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S.C. Massey, J.J. O'Brien, W. Li, J. Keung, J. O'Brien; Identification of an A–Type Horizontal Cell Connexin in the Rabbit Retina. . Invest. Ophthalmol. Vis. Sci. 2004;45(13):1320.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: There are many examples of neuronal coupling via gap junctions in the retina. Of these, perhaps the best known is the extensive coupling in the horizontal cell network. However, the connexins composing these gap junctions are unknown. In the rabbit retina, there are two types of horizontal cells, A–type and B–type, both of which are independently coupled. Here, we identify connexin50 as a gap junction protein in A–type horizontal cells. Methods: Rabbit retina cDNA was isolated from total rabbit retina, and RT–PCR was performed using specific Cx50 primers. Single cell RT–PCR was performed on isolated horizontal cells using the Cx50 primers. Retinal whole mounts were labeled with calbindin and connexin50 antibodies. The Cx50 antibodies recognized either intracellular loop or carboxyl terminus. Western blots were performed to evaluate specificity of the Cx50 antibody. Individual A– and B–type horizontal cells were injected with Lucifer Yellow, Neurobiotin or Alexa 568 to visualize the connexin50 plaques on individual horizontal cells within the network. Results: Connexin50 cDNA was successfully isolated from total rabbit retina, encoding a 440 amino acid protein. Rabbit Cx50 shares 80.1% and 83.4% amino acid homology with human and mouse Cx50, respectively. Cx50 was also obtained from isolated A–type horizontal cells by single cell RT–PCR. A– and B–type horizontal cells were visualized by intracellular dye injection or with an antibody to calbindin. Confocal analysis revealed all Cx50 labeling occurred on the A–type horizontal cell matrix, typically at dendritic intersections. The Cx50 plaques varied in size, from punctate signals where fine dendrites cofasciculated to plaques as large as 50 µm2 where large dendrites crossed. Although B–type horizontal cells also form a coupled network, they did not contain Cx50. Conclusions: Our results suggest Connexin50 is the major connexin at gap junctions in the rabbit A–type horizontal cell matrix. The extensive Cx50 plaques between A–type horizontal cells form a low resistance pathway, which adequately accounts for the extensive coupling observed in this network. Moreover, the absence of Cx50 in the B–type horizontal cells suggests the presence of additional connexins in the retina. These results indicate that multiple neuronal connexins are expressed in the mammalian retina and that different cell types express different connexins.
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