Optogenetic manipulation of Gq- and Gi/o-coupled receptor signaling in neurons and heart muscle cells.

G-protein-coupled receptors (GPCRs) transmit signals into cells depending on the G protein type. To analyze the functions of GPCR signaling, we assessed the effectiveness of animal G-protein-coupled bistable rhodopsins that can be controlled into active and inactive states by light application using zebrafish. We expressed Gq- and Gi/o-coupled bistable rhodopsins in hindbrain reticulospinal V2a neurons, which are involved in locomotion, or in cardiomyocytes. Light stimulation of the reticulospinal V2a neurons expressing Gq-coupled spider Rh1 resulted in an increase in the intracellular Ca2+ level 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 zebrafish neurons and cardiomyocytes.

Optogenetic manipulation of neuronal and cardiomyocyte functions in zebrafish using microbial rhodopsins and adenylyl cyclases.

Even though microbial photosensitive proteins have been used for optogenetics, their use should be optimized to precisely control cell and tissue functions in vivo. We exploited GtCCR4 and KnChR, cation channelrhodopsins from algae, BeGC1, a guanylyl cyclase rhodopsin from a fungus, and photoactivated adenylyl cyclases (PACs) from cyanobacteria (OaPAC) or bacteria (bPAC), to control cell functions in zebrafish. Optical activation of GtCCR4 and KnChR in the hindbrain reticulospinal V2a neurons, which are involved in locomotion, induced swimming behavior at relatively short latencies, whereas activation of BeGC1 or PACs achieved it at long latencies. Activation of GtCCR4 and KnChR in cardiomyocytes induced cardiac arrest, whereas activation of bPAC 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 function and regulation of zebrafish neurons and cardiomyocytes.

Involvement of Cerebellar Neural Circuits in Active Avoidance Conditioning in Zebrafish.

When animals repeatedly receive a combination of neutral conditional stimulus (CS) and aversive unconditional stimulus (US), they learn the relationship between CS and US, and show conditioned fear responses after CS. They show passive responses such as freezing or panic movements (classical or Pavlovian fear conditioning), or active behavioral responses to avoid aversive stimuli (active avoidance). Previous studies suggested the roles of the cerebellum in classical fear conditioning but it remains elusive whether the cerebellum is involved in active avoidance conditioning. In this study, we analyzed the roles of cerebellar neural circuits during active avoidance in adult zebrafish. When pairs of CS (light) and US (electric shock) were administered to wild-type zebrafish, about half of them displayed active avoidance. The expression of botulinum toxin, which inhibits the release of neurotransmitters, in cerebellar granule cells (GCs) or Purkinje cells (PCs) did not affect conditioning-independent swimming behaviors, but did inhibit active avoidance conditioning. Nitroreductase (NTR)-mediated ablation of PCs in adult zebrafish also impaired active avoidance. Furthermore, the inhibited transmission of GCs or PCs resulted in reduced fear-conditioned Pavlovian fear responses. Our findings suggest that the zebrafish cerebellum plays an active role in active avoidance conditioning.