOA) neurons. Furthermore, a novel exosome-based strategy targeting the central nervous system to deliver irisin, a protective peptide generated by HA, was established to protect against EHS. Results: HA-related neurons in the mPOA expressing transient receptor potential vanilloid-1 (TRPV1) were identified as a population whose activation reduces Tb; inversely, dysfunction of these neurons contributes to hyperthermia and EHS. mPOATRPV1 neurons facilitate vasodilation and reduce adipose tissue thermogenesis, which is associated with their inhibitory projection to the raphe pallidus nucleus (RPa) and dorsal medial hypothalamus (DMH) neurons, respectively. Furthermore, HA improves the function of preoptic heat-sensitive neurons by enhancing TRPV1 expression, and Trpv1 ablation reverses the HA-induced heat tolerance. A central nervous system-targeted exosome strategy to deliver irisin, a protective peptide generated by HA, can promote preoptic TRPV1 expression and exert similar protective effects against EHS. Conclusions: Preoptic TRPV1 neurons could be enhanced by HA, actively contributing to heat defense through the mPOA"DMH/RPa circuit during EHS, which results in the suppression of adipose tissue thermogenesis and facilitation of vasodilatation. A delivery strategy of exosomes engineered with RVG-Lamp2b-Irisin significantly improves the function of mPOATRPV1 neurons, providing a promising preventive strategy for EHS in the future.
... The center responsible for development of postoperative fever, particularly after neuroendoscopic procedures, is the hypothalamus, which regulates body temperature [3,9]. Current studies focus on the role of the anterior preoptic and dorsomedial nuclei in this regulation but largely overlook the contribution of the inferior hypothalamus, particularly the floor of the third ventricle [9,12,23]. Studies revealed that heat loss center is located in the anterior hypothalamus while heat gain center is located in the posterior hypothalamus [6], see Fig. 1. Normal body temperature is regulated by the thermoregulatory center in the anterior hypothalamus. ...
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Postoperative fever following neuroendoscopic procedures has been well-documented, yet specific differentiation based on the nature and site of the procedure remains lacking. Given the anatomical involvement of the hypothalamus in temperature regulation, we propose that endoscopic third ventriculostomy (ETV) may have a distinct impact on postoperative fever. This study aims to investigate this phenomenon. This retrospective comparative analysis includes all patients who underwent neuroendoscopic procedures between January 2017 and September 2023. Patients were divided into ETV and non-ETV groups, and comparisons were made regarding postoperative body temperature during the initial 7 days after surgery. Comprehensive data were collected on case numbers, surgical duration, symptoms, treatments, and outcomes. Body temperature was measured postoperatively in the morning and evening for 7 days, with elevated temperature categorized as sub-fever (37.5 to 38.2 °C) and fever (≥ 38.3 °C). 207 patients underwent neuroendoscopic procedures in our neurosurgical centers (median age19.1 ± 21.7 years, 50.7% male), primarily for aqueduct stenosis (25.6%) and intra- and periventricular tumors (25.1%). Among them, 104 (50.2%) patients underwent ETV, while 103 (49.8%) underwent other neuroendoscopic procedures (43.7% intracranial cysts fenestrations, 39.8% placement of intraventricular catheters, 3.9% intraventricular lavage, 4.9% septostomy, and 5.8% tumor biopsy). All postoperative infections were excluded. No significant differences were observed in preoperative symptoms, laboratory findings, or postoperative antibiotic usage between the two groups. The ETV group exhibited significantly more postoperative fever (37.5% vs. 19.4%, p = 0.005), particularly from the first night to the third night after the operation. This study substantiates the hypothesis that manipulation of the floor of third ventricle through endoscopic ventriculostomy may contribute to postoperative fever, rather than the neuroendoscopic procedure. Elevated temperatures were observable from the first night post-surgery and typically normalized by third day without necessitating specific treatment. Further prospective studies are warranted to elucidate the precise mechanisms underlying intraoperative manipulation.
... To identify novel targets related to fighting behaviors, we performed enrichment analysis [48] by calculating fold enrichment (RI-IP/Ctr-IP) and highlighting the top candidate genes ( Figure 6G-H). Among the most significantly enriched genes, several well-established immediate-early genes, including Arc, Egr1, Fgf2, and Fosb, were upregulated. ...
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Thermal cooling mechanisms of human exposed to electromagnetic (EM) radiation are studied in detail. The electromagnetic and thermal co-simulation method is utilized to calculate the electromagnetic and temperature distributions. Moreover, Pennes' bioheat equation is solved to understand different thermal cooling mechanisms including blood flow, convective cooling and radiative cooling separately or jointly. Numerical results demonstrate the characteristics and functions for each cooling mechanism. Different from the traditional view that the cooling effect of blood is usually reflected by its influence on sweat secretion and evaporation, our study indicates that the blood flow itself is an important factor of thermal cooling especially for high-intensity EM radiation. This work contributes to fundamental understanding of thermal cooling mechanisms of human.
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Heat acclimation is an adaptive process that improves physiological performance and supports survival in the face of increasing environmental temperatures, but the underlying mechanisms are not well understood. Here we identified a discrete group of neurons in the mouse hypothalamic preoptic area (POA) that rheostatically increase their activity over the course of heat acclimation, a property required for mice to become heat tolerant. In non-acclimated mice, peripheral thermoafferent pathways via the parabrachial nucleus activate POA neurons and mediate acute heat-defense mechanisms. However, long-term heat exposure promotes the POA neurons to gain intrinsically warm-sensitive activity, independent of thermoafferent parabrachial input. This newly gained cell-autonomous warm sensitivity is required to recruit peripheral heat tolerance mechanisms in acclimated animals. This pacemaker-like, warm-sensitive activity is driven by a combination of increased sodium leak current and enhanced utilization of the NaV1.3 ion channel. We propose that this salient neuronal plasticity mechanism adaptively drives acclimation to promote heat tolerance.
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Avoidance of noxious ambient heat is crucial for survival. A well-known phenomenon is that animals are sensitive to the rate of temperature change. However, the cellular and molecular underpinnings through which animals sense and respond much more vigorously to fast temperature changes are unknown. Using Drosophila larvae, we found that nociceptive rolling behavior was triggered at lower temperatures and at higher frequencies when the temperature increased rapidly. We identified neurons in the brain that were sensitive to the speed of the temperature increase rather than just to the absolute temperature. These cellular and behavioral responses depended on the TRPA1 channel, whose activity responded to the rate of temperature increase. We propose that larvae use low-threshold sensors in the brain to monitor rapid temperature increases as a protective alert signal to trigger rolling behaviors, allowing fast escape before the temperature of the brain rises to dangerous levels.
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Thermoregulation is one of the most vital functions of the brain, but how temperature information is converted into homeostatic responses remains unknown. Here, we use an unbiased approach for activity-dependent RNA sequencing to identify warm-sensitive neurons (WSNs) within the preoptic hypothalamus that orchestrate the homeostatic response to heat. We show that these WSNs are molecularly defined by co-expression of the neuropeptides BDNF and PACAP. Optical recordings in awake, behaving mice reveal that these neurons are selectively activated by environmental warmth. Optogenetic excitation of WSNs triggers rapid hypothermia, mediated by reciprocal changes in heat production and loss, as well as dramatic cold-seeking behavior. Projection-specific manipulations demonstrate that these distinct effectors are controlled by anatomically segregated pathways. These findings reveal a molecularly defined cell type that coordinates the diverse behavioral and autonomic responses to heat. Identification of these warm-sensitive cells provides genetic access to the core neural circuit regulating the body temperature of mammals. PaperClip Download audio (3MB)Help with mp3 files
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Central neural circuits orchestrate the behavioral and autonomic repertoire that maintains body temperature during environmental temperature challenges and alters body temperature during the inflammatory response and behavioral states and in response to declining energy homeostasis. This review summarizes the central nervous system circuit mechanisms controlling the principal thermoeffectors for body temperature regulation: cutaneous vasoconstriction regulating heat loss and shivering and brown adipose tissue for thermogenesis. The activation of these thermoeffectors is regulated by parallel but distinct efferent pathways within the central nervous system that share a common peripheral thermal sensory input. The model for the neural circuit mechanism underlying central thermoregulatory control provides a useful platform for further understanding of the functional organization of central thermoregulation, for elucidating the hypothalamic circuitry and neurotransmitters involved in body temperature regulation, and for the discovery of novel therapeutic approaches to modulating body temperature and energy homeostasis.
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Unlabelled: The preoptic area (POA) regulates body temperature, but is not considered a site for body weight control. A subpopulation of POA neurons express leptin receptors (LepRb(POA) neurons) and modulate reproductive function. However, LepRb(POA) neurons project to sympathetic premotor neurons that control brown adipose tissue (BAT) thermogenesis, suggesting an additional role in energy homeostasis and body weight regulation. We determined the role of LepRb(POA) neurons in energy homeostasis using cre-dependent viral vectors to selectively activate these neurons and analyzed functional outcomes in mice. We show that LepRb(POA) neurons mediate homeostatic adaptations to ambient temperature changes, and their pharmacogenetic activation drives robust suppression of energy expenditure and food intake, which lowers body temperature and body weight. Surprisingly, our data show that hypothermia-inducing LepRb(POA) neurons are glutamatergic, while GABAergic POA neurons, originally thought to mediate warm-induced inhibition of sympathetic premotor neurons, have no effect on energy expenditure. Our data suggest a new view into the neurochemical and functional properties of BAT-related POA circuits and highlight their additional role in modulating food intake and body weight. Significance statement: Brown adipose tissue (BAT)-induced thermogenesis is a promising therapeutic target to treat obesity and metabolic diseases. The preoptic area (POA) controls body temperature by modulating BAT activity, but its role in body weight homeostasis has not been addressed. LepRb(POA) neurons are BAT-related neurons and we show that they are sufficient to inhibit energy expenditure. We further show that LepRb(POA) neurons modulate food intake and body weight, which is mediated by temperature-dependent homeostatic responses. We further found that LepRb(POA) neurons are stimulatory glutamatergic neurons, contrary to prevalent models, providing a new view on thermoregulatory neural circuits. In summary, our study significantly expands our current understanding of central circuits and mechanisms that modulate energy homeostasis.
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In the face of starvation, animals will engage in high-risk behaviors that would normally be considered maladaptive. Starving rodents, for example, will forage in areas that are more susceptible to predators and will also modulate aggressive behavior within a territory of limited or depleted nutrients. The neural basis of these adaptive behaviors likely involves circuits that link innate feeding, aggression and fear. Hypothalamic agouti-related peptide (AgRP)-expressing neurons are critically important for driving feeding and project axons to brain regions implicated in aggression and fear. Using circuit-mapping techniques in mice, we define a disynaptic network originating from a subset of AgRP neurons that project to the medial nucleus of the amygdala and then to the principal bed nucleus of the stria terminalis, which suppresses territorial aggression and reduces contextual fear. We propose that AgRP neurons serve as a master switch capable of coordinating behavioral decisions relative to internal state and environmental cues.
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The dorsal raphe nucleus (DRN) is involved in organizing reward-related behaviours; however, it remains unclear how genetically defined neurons in the DRN of a freely behaving animal respond to various natural rewards. Here we addressed this question using fibre photometry and single-unit recording from serotonin (5-HT) neurons and GABA neurons in the DRN of behaving mice. Rewards including sucrose, food, sex and social interaction rapidly activate 5-HT neurons, but aversive stimuli including quinine and footshock do not. Both expected and unexpected rewards activate 5-HTneurons. After mice learn to wait for sucrose delivery, most 5-HT neurons fire tonically during waiting and then phasically on reward acquisition. Finally, GABA neurons are activated by aversive stimuli but inhibited when mice seek rewards. Thus, DRN 5-HT neurons positively encode a wide range of reward signals during anticipatory and consummatory phases of reward responses. Moreover, GABA neurons play a complementary role in reward processing.
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Light-gated rhodopsin cation channels from chlorophyte algae have transformed neuroscience research through their use as membrane-depolarizing optogenetic tools for targeted photoactivation of neuron firing. Photosuppression of neuronal action potentials has been limited by the lack of equally efficient tools for membrane hyperpolarization. We describe Anion Channel Rhodopsins (ACRs), a family of light-gated anion channels from cryptophyte algae that provide highly sensitive and efficient membrane hyperpolarization and neuronal silencing through light-gated chloride conduction. ACRs strictly conducted anions, completely excluding protons and larger cations, and hyperpolarized the membrane of cultured animal cells with much faster kinetics at less than one-thousandth of the light intensity than required by the most efficient currently available optogenetic proteins. Natural ACRs provide optogenetic inhibition tools with unprecedented light sensitivity and temporal precision. Copyright © 2015, American Association for the Advancement of Science.
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Brown adipose tissue (BAT) plays a critical role in cold- and diet-induced thermogenesis. Although BAT is densely innervated by the sympathetic nervous system (SNS), little is known about the central nervous system (CNS) origins of this innervation. The purpose of the present experiment was to determine the neuroanatomic chain of functionally connected neurons from the CNS to BAT. A transneuronal viral tract tracer, Bartha's K strain of the pseudorabies virus (PRV), was injected into the interscapular BAT of Siberian hamsters. The animals were killed 4 and 6 days postinjection, and the infected neurons were visualized by immunocytochemistry. PRV-infected neurons were found in the spinal cord, brain stem, midbrain, and forebrain. The intensity of labeled neurons in the forebrain varied from heavy infections in the medial preoptic area and paraventricular hypothalamic nucleus to few infections in the ventromedial hypothalamic nucleus, with moderate infections in the suprachiasmatic and lateral hypothalamic nuclei. These results define the SNS outflow from the brain to BAT for the first time in any species.
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Body temperature homeostasis is critical for survival and requires precise regulation by the nervous system. The hypothalamus serves as principal thermostat that detects and regulates internal temperature. We demonstrate that the ion channel TRPM2 is a temperature sensor in a subpopulation of hypothalamic neurons. TRPM2 limits the fever response, and may detect increased temperatures to prevent overheating. Furthermore, chemogenetic activation or inhibition of hypothalamic TRPM2-expressing neurons in vivo decreased and increased body temperature, respectively. Such manipulation may allow analysis of the beneficial effects of altered body temperature on diverse disease states. Identification of a functional role for TRP channels in monitoring internal body temperature should promote further analysis of molecular mechanisms governing thermoregulation and foster the genetic dissection of hypothalamic circuits concerned with temperature homeostasis.