Abstract
Abstract: :
Purpose:Connexin (Cx) hemichannels and gap junctions in retinal horizontal cells are believed to play a vital role in visual information processing. Because it is uncertain which of the many subtypes of zebrafish Cx's are involved in this processing, we investigated the distribution of two Cx's believed to be retina–specific, Cx52.6 and Cx55.5. Methods:We performed promoter expression and immunohistochemical studies to investigate the distribution Cx52.6 and Cx55.5. Constructs containing either the Cx55.5 or Cx52.6 promoters and an EGFP reporter were injected into one to two cell stage zebrafish embryos, which were subsequently examined for EGFP expression by fluorescence, confocal, and immuno–electron microscopy. Additionally, we generated antibodies against Cx52.6 and studied its labeling of the retina alone and in combination with other cell–specific markers. Results: Confocal fluorescence and immuno–electron microscopy studies revealed that both the Cx52.6 and Cx55.5 promoters drove EGFP expression in retinal horizontal cells, but not in any other cell types in the zebrafish. Immunohistochemical studies using two distinct antibodies directed against Cx52.6 confirmed that horizontal cells, but not other retinal neurons, express this Cx subtype. Light microscopy showed Cx52.6 staining in the outer plexiform layer (OPL), but not in other synaptic or cellular layers of the retina. Double labeling for Cx52.6 and tyrosine hydroxylase (TH), the G protein Go–α, the ionotropic glutamate receptor GluR2, the GABAC receptor rho subunit, or protein kinase C (PKC) suggested that Cx52.6 protein was confined to horizontal cells. Pre–embedding immuno–electron microscopy confirmed the specific expression of Cx52.6 in horizontal cells. Furthermore, electron microscopy demonstrated the presence of Cx52.6–containing hemichannels in horizontal cell spinules and lateral dendritic elements and of Cx52.6–containing gap junctions between horizontal cells. Conclusions: Our results provide clear evidence that Cx52.6 and Cx55.5 are expressed specifically in zebrafish retinal horizontal cells. Furthermore, our data indicate that Cx52.6 is the Cx that forms gap junctions between horizontal cells.
Keywords: retina: distal (photoreceptors, horizontal cells, bipolar cells) • gap junctions/coupling • retinal connections, networks, circuitry