Orexins (also called hypocretins) are hypothalamic neuropeptides that carry out essential functions in the central nervous system; however, little is known about their release and range of action in vivo owing to the limited resolution of current detection technologies. Here we developed a genetically encoded orexin sensor (OxLight1) based on the engineering of circularly permutated green fluorescent protein into the human type-2 orexin receptor. In mice OxLight1 detects optogenetically evoked release of endogenous orexins in vivo with high sensitivity. Photometry recordings of OxLight1 in mice show rapid orexin release associated with spontaneous running behavior, acute stress and sleep-to-wake transitions in different brain areas. Moreover, two-photon imaging of OxLight1 reveals orexin release in layer 2/3 of the mouse somatosensory cortex during emergence from anesthesia. Thus, OxLight1 enables sensitive and direct optical detection of orexin neuropeptides with high spatiotemporal resolution in living animals.

Neurochemicals have a large impact on brain states and animal behavior but are notoriously hard to detect accurately in the living brain. Recently developed genetically encoded sensors obtained from engineering a circularly permuted green fluorescent protein into G-protein coupled receptors (GPCR) provided a vital boost to neuroscience, by innovating the way we monitor neural communication. These new probes are becoming widely successful due to their flexible combination with state of the art optogenetic tools and in vivo imaging techniques, mainly fiber photometry and 2-photon microscopy, to dissect dynamic changes in brain chemicals with unprecedented spatial and temporal resolution. Here, we highlight current approaches and challenges as well as novel insights in the process of GPCR sensor development, and discuss possible future directions of the field.

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The glucagon-like peptide-1 receptor (GLP1R) is a broadly expressed target of peptide hormones with essential roles in energy and glucose homeostasis, as well as of the blockbuster weight-loss drugs semaglutide and liraglutide. Despite its large clinical relevance, tools to investigate the precise activation dynamics of this receptor with high spatiotemporal resolution are limited. Here we introduce a novel genetically-encoded sensor based on the engineering of a circularly-permuted green fluorescent protein into the human GLP1R, named GLPLight1. We demonstrate that fluorescence signal from GLPLight1 accurately reports the expected receptor conformational activation in response to pharmacological ligands with high sensitivity (max ΔF/F0 = 528%) and temporal resolution (τON = 4.7 sec). We further demonstrated that GLPLight1 shows comparable responses to GLP-1 derivatives as observed for the native receptor. Using GLPLight1, we established an all-optical assay to characterize a novel photocaged GLP-1 derivative (photo-GLP1) and to demonstrate optical control of GLP1R activation. Thus, the new all-optical toolkit introduced here enhances our ability to study GLP1R activation with high spatiotemporal resolution.