Real time electrochemical investigation of the release, distribution and modulation of nitric oxide in the intestine of individual zebrafish embryos

Nitric oxide (NO) is an important signaling molecule that has been implicated in a variety of physiological and pathophysiological processes in living organisms. NO plays an important role in embryonic development in vertebrates and has been reported to influence early organ development and plasticity. Quantifying NO in single embryos and their developing organs is challenging because of the small size of the embryos, the low dynamically changing concentration and the short life-time of NO. Here, we measured the distribution of NO in the intestine of live zebrafish (Danio rerio) embryos in physiological conditions and under the influence of therapeutic agents. NO measurements were performed using a miniaturized electrochemical sensor fabricated on a single carbon fiber (CF) which enables quantitative real time in vivo monitoring, and by fluorescence imaging using the 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (OAF-FM-DA) dye. NO production was detected in the middle segment the intestine at a level of 3.78 (+/- 0.64) mu M, and at lower levels in the anterior and posterior segments, 1.08 (+/- 0.22) and 1.00 (+/- 0.41) mu M respectively. In the presence of resveratrol and rosuvastatin, the intestinal NO concentration decreased by 87% and 84%, demonstrating a downregulating effect. These results indicate the presence of variable micromolar concentrations of NO along the intestine of zebrafish embryos and demonstrate the usefulness of CF microelectrodes to measure quantitatively the NO release at the level of a single organ in individual zebrafish embryos. This work provides a unique approach to study in real time the modulatory role of NO in vivo and contributes to further understanding of the molecular basis of embryonic development for developmental biology and drug screening applications.