inHR,MAP,andTcore(HR: 63% inhibition,
MAP: 52% inhibition,Tcore:66%inhibition;
Fig. 4G) as compared with illumination of
palGFP-expressing nerves. Commensurate
with these inhibitions, iChloC-mCherry was
expressed in 70 ± 2% of Cre-expressing (DMH-
projecting) DP/DTT neurons (counted in three
rats in which Cre immunostaining was successful;
Fig. 4E). Photoinhibition of DP/DTT→DMH
transmission did not reduce HR, MAP, orTcore
below baseline levels, which indicates that this
pathway is not involved in basal maintenance
of these parameters.
Photoinhibition of DP/DTT→DMH trans-
mission also suppressed BAT thermogenesis
evoked by cage-exchange stress, an anticipa-
tory anxiety model, in males and females (fig.
S6), indicating that DP/DTT→DMH transmis-
sion mediates sympathetic responses to broader
types of psychological stressors in both sexes.
In contrast, photoinhibition of IL→DMH trans-
mission did not impede SDS-induced stress
responses (fig. S7).
The DP/DTT→DMH pathway mediates stress
behaviors and skin vasoconstriction
Because the DP/DTT→DMH pathway is es-
sential for driving the repertoire of sympa-
thetic stress responses, we sought to examine
its involvement in stress behavior. We thus
investigated the effect of optogenetic inhibition
of DP/DTT→DMHtransmissiononavoidance
behavior from psychosocial stressors ( 25 ). A
male Wistar rat in which DP/DTT→DMH neu-
rons were selectively transduced with iChloC-
mCherry or palGFP (Fig. 4E) was subjected to
SDS and moved to an open field for habituation
(Fig. 5A). Subsequently, the dominant male
Long-Evans rat used in the SDS episode was
caged and placed in the open field (Fig. 5A).
All of the stressed Wistar rats that did not
undergo illuminationexhibited avoidance
behavior by staying away from the social in-
teraction zone surrounding the cage of the
Long-Evans rat (Fig. 5, B and C). By contrast,
naïve Wistar rats, which had not experienced
SDS, exhibited active social interactions (Fig.
5C). The stress-induced avoidance behavior was
suppressed by photoinhibition of DP/DTT→
DMH transmission in iChloC-mCherry rats,
and their social interactions were restored
to the level exhibited by naïve rats, whereas
illumination in palGFP rats had no effect (Fig.
5, B and C). Illumination administered when
iChloC-mCherry rats were lying down in a
relaxed state in the open field without the
presence of a Long-Evans rat did not elicit
locomotion (trials in four rats, five illumina-
tion episodes per rat). Therefore, it is unlikely
that the behavioral effect of photoinhibition
resulted from stimulation of any motivation
or vigilance circuit system. In addition, photo-
inhibition did not alter social interactions ex-
hibited by naïve rats (naïve ON; Fig. 5C).
Thermography revealed reduction of tail skin
temperature in palGFP rats during the social
interaction test (with illumination) compared
with temperature during habituation (Fig. 5, D
and E). This outcome represents stress-induced
cutaneous vasoconstriction, a sympathetic re-
sponse that contributes to stress-induced hy-
perthermia by reducing heat loss ( 26 ). Tail skin
vasoconstriction was absent in iChloC-mCherry
rats with photoinhibition of DP/DTT→DMH
transmission (Fig. 5, D and E).The DP/DTT receives stress-driven inputs from
thalamic and cortical regions
To explore the forebrain regions that provide
stress inputs to the DP/DTT, we combined
retrograde tracing from the DP/DTT with
detection of stress-induced Fos expression. Rats
that received a CTb injection into the DP/DTT
(fig. S8, A and B) underwent SDS or sham
handling. Substantialnumbers of neurons were
labeled with CTb in several regions of the thal-
amus, insular and piriform cortices, and amyg-
dala. SDS significantly increased Fos expression
in CTb-labeled neurons in the mediodorsal (MD)
and paraventricular (PVT) thalamic nuclei, the
posterior part of the agranular insular cortex,
andlayerIIofthepiriformcortex(fig.S8,Cto
F). This observation suggests that the DP/DTT
receives and integrates stress-driven inputs
from these thalamic and cortical regions and
provides the integrated signals to the DMH
to drive sympathetic and behavioral stress re-
sponses (Fig. 5F).Discussion
By using the rodent model of psychosocial
stress, we discovered a prefrontal cortex–
hypothalamus excitatory pathway that drives
sympathetic and behavioral stress responses,
in which the DP/DTT was crucial for stress sig-
naling to the DMH. Our anatomical, physiolog-
ical, and optogenetic experiments revealed that
VGLUT1-positive pyramidal neurons in the
DP/DTT transmit psychological stress–driven
glutamatergic signals to the DMH to elicit a va-
riety of stress responses. Most notably, selective
ablation or inhibition of the DP/DTT→DMH
monosynaptic pathway abolished BAT ther-
mogenic, skin vasoconstrictor, cardiovascular,
and behavioral responses to SDS without af-
fecting basal thermoregulatory or cardiovascular
homeostasis. Our presentfindings demonstrate
that the DP/DTT→DMH excitatory transmis-
sion of psychological stress signals is a master
driver of the wide range of sympathetic and
behavioral stress responses (Fig. 5F).
The DP/DTT is an unexplored brain area
located at the ventral limit of the mPFC. In
contrast, the PrL and IL have been a central
focus of stress research and have been shown
to provide signals to inhibit stress responses.
In this study, we discovered a group of neurons
that drive stress responses in the DP/DTT.Photostimulation of DP/DTT→DMH trans-
mission, but not that of IL→DMH, elicited
sympathetic responses that mimic stress re-
sponses. Photoinhibition of DP/DTT→DMH
transmission, but not that of IL→DMH, sup-
pressed sympathetic stress responses. Also, in-
activation of DP/DTT neurons suppressed stress
responses, in contrast to subtle effects of in-
activation of IL neurons. Thus, we propose that
there are two functional units in the mPFC: the
ventral (DP/DTT) unit that drives stress re-
sponses and the dorsal (PrL/IL) unit that in-
hibits these responses. The inhibitory unit may
constitute the negative feedback mechanism in
which the stress hormones, glucocorticoids,
act in the PrL and IL to mitigate or terminate
stress responses ( 11 ). This feedback inhibition
might involve stress-activated PrL/IL→DMH
neurons (Fig. 1) and/or local inhibition of
DP/DTT neurons from the PrL/IL.
DP/DTT→DMH stress signaling activates
DMH→rMR sympathoexcitatory neurons to
drive stress responses (Fig. 5F). The DMH→rMR
pathway also serves as the trunk pathway that
controls body temperature and develops in-
flammatory fever, whose activity level is con-
tinuously controlled by descending inputs from
the thermoregulatory center, the preoptic area
( 27 ). Because the DP/DTT→DMH pathway
does not contribute to basal thermoregulation
or cardiovascular control, the stress-driven ad
hoc inputs from the DP/DTT are likely inte-
grated in the DMH with the homeostatic tonic
inputs from the preoptic area by impinging on
the DMH→rMR efferent neurons. In addition,
stress-induced visceral and cutaneous vasocon-
strictionmayalsobemediatedinpartbyDMH
neurons innervating the rostral ventrolateral
medulla ( 28 , 29 )(Fig.5F).
TheDP/DTT→DMH pathway appears to
constitute a key psychosomatic connection
through which stress and emotions affect the
autonomic and behavioral motor systems. Al-
though the corticolimbic circuits that process
stress and emotions are undetermined, the PVT
and MD thalamic nuclei, which provide stress
inputs to the DP/DTT, constitute a fear stress
circuit involving the amygdala ( 30 , 31 ). Thus,
the stress and emotion signals processed by
forebrain circuits are likely integrated at the
DP/DTT and then transmitted to the DMH.
In panic disorder, glutamatergic inputs to the
DMH to develop the panic-prone state ( 32 )may
be provided from the DP/DTT. The DP/DTT→
DMH pathway is a potential target for treating
psychosomatic disorders that involve aberrant
physiological responses, particularly because
this pathway does not contribute to basal
autonomic homeostasis.REFERENCES AND NOTES- T. Oka,Temperature 2 , 368–378 (2015).
- B. Lkhagvasuren, Y. Nakamura, T. Oka, N. Sudo, K. Nakamura,
Eur. J. Neurosci. 34 , 1442–1452 (2011).
Kataokaet al.,Science 367 , 1105–1112 (2020) 6 March 2020 7of8
RESEARCH | RESEARCH ARTICLE