(National Institute of Biological Sciences, Beijing, China). DIO-eYFP,
DIO-hM3D(Gq)-mCherry, and DIO-hM4D(Gi)-mCherry were from
Vigenebio Inc. Surgical procedures were as previously described^20.
In brief, mice were anaesthetized by intraperitoneal injection of
pentobarbital (100 mg kg−1), kept warmed with an electric heating
pad (BrainKing Biotech), and mounted on a stereotaxic apparatus
to adjust the skull in parallel to the reference panel. Using a microsy-
ringe pump (Nanoject III #3-000-207, DRUMNOND), AAV virus was
slowly injected (20 nl min−1) into PVN (Bregma coordinates: −0.6 mm
AP, ±0.2 mm ML, −4.8 mm DV) and CeA (Bregma coordinates: −1.23
mm AP, ±2.75 mm ML, −4.5 mm DV), unilaterally for fibre photome-
try or bilaterally for pharmacogenetic experiments. The pump was
maintained in position for an additional period of 10 min before slow
withdrawal to allow viral particles to diffuse and be absorbed at the
injection site. For fibre photometry, coordinates for fibre implanta-
tion were offset relative to the viral injection sites (Bregma for PVN:
−0.6 mm AP, ±0.2 mm ML, −4.5 mm DV; bregma for CeA: −1.23 mm
AP, ±2.75 mm ML, −3.9 mm DV). Specific volumes of AAV injected to
each site were as follow: AAV2/9-CAG-DIO-taCasp3-TEVp (1.72 × 10^13
IFU/ml; 100 nl to PVN and CeA), AAV2/9-hsyn-DIO-GCaMP6m (1–5 × 10^12
IFU/ml; 300 nl to PVN and CeA), AAV9-EF1α-DIO-hM3D(Gq)-mCherry
(1.85 × 10^13 vector genomes (v.g.)/ml; 120 nl to PVN and 150 nl to CeA),
AAV9-EF1α-DIO-hM4D(Gi)-mCherry (1.53 × 10^13 v.g./ml; 120 nl to PVN
and 150 nl to CeA), AAV9-EF1α-DIO-eYFP (5.23 × 10^13 v.g./ml; 120 nl to
PVN and 150 nl to CeA), AAV2/9-EF1α-DIO-ChR2-mCherry (1–5 × 10^12
v.g./ml; 300 nl to PVN and 300 nl to CeA).
PRV retrograde tracing from the spleen
Pseudorabies virus (PRV-CMV-mRFP or PRV-CMV-GFP) was prepared as
stock of 10^9 genomic copies per ml. Adult B6 mice were anaesthetized
with 75 mg/kg sodium pentobarbital. The spleen was accessed through a
midline opening to the peritoneal cavity. One microlitre of the viral stock
solution was injected into each of the upper and lower tips of the spleen
using a syringe fitted with a needle 30 μm in diameter. The surgical wound
was closed by standard sutures and the indicated numbers of mice were
killed 24, 48, 72 and 96 h later to retrieve the spinal cord and brain. Sec-
tions were prepared and processed for identification of PRV–mRFP+ neu-
rons in different regions. In brief, brains were fixed in 4% PFA overnight
and dehydrated by 30% sucrose at 4 °C for 2–3 days. Coronal sections
(35 μm) were cut with a cryostat microtome (Leica). Tissue sections were
blocked with PBS containing 5% BSA and 0.3% Triton X-100, stained with
rabbit polyclonal anti-dsRed (632496, Clontech; 1:1,000) for 48 h at 4 °C,
washed three times, and then stained with secondary antibody AF594
donkey anti-rabbit IgG (1:1,000) for 2 h at room temperature.
Images were then acquired with an Olympus VS120 microscope and
analysed using Nikon A1R software. Different brain regions were identi-
fied using the Allen Mouse Brain Atlas (https://mouse.brain-map.org/)
and the Mouse Brain in Stereotaxic Coordinates^21.
Recording of splenic-nerve activity during optogenetic
stimulation of CRH neurons
CRH-IRES-Cre mice were stereotactically infected with
AAV2/9-EF1α-DIO-ChR2-mCherry at the CeA and PVN. Fibre implanta-
tion was offset relative to the viral injection sites (bregma for PVN: −0.6
mm AP, ±0.2 mm ML, −4.5 mm DV; bregma for CeA: −1.23 mm AP, ±2.75
mm ML, −3.9 mm DV). Two weeks later, these mice were anaesthetized
by an intraperitoneal injection of 1% pentobarbital sodium. The splenic
nerve was exposed in a similar manner as described for denervation
surgery above. The electrode was custom-made by CorTec GmbH
(1041.2406.51 - 2 Micro Cuff Sling 100 /Pt-Ir/ 1mm long /0.35mm C2C
/0.3 × 0.5mm Opening / Cable entry top – welded) and was implanted
around the splenic nerve so that the nerve could sit atop the metallic
surface of the electrode probe. The implanted electrode was connected
to a multichannel recording and signal processing system (LabChart,
ADInstruments). To activate CRH neurons expressing ChR2 for each
trial, a 473-nm laser light was delivered as 2-ms pulses at 50 Hz for 200
ms every second. Light intensity was adjusted with an optical power
meter (Sanwa) to reach 10 mW at the end of the implanted fibre stub.
Recording signals were sampled at 1,000 Hz. For data analysis, all sig-
nals were digitally bandpass-filtered between 10 and 60 Hz to reduce
noise. Firing spikes were automatically called by Matlab (R2016b) func-
tion findpeaks with a threshold set at 120 μV. Average firing rates during
20 s before and 20 s after optogenetic stimulation of CRH neurons were
calculated and statistically analysed by paired t-test.Pharmacogenetic studies
For activation of CRH neurons in PVN and CeA, groups of male
CRH-IRES-Cre mice were stereotactically injected with DIO-hM3D(Gq)-
or control AAV into the PVN and CeA regions. Under anaesthesia by
1.5% isoflurane, these mice were intraperitoneally injected with 1 mg/
kg CNO (Enzo) twice daily, ~10 h apart, between days 8 and 12 after
NP-KLH immunization. For inhibition of CRH neurons in PVN and CeA,
male CRH-IRES-Cre or control B6 mice were maintained with drinking
water that was supplemented with CNO to achieve a daily intake of ~5
mg/kg CNO, assuming water consumption of 5 ml per day per mouse.
The CNO-supplemented water was given from 2–3 days before immu-
nization to 12 days after immunization.The EPS behaviour regimen
Mice were individually placed on a transparent plastic platform of 10 cm
in diameter and raised 150 cm above the ground. For each session, the
animal was acoustically and visually isolated for the indicated amount
of time. To test the effect on GC and plasma cell formation, mice were
subjected to 3 min EPS twice daily for the indicated number of days,
one administered between 10:00 and 12:00 and the other between
22:00 and 24:00 each day.The PBR behaviour regimen
Mice were individually placed into a Mouse DecapiCone disposable
restrainer (Braintree Scientific Inc.), with the open end of the restrainer
cable-tied near the base of the mouse tail. For each session, the animal
was acoustically and visually isolated for 90 min. To test the effect on
GC and plasma cell formation, mice were subjected to the PBR twice
daily as scheduled for EPS.Fibre photometry
Following AAV-DIO-GCaMP6m or control AAV-DIO-GFP virus injection,
an optical fibre with an outer diameter of 200 μm and 0.37 numerical
aperture (Anilab) was placed in a ceramic ferrule and inserted towards
the PVN through the craniotomy. Mice were individually housed for at
least 2 weeks to recover. Fluorescence signals were acquired with a fibre
photometric system equipped with a 488 nm excitation laser, 505–544
nm emission filter and a photomultiplier tube (R3896, Hamamatsu). The
analogue voltage signals were digitalized at 100 Hz and recorded using
a Power 1401 digitizer and Spike2 software (CED, Cambridge, UK). To
connect the implanted fibre and the photometric system, an optical fibre
(RJPSF2, Thorlabs) with integrated rotary joint was used to prevent fibre
damage as a result of animal movement. The laser power was adjusted at
the tip of the optical fibre to a low level of 20–40 μW in order to minimize
bleaching. Acquired photometry data were exported to Matlab R2016b
mat files from Spike2 for further analyses. Data were segmented accord-
ing to EPS behavioural events within individual trials. The normalized
change in fluorescence signal (ΔF/F) was calculated as (F−F 0 )/F 0 , where
F 0 is the average baseline fluorescent signal before EPS. ΔF/F values were
plotted as an average trace bracketed by a shaded area indicating s.e.m.Preparation of brain slices
Adult (8–12-week-old) male CRH-IRES-Cre mice:Rosa26-Ai3 mice were
anaesthetized by intraperitoneal injection of pentobarbital (100 mg kg−1)
and then perfused transcardially with ice-cold, oxygenated (95% O 2 /5% CO 2 )