Tracking tagged molecules in single neurons in intact zebrafish

INTRODUCTIONThis protocol describes an approach for monitoring the movement of tagged molecules in single neurons in intact embryonic and larval zebrafish. The intact preparation provides a meaningful context for the physiological event being studied. Other advantages offered by the young zebrafish include direct in vivo imaging, the ability to produce large numbers of labeled embryos easily using microinjection, and the existence of identified sensory circuits that can be exploited to activate a particular cell type. One limitation of this system is the fragility of 2- to 3-d-old embryos, which demands delicate physical manipulation of the fish during all stages preceding and during the experiment. In contrast to brain slices or isolated cells, nearly all original neural connections and sensory components are maintained in the intact preparation, so the occurrence of a downstream event may be precluded (or its manifestation enhanced) by some complex interplay of biological processes that are not fully understood.