Labeling blastomeres with a calcium indicator: a non-invasive method of visualizing neuronal activity in zebrafish

Injections of the calcium indicator calcium green dextran (CGD) into zebrafish embryos at the 1-4 cell stages were used to monitor the activity of neurons in larval fish. Dye was pressure injected into a single cell and the fish allowed to develop until post-hatching, when they were embedded in agar and viewed under a confocal microscope. Labeled larval cells, including identifiable neuronal classes such as Rohon-Beard cells and olfactory neurons, were clearly visible with extensive labeling of the whole fish following injections at the one cell embryonic stage, and a mosaic labeling pattern following injections at the 2 or 4 cell stages. Activity of neurons in the spinal cord, as indicated by intracellular calcium concentration changes, was observed directly by monitoring fluorescence changes of individual spinal neurons and groups of spinal neurons on a confocal microscope. Fluorescence increases of between 9 and 55% in spinal neurons were seen during escape responses produced when the fish was tapped on the tail. This technique can potentially be used to monitor the activity of any neuron or group of neurons with respect to behavior non-invasively in intact living zebrafish.