Imaging neuropeptide release in the Drosophila neuromuscular junction (NMJ)

Electrophysiological studies of synaptic function cannot directly reveal the internal workings of the nerve terminal and do not robustly report release of neuropeptides and neurotrophins. These limitations can now be overcome with the presynaptic expression of green fluorescent protein (GFP) indicators of vesicle motion, release, and signaling. This protocol describes how to image single wavelength and ratiometric fluorescence resonance energy transfer (FRET)-based GFP indicators with fluorescence microscopy in living synaptic boutons of the Drosophila neuromuscular junction (NMJ). The steps for setting up the imaging equipment for epifluorescence microscopy are given, followed by special considerations for preparing the larval NMJ for peptide release studies.