Male C57BL/6 J wild type and MyD88KO (Stock# 009088) mice were purchased from The Jackson Labs (Bar Harbor, ME, USA). Nestin-cre mice (Stock# 003771) and MyD88 Flox mice (Stock# 008888) were obtained from The Jackson Labs, and crossed to obtained Nes-cre+/#8722;/MyD88 Flox+/+ mice. These mice were then crossed back to the original MyD88 Flox+/+ line to obtain MyD88ΔCNS and MyD88Flox littermate controls. Nestin-cre mice were also bred to mice harboring a Flox-Stop-Flox tdTomato behind a CAG promoter knocked into the ROSA locus (The Jackson Labs, Stock# 007908) or nuclear-localized green fluorescent protein/beta-galactosidase fusion protein (GNZ) (The Jackson Labs, Stock# 008606) to examine the cellular specificity of cre-mediated recombination. All mice were genotyped using standard protocols from The Jackson Labs. All animals were maintained on a normal 12:12 hr light/dark cycle and provided ad libitum access to water and food (Purina rodent diet 5001; Purina Mills, St. Louis, MO, USA). Mice were used for experiments at between 6 and 10 weeks of age. Experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and approved by the Animal Care and Use Committee of Oregon Health and Science University.
26-gauge lateral ventricle cannulas were placed (PlasticsOne, Roanoke, VA, USA) under isofluorane anesthesia, using a stereotactic alignment instrument (Kopf, Tujunga, CA, USA) at the following coordinates relative to bregma: -1.0 mm X, -0.5 mm Y and −2.25 mm Z. Ten ng mouse IL-1β or 500 ng mouse tumor necrosis factor (TNF, R&D, Minneapolis, MN, USA) injections were given in 1 μL total volume. IL-1β and TNF were dissolved in artificial cerebrospinal fluid (aCSF, 150 mM NaCl, 3 mM KCl, 1.4 mM CaCl2, 0.8 mM MgCl2, 1.0 mM NaPO4) with 0.1% endotoxin free BSA.
LPS (Sigma, St. Louis, MO, USA) was dissolved at 62.5 μg/mL in 0.9% saline/0.5% endotoxin free BSA, and injected intraperitoneally at 4 μL/g body weight (250 μg/kg).
Locomotor activity measurement
Voluntary home cage LMA was measured using implantable telemetric transponders (MiniMitter, Bend, OR, USA). Animals were anesthetized using 2% isoflurane, a small midline incision was made in the abdominal wall, and transponders were implanted adjacent to the abdominal aorta in the retroperitoneal space. Transponders were implanted during the lateral ventricle cannulation surgery. Mice were individually housed and allowed to acclimate for at least five days before temperature and net movement in x-, y- and z-axes was recorded in one minute intervals (Vital View, MiniMitter).
Overnight feeding studies
Animals were transferred to clean cages and injected with i.c.v. IL-1β (10 ng), i.c.v. TNF (500 ng) or i.p. LPS (250 μg) 1 h prior to lights off. At 2, 6, 13, 24, 37 and 48 h after the onset of the dark cycle, food was weighed and returned to the cage. Body weight was recorded at 13, 24 and 48 h.
Fast-refeed feeding studies
At lights out, food was removed from the cages and animals were fasted for 12 h. At the start of the light cycle, animals were injected i.c.v with IL-1β (10 ng) and food was returned to the cages 15 minutes later. Food intake was measured at 1, 2, 5, 8, 12 and 24 h after the return of food to the cages.
For histology experiments, mice were deeply anesthetized using a ketamine cocktail and sacrificed by transcardial perfusion fixation with 15 mL ice cold 0.01 M PBS + heparin sodium (15,000 U/L) followed by 25 mL 4% paraformadehyde (PFA) in 0.01 M PBS. Brains were post-fixed in 4% PFA overnight at 4°C and cryoprotected in 20% sucrose for 24 h at 4°C before being stored at −80°C until used for immunohistochemistry (IHC). Dual-immunofluorescence histochemistry was performed as described below. Free-floating sections were cut at 30 μm from perfused brains using a sliding microtome (Leica SM2000R, Leica Microsystems, Bannockburn, IL, USA). Hypothalamic sections were collected from the division of the optic chiasm (bregma −1.0 mm) caudally through the mammillary bodies (bregma −3.0 mm). The sections were incubated for 1 h at room temperature in blocking reagent (5% normal donkey serum in 0.01 M PBS and 0.1% Triton X-100). After the initial blocking step, the sections were incubated in mouse anti-NeuN (1:1,000, Millipore, Billerica, MA, USA), rabbit anti-GFAP (1:500, DAKO, Carpinteria, CA, USA), rabbit anti-IbaI (1:500, DAKO), rat anti-CD31 (1:100, BD Pharmingen, Sparks, MD, USA) in blocking reagent for 72 h at 4°C, followed by incubation in donkey anti-rabbit Alexa 488 (1:500), donkey anti-mouse Alexa 488 or 647 (1:500), goat anti-rat Alexa 488 or 633 (1:500, Invitrogen) for 2 h at room temperature. Between each stage, the sections were washed thoroughly with 0.01 M PBS. Incubating the sections in the absence of primary antisera was used to ensure specificity of the secondary antibodies. Sections were mounted onto gelatin-coated slides, coverslipped using Vectashield mounting media (Vector Laboratories, Burlingame, CA, USA), and viewed on a Nikon A1 inverted laser-scanning confocal microscope (Nikon Instruments, Melville, NY, USA). Sections from Nestin-cre x GNZ animals were cut at 75 μm on a vibratome (Leica Microsystems, Buffalo Grove, IL, USA). Sections were incubated for 30 minutes in 98°C pH 6 sodium citrate buffer, then blocked as above. Sections were then incubated overnight at 4°C in 1:500 chicken anti-GFP antibody (Aves, Tigard, OR, USA). Sections were then washed as above, and incubated for 1 h in goat anti-chicken Alexa 568 and 647. Images were obtained as above, and both red and far red channels superimposed to generate higher anti-GFP signal.
Embryonic Day 14.5 (E14.5) timed pregnant female Nestin-cre/tdTomato mice were sacrificed by cervical dislocation and embryos were collected in cold complete HBSS. Heads were removed and cortices and hypothalami dissected and collected in cold complete HBSS. Tails were collected to confirm genotyping. Cortical and hypothalamic neurons were dissociated and were cultured for five days on Poly-D-Lysine-Laminin-coated glass coverslips in serum-free Gibco Neurobasal Medium containing Invitrogen penicillin-streptomycin and Invitrogen B-27 Supplement. Neurons were fixed in 4% paraformaldehyde in 1X PBS for 20 minutes at room temperature, then blocked and immunostained in a 1% BSA, 0.1% cold fish skin gelatin, 0.5% Triton-X 100, 10 mM TBS solution (blocking buffer). Neurons were immunostained overnight for MAP2 (1:1,000, Aves), Tau (1:10,000, Chemicon, Billerica, MA), NeuN (1:1,000, Chemicon) and βIII Tubulin (1:1,000, Chemicon) to confirm polarity and neuronal identity, respectively. Primary antibodies were applied overnight at 4°C and were washed off the next day 3x with 1XPBS. Hoechst and Alexa-Fluor secondary antibodies were used at 1:1,000 for one hour at room temperature in blocking buffer. Coverslips were mounted on slides and imaged on a Nikon A1 inverted laser-scanning confocal microscope. Neurons were scored for the presence of TdTomato using ImageJ software is open source free software from the National Institutes of Health. It is available at
http://rsbweb.nih.gov/ij/download.html to determine efficiency of recombination.
Muscles were homogenized in cell lysis buffer (Cell Signaling, Danvers, MA, USA): 20 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA 1% Triton X, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM sodium orthovanadate, 1 ug/mL leupeptin, and supplemented with Complete protease inhibitors (Roche, Indianapolis, IN, USA), 1 mM PMSF (Sigma), and 5 μL/mL phosphatase inibitor cocktail II (Sigma). Samples were homogenized for 30 s using a Polytron homogenizer (Kinematica, Bohemia, NY, USA), then were sonicated 2 x 10s. Samples were then centrifuged at 13,000 RPM for 10 minutes at 4°C. For Western blots, 100 ug of total protein per lane was run on Criterion 4 to 15% gradient polyacrylamide gels (Biorad, Hercules, CA, USA), and transferred to Immobilon PVDF membranes (Millipore. Proteins were detected using a Lumi-Imager (Roche) and SuperSignal West Pico Chemiluminescent Substrate (Thermo, Rockford, IL, USA). The following antibodies were used: rabbit anti-MyD88 (Cell Signaling) and an anti-rabbit HRP conjugate (Promega, Madison, WI, USA).