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.