The co-existence and co-transmission of neuropeptides and small molecule neurotransmitters within individual neuron represent a fundamental characteristic observed across various species. However, the differences regarding their in vivo spatiotemporal dynamics and underlying molecular regulation remain poorly understood. Here, we develop a GPCR-activation-based (GRAB) sensor for detecting short neuropeptide F (sNPF) with high sensitivity and spatiotemporal resolution. Furthermore, we investigate the in vivo dynamics and molecular regulation differences between sNPF and acetylcholine (ACh) from the same neurons. Interestingly, our findings reveal distinct spatiotemporal dynamics in the release of sNPF and ACh. Notably, our results indicate that distinct synaptotagmins (Syt) are involved in these two processes, as Syt7 and Sytα for sNPF release, while Syt1 for ACh release. Thus, this high-performance GRAB sensor provides a robust tool for studying neuropeptide release and shedding insights into the unique release dynamics and molecular regulation that distinguish neuropeptides from small molecule neurotransmitters.
© 2025. The Author(s).