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Annie Espinal, JorgeAlberto Perez-Leon, Manuel Miranda-Arango; The study of retinal neurons in the GlyT2-EGFP transgenic mouse. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2505.
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Glycine plays two main roles in the nervous system; in pathways mediated by the strychnine-sensitive receptor (GlyR) and as co-agonist of N-methyl-D-aspartate receptor (NMDAr). Glycine is cleared from the synaptic cleft by the two transporters, GlyT1 which has been associated with the localization of the NMDAr in glutamatergic synapses, and GlyT2, that takes glycine up into presynaptic neurons to the GlyR. It has been demonstrated the expression of the two transporters in the mammalian retina, however only the GlyT1 protein has been found, restricted to the amacrine cell population. The presence of the GyT2 protein in the mammalian retina remains elusive, though several studies indicated that the glycine transport does not entirely fit the GyT1 features. Based on these findings, we decide to evaluate the expression of GlyT2 at the cell level with a transgenic mouse model.
We use the transgenic mice that express the enhanced green fluorescent protein under the control of the GlyT2 promoter. The generation of the mice and the description of the EGFP expressing neurons overlapping with GlyT2 in the brain stem have been published. Retinae from 2 month old male mice were fixed, cryoprotected and sectioned at 20 μm, the slides obtained were frozen until use. Immunoflourescence was used combining antibodies recognizing several synaptic proteins, slides were analyzed by laser scanning microscopy.
Neurons expressing EGFP corresponding to the amacrine cell glycinergic population were characterized in this study. These cells showed immunoreactivity to the GlyT1 and the amino acid glycine. Numerous punctae corresponding to the GlyR subunits, gephyrin and the VIIAT were found colocalizing with the EGFP processes. Some EGFP expressing somata were positive for GAD67 and the GABA transporters GAT1 and GAT2. In cultures from neurons of the transgenic retinae, the EGFP cells were marked for MAP1; by contrast, GFAP immunostaining did not detect GFP expression in glia cells.
The EGFP-GlyT2 mouse retinal cells correspond to the glycinergic amacrine cells. Thus GlyT2 can also be an active player regulating the glycine transmission within the retina. The data suggest the existence of amacrine cells which use glycine and GABA as co-transmitters. This transgenic mouse provides a tool to the study of the retinal glycinergic system by physiological approaches.
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