206 | Nature | Vol 581 | 14 May 2020
Article
toxin daily for 6 days to ablate DTR-expressing CD4 T cells. As shown
in Extended Data Fig. 5d, e, significantly fewer SPPCs were generated
in the ChAT-IRES-Cre:Rosa26DTR group. GC responses were not signifi-
cantly altered, indicating that the deletion procedure did not cripple
the helper T cell compartment. These data support the possibility
that ChAT-expressing T cells serve as a relay between the sympathetic
splenic nerve and the acetylcholine-responsive process of SPPC forma-
tion. Other cell types may also contribute acetylcholine in this regard,
although we did not find evidence to implicate B-cell-derived acetyl-
choline (Extended Data Fig. 5f–h).
To trace the brain origin of splenic nerve-dependent immuno-
stimulatory neural activity, we conducted retrograde tracing by inject-
ing fluorescent protein-expressing recombinant pseudo-rabies virus
(PRV) Bartha strain into the spleen^5. Over 96 h, PRV ascended from the
spleen up into the spinal cord, brain stem and hypothalamus (Sup-
plementary Tables 1, 2, Extended Data Fig. 6a–d), similar to findings
in rats^6. In the forebrain, the CeA and PVN were prominently labelled
(Fig. 3a). The CeA orchestrates physiological and behavioural responses
to threats and fears^7 , while the PVN processes inputs about stress and
controls the hypothalamo-pituitary-adrenocortical (HPA) response^8.
Both CeA and PVN harbour abundant neurons that produce CRH^9 , the
upstream hormone that drives the HPA axis to produce glucocorti-
coids^10 ,^11. We hypothesized that, in contrast to the blood-borne effects
of the HPA axis, the activity of CRH neurons might also be transmitted as
efferent outputs through the splenic nerve to promote SPPC formation.
To test for a connection between CRH neurons in the CeA and PVN
and the splenic nerve, we stereotactically injected an adeno-associated
virus (AAV) that conditionally expresses Channelrhodopsin-2 (ChR2)
and mCherry into the CeA and PVN regions of CRH-IRES-Cre knock-in
mice (Fig. 3b, optogenetic setup). When triggered by light, ChR2 acti-
vates neurons in a Cre-dependent manner. After verification of correct
stereotactic targeting (Fig. 3c), we conducted direct electrophysiologi-
cal recordings from the splenic nerve during optogenetic activation
of CRH neurons in the CeA or PVN (Extended Data Fig. 6e–g). Light
stimulation of CRH neurons induced markedly increased firing through
the splenic nerve (Fig. 3d, e). Therefore, CRH neurons in the CeA and
PVN are connected to and can stimulate the splenic nerve.
To test the functional effects of CRH neuron activity on SPPC forma-
tion, we stereotactically injected an AAV that conditionally expresses
death-inducing active caspase 3 into the CeA and PVN of CRH-IRES-Cre
mice (Fig. 3b, ablation setup). As assessed with the Rosa26Ai3 reporter
line, our targeting and ablation efficiency of CRH neurons was about
80% in both the CeA and the PVN (Extended Data Fig. 7a–c). Follow-
ing immunization, SPPC formation was significantly impaired in the
CRH ablation group as compared to the control group (Fig. 3f, g).
Next, we sought to inhibit the activity of CRH neurons by using a
recombinant AAV that conditionally expresses the inhibitory receptor
hM4D(Gi) (Fig. 3b, inhibition setup). When activated by the designer
drug clozapine-N-oxide (CNO), hM4D(Gi) suppresses neuronal
firing^12. Inhibition of CRH-IRES-Cre neurons in the CeA and PVN wasPVN CeAa bcd(^200) 20 s
μV
Light OFF Light ON Light OFF
(^200) 0.5 s
μV
DREADD CRH neuron inhibition
DREADD CRH neuron activation
Control for DREADD neuron activation
Optogenetic CRH neuron activation
CRH neuron ablation
(^0) BeforeAfter
20
40
60
80
Firing rate (Hz)
e
Control Ablation
0.6 0.4
2.0 1.8
B220
CD138
GL7
FAS
SPPC (%)
0
0.5
1.0
Cont
ro l
Ablation
GC (%)
0
1
2
3
4
Cont
ro l
Ablation
f
AAV to PVN + CeA
–14
NP-KLH immunization
0 13 (day)
Analysis
B6 control mice: control group
CRH-IRES-Cre mice: inhibition group
g
hi
AAV to PVN + CeA
–14
NP-KLH immunization
0 13 (day)
Analysis
1 12
CNO (5 mg kg–1 per day, drinking water)
0.6 0.4
1.0 1.1
B220
CD138
GL7
FA
S
Control Inhibition
0
0.5
1.0
SPPC (%)
Cont
ro l
Inhibition
0
1
2
GC (%)
Cont
ro l
Inhibition
AAV to PVN + CeA
–14
NP-KLH immunization
0 13 (day)
Analysis
812
CNO (i.p. 1 mg kg–1 b.i.d.)
CRH-IRES-Cre mice
jk
0
0.5
1.0
SPPC (%)
Cont
ro l
Activation
0
1
2
3
4
GC (%)
Cont
ro l
Activation
CAG taCaspase3
CAG taCaspase3
CRH-IRES-Cre
EF1α ChR2-mCherry
EF1α ChR2-mCherry
CRH-IRES-Cre
EF1α hm4D(Gi)-mCherry
EF1α hm4D(Gi)-mCherry
CRH-IRES-Cre
EF1α hm3D(Gq)-mCherry
EF1α hm3D(Gq)-mCherry
CRH-IRES-Cre
EF1α eYFP
EF1α eYFP
CRH-IRES-Cre
1.0 1.0
0.5 0.6
B220
CD138
GL7
FAS
Control Activation
P < 0.0001
P = 0.0007 P = 0.1
P = 0.03 P = 0.8
P = 0.01 P = 0.5
B6 control mice: control group
CRH-IRES-Cre mice: inhibition group
Fig. 3 | CRH neurons in CeA and PVN promote SPPC formation.
a, Representative coronal brain sections showing mRFP-labelled PVN (top) and
CeA (bottom) from mice injected with PRV–mRFP in the spleen 96 h previously.
Left, whole-brain views overlaid on the mouse brain atlas (scale bar, 1 mm);
right, magnified views of the circled PVN and CeA regions (scale bar, 100 μm).
White, PRV–mRFP; blue, DAPI. b, A AV constructs used to manipulate CRH
neurons. DIO-ChR2 for optogenetic activation, DIO-Caspase 3 for cell ablation,
DIO-hM4D(Gi) for pharmacogenetic inhibition, DIO-hM3D(Gq) for
pharmacogenetic activation, and DIO-fluorescent protein as infection control.
c, Representative images of PVN and CeA regions from a CRH-IRES-Cre mouse
injected with A AV-DIO-eYFP; white, YFP; blue, DAPI. Scale bars, 100 μm. d, A
representative trace of electrical activity in the splenic nerve before and during
ChR2-mediated activation of CeA or PVN CRH neurons. Spikes >120 μV are
marked in red. e, Firing rates of the splenic nerve before and after optogenetic
stimulation of CeA and PVN CRH neurons (n = 10 trials, 5 male mice, 3
experiments; two-tailed paired t-test). f, g, Immunization following ablation of
PVN and CeA CRH neuron ablation. f, Protocol (NP-KLH immunization is
intraperitoneal (i.p.)); g, representative contour plots and summary data of
SPPC and GC formation. Each symbol denotes one mouse; lines denote means;
pooled data from three experiments. h, i, Immunization while PVN and CeA
CRH neurons are inhibited. h, Protocol; i, representative contour plots and
summary data of SPPC and GC formation. Each symbol denotes one mouse;
lines denote means; pooled data from four experiments. j, k, Immunization
while PVN and CeA CRH neuron activity is enhanced. j, Protocol; k,
representative contour plots and summary data of SPPC and GC formation.
Each symbol denotes one mouse; lines denote means; pooled data from four
experiments. Two-tailed unpaired t-test (g, i, k).