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J. Pearring, J.O. Deshler, S.J. Heflin, P.B. Cook; Molecular Characterization and Localization of Voltage–Gated Sodium Channels in the Tiger Salamander Retina . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1713.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Electrophysiology evidence indicates the presence of voltage–gated sodium channels (NaVs) in some bipolar, most amacrine and all ganglion cells of the tiger salamander, but the genetic sequence and location of mRNA for NaVs has not been determined. In the present study, we identified the partial genetic sequence of the NaV and used this to localize mRNA with in situ hybridization. Methods: A partial NaV sequence of 300bp was extracted from the cDNA library of the salamander (originally created by Jeff Arriza). The gene was transcribed to create a digoxigenin labeled riboprobe for non–radioactive in situ hybridization. A partial salamander opsin DNA segment served as a positive control, while an e.coli pTZ18U plasmid riboprobe was created as a negative control along with the sense and no RNA probes. Results: The partial NaV encoded 305 base pairs and 101 amino acids. Sequence identity was found with mouse, newt, and xenopus NaVs at the nucleic acid (81% to 82%) level. In situ hybridization of antisense RNA showed weak isolated staining in the outer retina, possibly in bipolar cells or processes of interplexiform cells, and heavy staining in the inner retina. In the inner nuclear layer some somas adjacent to the IPL had large, unstained nuclei, and dense, punctate mRNA staining in the cytoplasm, indicating that these cells could express NaV channels. The entire inner plexiform layer was strongly stained, but individual processes could not be identified with light microscopy. Somas in the ganglion cell layer had large, unstained nuclei, surrounded by dark stain as evidence of NaV mRNA. Conclusions: NaV sequence is highly conserved across many species, consistent with the significance of this protein in signal propagation. Localization of mRNA expression in a high proportion of amacrine cells and all ganglion cells is consistent with previous electrophysiological evidence of NaV action potentials in these cell classes.
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