Cellular source and circuit context organize functional specificity in the Drosophila NPF system.

Neuropeptides regulate diverse and sometimes opposing functions, yet how a single peptide achieves functional specificity remains unclear. Receptor diversity can provide specificity, but not in systems with only one receptor. Drosophila neuropeptide F (NPF) acts through a single receptor, providing a model to test whether specificity is organized by the peptide's cellular source. Using the whole-brain connectome, we defined four adult NPF cell types with no shared first-order synaptic partners and separate networks. These cell types made distinct and partly opposing contributions to reinforcement, feeding, mating, and energy storage. Most peptide-dependent effects were undetectable after population-level NPF reduction, showing that global perturbation can obscure source-specific functions. Changing neuronal activity and reducing NPF in the same cells produced different, sometimes opposing, phenotypes, so neuronal activation cannot be equated with peptide action. Thus, cellular source and circuit context generate functional specificity that population-level perturbation conceals.