Discrete populations of cells secrete neuropeptides that subsequently interact with G-protein-coupled receptors (GPCRs) to regulate various behavioral and physiological processes. However, detecting neuropeptide release with spatiotemporal resolution remains difficult. GPCR activation-based (GRAB) sensors have previously been developed to detect the release of neuropeptides such as orexin and oxytocin. The design of GRAB sensors requires the insertion of circularly permuted green fluorescent protein (cpGFP) into the third intracellular loop (ICL3) of a particular neuropeptide receptor. Ligand-induced changes in GPCR conformation then provide a corresponding fluorescence readout. However, given the sequence variation in the ICL3 between different receptors, insertion of cpGFP into the ICL3 to obtain optimal fluorescence requires constant trial and error. Wang et al. developed a strategy to expedite the design of GRAB sensors for multiple neuropeptides by transplanting the entire cpGFP-containing ICL3 with an optimized linker length from existing GRAB sensors into the neuropeptide receptor of interest. This approach enabled the development of GRAB sensors for the somatostatin receptor and the corticotropin-releasing factor receptor with high sensitivity and selectivity ex vivo and in vivo. In particular, the somatostatin receptor sensor detected the release of endogenous somatostatin in mouse cortical neurons and pancreatic islets and from the basolateral amygdala in response to conditioned learning. Viral-mediated expression of the corticotropin-releasing factor receptor sensor produced increased fluorescence in the central amygdala with electric stimuli that corresponded to increased levels of corticotropin-releasing factor. Overall, the strategy from Wang et al. will permit the rapid synthesis of neuropeptide sensors and will enable new insights into neuropeptide action and function.
Original reference: Science 382, 786 (2023)
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