Abstract
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.
Highlights
Orexins are hypothalamic neuropeptides that carry out essential functions in the central nervous system; little is known about their release and range of action in vivo owing to the limited resolution of current detection technologies
Developed genetically encoded sensors derived from the engineering of a circularly permutated green fluorescent protein into G protein-coupled receptors (GPCRs) started shedding light on the release of neurotransmitters[23,24,25,26,27,28] in the brain and offer an attractive solution to elucidate neuropeptide release. Using this approach we developed a genetically encoded neuropeptide sensor (OxLight1) that accurately reports the dynamics of endogenous orexins in the mouse brain under both one-photon and two-photon illumination
To develop a genetically encoded orexin sensor we inserted a circularly permutated green fluorescent protein (cpGFP) module from the dopamine sensor dLight[1] into each of the two human orexin receptors (OX1R and OX2R) at a site selected on the basis of sequence alignment with the dopamine receptor D1 (Extended Data Fig. 1a and Supplementary Note)
Summary
Orexins ( called hypocretins) are hypothalamic neuropeptides that carry out essential functions in the central nervous system; little is known about their release and range of action in vivo owing to the limited resolution of current detection technologies. Orexins ( known as hypocretins) are two neuropeptides with conserved structures and functions across mammals[1,2]; they are necessary for stable wakefulness and sleep–wake cycles, as illustrated by the cataplexy and sleep fragmentation displayed by orexin or orexin type-2 receptor or orexin cell knockout mice[3,4,5], orexin type-2 receptor-deficient dogs[6] and orexin-deficient humans[7,8,9] Both endogenous orexins (orexin-A and orexin-B) are produced by a subpopulation of neurons exclusively located in the lateral hypothalamus (LH)[10]. In spite of current knowledge, fundamental aspects of the orexin system such as the mechanisms controlling extracellular orexin degradation in the brain and the natural dynamics of orexins across behaviors, brain areas and timescales, remain difficult to study[17,22]
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