Science_-_6_March_2020

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protein (GFP) ( 19 ), using adeno-associated virus
(AAV) (Fig. 3A) allowed us to visualize their
numerous axons projecting to the DMH. We
found close association of these axons with
DMH neurons retrogradely labeled with CTb
from the rMR (Fig. 3, B and C, and fig. S3A),
indicating putative synapses formed by DP/DTT-
derived axons on DMH→rMR projection
neurons. Confocal imaging revealed that the
swellings of DP/DTT-derived axons apposed
to rMR-projecting DMH neurons contained
VGLUT1 (vesicular glutamate transporter 1),
a marker for cortical glutamatergic neurons,
but not VGLUT2, a subcortical glutamatergic
neuron marker, or VGAT (vesicular GABA trans-
porter), a GABAergic neuron marker (Fig. 3, D
to F, and fig. S3B).
Consistent with the view that glutamatergic
inputs to the DMH mediate stress responses,
blockade of glutamatergic synapses in the DMH
with nanoinjections of a mixture of 2-amino-5-
phosphonovaleric acid and cyanquixaline (AP5/
CNQX) inhibited SDS-induced sympathetic re-
sponses for the first 20 min of stress exposure


(TBAT: 88% inhibition,Tcore: 80% inhibition, HR:
56% inhibition, MAP: 41% inhibition; fig. S3,
C to F), after which the antagonistic effects
ofthedrugswaned.Thedifferenceinthein-
hibitory effects of AP5/CNQX on the thermal
and cardiovascular responses may be because
the distribution of cardiovascular neurons in
the DMH is broader than that of thermoreg-
ulatory neurons ( 20 ).

In vivo optogenetic stimulation of DP/
DTT→DMH glutamatergic transmission elicits
sympathetic responses
To directly examine the sympathoexcitatory role
of the DP/DTT→DMH pathway, we selectively
stimulated DP/DTT→DMH monosynaptic trans-
mission by using an in vivo optogenetic tech-
nique (Fig. 3G). ChIEF, a channelrhodopsin
variant ( 21 ), was used to induce membrane
depolarization and action potentials in neurons
with exposure to light. AAV transduction of
DP/DTT neurons with ChIEF-tdTomato or
palGFP resulted in the localization of the ex-
pressed proteins in their cell bodies in the

DP/DTT and nerve endings densely distributed
in the DMH (Fig. 3, H and I, and fig. S4, A and B).
Illumination of ChIEF-tdTomato–containing
nerve endings, but not palGFP-containing
nerves, in the DMH with pulsed blue laser
light for 30 s consistently increased BAT SNA,
TBAT, HR, and MAP in anesthetized rats (Fig.
3, J and K, and fig. S4C). Longer illumination
(180 s) elicited larger increases, and the photo-
stimulated BAT thermogenesis was as intense
as that induced by SDS (fig. S4, D and E, and
table S1). Photostimulation of DP/DTT→DMH
transmission activated the DMH→rMR path-
way (fig. S4, G to I). Consistent with the view
that DP/DTT→DMH transmission driving the
responses is glutamatergic, antagonizing glu-
tamate receptors in the DMH eliminated all
sympathetic responses elicited by photostimu-
lation of DP/DTT→DMH transmission (Fig. 3,
L to O, and fig. S4F).
DP/DTT-derived nerves with ChIEF-tdTomato
were also distributed in the lateral hypo-
thalamic area (LH) (Fig. 3I), which contains
orexin neurons proposed to participate in

Kataokaet al.,Science 367 , 1105–1112 (2020) 6 March 2020 2of8


f

mt
3V
DMH

opt

DTT Acb

PrL

IL
DP

3V

DMH

VMH

Control
Stress

Control

fmi

DTT

PrL

IL
DP

Cg

Nv

E/OVac

Acb

M2



**




% of Fos expressionin CTb-labeled cells

Stress

Control

E Control Stress

F

fmi

LV

C

A

B

DTT Acb

PrL

IL
DP

LV

E

DTT

PrL

IL
DP

DTT

PrL

IL
DP

LV

E

DTT

PrL

IL
DP

D

Rostral Caudal
Bregma: +2.8 mm +2.4 mm

:CTb+ :CTb+, Fos+

DTT

PrL

IL

DP

Stress

DTT

PrL

IL
DP

Cg

Nv
Acb

fmi

E/OV
ac

M2

0

4

8

12

16

20

PrL IL DP DTT





DP DP

IL IL

DTT DTT

Fig. 1. SDS activates DMH-projecting neurons in the mPFC.(A) CTb injection
into the DMH. 3V, third ventricle; f, fornix; mt, mammillothalamic tract; opt, optic
tract. Scale bar, 500mm. (B) CTb injection sites in the rats for which data are
plotted in (F). VMH, ventromedial hypothalamic nucleus. (C) CTb-labeled neurons
(arrowheads) in the mPFC. fmi, forceps minor of the corpus callosum; LV, lateral
ventricle. Scale bars, 500mm (main panel); 30mm (inset). (D) Distribution of
CTb-labeled cells with or without Fos immunoreactivity in the mPFC after sham
handling (Control) or SDS (Stress). ac, anterior commissure; Acb, nucleus


accumbens; Cg, cingulate cortex; E, ependymal and subependymal layer; M2,
secondary motor cortex; Nv, navicular nucleus; OV, olfactory ventricle.
(E) Stress-induced Fos expression (blue-black) in CTb-labeled (brown) cells in
the DP (arrows). Note the typical pyramidal neuron morphology. Scale bars,
200 mm (main panel); 30mm (inset). (F) Percentages of Fos-immunoreactive
cells in CTb-labeled populations in mPFC subregions (n= 4 rats per group).
*P< 0.05; **P< 0.01; ***P< 0.001 (unpairedttest; PrL,t 6 = 6.16; IL,t 6 = 5.93;
DP,t 6 = 7.41; DTT,t 6 = 2.47). Error bars indicate SEM.

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