Science 6.03.2020

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

0

100

150

200

250

300

Δ

BAT SNA power(% baseline)

******

ΔHR ( bpm)

8

2

4

6

10

0

Δ

MAP (mmHg)

P>0.9*

2

0

1

–1

ΔT

BAT

(°C)

*********

0.3

0.2

0

0.1

palGFP

DP/DTT DMH

IL DMH

DP/DTT DMH

DP/DTT LH

ChIEF-tdTomato

DP

IL

DTT

DMH

Brain

rMR

Spinal

cord

Effector

Virus

ChIEF-tdTomato- or

palGFP-expressing

neuron Sympathetic premotor neuron

DP/DTT

G Optical fiber

3V

f

mt

DMH

VMH

LHLH

HI

Laser ON

BAT SNA

(power / 4 s)

BAT SNA

TBAT

(°C)

Tcore

(°C)36.0

37.0

HR

(bpm)

AP

(mmHg)

Laser ON

1.0

0

35.3

360

340

150

50

34.3

100

μV

ChIEF-tdTomato (DP/DTT DMH)

30 s

JK

Left RightLaser ON

AP5/CNQX

170

70

38.0

37.0

0.75

0

34.5

33.5

370

340

30 s

100

μV

ChIEF-tdTomato

(DP/DTT DMH)

BAT SNA

(power / 4 s)

BAT SNA

TBAT

(°C)

Tcore

(°C)

HR

(bpm)

AP

(mmHg)

M N

DMH

f

mt

VMH

3V

Bregma –3.1 mm

O

0

50

100

150

200

250

AP5/CNQX – +

–0.1

0

0.1

0.2

0.3

*

Δ

T

BAT

(°C)

• 
  
  
  
  • 
    
  
  
  
  

0

2

4

6

8

*

Δ

HR (bpm

)

• 
  
  
  
  • 
    
  
  
  
  

0

1

2 *

–1

–2

Δ

MAP(mmHg

)

• 
  
  
  
  • 
    
  
  
  
  

***

Δ

BAT SNA power(% baseline)

DMH

Brain

rMR

Spinal

cord

Effector

AP5/CNQX

or saline

Sympathetic premotor neuron

DP/DTT

Optical fiber

ChIEF-tdTomato-

expressing neuron

L

DMH

Laser ON

ppaallGGFFPP

VVGGLLUUTT11

CCTTbb

• 
  

D

• 
  

E VVGGLLUUTT22ppaallGGFFPP

CCTTbb

• 
  

F ppaallGGFFPPVVGGAATT

CCTTbb

ppaallGGFFPP

C CCTTbb

RMg

rRPa

py

A B

–50

10

–2 –3

–0.1

0.4

12

–2 –3

–2

3

0.4

RESEARCH | RESEARCH ARTICLE