Retinal horizontal cell-specific promoter activity and protein expression of zebrafish connexin 52.6 and connexin...

Connexins in retinal horizontal cells (HC) function in the processing of visual information. For example, gap junction-forming connexins may contribute to the spatial integration of visual stimuli. Additionally, connexin hemichannels have been hypothesized to participate in the feedback pathway from HCs to cones. To verify the identities of the zebrafish HC connexins, we performed promoter expression and immunohistochemical studies of connexin 52.6 (Cx52.6) and Cx55.5. Zebrafish embryos were microinjected with Cx52.6 or Cx55.5 promoter sequences and a green fluorescent protein reporter construct. Light and electron microscopic (EM) analysis showed green fluorescent protein expression exclusively in retinal HCs. Immunohistochemistry confirmed that HCs express Cx52.6 and Cx55.5 proteins. Light microscopy revealed Cx52.6 and Cx55.5 in the retinal inner nuclear and outer plexiform layers. Double labeling for Cx55.5 or Cx52.6 and cell-specific markers (tyrosine hydroxylase, protein kinase C-alpha, or GluR2) demonstrated that these connexins do not localize to interplexiform or ON bipolar cells, but most likely are present in HCs. Preembedding immuno-EM confirmed the HC-specific expression of Cx52.6 and Cx55.5 and illustrated the presence of these two connexins in gap junctions between HCs. The EM data also revealed robust labeling for Cx55.5 in hemichannels on HC dendrites in photoreceptor synaptic terminals. Voltage-clamp experiments in cultured cells demonstrated that Cx55.5-containing hemichannels can open at physiological membrane potentials. These results offer the first in vivo demonstration of the HC-specific activities of the Cx52.6 and Cx55.5 promoters. Furthermore, these data provide the first proof at the protein level for retinal HC-specific connexins in the zebrafish.