Biochemical analysis of phototransduction and visual cycle in zebrafish larvae.

Publisher Summary The zebrafish ( Danio rerio ) has received considerable attention as a model organism for the study of vertebrate development. Its visual system has been well characterized and is similar to that of other vertebrates. The retina contains rods and four types of cones, making it possible to study both scotopic and photopic visual responses. Zebrafish larvae develop rapidly, allowing behavioral analysis of visual function by 4 days postfertilization. Zebrafish with recessive mutations that cause defective visual responses have been isolated by a behavioral screening strategy. These mutants have been characterized by examining retinal histology and electroretinogram (ERG) responses. In this way, the visual defects in several mutants were localized to the outer layers of the retina. To further characterize mutants with vision defects, biochemical assays were adapted to analyze the phototransduction cascade and the visual cycle in zebrafish. These assays must be performed on larvae because mutants with visual defects do not survive to adulthood. This chapter presents methods that provide valuable tools for characterizing the phenotypes of an increasing number of zebrafish vision mutants. They include assays for transducin activation, phosphodiesterase (PDE) activity, guanylyl cyclase (GC) activity, rhodopsin and cone opsin phosphorylation, and the analysis of retinoids in the visual cycle. For each assay, the general principles are discussed, the necessary materials listed, and the procedure outlined.