The transgenic mouse strains including RIP-Cre, R26R-LacZ, R26R-GFP, mT/mG
, were originally obtained from Jackson Laboratory. The Pcdh-γfC3
transgenic mice were recently described (Lefebvre et al., 2008
; Prasad et al., 2008
; Xu et al., 2005b
). The Pcdh-γfC3
conditional allele was originally generated in 129S7 ES cells. After germline transmission, the allele was backcrossed with C57BL/6-Tyr c-Brd
for 4 generations. All experiments were carried out on 129S7/C57BL/6-Tyr c-Brd
F4 hybrids. Both RIP-Cre; Pcdh-γfC3/fC3
and POMC-Cre; Pcdh-γfC3/fC3
conditional mutants were generated from double heterozygous mating. All mice were fed TEKLAD mouse diet 7904 (minimal 17% protein and 11% fat, Harlan TEKLAD, Madison WI) and water ad libitum unless otherwise stated. All procedures were reviewed and approved by the Northwestern University Institutional Animal Care and Use Committee.
Body weight, body composition and feeding behavior tests
Body weights were measured weekly. Food intake was determined in single-housed mice by weighing remaining diet. Data was discarded in the event of food spillage in the cage. Fat composition and lean mass were measured using an MRI whole body composition analyzer (Echo medical Systems, Houston, TX). The compensatory refeeding experiments were performed as previously described (Xu et al., 2005a
Oxygen consumption was measured by an indirect calorimeter chamber (AccuScan Instruments Inc., Columbus, OH) at the University of Cincinnati Mouse Metabolic Phenotyping Center. Male mice, 10-12 weeks old, were placed in the chambers 3 hours before the dark cycle and energy expenditure measurements were collected for 24 hours.
Blood hormone measurements
Blood samples were collected from cardiac puncture on deeply anesthetized mice between 2 pm and 4 pm. Serum samples were collected by centrifugation and stored at -80°C. Glucose, triglycerides, and cholesterol were assayed by using commercial kits from Analox Instruments LTD (GMRD-002A, GMRD-195, GMRD-084 respectively). Insulin was measured by Mouse Insulin ELISA (Crystal Chem Inc), and leptin was assayed using mouse endocrine mutiplex kit from Linco (Kohsaka et al., 2007
). LH was measured by radioimmunoassay. T3, T4 and corticosterone levels were assayed by the Hormone Assay & Analytical Services Core at Vanderbilt University.
Glucose tolerance test and islet glucose-responsive insulin secretion
Glucose tolerance tests were performed between 10 am to 11 am after a 17-hour fast. Blood samples were harvested from tail vein bleed. Glucose levels were determined using a Precision Xtra glucose monitor (Abbott Laboratories) at 0, 5, 15, 30, 60 and 120 minutes after intraperitoneal injection of glucose (2 g/kg body weight). Islet glucose-responsive insulin secretion was examined in static incubation studies. Briefly, pancreatic islets were isolated via collagenase digestion and left to recover overnight in RPMI-based media. Islets were then incubated for 1h in Krebs Ringer Buffer with 2 mM glucose to reach basal levels of insulin secretion. Groups of five size-matched islets were hand-picked and statically incubated in 2 mM, 12 mM or 20 mM of glucose for 1h. Supernatant was collected, and islets were then sonicated in acid-ethanol solution to obtain total insulin content. Amounts of secreted insulin and insulin content were determined by ELISA as described above.
RNA isolation and quantitative PCR analysis
The hypothalamus was dissected out using the optic chiasm as the anterior boundary, the mammillary bodies as the posterior border, and anterior commissure as the horizontal limit. Tissues were snap frozen in liquid nitrogen and stored at −80°C for subsequent analysis. Total RNAs were purified using RNeasy mini kits (Qiagen). Reverse transcription and quantitative PCR were performed with the ABI Prism 7700/7900 PCR instrument (Applied Biosystems) using SYBR green PCR Master Mix reagent kit (Applied Biosystems) as previously described (Kohsaka et al., 2007
Immunohistochemistry and in situ hybridization
Mice were anesthetized with i.p. injection of Avertin (2.5% tribromoethanol) and perfused with PBS followed by 4% PFA. The brain and pancreas were post-fixed with 4% PFA for 4 hours at 4°C, cryoprotected with 30% sucrose and then frozen in Tissue-Tek O.C.T. 12 μm- or 20 μm- sections were collected for various antibody staining. The following primary antibodies were used in this studies: guinea pig anti-insulin (1:100, DAKO), monoclonal rat anti-BrdU (1:1500, AbD Serotec); mouse monoclonal anti-class III β-tubulin (TuJ1, 1:1000, Covance); mouse monoclonal anti-GFAP (1:500, Chemicon); rabbit polyclonal anti-pSTAT3 (1:200, Cell Signaling Technology), rabbit anti-POMC (1:400, Phoenix pharmaceuticals), rabbit anti-AgRP (1:2000, Phoenix pharmaceuticals), sheep anti-a MSH (1:4000, Millipore), rat-anti-GFP (1:5000, MBL), guinea pig anti-VGLUT2 (1:3000, Millipore), rabbit anti-GFP (1:10,000, Invitrogen), rabbit anti-LacZ (a gift from Dr. Holmgren). Single and double staining were visualized with FITC-, DyLight488, DyLight 594- and Cy3-conjugated secondary antibodies (1: 500 and 1: 1000, Jackson ImmunoReseach) and counter stained with DAPI (Sigma-Aldrich). Images were collected with Zeiss Axiovision 4.6 using a Zeiss Axiovert 200M with Apotome module.
For BrdU labeling, pregnant mice at E12.5 or E14.5 were injected intraperitoneally with 50mg/kg body weight BrdU solution (BD Biosciences), and sacrificed one hour after injection. To evaluate leptin-induced STAT3 phosphorylation, 15 days old sex matched mice were intraperitoneally injected with leptin (National Hormone and Peptide Program; 10.0 mg/kg body weight) or a similar volume of saline at 10 a.m. 45 minutes after leptin injection, the mice were anesthetized and perfused with 4% PFA in PBS. For pSTAT3 single or double labeling, sections were sequentially processed with 1% NaOH, 0.3% glycine and 0.03% Sodium dodecyl sulfate, followed by antibody staining as described above. To colocalize POMC expressing neurons, mice were pre-treated with i.c.v. injection of colchicine (Sigma) 24 h before euthanasia to block axonal transport. In brief, deeply anesthetized mice received 1 μl colchicine solution (20μg/μl in 0.9% sterile saline, 0.75 μg/g body weight) at 1 mm lateral and 0.3 mm posterior to bregma and 2 mm ventral to the surface of the skull using standard sterile surgical procedures. Brains were harvested 24 hours later for immunohistochemistry as described above.
LacZ staining was performed as described before (Wang et al., 2002a
). For in situ
hybridization, a GFP riboprobe was generated by in vitro
transcription with T3 polymerases and the sample processing, hybridization and development of fluorescent signals were performed as previously described (Wang et al., 2002a
Estimate of active caspase-3 positive cell count
For quantification of active caspase 3 positive cells, immunohistochemistry staining was processed as described above. Pictures were taken every 3rd sections using a 10× objective with ARC positioned in the midline, starting from where ARH and VMH were first visible in a caudal to rostral manner throughout the entire ARC. Active caspase 3-positive cells were found in the ARC, the DMH, the VMH, the LH and PVH, and were counted using Zeiss Axiovision 4.6 software by an observer blinded to the genotype. Cell counts were performed every 3rd section in 3-5 animals per group (12 sections/animal) and represent the average total number of cells counted.
Anesthetized animals were first perfused transcardially with saline, followed by a fixative containing 4% PFA and 0.1% Glutaraldehyde in 0.1M phosphate buffer (PB, pH 7.2-7.4). After an overnight fixation with 4% PFA, the brain tissues that included the hypothalamus were cut into 50 μm coronal sections using a vibrotome (Leica VT1000S). The vibrotome sections were then extensively washed with PB, blocked with 50 mM Glycine, and permeabilized with 0.05% Triton X-100/PB for 30 minutes. After blocking with 5% bovine serum albumin/2% goat serum / PBS for 30 minutes, the sections were incubated with 1% goat serum/0.1M PBS containing rabbit anti-LacZ antibody (1:200) overnight at 4°C. After multiple washes, the samples were incubated with NanaGold-conjugated Fab' anti-rabbit secondary antibody (1:250, Nanoprobes, NY)) at room temperature for 1 hour and at 4°C for overnight. The brain slices were subsequently washed with 0.1M PBS and fixed with 2% glutaraldehyde in PBS for one and half hours. After washes with PBS and distilled water, silver enhancement (HQ Silver enhancement kit, Nanoprobes, NY) was performed in the dark for 12 min. Finally, the samples were fixed in 0.2% OsO4 for 30 min, block stained with uranyl acetate, dehydrated in ethanol and flat embedded in LX112. Semi-thin sections were cut at 1 μm and stained with 1% Toluidine Blue to evaluate the quality of preservation. Ultra-thin sections (50 nm) were cut, mounted on formvar-coated single-slot copper grids and stained with uranyl acetate and lead citrate by standard methods. Stained grids were examined under Philips CM-12 electron microscope (FEI; Eindhoven, The Netherlands) and photographed with a Gatan (4k ×2.7k) digital camera (Gatan, Inc., Pleasanton, CA) at different magnifications. After obtaining digital montages with Adobe Photoshop, symmetric and asymmetric synapses were counted on all lacZ-positive cells only if the pre- and/or postsynaptic membrane specializations were clearly seen. Synapses without clearly symmetric or asymmetric membrane specializations were excluded from the assessment.
Data and statistical analysis
All data are presented as means with standard error of the means. Detailed information on sample sizes, genotype and p values are marked on the individual figures or described in the corresponded legends. Comparisons between two groups are analyzed using two-tailed unpaired student's t-test or two-way ANOVA. For two-way ANOVA, if the overall F value was significant, Bonferroni post-hoc test were performed using GraphPad.