Abstract Synaptic transmission is mainly mediated by classical neurotransmitters such as glutamate and g-amino butyric acid (GABA) which transduce fast information flow in the brain. This process is tightly regulated by neuromodu- lators including monoamines and neuropeptides. Among neuromodulators, neuropeptides in particular, have been difficult to study because they are often chemically inert (non-oxidizable) and typically activate 7 transmem- brane G-protein coupled receptors (GPCRs) which rely on delayed (seconds to minutes) second messenger signaling, precluding their study by conventional electrophysiology or with oxidizable probes. Moreover, endog- enous neuropeptides are typically sorted from large polyprotein precursors into specific pools of dense core vesicles (DCVs), suggesting the need for a detection strategy that does not interfere with the biogenesis and native sorting of endogenous DCVs. As a result, there is currently a lack of suitable tools for studying the spatial and temporal dynamics of neuropeptide release and peptidergic neurocircuitry. Toward addressing this need, we propose to develop a new family of genetically-encoded optical sensors, termed Chimeric Detectors for Neu- ropeptide Release (CDNRs), by harnessing the unique structural signatures of Class B (Secretin-like) GPCRs that exclusively recognize peptides as their native ligands. Based on recently solved protein structures and the availability of new robust genetic models to apply and validate our approach, we will specifically focus on gluca- gon-like peptide-1 (GLP-1) and corticotropin-releasing hormone/factor (CRH/CRF) which have important func- tions in complex neurobehaviors such as feeding and stress. To validate CDNRs for detection of neuropeptide release, we will express CDNRs in defined GLP-1 and CRF circuitry using state-of-the-art viral mediated gene transduction in specific genetically modified mouse models and perform high-resolution optical recording to de- tect release of endogenous GLP-1 and CRF, both ex vivo in brain slices and in vivo in behaving animals. Suc- cessful implementation of this work will deliver a set of novel and well validated genetically-encoded CDNRs that can be immediately applied to dissect the peptidergic circuitry of GLP-1 and CRF in the brain. Moreover, this will also lay a conceptual and technical foundation for the future development of detectors for several other neuro- peptides (PACAP, VIP, CGRP and secretin) included among peptide-binding class B GPCRs. !

Narrative Neuropeptides critically modulate synaptic transmission and neural activity in the brain. However, existing tools for detecting and studying neuropeptide release are lacking. We propose to develop and apply optical biosensors for real time imaging of neuropeptide release, focusing on glucagon-like peptide-1 (GLP-1) and corticotrophin- releasing factor (CRF). !