Shigekazu Oda

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.

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.

The Temporal Pattern of Stimulation Determines the Extent and Duration of MAPK Activation in a Caenorhabditis elegans Sensory Neuron

Live-cell imaging in nematodes reveals the complex responses of a mitogen-activated protein kinase to neuronal stimulation. Live Dynamics of Cell Signaling Tomida et al. describe the development of a fluorescent sensor that reported the activity of a specific kinase, ERK, and its application to the analysis of cell signaling in neurons of living nematode worms. By expressing this ERK sensor in a specific neuron that responds to changes in extracellular salt concentration, they found that the activity of the kinase exhibited complex, nonlinear kinetics in response to pulsatile changes in NaCl. The nonlinear dynamics appeared to result from nonlinear calcium signals triggered by the different stimulation paradigms, and simulations were consistent with this hypothesis. Thus, tracking signaling in single cells of living organisms reveals the dynamic complexity of cellular responses. 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.

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

Significance Although cGMP is a prominent second messenger in neurons, it has proven difficult to follow its dynamics in an intact nervous system in real time. Here, we use a genetically encoded cGMP sensor, cGi500, to visualize and genetically dissect endogenous cGMP dynamics in vivo. As a model, we use a Caenorhabditis elegans O2-sensing neuron. We uncover mutually regulating negative feedback mechanisms mediated by cGMP and Ca2+ that involve PDE-1 and PDE-2 phosphodiesterases, protein kinase G, and cyclic nucleotide-gated channels. By simultaneously imaging Ca2+ and cGMP, we provide evidence of compartmentalization of cGMP signaling in different cellular domains. 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 Ca2+ 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 Ca2+ response in the neuron that depends on cGMP-gated ion channels. Elevated levels of cGMP and Ca2+ stimulate competing negative feedback loops that shape cGMP dynamics. Ca2+-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 Ca2+-dependent negative feedback loops. Simultaneous imaging of Ca2+ and cGMP suggests that cGMP levels can rise close to cGMP channels while falling elsewhere. O2-evoked cGMP and Ca2+ 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.

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)P3 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 Ca2+ 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.

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 Ca2+ responses in the URX O2 sensors, and GLB-5 enables rapid recovery of these responses upon re-oxygenation. Forward genetic screens indicate that GLB-5s 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 neurons 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.

Modulation of sensory information processing by a neuroglobin in Caenorhabditis elegans

Significance Sensory neurons encode environmental stimuli in their electrical activity and alter behavior and physiology by transmitting this information to downstream circuits. Their response properties can be characterized by tuning curves that relate stimulus parameters to neural responses. Tuning curves identify the response threshold, the stimulus features at the tuning curve peak, and high-slope regions that give maximum stimulus discrimination. Here we show that two antagonistically acting molecular oxygen sensors, a neuroglobin and a soluble guanylate cyclase, sculpt a sharp sigmoidal tuning curve in the URX oxygen sensing neurons of Caenorhabditis elegans. By combining experiments with computational modelling, we show that these changes in stimulus-encoding properties broaden C. elegans’s O2 preference. 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.

Coordinated change of acting sensory neurons is important for olfactory preference change depending upon odor concentration

Previous effective Granger causality analyses of functional magnetic resonance imaging (fMRI) have revealed dynamic causal flows along the dorsal attention network (DAN) during voluntary attentional control in the human brain. During resting state, however, fMRI studies have shown that the DAN is also intrinsically configured by functional connectivity, even in the absence of explicit task demands, and that may conflict with studies using Granger causality on visual attention. To resolve this contradiction, we performed a partial Granger causality (pGC) on event-related fMRI data during an attentional cueing paradigm while optimizing experimental and imaging parameters for pGC analysis. Analysis by pGC can factor out exogenous or latent influences from results of the analysis due to unmeasured variables. Typical regions along the DAN with greater activation during orienting (spatial cue) than withholding of attention (neutral cue) were selected as regions of interest (ROIs). pGC analysis on fMRI data from the ROIs showed that frontal-to-parietal top-down causal flows along the DAN appeared during (voluntary) orienting, but not during other, less-attentive and/or resting-like conditions. These results demonstrate that these causal flows along the DAN exclusively mediate voluntary covert orienting. These findings suggest that neural representations of attention in frontal regions are at the top of the hierarchy of the DAN for embodying voluntary attentional control. Research fund: MEXT Grant-in-Aid for Young Scientists (B) (22700281).