Takagaki, Natsune et al. (2015) International Worm Meeting "Plasticity of cold habituation and isolation of genes involved in temperature experience storage."

Minakuchi, Yohei, Ishiwari, Tomohiro, Takagaki, Natsune, Ohta, Akane, Kuhara, Aysushi, Toyoda, Atsushi

[International Worm Meeting, 2015]

Animals can survive and proliferate under continual environmental temperature changes, by receiving temperature information and processing to previous temperature experience. We are aiming at the elucidation of temperature habituation mechanism by using cold tolerance. Wild-type animals can survive at 2degC for 48 hours after cultivation at 15degC (15degC > 2degC(48h)), however, 25degC-cultivated animals can not survive at 2degC for 48 hours (25degC > 2degC(48h)). We previously reported that ASJ sensoryneurons sense temperature and regulate temperature habituation through insulin signaling (Ohta, Ujisawa et al., Nature Commun, 2014). We also previously found that 25degC-cultivated animals are shifted to and stayed at 15degC for 3 hours, and then these animals can survive at 2degC (25degC > 15degC(3h) > 2degC(48h)). This data indicates that temperature experience for cold habituation has been established within 3 hours. To analyze whether this habituation has a plasticity aspect, we utilized multiple steps temperature shift assay. For example, 15degC-cultivated animals were exposed to 2degC for 12 hours, and then transferred to and stayed at 25degC for 8 hours, finally exposed to 2degC for 48 hours again (15degC > 2degC("12h") > 25degC(8h) > 2degC(48h)). We found that about 60% of animals survived after this multiple steps temperature shift. By contrast, most of animals did not survive after 15degC > 2degC("0h") > 25degC(8h) >2degC(48h). These results suggest that previous cold experience is memorized and plastically affects cold habituation. Similarly, after 25degC > 15degC(12h) > 25degC(5h) > 2>(48h) shift, only 5% of wild-type can survive. By contrast, without first 25degC experience (15degC > 25degC(5h) > 2degC(48h)), the survival rates of wild-type animals were 30%. These results suggest that previous temperature experiences affects the time needed for habituation to new temperature. We found that crh-1/CREB mutants showed abnormality in the assay. In order to isolate molecules involved in formation of temperature experience in the cold habituation, we performed DNA microarray analysis. The expression levels of 112 genes were significantly changed 3 hours after temperature shift from 15 to 25degC. Of these, mutants defective in a transmembrane glycoprotein and a nuclear hormone receptor at least showed abnormal temperature habituation speed. We are also going on another study. flp-17 gene was isolated by our DNA microarray analysis. Although flp-17(ok3587) null mutant showed abnormal cold habituation, another null mutant flp-17(n4894) showed normal phenotype. We speculated that background mutation causes this abnormality. We are now performing SNP analysis of this mutation based on whole genome DNA sequence of flp-17(ok3587) mutant, which was decoded by next generation DNA sequencer. .

Fumiya Nakamura et al. (2010) East Asia Worm Meeting "Elucidation of mechanism underlying information processing in the neural circuit for thermotaxis"

Noriyuki Ohnishi, Ikue Mori, Atsushi Kuhara, Fumiya Nakamura

[East Asia Worm Meeting, 2010]

Behavior is an ultimate consequence of processing huge information in neural circuits. In order to dissect the mechanism of information processing in the neural circuit, we focused on the simple neural circuit for thermotaxis behavior. Temperature is sensed and remembered by AFD and AWC sensory neurons, thermal information from AFD and AWC is transmitted to AIY interneuron, and the subsequent information from AIY is further transmitted to AIZ and RIA interneurons for further neural information processing (1,2). Neuronal signaling between neurons is mainly regulated through synapses. Recent studies in our laboratory revealed that regulation of neuron-to-neuron signaling in the synapses is rather complex, involving multiple neurotransmitters and the receptors (3,4). To further investigate the information processing mechanism in the synapses of the circuit, we tested themotaxis of 54 mutants defective in the genes related to synaptic neurotransmission. Our results found that several genes encoding Cl- channel-type glutamate receptor, nicotinic acetylcholine receptor or neuropeptide are needed for thermotaxis behavior. We are currently trying to identify cells and synapses where these genes function.(1) Kuhara et al., 2008, Science 320, 803-807. (2) Mori and Ohshima, 1995, Nature 376, 344-348.(3) Kuhara et al., (submitted)(4) Ohnishi et al., (submitted)

Kuhara, Atsushi et al. (2009) International Worm Meeting "Exploring the neural code in the neural circuit for thermotaxis behavior."

Mori, Ikue, Shimowada, Tomoyasu, Kuhara, Atsushi, Ohnishi, Noriyuki

[International Worm Meeting, 2009]

Behavior is an ultimate consequence of orchestrated neural calculation. Thermotaxis of C. elegans is an ideal system for comprehensively understanding how a neural circuit encodes a behavioral output (1). In thermotaxis neural circuit, temperature is mainly sensed by AFD neuron, and its information is conveyed to AIY interneuron (1, 2). To elucidate neural calculation, we aimed to inactivate neuronal activity by employing light-activated chloride pump halorhodopsin (HR) (3). Millisecond scale pulsed-light system, which allows Hz-controllable deliveries of light, was developed and equipped in both calcium imaging microscope and C. elegans auto-tracking microscope. Calcium imaging analysis demonstrated that pulsed-excitation of HR in AFD partially reduced calcium influx in AFD for thermal stimuli. Since AFD-ablated animals move randomly or migrate to lower temperature than the cultivation temperature on a thermal gradient (1), we speculated that exciting HR in AFD induces cryophilic or athermotactic abnormalities. We unexpectedly found that pulsed-excitation of HR in AFD induced thermophlic abnormality. Similar abnormality was observed in the mutant exhibiting an abnormal glutamate synaptic transmission in AFD due to AFD-specific defect in EAT-4 (vesicular glutamate transporter) (N. O., A. K. and I. M., this meeting). Previous report (4) and our calcium imaging analysis revealed that calcium influx in AFD for thermal stimuli induced calcium influx in AIY. This suggests that temperature information of AFD is conveyed to AIY through "excitatory" connection. We however found that pulsed-excitation of HR in AFD notably enhanced calcium influx of AIY for thermal stimuli, despite AFD activity itself was partially reduced. Similar abnormal thermal responses of both AFD and AIY were observed in thermophilic mutant tax-6 (calcineurin) that is defective in AFD thermosensory signaling (5, 6), implicating "inhibitory" connection from AFD to AIY. Altogether, these physiological and behavioral results are consistent with the notion that temperature signal in AFD dynamically affects AIY activity through both "inhibitory" and "excitatory" transmissions, which as a consequence likely generates opposite thermotactic behaviors, thermophilic and cryophlic migration on a temperature gradient. (1) Mori and Ohshima, Nature, 1995 (2) Kuhara, Okumura, et al., Science, 2008 (3) Zhang et al., Nature, 2007 (4) Biron et al., Nature Neuroscience, 2006 (5) Kuhara et al., Neuron, 2002 (6) Kuhara and Mori, J. Neurosci, 2006.

Kano, Amane et al. (2019) International Worm Meeting "Toward understanding neural computations in an interneuron AIY through optogenetical manipulation of its presynaptic sensory neurons AFD and AWC."

Kano, Amane, Tsukada, Yuki, Mori, Ikue

[International Worm Meeting, 2019]

How an interneuron integrates environmental cues transformed by sensory neurons is an important problem in neuroscience. The simplest model to elucidate this problem is a neural circuit consisting of two sensory neurons and their postsynaptic interneuron. One such model in C. elegans is a circuit consisting of thermosensory neurons AFD and AWC, and their postsynaptic interneuron AIY, which has been identified as a part of the circuit for thermotaxis (Mori & Ohshima, Nature, 1995; Kuhara, Okumura et al., Science, 2008). Previously, one of our studies shows that AFD either excited or inhibited AIY activity depending on thermal stimuli (Kuhara et al., Nat. Commun., 2011). Similarly, other study reports that activation of AWC suppressed AIY in response to thermal stimulus (Kuhara, Okumura et al., Science, 2008). However, how AIY responses depend on states of both AFD and AWC activities remains unclear. In this study, we investigated how AIY activity is affected by optogenetically controlling states of AFD and AWC activities. We first introduced GCaMP6f and Chrimson in either AFD or AWC, and confirmed that AFD and AWC activity could be excited by Chrimson. We next quantified AIY activity under controlling AFD, AWC, or both AFD and AWC was evoked by Chrimson by scoring spike-like AIY activity peaks to define as the frequency of AIY activity. We found that when AFD was excited by Chrimson, the frequency of AIY activity also increased. By contrast, when AWC was excited by Chrimson, the frequency of AIY activity was unchanged. However, when both AFD and AWC were simultaneously evoked, we observed two states, increment or decrement, of the frequency of AIY activity. Our results suggest that AIY responses are indeed dependent on the state of both AFD and AWC. Based on this result on simultaneous evoking of AFD and AWC activity, we are currently seeking the critical time window between excitation of AFD and AWC, which may affect AIY responses. To reveal critical time window, we use projection mapping system that enables illumination of pinpointed areas. We so far established our projection mapping system to illuminate AFD soma and AWC soma, both of which are about 10 m close to each other. We are also improving the software aiming to detect the increment of calcium signal and feedback it to excitation timing and illumination areas. By using this feedback software, we hope to reveal how states of AFD and AWC dynamically affect AIY activity, thereby contributing to clarify how sensory signals integrate within an interneuron.

Atsushi Kuhara et al. (2002) Japanese Worm Meeting "CaM kinase II UNC-43 positively regulates neuronal activity of AFD thermosensory neurons"

Ikue Mori, Atsushi Kuhara

[Japanese Worm Meeting, 2002]

Calcium/calmodulin dependent protein phosphatase calcineurin TAX-6 negatively regulates sensory neuronal signaling in C. elegans (Kuhara et al., 2002). How TAX-6 calcineurin is involved in sensory signal transduction pathway is still unclear. In mammalian hippocampus, calcium/calmodulin dependent protein kinase II (CaMKII) is assumed to be a candidate of counter partner of calcineurin (Malenka, 1994). In C. elegans , unc-43 gene encodes CaMKII and is essential for pleiotropic aspects, such as muscle contraction, defecation, and gene expression (Reiner et al., 1999, Troemel et al., 2001). unc-43 loss-of-function(lf) mutants have many defects in these biological processes, but sensory behavioral abnormality has not been reported. In this study, we show that CaMKII UNC-43 functions cell-autonomously, and positively regulates neuronal activity in thermosensory neuron AFD. unc-43(lf) mutants showed cryophilic phenotype, which resembles the phenotype of AFD defective mutants, and this phenotype is opposite to AFD hyper-activated thermophilic phenotype of tax-6(lf) mutants. unc-43(lf) mutation also lead to defective chemotaxis to AWC-sensed odorants, although chemotaxis to NaCl and AWA-sensed odorants are normal. The cryophilic phenotype of unc-43(lf) mutants was rescued by AFD specific expression of unc-43 cDNA, but this transgenic animals remained to be defective AWA-mediated olfactory responses. This results suggest that unc-43 acts cell-autonomously in thermosensory neuron AFD. Thus, the cryophilic phenotype of unc-43(lf) mutants are caused by reduced activity of AFD neurons. Reversibly, overexpression of unc-43 cDNA in AFD neurons caused thermophilic phenotype in approximately 40% of wild-type animals. Altogether, we suggest that UNC-43 CaMKII positively regulates neuronal activity in AFD neurons. references Kuhara et al (2002), Neuron 33, 751-763. Malenka (1994), Cell 78, 535-538. Reiner et al (1999), Nature 402; 199-203. Sagasti et al (2001), Cell 105; 221-232.

Atsushi Kuhara et al. (2004) East Asia Worm Meeting "TAX-6 calcineurin is required in RIA and AIZ interneurons for associative learning between temperature and starvation"

Atsushi Kuhara, Ikue Mori

[East Asia Worm Meeting, 2004]

Learning and memory are highly complex processes whose mechanisms are extremely intricate. In mammalian nervous system, the amount of information that is constantly received by the sensory neurons is processed and stored in complicated downstream neural circuits. We show here the simplest neural circuit regulating associative learning in C. elegans , where calcineurin, the Ca 2+ activated protein phosphatase, plays a critical role as a modulator of learning and memory. The calcineurin encoded by tax-6 gene is expressed in nearly all neurons including interneurons (Kuhara et. al., 2002), in which the learning and memory processes should occur. In order to investigate the function of TAX-6 calcineurin in the aspect of learning and memory, we made use of the interneuronal TAX-6 deficient transgenic mutants, tax-6;Ex[sensory+, inter-] , in which TAX-6 is expressed only in sensory neurons of tax-6 null mutants. We found that tax-6;Ex[sensory+, inter-] mutant animals showed defects in associative learning in two paradigms, (1) associative learning between temperature and starvation (Mohri et. al., 2003) and (2) associative learning between NaCl and starvation (Saeki et. al., 2001), although their capability of sensation and integration of two sensory inputs were normal. Defective associative learning between temperature and starvation of tax-6;Ex[sensory+, inter-] mutants were rescued by the expression of tax-6 cDNA in two pairs of interneurons, AIZ and RIA. We also found that in these neurons, TAX-6 calcineurin cell autonomously functions and its phosphatase activity is essential for associative learning. The AIZ-RIA mediated neural pathway is a critical wiring of thermotaxis neural circuit (Mori and Ohshima, 1995), suggesting that associative learning is regulated at neural circuit level. Thus, our results allow us to demonstrate the essential neural circuit regulating calcineurin-mediated associative learning in vivo. Kuhara et. al., 2002, Neuron 33, 751. Mohri et. al., 2003, IWM abstract. Saeki et. al., 2001, The Journal of Experimental Biology 204, 1757. Mori and Ohshima, 1995, Nature 376, 344.

Atsushi Kuhara et al. (2007) International Worm Meeting "Calcium imaging of the thermotaxis neural circuit: toward combinatorial feed back analysis between in vivo neurobiology and in silico computation."

Atsushi Kuhara, Noriyuki Ohnishi, Eiji Kodama, Ikue Mori

[International Worm Meeting, 2007]

We are using thermotaxis behavior as a model to investigate the essence of neural mechanism. Over the years, many molecular, cellular, neural circuit, and behavioral information underlying thermotaxis have been revealed out of reductionistic experimental approach, which will continue to be powerful for novel findings. We realized however that it would be still hard to fully reconstruct the living behavior, even if all molecules and functional neural circuits were determined. We then considered that one way to advance to the next stage of neurobiology is to conduct the combinatorial analysis based on various scientific disciplines, such as feed back analysis between in vivo neurobiology and in silico computation. Our strategy: 1. Measuring the neural property of thermotaxis neural circuit using calcium indicator. Measuring thermotactic output using tracking system. 2. Mathematically expressing individual neuronal property and thermotactic output. 3. Mathematically expressing neural circuit property by combining the in silico mathematical expressions of individual neurons and in vivo functional neural connections. 4. Predicting novel neural property, functional wiring, and behavioral output by in silico calculation 5. Judging the in silico prediction by using in vivo biological techniques, such as gene manipulation, calcium imaging, and behavioral test. 6. Circular feed back analysis between in vivo and in silico studies. We are now on the first stage. We have succeeded in measuring the thermal responses of individual thermosensory- and inter-neurons on thermotaxis neural circuit in wild type and various thermotaxis mutants by calcium imaging (a, b, c). We also succeeded in measuring the changes in neural circuit activity in thermotaxis mutants. For example, hyper-activation of AWC thermnosensory neuron caused by RGS eat-16 mutation down-regulated the activity of its downstream AIY interneuron. For quantifying thermotactic output, we focused on head swing since most of pre-synapses in RIA, a key integrating interneuron in thermotaxis circuit, connect to SMD and RMD motor neurons that control head muscles. We are trying to measure the head swing angle and frequency in animals behaving on thermal gradient by using the tracking system (d). After getting the in vivo parameters for computation, we are going on to mathematical expression. Through our analysis, we are hoping to find the novel neural mechanisms, such as unidentified neural property and wiring. (a) Kimura et al., Curr Biol, 2004, (b) Kuhara & Mori, J. Neurosci, 2006, (c) Kuhara et al., this meeting, (d) Ohnishi et al., this meeting.

Hiroyuki Sasakura et al. (2007) International Worm Meeting "Analysis and screening of genes involved in thermosensory signaling."

Keita Suzuki, Hiroko Ito, Hiroyuki Sasakura, Ikue Mori, Takuma Sugi

[International Worm Meeting, 2007]

C. elegans senses the environmental temperature by AFD and AWC sensory neurons. AFD neurons are specialized sensory neurons for temperature reception. AWC, known as olfactory neurons, also senses the temperature (Kuhara et al., this meeting). Genetic analysis suggested that signal transduction of temperature is similar to that of olfactory and visual system in C. elegans and other animals. Namely, cGMP produced by guanylate cyclases is used as second messenger and cGMP dependent cation channel TAX-2 and TAX-4 are crucial for activation of AFD and AWC. Although thermoreceptor itself is not identified, it is plausible that GPCRs sense the environmental temperature based on analogy to olfactory and visual system. Consistent with this possibility, G alpla ODR-3 and negative regulator of G protein EAT-16 are important for thermosensation in AWC sensory neurons (Kuhara et al., this meeting). GPCRs expressed in AFD and/or AWC become good candiates for thermoreceptors. It has been reported that B0496.5 encodes GPCR and expressed in sensory ending of both AFD and AWC (Colosimo et al., 2004). To examine B0496.5 gene function, we isolated two deletion mutants, nj62 and nj63. From deletion sites, both alleles are likely to be null. When nj62 and nj63 mutants are cultivated in 20 degree, they exhibited significantly smaller isothermal tracking than wild type. We also tested these mutants on linear thermal gradient. When nj62 and nj63 mutants are cultivated in 20 or 23 degree, they migrated to the lower temperature than wild type animals. From behavioral tests, B0496.5 gene likely has an important role on thermosensation. Overexpression experiments and physiological analysis are in progress. We also performed forward genetic screening to isolate new genes involved in AFD thermosensory signaling. The gene expression of nhr-38 is AFD specific and regulated by temperature stimulus. For example, the mutations of tax-4 or cmk-1 genes induce the downregulation of nhr-38::GFP (Satterlee et al., 2004; H.S., I.M., A.Kuhara, unpublished results). We utilized this phenomenon and isolated 12 suppressor strains that recover the gene expression of nhr-38::GFP in cmk-1 mutants. The behaviors of 11 strains are identical to athermotactic phenotype of cmk-1 mutants, while one suppressor strain IK673 exhibited neomorpholic, cryophilic phenotype. The gene responsible for this mutation was mapped on Ch I -0.69 to + 2.98. We thank T. Ishihara for nhr-38::GFP(H13::GFP), H. Inada for TMV-UV library, and CGC for cmk-1 mutants.

Paola Jurado et al. (2009) European Worm Neurobiology Meeting "Insights into the molecular mechanisms of Caenorhabditis elegans memory"

Paola Jurado, Ikue Mori

[European Worm Neurobiology Meeting, 2009]

Caenorhabditis elegans responds to a variety of environmental signals (volatile and water soluble chemicals, temperature, etc). The animals memorize these cues and are able to associate them with other existent conditions. Thermotaxis is a well characterized behavior in which C. elegans associates its cultivation temperature with food (Escherichia coli). This association drives the worms to seek their memorized temperature even in the absence of bacteria (1,2). This presents an ideal situation to investigate the molecular and cellular bases of sensory integration leading to complex processes such as memory and learning. We are trying to thoroughly investigate how the assimilation of thermo-sensory cues leads to memory, and how this thermal memory is modified over time. We have performed a screen to look for mutants with an abnormal memory or an altered decission making balance over thermal conditioning. We searched for animals conditioned to find E. coli at a certain temperature that take longer to leave it, in the absence of food, than wild type animals. These animals display a longer lasting memory or a slower decission making when thermal memory is confronted with hunger or the absence of food. One of the mutants isolated from this screening has been mapped to chromosome I and is currently under characterization. 1. I. Mori and Y. Ohshima (1995). "Neural regulation of thermotaxis in C. elegans." Nature 376(6538): 344-8. 2. I. Mori, H. Sasakura and A. Kuhara (2008). "Worm thermotaxis: a model system for analyzing thermosensation and neural plasticity." Curr Opin Neubiol. 2007 Dec 17(6):712-9.

Ikeda, Shingo et al. (2011) International Worm Meeting "Integration of temperature signals in interneurons of C. elegans."

Kimata, Tsubasa, Ikeda, Shingo, Mori, Ikue

[International Worm Meeting, 2011]

Animals show complex behaviors as a consequence of integrating environmental information through neural circuits. In order to reveal the mechanism of integration, we are focusing on a neuronal circuit of C. elegans, composed of three interneurons, AIY, AIZ and RIA, which play an important role in thermotaxis behavior. The RIA neuron receives two upstream signals, one from AIY for thermophilic movement and the other from AIZ for cryophilic movement, and is supposed to integrate them to transmit to downstream motorneurons SMD and RMD, which connect with neck muscles directly (Mori and Ohshima, Nature, 1995; White et al., Phil. Trans. R. Soc. London, 1986). To understand how RIA integrates two opposite signals from AIY and AIZ, thus reflecting in the behavior, we tried to monitor the activity of AIY, AIZ and RIA with Ca2+ imaging using calcium sensor, GCaMP3 (Tian et al., Nat Methods, 2009). We so far observed significant responses of AIY and RIA to thermal stimuli, some of which were temperature-independent. The previous study using Cameleon showed that AIY responded significantly to thermal stimuli, while the response of RIA to thermal stimuli was minimal (Kuhara and Mori, J. Neurosci.,2006). Further, temperature-independent responses of AIY and RIA have not been reported, suggesting that GCaMP3 can detect different concentration range of intracellular Ca2+ compared to Cameleon YC2.12 or YC3.60. To further analyze how the circuit functions in integrating neural signals, we are currently trying to image the activity of AIZ and will perform simultaneous imaging of AIY, AIZ and RIA.