Bioassay of Vitamin A2 in the Retinal Tissue of the zebrafish, Danio rerio, Using Microspectrophotometry and 2D-Imaging

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Zebrafish have been shown to utilize the vitamin A1 chromophore in synthesizing their visual pigments. Recent evidence suggests that they may also be able to utilize vitamin A2 under both natural conditions and after treatment with thyroxine. To test for the presence of vitamin A2 in the retinae of zebrafish and map its location, I combined microspectrophotometric (MSP) analysis with 2D-imaging. MSP measures the absorbance spectra of visual pigments within single photoreceptors, while 2D-imaging uses differential bleaching to attempt differentiation of those regions rich in A1 pigment from those containing A2. Previous MSP has found the occasional A2 cell in some fish, but failed to demonstrate a specific area or island utilizing the A2 chromophore in zebrafish, possibly due to sampling problems of the MSP technique. Unfortunately, the 2D-imaging failed to show enough contrast between bleached and unbleached retinal regions to determine if, in fact, bleaching occurred at all. In an attempt to enrich the retina in vitamin A2, thyroxine treated eyecup cultures were used. In this case the hope was to stimulate A2 synthesis in cultured eyecup preparations. MSP was used to monitor changes in vitamin A2-based pigment. There was a failure to observe A2 synthesis in culture. While the most likely culture conditions were tried first, it is believed that these were NOT conducive to zebrafish retinal culture. This study has demonstrated methodological challenges that need to be solved before the original question can be satisfactorily addressed.
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