monosynaptic pathway for driving sympathetic
stress responses. First, DP/DTT→DMH pro-
jection neurons were selectively lesioned. An
AAV for Cre recombinase–dependent expression
of taCasp3, which induces cell-autonomous
apoptosis ( 23 ), and a retrograde AAV (AAVrg)
for Cre expression were bilaterally injected
into the DP/DTT and DMH, respectively, to
selectively lesion double-infected neurons (i.e.,
DP/DTT→DMH projection neurons) (Fig. 4A).
Figure 4B shows an example of lesions of
DP/DTT→DMH neurons: taCasp3 expression
eliminated 88% of Cre-expressing (i.e., DMH-
projecting) DP/DTT neurons in one represent-
ative rat as compared with palGFP-transduced
control rats. This lesioned rat failed to exhibit
BAT thermogenic and hyperthermic responses
to SDS (Fig. 4C). Group data (Fig. 4D and fig.
S5A) showed that stress-induced BAT thermo-
genesis was inhibited in proportion to the
extent of lesions of DP/DTT→DMH neurons,
and the stress responsewas totally eliminated
when all the neurons were lesioned. However,
the lesions of DP/DTT→DMH neurons did not
affect diurnal control ofTBATorTcore(fig. S5B).
To exclude the possible influence of long-
term alterations in the circuits after neuronal le-
sions, we optogenetically suppressed DP/DTT→DMH transmission in free-moving rats. The
Cre-dependent expression system with an-
terograde and retrograde AAVs was used to
selectively transduce DP/DTT→DMH projection
neurons with iChloC, a chloride-conducting
channelrhodopsin shown to photoinhibit neu-
ronal activity ( 24 )(Fig.4E).ExpressediChloC-
mCherry proteins (or palGFP for control) were
bilaterally detected in cell bodies in the
DP/DTT and their nerve endings in the DMH
(Fig.4F).InvivobilateralilluminationofiChloC-
mCherry–containing nerve endings in the DMH
with pulsed blue light exerted potent, long-
lasting inhibition on SDS-induced increasesKataokaet al.,Science 367 , 1105–1112 (2020) 6 March 2020 6of8
Fig. 5. Photoinhibition of the DP/
DTT→DMH pathway abolishes stress-
induced avoidance behavior and skin
vasoconstriction.(A)Wistarratsin
which DP/DTT→DMH neurons were
transduced with iChloC-mCherry or
palGFP (Fig. 4E) underwent a social
interaction test after SDS. (B)Repre-
sentative examples of behavior
tracking of palGFP- and iChloC-
mCherry–transduced rats during the
social interaction test. (C) Behaviors
during the social interaction test (n=5
per group). Naïve rats did not undergo
SDS before habituation and social inter-
action test. Data were analyzed by one-
way ANOVA (Entries:F5,24=12.87,P<
0.001; Time spent:F5,24=15.49,
P< 0.001; Moving distance:F5,24=9.15,
P< 0.001) followed by Bonferroni’s
post hoc test. ns, not significant;
**P< 0.01; **P< 0.001. Error
bars indicate SEM. (DandE)Thermo-
graphic measurements at proximal and
distal parts [arrowheads in (D)] on the
tail skin (n=5pergroup)wereanalyzed
by repeated measures two-way ANOVA
(Proximal: treatment:F1,4= 17.44,P=
0.014; group:F1,4= 2.11,P= 0.220;
interaction:F1,4=9.01,P= 0.040; Distal:
treatment:F1,4=12.13,P= 0.025; group:
F1,4=10.74,P=0.031;interaction:
F1,4=11.36,P=0.028)followedby
Bonferroni’s post hoc test. ns, not signif-
icant; P< 0.05 (versus habituation).
Error bars indicate SEM. (F) Model of the
central psychosomatic pathways that
drive stress responses. The DP/DTT
integrates signals from multiple forebrain
regions processing stress and emotion
and then provides a glutamatergic (Glu)
master signal to the DMH to excite
neuronal groups controlling different
effectors. The excited DMH neurons drive
sympathetic outflows to BAT, the heart,
and cutaneous and visceral blood vessels
through the rMR and partly through the rostral ventrolateral medulla (RVLM). Another subset of DMH neurons drivesavoidance behavior through as-yet-unknown circuit
mechanisms. AIP, posterior part of the agranular insular cortex; Pir2, layer II of the piriform cortex.
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