LABELING BLASTOMERES WITH A CALCIUM INDICATOR - A NONINVASIVE METHOD OF VISUALIZING NEURONAL-ACTIVITY IN ZEBRAFISH

Injections of the calcium indicator calcium green dextran (CGD) into z ebrafish embryos at the 1-4 cell stages were used to monitor the activ ity of neurons in larval fish. Dye was pressure injected into a single cell and the fish allowed to develop until post-hatching, when they w ere embedded in agar and viewed under a confocal microscope. Labeled l arval cells, including identifiable neuronal classes such as Rohon-Bea rd cells and olfactory neurons, were clearly visible with extensive la beling of the whole fish following injections at the one cell embryoni c stage, and a mosaic labeling pattern following injections at the 2 o r 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 gr oups of spinal neurons on a confocal microscope. Fluorescence increase s of between 9 and 55% in spinal neurons were seen during escape respo nses 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 behaviour non-invasively in intact living zeb rafish.