Optical Control of Cell Signaling with Red/Far-Red Light-Responsive Optogenetic Tools in Caenorhabditis elegans.

Optogenetic techniques have been intensively applied to the nematode Caenorhabditis elegans to investigate its neural functions. However, as most of these optogenetics are responsive to blue light and the animal exhibits avoidance behavior to blue light, the application of optogenetic tools responsive to longer wavelength light has been eagerly anticipated. In this study, we report the implementation in C. elegans of a phytochrome-based optogenetic tool that responds to red/near-infrared light and manipulates cell signaling. We first introduced the SynPCB system, which enabled us to synthesize phycocyanobilin (PCB), a chromophore for phytochrome, and confirmed the biosynthesis of PCB in neurons, muscles, and intestinal cells. We further confirmed that the amount of PCBs synthesized by the SynPCB system was sufficient for photoswitching of phytochrome B (PhyB)-phytochrome interacting factor 3 (PIF3). In addition, optogenetic elevation of intracellular Ca2+ levels in intestinal cells induced a defecation motor program. These SynPCB system and phytochrome-based optogenetic techniques would be of great value in elucidating the molecular mechanisms underlying C. elegans behaviors.

OFF-responses of interneurons optimize avoidance behaviors depending on stimulus strength via electrical synapses.

Optimization of the types and timing of avoidance behaviors depending on the intensity of a noxious stimulus is essential for survival; however, processing in the central nervous system and its developmental basis are largely unknown. Here, we report that Caenorhabditis elegans preferentially selects one of three different types of avoidance behaviors depending on the strength of the noxious stimulus. We screened 210 neuronal transcription factors using a combination of optogenetics and RNA interference methods and identified 19 candidates required for avoidance behaviors. One candidate, gene lin-32 (abnormal cell LINeage 32), which encodes an atonal homolog, is required for the neural fate determination of AIB interneurons and functions by regulating the expression of electrical and chemical synapse genes, namely, inx-1 (innexin 1) and AMPA-type ionotropic glutamate receptor glr-1. When examined by Ca imaging, AIB showed an OFF calcium increase to the noxious stimulus. The OFF calcium increase was provoked only by strong stimulation, suggesting a role for optimization of the avoidance behavior. However, lin-32 mutants showed an impaired AIB OFF calcium increase, concomitant with a reduced occurrence of the dynamic avoidance behavior called the "omega turn". The AIB neural responses may be transferred to downstream inter/motor neurons projecting to the neck muscles via electrical synapses comprising inx-1. Finally, we found a correlation between powerful contractions of the neck muscles and omega turns. Thus, the central regulation of the magnitude and timing of activation of the AIB interneurons optimizes the probability of omega turn depending on the stimulus context.

Efficient synthesis of phycocyanobilin in mammalian cells for optogenetic control of cell signaling.

Optogenetics is a powerful tool to precisely manipulate cell signaling in space and time. For example, protein activity can be regulated by several light-induced dimerization (LID) systems. Among them, the phytochrome B (PhyB)-phytochrome-interacting factor (PIF) system is the only available LID system controlled by red and far-red lights. However, the PhyB-PIF system requires phycocyanobilin (PCB) or phytochromobilin as a chromophore, which must be artificially added to mammalian cells. Here, we report an expression vector that coexpresses HO1 and PcyA with Ferredoxin and Ferredoxin-NADP+ reductase for the efficient synthesis of PCB in the mitochondria of mammalian cells. An even higher intracellular PCB concentration was achieved by the depletion of biliverdin reductase A, which degrades PCB. The PCB synthesis and PhyB-PIF systems allowed us to optogenetically regulate intracellular signaling without any external supply of chromophores. Thus, we have provided a practical method for developing a fully genetically encoded PhyB-PIF system, which paves the way for its application to a living animal.

Modulation of sensory information processing by a neuroglobin in Caenorhabditis elegans.

Sensory receptor neurons match their dynamic range to ecologically relevant stimulus intensities. How this tuning is achieved is poorly understood in most receptors. The roundworm Caenorhabditis elegans avoids 21% O2 and hypoxia and prefers intermediate O2 concentrations. We show how this O2 preference is sculpted by the antagonistic action of a neuroglobin and an O2-binding soluble guanylate cyclase. These putative molecular O2 sensors confer a sigmoidal O2 response curve in the URX neurons that has highest slope between 15 and 19% O2 and approaches saturation when O2 reaches 21%. In the absence of the neuroglobin, the response curve is shifted to lower O2 values and approaches saturation at 14% O2 In behavioral terms, neuroglobin signaling broadens the O2 preference of Caenorhabditis elegans while maintaining avoidance of 21% O2 A computational model of aerotaxis suggests the relationship between GLB-5-modulated URX responses and reversal behavior is sufficient to broaden O2 preference. In summary, we show that a neuroglobin can shift neural information coding leading to altered behavior. Antagonistically acting molecular sensors may represent a common mechanism to sharpen tuning of sensory neurons.

GLOBIN-5-dependent O2 responses are regulated by PDL-1/PrBP that targets prenylated soluble guanylate cyclases to dendritic endings.

Aerobic animals constantly monitor and adapt to changes in O2 levels. The molecular mechanisms involved in sensing O2 are, however, incompletely understood. Previous studies showed that a hexacoordinated globin called GLB-5 tunes the dynamic range of O2-sensing neurons in natural C. elegans isolates, but is defective in the N2 lab reference strain (McGrath et al., 2009; Persson et al., 2009). GLB-5 enables a sharp behavioral switch when O2 changes between 21 and 17%. Here, we show that GLB-5 also confers rapid behavioral and cellular recovery from exposure to hypoxia. Hypoxia reconfigures O2-evoked Ca(2+) responses in the URX O2 sensors, and GLB-5 enables rapid recovery of these responses upon re-oxygenation. Forward genetic screens indicate that GLB-5's effects on O2 sensing require PDL-1, the C. elegans ortholog of mammalian PrBP/PDE6δ protein. In mammals, PDE6δ regulates the traffic and activity of prenylated proteins (Zhang et al., 2004; Norton et al., 2005). PDL-1 promotes localization of GCY-33 and GCY-35, atypical soluble guanylate cyclases that act as O2 sensors, to the dendritic endings of URX and BAG neurons, where they colocalize with GLB-5. Both GCY-33 and GCY-35 are predicted to be prenylated. Dendritic localization is not essential for GCY-35 to function as an O2 sensor, but disrupting pdl-1 alters the URX neuron's O2 response properties. Functional GLB-5 can restore dendritic localization of GCY-33 in pdl-1 mutants, suggesting GCY-33 and GLB-5 are in a complex. Our data suggest GLB-5 and the soluble guanylate cyclases operate in close proximity to sculpt O2 responses.

In vivo genetic dissection of O2-evoked cGMP dynamics in a Caenorhabditis elegans gas sensor.

cGMP signaling is widespread in the nervous system. However, it has proved difficult to visualize and genetically probe endogenously evoked cGMP dynamics in neurons in vivo. Here, we combine cGMP and Ca(2+) biosensors to image and dissect a cGMP signaling network in a Caenorhabditis elegans oxygen-sensing neuron. We show that a rise in O2 can evoke a tonic increase in cGMP that requires an atypical O2-binding soluble guanylate cyclase and that is sustained until oxygen levels fall. Increased cGMP leads to a sustained Ca(2+) response in the neuron that depends on cGMP-gated ion channels. Elevated levels of cGMP and Ca(2+) stimulate competing negative feedback loops that shape cGMP dynamics. Ca(2+)-dependent negative feedback loops, including activation of phosphodiesterase-1 (PDE-1), dampen the rise of cGMP. A different negative feedback loop, mediated by phosphodiesterase-2 (PDE-2) and stimulated by cGMP-dependent kinase (PKG), unexpectedly promotes cGMP accumulation following a rise in O2, apparently by keeping in check gating of cGMP channels and limiting activation of Ca(2+)-dependent negative feedback loops. Simultaneous imaging of Ca(2+) and cGMP suggests that cGMP levels can rise close to cGMP channels while falling elsewhere. O2-evoked cGMP and Ca(2+) responses are highly reproducible when the same neuron in an individual animal is stimulated repeatedly, suggesting that cGMP transduction has high intrinsic reliability. However, responses vary substantially across individuals, despite animals being genetically identical and similarly reared. This variability may reflect stochastic differences in expression of cGMP signaling components. Our work provides in vivo insights into the architecture of neuronal cGMP signaling.

The temporal pattern of stimulation determines the extent and duration of MAPK activation in a Caenorhabditis elegans sensory neuron.

The Caenorhabditis elegans ASER sensory neuron is excited when environmental NaCl concentration is decreased. The mitogen-activated protein kinase (MAPK) MPK-1, a homolog of ERK (extracellular signal-regulated kinase), is activated during excitation of ASER sensory neurons. We created and expressed a fluorescence resonance energy transfer (FRET)-based MAPK activity probe in ASER neurons and then exposed the worms to various cyclic patterns of stimulation (changes in NaCl concentration) to monitor the dynamics of MPK-1 activity. We found that the intensity and duration of MPK-1 activity were determined by the temporal pattern of stimulation, namely, a combination of stimulation period length, stimulation duration, and time between stimuli. The complex, nonlinear relationship between stimulation and MPK-1 activation was explained by the properties of intracellular calcium responses upstream of MPK-1. Thus, we visualized the dynamics of MAPK activation in a sensory neuron in living nematodes in response to complex stimuli and present a reporter that can be used in higher eukaryotes to test in silico predictions regarding the MAPK pathway.

Odour concentration-dependent olfactory preference change in C. elegans.

The same odorant can induce attractive or repulsive responses depending on its concentration in various animals including humans. However, little is understood about the neuronal basis of this behavioural phenomenon. Here we show that Caenorhabditis elegans avoids high concentrations of odorants that are attractive at low concentrations. Behavioural analyses and computer simulation reveal that the odour concentration-dependent behaviour is primarily generated by klinokinesis, a behavioural strategy in C. elegans. Genetic analyses and lesion experiments show that distinct combinations of sensory neurons function at different concentrations of the odorant; AWC and ASH sensory neurons have critical roles for attraction to or avoidance of the odorant, respectively. Moreover, we found that AWC neurons respond to only lower concentrations of the odorant, whereas ASH neurons respond to only higher concentrations of odorant. Hence, our study suggests that odour concentration coding in C. elegans mostly conforms to the labelled-line principle where distinct neurons respond to distinct stimuli.

Neuronal plasticity regulated by the insulin-like signaling pathway underlies salt chemotaxis learning in Caenorhabditis elegans.

Quantification of neuronal plasticity in a living animal is essential for understanding learning and memory. Caenorhabditis elegans shows a chemotactic behavior toward NaCl. However, it learns to avoid NaCl after prolonged exposure to NaCl under starvation conditions, which is called salt chemotaxis learning. Insulin-like signaling is important for this behavioral plasticity and functions in one of the salt-sensing sensory neurons, ASE right (ASER). However, how neurons including ASER show neuronal plasticity is unknown. To determine the neuronal plasticity related to salt chemotaxis learning, we measured Ca(2+) response and synaptic release of individual neurons by using in vivo imaging techniques. We found that response of ASER increased whereas its synaptic release decreased after prolonged exposure to NaCl without food. These changes in the opposite directions were abolished in insulin-like signaling mutants, suggesting that insulin-like signaling regulates these plasticities in ASER. The response of one of the downstream interneurons, AIB, decreased profoundly after NaCl conditioning. This alteration in AIB response was independent of the insulin-like signaling pathway. Our results suggest that information on NaCl is modulated at the level of both sensory neurons and interneurons in salt chemotaxis learning.

Roles for class IIA phosphatidylinositol transfer protein in neurotransmission and behavioral plasticity at the sensory neuron synapses of Caenorhabditis elegans.

Growing evidence suggests that sensory neuron synapses not merely pass, but actively encode sensory information and convey it to the central nervous system. The chemosensory preferences of Caenorhabditis elegans, as manifested in the direction of chemotaxis, are reversibly regulated by prior experience at the level of sensory neurons; the attractive drive is promoted by diacylglycerol (DAG) signaling, whereas the counteracting repulsive drive requires PtdIns(3,4,5)P(3) signaling. Here we report that the two opposing drives require a class IIA phosphatidylinositol transfer protein (PITP), PITP-1, which localizes to the sensory neuron synapses. In pitp-1 mutants, attraction behavior to salt is reduced, whereas conditioned repulsion from salt is eliminated: the mutants inflexibly show weak attraction behavior to salt, irrespective of prior experience. To generate flexible behavioral outputs, attraction and repulsion, PITP-1 acts in the gustatory neuron ASER and likely regulates neurotransmission from ASER, as pitp-1 mutations do not affect the ASER Ca(2+) response to sensory stimulus. Furthermore, full attraction to salt is restored in pitp-1 mutants by expression of the phosphatidylinositol transfer domain alone, and also by mutations of a DGK gene that cause accumulation of DAG, suggesting that PITP-1 serves for DAG production via phosphatidylinositol transport and, hence, regulates synaptic transmission. In addition to gustatory behavior, olfactory behaviors and osmotic avoidance are also regulated by PITP-1 in the sensory neurons that detect each sensory stimulus. Thus, PITP-1-dependent phosphatidylinositol transport is essential for sensory neuron synapses to couple sensory inputs to effective behavioral responses.