GFP and FAP Imaging of Neuropeptide Release in Drosophila

Genetics in Drosophila have revealed the role of neuropeptides in development and behavior. However, determining when and where neuropeptides are released has been challenging. Furthermore, the cell biology underlying neuropeptide release has largely been unexplored. Thus, it has not been possible to determine whether changes in neuropeptide immunofluorescence reflect traffic and/or release, and in neurons where such changes are not detectable, conclusions about neuropeptide release have been formulated based on the assumption that electrical and Ca²⁺ recordings are accurate and quantitative predictors of release. Recently, the advent of optical detection of neuropeptides tagged with fluorescent proteins and fluorogen-activating proteins (FAPs) has made it feasible to directly image vesicle traffic and exocytosis that mediates neuropeptide release in peripheral synapses and in the brain. In fact, these approaches have led to the discovery of unexpected insights concerning neuropeptide release. Here procedures are presented for optimizing fluorescence imaging of neuropeptides tagged with green fluorescent protein or a FAP.