Science - 06.03.2020

sympathetic stress responses ( 22 ). However,
photostimulation of DP/DTT→LH transmis-
sion elicited no or subtle changes in the phys-
iological variables (Fig. 3K and fig. S4J).

Additionally, no response was elicited by photo-

stimulation of IL→DMH axonal projections

with ChIEF-tdTomato (Fig. 3K and fig. S4,

KtoM).

DP/DTT→DMH transmission is essential for

sympathetic responses to psychosocial stress

We next employed genetic approaches to deter-

mine the importance of the DP/DTT→DMH

Kataokaet al.,Science 367 , 1105–1112 (2020) 6 March 2020 5of8

*** ***

***

Laser

–50

0

50

100

150

200

–60 –30 0 30 60 90 120

Time (min)

ΔHR (bpm)

250

0

2,000

4,000

6,000

8,000

ΔHR A

UC(bp

m·m in)

pa lGFPiChlo

C

palGFP

iChloC

palGFP

taCasp3

–60 –30 0 30 60 90 120

Time (min)

–1.5

–1.0

–0.5

0

0.5

1.0

1.5

2.0

–60 –30 0 30 60 90 120

Time (min)

0

20

40

60

80

100

120

0 100 200300 400 500

taCasp3

palGFP

Cre+ cell bodies in DP/DTT

taCasp3 AVG

palGFP AVG

–20

0

20

40

60

80

100

120

0 100 200 300 400 500

ΔT

BAT

(°C)

ΔT

BAT

AUC (°C·min)

ΔT

core

(°C)

ΔT

core

AUC (°C·min)

Cre+ cell bodies in DP/DTT

r=0.96

P=0.01

r=0.74

P=0.16

**

palGFP

taCasp3

–1.5

–1.0

–0.5

0

0.5

1.0

1.5

2.0

***

taCasp3-expressing

neuron

AAV-Ef1α-flex

-taCasp3-TEVp AAVrg-pgk-Cre

Brain

rMR

Spinal

cord

DP/DTT Effector

DMH

taCasp3-TEVp

ITR Ef1α taCasp3-TEVp WPREbGH polyAITR

Apoptosis

ITR Ef1α WPREbGH polyAITR

+Cre

A

>3weeks

ITR Ef1α iChloC-mCherry

ITR Ef1α iChloC-mCherry

WPREbGH polyAITR

WPREbGH polyAITR

AAV-Ef1α-DIO

-iChloC-mCherry AAVrg-pgk-Cre

Brain

rMR

Spinal

cord

DP/DTT Effector

DMH

iChloC-mCherry-

expressing neuron

Brain

rMR

Spinal

cord

DP/DTT Effector

DMH

Optical fiber

+Cre

iChloC-mCherry

Cre

IL fmi

DP

DTT

E

palGFP iChloC-mCherry

DP/DTT

IL fmi

DP

DTT

E

DDMMMHHHDDDMMMHHH

Laser

0

10

20

30

–60 –30 0 30 60 90

–10

Time (min)

120

Δ

MAP (mmHg)

0

500

1,000

1,500

ΔMAP AUC (mm

Hg·min)

palG FPiChloC

2.5

2.0

1.5

1.0

0.5

0

–0.5

–60 –30 0 30 60 90

Time (min)

120

Laser palGFP

iChloC

100

60

20

40

0

80

palG FPiChloC

**

ΔT

core

AUC (°C·min)

ΔT

cor e

(°C)

IIL

DTT

DP

IL

DTT

DP

ILL

fmi

DDTTTT

DDPP

E/OV

fmi

IILL

DTT

DP

E/OV

palGFP taCasp3

BCD

E F

G

Cre-expressing

(DMH-projecting)

neurons

120

–20

*** ** ***

***

palGFP

iChloC

Stress Stress Stress

Fig. 4. Selective lesion or inhibition of the DP/DTT→DMH pathway
abolishes sympathetic stress responses.(A) Scheme for genetic lesions
of DP/DTT→DMH neurons. ITR, inverted terminal repeat; WPRE, woodchuck
hepatitis virus posttranscriptional regulatory element; bGH polyA, bovine growth
hormone polyadenylation signal. (BandC) Representative examples of palGFP
or taCasp3 transduction of DP/DTT→DMH neurons from a single rat. (B) Cre-
immunopositive (DMH-projecting) neurons were eliminated exclusively in the DP/DTT
after taCasp3 transduction. Arrowheads indicate deep layers ofthe DP/DTT. Scale
bar, 500mm. (C) This lesioned rat failed to exhibit thermal responses to SDS
(gray zone). (D) Relationship between the number of Cre-positive neurons remaining
in the DP/DTT and stress responses (AUC ofDTBATandDTcoreduring stress; see
also time-course data in fig. S5A). Datafrom taCasp3 rats were subjected to linear
regression analysis (Pearson’s correlation test). The averages (AVG) of AUC were
compared for the palGFP and taCasp3 groups (unpairedttest;n=5pergroup;DTBAT:
t 8 =4.30;DTcore:t 8 =5.81).**P< 0.01; ***P< 0.001. Arrows indicate the cases
plotted in (C). (E) In vivo optogenetic inhibition of DP/DTT→DMH neurons. Many

Cre-immunopositive (DMH-projecting) DP/DTT neurons expressed iChloC-mCherry.

Scale bar, 20mm. (F) DP/DTT neurons were transduced with palGFP or iChloC-

mCherry (top), and their axons with the proteins were densely distributed in the DMH

(bottom; immunoperoxidase staining). Scale bars, 500mm(top);20mm(bottom).

(G) Illumination of DP/DTT→DMH nerve endings with iChloC-mCherry for the first

10 min (blue zone) of SDS inhibited cardiovascular and hyperthermic stress responses.

Increases in HR and MAP after SDS were caused by stress from returning to the home

cage. Time-course changes in the variables were analyzed by repeated measures

two-way ANOVA (n=5pergroup;DHR: group:F1,8=23.7,P= 0.001, time:F180,1440=

23.55,P< 0.001, interaction:F180,1440= 5.68,P< 0.001;DMAP: group:F1,8= 5.98,

P= 0.040, time:F180,1440=11.82,P< 0.001, interaction:F180,1440=2.24,P< 0.001;

DTcore:group:F1,8=9.69,P= 0.014, time:F180,1440=31.83,P< 0.001, interaction:

F180,1440=7.69,P< 0.001) followed by Bonferroni’s post hoc test (red bars with

asterisks indicate time points with significant differences). The difference in AUC

during the stress period was analyzed by unpairedttest (DHR:t 8 =8.21;DMAP:t 8 =

4.91;DTcore:t 8 =3.91).*P<0.05;**P<0.01;***P< 0.001. Error bars indicate SEM.

RESEARCH | RESEARCH ARTICLE