Kataokaet al.,Science 367 , 1105–1112 (2020) 6 March 2020 4of8
Fig. 3. Optogenetic stimu-
lation of DP/DTT→DMH
glutamatergic transmis-
sion elicits sympathetic
responses.(AandB)
Injections of AAV-CMV-
palGFP into the DP/DTT
(A) and CTb into the rMR
(B). The inset in (A) shows
a palGFP-expressing neu-
ron. py, pyramidal tract;
RMg, raphe magnus nucleus;
rRPa, rostral raphe pallidus
nucleus. Scale bars, 500mm
(main panels); 20mm
(inset). (C)palGFP-labeled
axons closely associated
with a DMH neuron
retrogradely labeled with
CTb. Scale bar, 10mm.
(DtoF) Pseudocolored
confocal images showing
palGFP-labeled axon
swellings (arrowheads)
apposed to CTb-labeled cell
bodies (asterisks) in the
DMH. VGLUT1 (D) (filled
arrowheads), but not
VGLUT2 (E) or VGAT (F)
(hollow arrowheads), was
detected in these axon
swellings. See also fig. S3.
Scale bars, 5mm. (G)In
vivo optogenetic stimulation
of DP/DTT→DMH nerve
endings. (HandI) DP/DTT
neurons transduced with
ChIEF-tdTomato (H) and
their axons containing
ChIEF-tdTomato in the DMH
[(I); immunoperoxidase
staining]. Scale bars,
500 mm. (J) Representative
example of BAT thermo-
genic and cardiovascular
responses elicited by
bilateral laser illumination of
DP/DTT→DMH nerve end-
ings with ChIEF-tdTomato.
AP, arterial pressure.
(K) Changes in physiologi-
cal variables induced by
illumination of selective
pathways (one-way ANOVA
followed by Bonferroni’s
post hoc test,n= 5 per group;DBAT SNA:F3,16= 28.74,P< 0.001;DTBAT:F3,16= 30.46,P< 0.001;DHR:F3,16= 17.27,P< 0.001;DMAP:F3,16= 8.29,P= 0.002).
P< 0.05; P< 0.01; P< 0.001. See also fig. S4. (L) Experiment to test the effects of blockade of glutamatergic synapses in the DMH on physiological
responses to photostimulation of DP/DTT→DMH transmission. (M) Each circle indicates a site of saline and AP5/CNQX injections made at the same location in
the DMH of each rat (saline was always injected first). The right side of symmetric bilateral injections is shown. (N) AP5/CNQX injections into the DMH eliminated
sympathetic responses elicited by photostimulation of DP/DTT→DMH nerve endings (compare with fig. S4F, which shows a result for the saline control). (O) Changes
in physiological variables elicited by photostimulation of the DP/DTT→DMH pathway after saline or AP5/CNQX injections into the DMH (n= 5 per group).
*P< 0.05; ***P< 0.001 (pairedttest;DBAT SNA:t 4 = 13.99;DTBAT:t 4 = 3.98;DHR:t 4 = 4.07;DMAP:t 4 = 2.86). Error bars indicate SEM.
*** ***********50
0100150200250300Δ
BAT SNA power(% baseline)******ΔHR ( bpm)8246100
Δ
MAP (mmHg)P>0.9*
201–1ΔTBAT(°C)*********0.3
0.200.1palGFP
DP/DTT DMHIL DMHDP/DTT DMH
DP/DTT LHChIEF-tdTomatoDPILDTTDMHBrainrMR
Spinal
cordEffectorVirusChIEF-tdTomato- or
palGFP-expressing
neuron Sympathetic premotor neuronDP/DTTG Optical fiber3VfmtDMHVMHLHLHHILaser ONBAT SNA
(power / 4 s)BAT SNATBAT
(°C)Tcore
(°C)36.037.0HR
(bpm)
AP
(mmHg)Laser ON
1.0035.3360
340
150
5034.3100
μVChIEF-tdTomato (DP/DTT DMH)30 sJKLeft RightLaser ONAP5/CNQX170
70
38.0
37.00.75034.533.5
37034030 s100
μVChIEF-tdTomato
(DP/DTT DMH)BAT SNA
(power / 4 s)BAT SNATBAT
(°C)Tcore
(°C)HR
(bpm)
AP
(mmHg)M NDMHfmtVMH3VBregma –3.1 mmO050100150200250AP5/CNQX – +–0.100.10.20.3*Δ
T
BAT(°C)02468*Δ
HR (bpm)012 *–1
–2
Δ
MA P(mmHg)***Δ
BAT SNA power(% baseline)DMHBrainrMR
Spinal
cordEffectorAP5/CNQX
or salineSympathetic premotor neuronDP/DTTOptical fiberChIEF-tdTomato-
expressing neuronL
DMH
Laser ONppaallGGFFPP
VVGGLLUUTT11
CCTTbbDE VVGGLLUUTT22ppaallGGFFPP
CCTTbbF ppaallGGFFPPVVGGAATT
CCTTbbppaallGGFFPP
C CCTTbbRMg
rRPa
pyA B–5010–2 –3–0.10.412–2 –3–230.4RESEARCH | RESEARCH ARTICLE