(A) Schematic illustrating the principle of NPRRs (Neuropeptide Release Reporters). NPRR molecules in the DCV lumen (low pH/low calcium, left) exhibit increased fluorescence when released by fusion into the extracellular space (neutral pH/high calcium, right). NPRR fluorescent signal is expected to decay following diffusion into the synaptic cleft. New NPRR-containing DCVs are produced by synthesis and transport from the soma, not by recycling. NP: Neuropeptide. DCV: Dense Core Vesicle. SV: Synaptic Vesicle. (B) Distinct motor neuron subtypes at the Drosophila NMJ (muscle 12/13) have different proportions of DCVs vs. SVs. The GAL4 driver R57C10-Gal4 (nsyb-GAL4) labels all subtypes, while R20C11-GAL4 selectively labels only Type III neurons, which lack SVs (‘Type III-GAL4’). Light gray circles, black lines and dark gray shading represent boutons, inter-bouton intervals and subsynaptic reticulum respectively. The studies in this paper focus on Type Ib neurons and Type III neurons (in red rectangles). (C) Triple immunolabeling for GFP (green), Bursicon (blue) and vGluT (red), in flies containing nsyb-GAL4 driving UAS-GCaMP6s (upper), or NPRRANP (lower). Type Ib and Type III boutons are indicated. Scale bar, 5 µm. Inset image (NPRRANP, a-GFP channel) shows details of puncta distribution of NPRRANP in Type Ib neuron. Scale bar, 2 µm. (D) TEM images of boutons immunolabeled with anti-GFP (5 nm gold particle-conjugated) to detect nsyb>NPRRANP-GFP, which has an identical structure to NPRRANP, but is a GFP rather than GCaMP6s fusion to improve antigenicity (see Figure 1—figure supplement 4). Note strong labeling in DCVs (arrows) and the neuronal plasma membrane (arrowheads). Scale bar, 200 nm. Lower panel shows representative images of labeled DCVs. Scale bar,100 nm. (E) Quantification for TEM images in (D).