ABSTRACT Neuromodulators, such as neuropeptides and biogenic amines are produced and released by neurons to communicate with each other. They act as neurotransmitters, as well as neuromodulators, to profoundly influence the function of neural circuits, thereby regulating critical brain functions, including arousal, feeding, metabolism, social behavior, fear/anxiety, learning/memory, reward, and sexual behaviors. Dysfunction in neuromodulator signaling is associated with many neurological and neuropsychiatric disorders. Therefore, understanding the mechanism of neuromodulator functions in the brain is important for understanding brain disorders. Among these, neuropeptides are by far the most diverse class of neuromodulators in the brain, and they are involved in a range of physiological, emotional, and cognitive processes in the brain. Although more than 100 neuropeptides and their downstream receptors have been discovered, circuit-based mechanisms of neuropeptide functions have not been adequately explored. This is primarily because the field lacks tools for monitoring the release of neuropeptides in a temporally precise manner as animals perform behaviors. To fill this void, the research proposed here will develop a novel quantitative technique for detecting neuropeptide release from the presynaptic terminals in awake behaving mice. This proposal will develop a sensor that detects the release of dense-core vesicles that specifically packages neuropeptides in the axonal terminals. This innovative approach will allow us to monitor the presynaptic release of essentially all neuropeptides, thereby addressing fundamental questions about the neuropeptidergic regulation of brain functions.