MATERIALS AND METHODS
Animals
Adult male 6- to 8-week-old wild-type C57BL/6 mice and ChAT-ChR2-EYFP
transgenic mice (subsequently referred to as ChAT mice) were used in our study.
C57BL/6 mice were purchased from the Animal Center of Zhejiang University,
Hangzhou, China. The ChAT mice were provided by Laboratory of Neurobiology,
Zhejiang University School of Medicine, Hangzhou, China. ChAT-ChR2-EYFP
transgenic mice were generated using a BAC clone containing the ChAT gene. ChR2-
EYFP was inserted into bacteria via homologous recombination into the ATG site of
the ChAT gene. Endogenous ChAT gene expression was disrupted to avoid ChAT
overexpression. ChAT-EYFP was stably expressed under the control of the choline-
acetyltransferase promoter (ChAT) in channelrhodopsin-2(ChR2)-enhanced
fluorescent protein (EYFP)-tagged neurons in the basal forebrain, habenula, and other
brain regions in all ChAT mice. All animal experiments were performed according to
procedures approved by the Animal Care and Use Committee of Zhejiang University.
Before starting experimental procedures, animals were housed in individual chambers
under standard conditions (room temperature, 22-23°C; humidity 40-60%; and a
12:12-h light/dark cycle) with access to regular food and water. All animals were
acclimated for at least 10 days.
Photostimulation of Basal Forebrain Cholinergic Neurons in ChAT Mice
Photostimulation was induced unilaterally on the right basal forebrain. An optical
fiber was inserted into an implanted cannula one day before the experiments. We
confirmed that ChR2 expression was selectively induced with high precision by
applying blue light in basal forebrain cholinergic neurons of ChAT mice (19, 21). The
optical fiber was coupled to a 473-nm laser under driver control. Light pulse-trains
(30 ms pulses at 20 Hz for 15 s once per minute for 30 min every 1.5 hr) were
controlled using digital commands and a Master-8 pulse stimulator.
Surgical Implantation
A guide cannula (RWD Life Science) was unilaterally surgically implanted in all. The
animals were anesthetized using chloral hydrate (400 mg/kg, i.p.) and mounted on a
small animal stereotaxic frame (Stoelting Corp.). A cannula was placed above the
right basal forebrain [antero-posterior (AP), 0.7 mm; mediolateral (ML), 1.6 mm;
dorso-ventral, 4.0 mm] according to the mouse brain in stereotaxic coordinates (2nd
edition, Keith B.J. Franklin and George Paxinos). Four skull screw-holes were drilled,
and tightly fitting screws were driven through the skull to the surface of the dura.
After surgery, the animals were allowed to recover in individual chambers for at least
7 days.
Polymicrobial Sepsis Model
A cecal ligation and puncture (CLP)–induced sepsis model was generated as
previously described (22). The mice were anesthetized using an injection of chloral
hydrate (400 mg/kg, i.p.). The cecum was exteriorized via a 1-cm abdominal midline
incision and ligated using a 4-0 silk ligature midway between the distal pole and base
of the cecum. The cecum was then punctured once through both surfaces using an 18-
gauge needle at the middle of the ligation and the tip of the cecum. The cecum was
replaced after a small amount of fecal material was extruded, and the abdomen was
then closed. All mice received 1 ml 0.9% normal saline subcutaneously after surgery.
Sham CLP mice underwent the same procedure described above but were not ligated
and punctured.
Left Cervical Vagotomy
A ventral cervical midline incision was used to expose the left cervical vagus trunk,
which was ligated using 4-0 silk sutures and excised at least 1 cm. The skin was then
closed using 3-0 sutures. In sham-operated mice, the left vagus nerve was exposed
and isolated from the surrounding tissue but not transected. All animals were
vagotomized three days before CLP.
Cytokine Measurements
Blood was collected at the indicated time points, allowed to clot for 2 h at room
temperature, and centrifuged at 14,000 rpm for 15 min at 4°C. The supernatants were
then collected for cytokine determination. Spleens were obtained from mice after
CLP, frozen in liquid nitrogen and grinded using a mortar and pestle. A 500 μl volume
of RIPA+1% PMSF was added. The mixture was then centrifuged at 14,000 rpm for
15 min at 4°C, and the supernatants were collected to determine cytokine levels.
Serum and tissue supernatants were used to analyze the protein levels of TNF-α, IL-6
and IL-10 using ELISA (R&D Systems, Minneapolis, MN) according to the
manufacturer’s recommendations.
Brain Slice Preparation
Adult ChAT mice were deeply anesthetized using 4% chloral hydrate (400 mg/kg,
i.p.) and transcardially perfused with cold normal saline followed by 4%
paraformaldehyde in 0.1 M PBS. Whole brains were post-fixed for 12 h and then
cryoprotected in 10%/20%/30% sucrose solutions until the brain sank to the bottom of
the centrifuge tube at 4°C. After the brains were embedded in optimal cutting
temperature compound, they were sectioned coronally or sagittally at 30 μm using a
freezing microtome (VTA-1000S; Leica). Brain slices were stored at -20°C.
Immunohistochemistry
Slices containing the basal forebrain were rinsed with 0.3% Triton X-100 in 0.1 M
PBS and blocked in 5% donkey serum for at least 2 h at 4°C. Sections were incubated
with c-fos (rabbit anti-c-fos, 1:1000, Synaptic Sytems, Inc.), TH (sheep anti-TH,
1:1000, Millipore, Inc.) and ChAT (goat anti-ChAT, 1:500, Millipore, Inc.) primary
antibodies in 0.1 M PBS containing 0.3% Triton X-100 for 48 h at 4°C. Up to two
compatible primary antibodies were added together. For c-fos labeling, we incubated
slices with Biotin-SP-conjugated Affinipure Donkey anti-Rabbit IgG for 12 h at 4°C
(1:500, Jackson Immunoresearch Laboratories, Inc.). Sections were then rinsed
repeatedly and incubated with streptavidin and Texas Red®-X conjugate (1:200,
Molecular Probes®, Labeling&Detection Technologies, Thermo Fisher
Scientific, Inc.) for 2 h at 4°C. When TH and ChAT were combined with c-fos
double-labeling, sections were incubated in Biotin-SP-conjugated Affinipure Donkey
anti-sheep IgG and Biotin-SP-conjugated Affinipure Donkey anti-goat IgG for 12 h at
4°C (1:500, Jackson Immunoresearch Laboratories, Inc.). After the sections were
washed three times (10 min each) with PBS, the sections were incubated for 2 h at
4°C with the following secondary antibodies: anti-rabbit IgG (H+L) Cross-Adsorbed,
Cyanine3 (1:500, Invitrogen™, Thermo Fisher Scientific, Inc.) and streptavidin, or
Alexa Fluor®-647-conjugated (1:200, Invitrogen™, Thermo Fisher Scientific, Inc.).
After immunofluorescence labelling, the sections were washed and immersed in
DAPI (1:1000, Sigma-Aldrich, St. Louis, MO) for 10 min on a shaker to stain cell
nuclei. Finally, the labelled sections were mounted and coverslipped in FluorSave
reagent (Calbiochem®, EMD Chemicals, Inc.). Immunostained neurons were
immediately observed using a fluorescence microscope (BX53, Olympus Life
Science).
Administration of 6-hydroxydopamine
Chemical splenic denervation was performed by injecting 6-hydroxydopamine (6-
OHDA, 60-120μg, Sigma-Aldrich, St. Louis, MO) or saline into exteriorized spleens
according to the method described by Joseph C. Gigliotti et al. (23). The mice were
then allowed to recover for 1 week before photostimulation.
Statistical Analysis
All statistical analyses were performed using SPSS16.0 for Windows (SPSS Inc.,
Chicago, IL) and GraphPad Prism 5.00 for Windows (GraphPad Software Inc., La
Jolla, CA). All data shown in the figures and text are presented as the mean ± SEM.
One-way analysis of variance (ANOVA) followed by a Bonferroni test were
performed to compare mean values between multiple groups. Survival rates were
analyzed using the Mantel–Cox test. The threshold for significance was set at
P<0.05.
Fig. S1. The survival rate between ChAT-lit mice and ChAT-unlit mice after CLP
Fig. S2. c-fos expression in the basal forebrain and the dorsal motor nucleus of the
vagus/ventral part of solitary nucleus neurons when basal forebrain cholinergic
neurons were not photostimulated. (A) Schematic drawing of the brain slice
experiment, shown in different coronal plates. The first plate shows a coronal section
in which the cannula was placed above the BF. The red box represents the area
containing ch-BF neurons. (B) The second plate shows a coronal section that contains
the DMN/SolV. The areas in the red line includes the DMN/SolV neurons. (C-
F) When no photostimulation was applied, very few c-fos-expressing neurons were
observed in either the BF or the DMN/SolV, and none of them were ChAT+ neurons.
Fig. S3. CHAT positive dopaminergic neurons occupy only 6.1% of c-fos-positive
neurons in the dorsal motor nucleus of the vagus/ventral part of solitary nucleus
activated by photostimulation of basal forebrain cholinergic neurons (A1-D1)
Immunofluorescent labelling showed that c-fos-positive neurons in the DMN/SolV
were not CHAT+. (A2-D2) Serial image projection.
Fig. S4. The serum concentrations of inflammatory cytokines in ChAT-lit septic mice
and ChAT-lit septic mice that were injected with 6-OHDA to the spleen. (A-B) TNF-α
and IL-6 levels were lower in the serum in the ChAT septic mice that were not
injected with 6-OHDA to the spleen. This effect was fully reversed in the ChAT mice
that were administered 6-OHDA. (C) IL-10 level were not significantly different
between ChAT-lit and ChAT-lit mice that were injected with 6-OHDA to the spleen.
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