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Merve Tekmen, Evanna Gleason; The Pattern of Nitric Oxide Production in the Chicken Retina. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1171.
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Here, we investigate the cell-autonomous nature of the retinal nitric oxide (NO) signal by comparing the pattern of NO production to the expression of the Ca2+-dependent NO-synthesizing enzymes, neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS).
Adult White Leghorn chickens were sacrificed by IP injections of sodium pentobarbital as approved by the Institutional Animal Care and Use Committee, Louisiana State University (Assurance # A3612-01). Retinae were loaded with the NO-sensitive fluorescent dye, DAF-FM diacetate (20 µM, 30 min). DAF-loaded retinae were then stimulated by NMDA (250 µM, 10 min). Retinae were then fixed in 4% paraformaldehyde, and processed to prepare 12-16 µm frozen sections. Polyclonal antibodies against nNOS and eNOS (Sigma-Aldrich) were used to identify NOS-expressing cells.
The nNOS antibody labeled all photoreceptor inner segments and a subset of photoreceptors were strongly labeled. Some amacrine cells in the inner nuclear layer (INL) and their processes in the inner plexiform layer (IPL) as well as distinctive IPL bands of labeling were also labeled with the nNOS antibody. Cells in the ganglion cell layer (GCL) were also strongly labeled. Some axons were labeled in the nerve fiber layer (NFL). The eNOS antibody strongly labeled photoreceptors, horizontal cells and most amacrine cell bodies. Similar population of cells in the GCL was labeled strongly with the eNOS antibody as with nNOS antibody although dual expression is not yet confirmed. Some eNOS labeling was observed in the NFL. NMDA stimulation of the retina promoted wide-spread NO production, mainly in photoreceptors, horizontal cells, and some amacrine cells. When retinal tissue from the same animal was co-incubated with a general NOS inhibitor L-NAME, the DAF signal was substantially reduced. Photoreceptors, distinctive populations of amacrine cells, some cells in the GCL, and some of axons in NFL were positive for either DAF and anti-nNOS or DAF and anti-eNOS. Horizontal cells were positive for DAF and anti-eNOS only.
The degree of overlap between the DAF signal and either anti-eNOS or anti-nNOS suggests that a large fraction of the NO signals are generated cell-autonomously.
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