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Sally A McFadden, Jose M Romero Hombrebueno, Guang Zeng, Dominique (Lena) Fuchs, Eun-Jin Lee; The distribution of nNOS amacrine cells in the guinea pig retina. Invest. Ophthalmol. Vis. Sci. 2019;60(9):539.
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Nitric Oxide (NO) acts as a neural modulator in the retina and is involved in light and dark adaptation, and modifies the coupling between horizontal cells. The synthesis of NO is catalyzed by one of three isoforms of nitric oxide synthase (NOS), one being neuronal NOS (nNOS) located ubiquitously in the retina. In the current study, we investigated the distribution of nNOS in amacrine cells (ACs) in the guinea pig eye.
Tri-coloured guinea pigs raised under white LEDs were euthanized in the middle of their light cycle at 2 weeks of age. For Immunohistochemistry (IHC), eyecups were fixed in PFA for 1 hr (vertical sections) or 2 hrs (whole mounts). Incubation with polyclonal nNOS antibody (Sigma Aldrich, 1:10000) was overnight for 25 μm vertical retinal sections and 3 days for whole mount retinas. Immunoreactive (IR) cells were counted in whole mounts along 8 radii every 1mm and isodensity maps constructed. nNOS gene expression was measured with qPCR in 3mm punches from ventral or dorsal retina. 1500 ng of each mRNA sample was reverse-transcribed to cDNA. Primers for nNOS were confirmed through meltcurve analysis, gel electrophoresis, and automated sequencing. The reactions were performed on a 7500 fast Real-Time PCR System. The mean normalized Expression (MNE) of nNOS mRNA was calculated using β-actin as the reference gene.
nNOS IR occurred in three different ACs (type-I, type-II and NOS-Displaced ACs) and in one type of cone bipolar cell as previously reported. Both nNOS displaced ACs and Type I ACs were expressed most densely in the center of the retina compared with the periphery (ratio of 2:3). Displaced AC cells expressing nNOS were more numerous than Type I cells (116 Vs. 27 cells/mm2). The former were denser in the ventral compared with the dorsal hemi-retina (140 Vs. 65 cells/mm2). The MNE*1000 of mRNA for nNOS was also significantly higher in ventral retina (10.8 Vs. 6.4, p < .0001).
nNOS expressing amacrine cells differ substantially in density in different sectors of the retina. The most numerous cell expressing nNOS was a Displaced AC with cell body located in the ganglion cell layer, and a marked asymmetry in density, with higher expression in ventral retina. This zone of the eye which normally views the sky is also characterized by greater numbers of blue cones and a hyperopic refractive error, suggesting the possibility that NO expression could be linked to light exposure.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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