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The Institutional Animal Care and Use Committees at the Veterans Affairs San Diego Healthcare Systems and University of California, San Diego approved the study protocol. All experiments were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals (National Institutes of Health, Bethesda, MD). Mice were scarificed by dislocation of cervical spine (for freshly tissue samples) or perfusing with 2 to 4% paraformaldehyde under deep anesthesia using a cocktail containing ketamine (3.75 ml, 100mg/ml), xylazine (0.4 ml, 100mg/ml), and acepromazine (0.75 ml, 10mg/ml) mixed with 15.1 ml distilled water. Mice hemizygous for ChAT-ChR2-YFP BAC transgene with expression of the mhChR2:YFP fusion protein directed by the choline acetyl transferase (ChAT) promoter/enhancer regions on the BAC transgene were purchased from the Jackson Lab, (stock#012355) and bred at UCSD. To construct mice with nNOS positive neurons, the Cre/LoxP technique of somatic recombination was used. Mice expressing Cre-ERT2 recombinase under the control of the nNOS promoter (knock-in mutation) were crossbred with Cre reporter mouse positive for red fluorescent protein, Td-Tomato. The Cre-ERT2 fusion gene activity was induced by administration of tamoxifen, so only cells that express Cre (i.e., nNOS positive cells) produced the fluorescent reporter. The two fluorescent reporter strains for ChAT and nNOS were cross bred to generate the double-reporter mice. The two fluorescent reporter proteins have different spectral properties, i.e., emission at 527 nm for YFP and 581 nm for Td-Tomato. The mice that confirmed positive for both ChAT and nNOS were treated with oral tamoxifen, 1 mg/day for 2 days, after which either fresh tissue was obtained from the animals or they were perfused with 2–4% paraformaldehyde under deep anesthesia. For fresh tissue, the entire gut, from the esophagus (at the level of cricoid cartilage) to the distal rectum was harvested. The mucosa of the harvested segments, i.e., entire esophagus, fundus of the stomach, small intestine (20mm proximal to the ileocecal valve) and colon (20mm distal to the ileocecal valve) was removed and the muscularis propria was mounted on a glass slide with 99% glycerol and examined under the microscope. The perfusion was performed though the left ventricle with 20 ml saline followed by 4% paraformadehyde in 0.1M phosphate buffer, pH 7.4. The entire gut was removed in a manner similar to the fresh tissue and examined under the microscope. For immunostaining, wild type mice were perfused and tissue harvested as described earlier, post-fixed overnight in 4% paraformaldehyde at 4°C. Samples were then soaked in 30% sucrose solution in 0.1M phosphate buffer overnight. Samples were treated with 0.3% TritonX-100/1%BSA/PBS over night at 4°C to make them permeable as well as block non-specific staining. Samples were placed on a shaker, washed 3 times with PBS, each time for 15 minutes and then primary antibodies (Table 1) were applied for 48 hours at 4°C, following which tissue was washed again 3 times (15 minutes each) before incubating with secondary antibodies (Table 1) for 1 hour at room temperature. Finally the samples were mounted on glass slides using Prolong Gold antifaded with DAPI and examined with multi-photon Leica SP5 microscope (UCSD School of Medicine Light Microscopy Facility). In few single reporter mice, we performed immunostaining and histochemical staining using the above methods as well. Tamoxifen treatment was also given in few wild type mice before staining with nNOS and ChAT antibodies to determine if it had any effect on the myenteric neurons. The ChAT and nNOS positive neurons were analyzed using maximum projection images, a volume rendering method for 3D data that projects in the visualized plane the voxels with maximum intensity that fall in the way of parallel rays traced from the viewpoint to plane of projection.
Table data removed from full text. Table identifier and caption: 10.1371/journal.pone.0171239.t001 Primary and secondary antibodies (made with 1% BSA/PBS) used for staining. NADPH-diaphorase activity was detected histochemically, as described previously [13]. Briefly, the tissue was placed in 0.1 M phosphate buffer pH 7.4, containing 0.3% Triton X-100, 0.1 mg/ml nitroblue tetrazolium and 1.0 mg/ml /I-NADPH, at 37°C for 30–60 min. The reaction was halted by placing the sample in 0.1 M phosphate buffer. Stained samples were examined under a bright-field microscope. Histochemically stained samples were counterstained with DAPI 1:5000 to visualize nuclei. Samples were whole-mounted with either Fluoro-mount or 99% glycerol and covered with a cover slip. Microscopic imaging was performed using a multi-photon, Leica SP5 confocal microscope. Z-stacks of myenteric plexuses were recorded using an upright laser scanning confocal microscope (SP5) with a water immersion 20x objective (0.9-μm-thick slices; z stack). Images were taken from the esophagus at 1, 2, 4, 6, 8, 10, 12, 14mm above the angle of HIS, intestine and colon. At least three images (high power field) were obtained from each animal at 20x magnification for cell counting. Tissue specimens were excited according to their specific excitation/emission characteristics. The detection pinhole was set for use with different objectives accordingly. Offset and gain settings were determined at the start of each experiment and kept constant throughout the image capturing time. The neurons were counted from these images manually using LAS AF software 4.3. Different magnification views were captured to visualize the neuronal process and cell interaction.
Overall quantitative evaluation of neurons (ChAT/nNOS): Neuron counting was performed manually on images taken under 20X magnification using Leica Application Suite Advanced Fluorescence Version 4.3. A 20x dry and/or water objective lens (numerical aperture 0.7 to 1) was used; the zoom factor was set at 1.72 in all scanning sessions. The neuron counts for only nNOS positive, only ChAT positive, both nNOS and ChAT positive and both nNOS and ChAT negative were obtained. Data are shown as mean ± SEM. One-way ANOVA was used to compare differences between groups. p<0.05 was considered significant.
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