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Even though microbial photosensitive proteins have been used for optogenetics, their use should be optimized to precisely control second messengersin vivo. We exploitedGtCCR4andKnChR, cation channelrhodopsins from algae,BeGC1, a guanylyl cyclase rhodopsin from a fungus, and photoactivated adenylyl cyclases (PACs) from cyanobacteria (OαPAC) or bacteria (bPAC), to control cell functions in zebrafish. Optical activation ofGtCCR4 andKnChR in the hindbrain reticulospinal V2a neurons, which are involved in locomotion, immediately induced swimming behavior, whereas activation ofBeGC1 or PACs was achieved at a short latency.KnChR had the highest locomotioninducing activity of all the channelrhodopsins examined. Activation ofGtCCR4 andKnChR in cardiomyocytes induced cardiac arrest, whereas activation ofbPAC gradually induced bradycardia.KnChR activation led to an increase in intracellular Ca2+in the heart, suggesting that depolarization caused cardiac arrest. These data suggest that these optogenetic tools can be used to reveal the roles of second messengers in various cell types in vertebrates.Impact statementWe identified efficient and useful microbial channelrhodopsin, guanylyl cyclase rhodopsin, and photoactivated adenylyl cyclase that regulate neural activity and cardiac function in zebrafish.Major subject areasNeuroscience, Cell biologyResearch organismZebrafish (Danio rerio)

G protein-coupled receptors (GPCRs) transmit extracellular signals into the cell depending on the type of G protein. To analyze the functions of GPCR signaling, we developed optogenetic tools using animal G protein-coupled bistable rhodopsins that can be controlled into active and inactive states by light irradiation. We expressed Gq- and Gi/o-coupled bistable rhodopsins in hindbrain reticulospinal V2a neurons, which are involved in locomotion, or in cardiomyocytes of zebrafish. Light stimulation of the reticulospinal V2a neurons expressing Gq-coupled spider Rh1 resulted in an increase in the level of cytoplasmic Ca2+and evoked swimming behavior. Light stimulation of cardiomyocytes expressing the Gi/o-coupled mosquito Opn3, pufferfish TMT opsin, or lamprey parapinopsin induced cardiac arrest, and the effect was suppressed by treatment with pertussis toxin or barium, suggesting that Gi/o-dependent regulation of inward-rectifier K+channels controls cardiac function. These data indicate that these rhodopsins are useful for optogenetic control of GPCR-mediated signaling in neurons and cardiomyocytesin vivo.Impact statementAnimal G protein-coupled bistable rhodopsins can regulate Gq and Gi-mediated signaling in a light-dependent manner in neurons and cardiomyocytes, making them useful for analyzing roles of GPCR signalingin vivo.