Article
NMDG artificial cerebrospinal fluid (ACSF) solution (93 mM NMDG,
93 mM HCl, 2.5 mM KCl, 1.25 mM NaH 2 PO 4 ,, 10 mM MgSO 4 ·7H 2 O,
30 mM NaHCO 3 , 25 mM glucose, 20 mM HEPES, 5 mM sodium ascorbate,
3 mM sodium pyruvate, 2 mM thiourea). After perfusion, the brain
was rapidly removed, immediately transferred into ice-cold, oxygen-
ated NMDG ACSF solution, and sectioned coronally into 280-μm slices
with a vibratome (VT1200 S, Leica). Brain slices containing the PVN
and CeA were incubated in oxygenated NMDG ACSF solution at 32 °C
for 15 min, and then transferred into a normal oxygenated ACSF solu-
tion (126 mM NaCl, 2.5 mM KCl, 1.25 mM NaH 2 PO 4 , 2 mM MgSO 4 ·7H 2 O,
10 mM glucose, 26 mM NaHCO 3 , 2 mM CaCl 2 ) for incubation at room
temperature for 1 h. All chemicals used in slice preparation were pur-
chased from Sigma-Aldrich (St. Louis, MO, USA).
Electrophysiological recording from brain slices
Slices were placed in the recording chamber, submerged and perfused
with ACSF at a rate of 3 ml min−1 at 28 °C. CRH+ positive neurons were
identified by differential interference contrast optics (DIC; Olympus
BX61WI). The recording pipettes (3–4 MΩ) were prepared using a micro-
pipette puller (P97, Sutter Instrument; USA). To analyse firing rates,
cell-attached or whole-cell recording was conducted. For cell-attached
recording, the pipette was filled with ACSF solution. Cells were held at
0 pA under a current-clamp mode to record spontaneous firing. CNO
(5 μM) was locally perfused to the cell through a drug perfusion system.
For each recording, the baseline of spontaneous firing was recorded
for at least 3 min before CNO was given. For whole-cell recording, the
pipette was filled with ACSF solution containing 133 mM potassium
gluconate, 18 mM NaCl, 0.6 mM EGTA, 10 mM HEPES, 2 mM Mg·ATP,
and 0.3 mM Na 3 ·GTP (pH 7.2, 280 mOsm). All recordings were acquired
using a Multiclamp 700B amplifier and signals were low-pass filtered
at 3 kHz and digitized at 10 kHz (DigiData 1550, Molecular Devices).
Data were analysed using Clampfit 10 software (Molecular Devices),
Mini Analysis Program (Synaptosoft) and Matlab R2016b programs.
Measurement of serum corticosterone
Mice were bled from the orbital sinus under pentobarbital anaesthesia.
Blood samples were left to coagulate at room temperature for 2 h before
serum harvesting by 10-min centrifugation at 3,000g. Sera were mixed
with 100% methanol and centrifuged for 10 min at 4 °C. The superna-
tants, containing free-state corticosterone, were then vacuum-dried
(Savant SpeedVac SPD121P, Thermo Fisher) and submitted for LC–MS.
Detection and quantification of corticosterone were carried out using a
Waters Acquity I Class UPLC system connected to an AB Sciex tripleTOF
mass spectrometer with electrospray ionization. The compound was
detected in positive ion mode. Calibration curves from 14.43 to 577.27
nmol/l were constructed using commercial corticosterone standard
(CAS No. 50-22-6, Cayman) and the peak area was linear to concentra-
tion over this range (R^2 > 0.99). The area-under-the-curve upon LC−MS
elution was used to quantify the corticosterone level in the samples.
Statistical data analysis
Statistics and graphing were conducted in Prism (Graphpad). Unless
specifically noted otherwise, two-tailed unpaired Student’s t-tests were
used to compare the endpoint means of two groups. Non-parametric,
two-tailed Mann–Whitney tests were used in cases of highly skewed
distributions.Reporting summary
Further information on research design is available in the Nature
Research Reporting Summary linked to this paper.Data availability
Data generated here are included within the paper (and its Supplemen-
tary Information files) or are available from the corresponding authors
upon reasonable request. Source data for Figs. 1–5 and Extended Data
Figs. 1–10 are provided with the paper.- Wang, Y. et al. Germinal-center development of memory B cells driven by IL-9 from
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receptor. Nature 504 , 101–106 (2013). - Li, Y. et al. Serotonin neurons in the dorsal raphe nucleus encode reward signals. Nat.
Commun. 7 , 10503 (2016). - Paxinos, G. & Franklin, K. B. J. Paxinos and Franklin’s the Mouse Brain in Stereotaxic
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Acknowledgements We thank S. Chavan for sharing technical information about
electrophysiological recording. The work was funded in part by the National Key R&D Program
of China (Ministry of Science and Technology, 2018YFE0200300 to H.Q.), National Natural
Science Foundation of China (grants 81621002, 31830023 to H.Q.; grants 61890951,
61890950, 31671086 to J.H.), the Tsinghua-Peking Center for Life Sciences, and the Beijing
Municipal Science & Technology Commission.Author contributions H.Q. conceptualized and supervised the study. X.Z. developed the
splenic denervation technique and, together with L.Z., the EPS regimen. X.Z., B.L. and Y.Y.
participated in designing the overall study together with H.Q., J.H., and Y.Z. X.Z. and L.Z.
conducted most of the immunological experiments. L.H. synthesized the acetylcholine
analogue under the supervision of X.L. Y.Y., S.J., and L.Z. conducted the PRV tracing
experiments under the supervision of F.X. and J.H. X.Z. and Y.Y. conducted
electrophysiological recording of the splenic nerve. X.Z., Y.Y. and L.Z. conducted CRH
neuron ablation and fibre photometry studies under the supervision of J.H., W.S. and H.Q.
B.L., X.Z., B.K. and L.Z. conducted CRH neuron inhibition and activation studies under the
supervision of Y.Z. and H.Q. All authors contributed to data interpretation. H.Q. and X.Z.
wrote the paper with input from all authors.Competing interests The authors declare no competing interests.Additional information
Supplementary information is available for this paper at https://doi.org/10.1038/s41586-020-
2235-7.
Correspondence and requests for materials should be addressed to Y.Z., J.H. or H.Q.
Peer review information Nature thanks Jonathan Kipnis and the other, anonymous, reviewer(s)
for their contribution to the peer review of this work.
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