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Iwasaki, Yuishi, Hashimoto, Koichi, Kawazoe, Yuya, Fujita, Kosuke, Busch, Karl Emanuel, Iino, Yuichi, Gengyo-Ando, Keiko, Nakai, Junichi, Fei, Xianfeng, Yamazaki, Shuhei, Tanimoto, Yuki, Miyanishi, Yosuke, Yamazoe, Akiko, Kimura, Kotaro
[
International Worm Meeting,
2013]
For survival and reproduction, animals navigate toward or away from certain stimuli, which requires the coordinated transformation of sensory information into motor responses. In worms, the pirouette and the weathervane strategies are considered the primary navigation strategies for responding chemosensory stimuli. We found, however, that worms use a novel navigation strategy in odor avoidance behavior: In a gradient of the repulsive odor 2-nonanone, worms efficiently avoid the odor, and ~80% of initiation of long, straight migrations ("runs") were away from the odor source, which cannot be simply explained by the two known major strategies. Direct measurement of local odor concentration suggested that pirouettes are efficiently switched to runs when worms sense negative dC/dt of 2-nonanone. To test whether runs are indeed caused by negative dC/dt, we established an integrated microscope system that tracks a freely moving worm during stimulation with a virtual odor gradient and simultaneously allows for calcium imaging and optogenetic manipulations of neuronal activity (Tanimoto et al., this meeting). Using this system, we found that a realistic temporal decrement in 2-nonanone concentration (~ 10 nM/sec) caused straight migration by suppressing turns. We also found that a pair of AWB sensory neurons were continuously activated during the odor decrement and that optogenetic activation or inactivation of AWB neurons suppressed or increased turning frequency, respectively. In addition, we found that ASH nociceptive neurons increased turning frequency during odor increment. Taken together, our data indicate that the counteracting turn-inducing and turn-suppressing sensory pathways can effectively transform temporal sensory information into spatial movement to select the right path leading away from potential hazards.
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[
International Worm Meeting,
2011]
Animals can maintain their behavioral response to environmental stimuli even under unstable environmental conditions and during various animal movements. To investigate neural mechanisms for such robust behavioral responses, it is necessary to quantitatively analyze the time-course changes in the correlation between the stimulus and behavioral response. For this, we quantitatively analyzed stimulus as well as behavior of worms' avoidance response to repulsive odor 2-nonanone. When animals migrate away from a source of repulsive signal, their avoidance response is likely weakened. In a previous study, however, we have shown that worms exhibited a constant average velocity of avoidance from 2-nonanone for 10 min (Kimura et al., J. Neurosci., 2010), suggesting a neural mechanism for such constant avoidance.
In addition to the quantitative analysis of avoidance response to 2-nonanone (Yamazoe & Kimura, CeNeuro, 2010), we recently developed a technique to measure the concentration of 2-nonanone at specific spatial and temporal points of gas phase in the assay plate. By using a highly sensitive gas chromatograph, we observed a clear gradient of 2-nonanone, of which concentration increased with time. Based on this measured gradient of 2-nonanone, we determined the 2-nonanone concentration that each worm experienced during the avoidance assay (Cworm) and observed the following: (1) During the first 2 min of the assay worms did not initiate avoidance response and migrated randomly, and Cworm increased continuously up to the order of mM at 2 min. (2) After 2 min, worms started to migrate farther away from the odor source, and Cworm was maintained around the concentration, despite increase in the concentration gradient. (3) Cworm decreased effectively during runs, while it increased and decreased largely during pirouettes. (4) When compared between the early and late phases of the assay, the maximum dCworm/dt in each run decreased several fold along with the avoidance behavior, even though the orientation directions did not change considerably; that is, even when the gradient of 2-nonanone became shallower, the accuracy of worm orientation appeared maintained. These results suggest that worms may increase sensitivity to dC/dt during exposure to a certain concentration of 2-nonanone. We are currently conducting computer simulation to test this hypothesis. Further analysis may help us uncover the mechanism of maintaining proper behavioral responses.
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[
East Asia Worm Meeting,
2010]
To understand the neural mechanisms underlying behavioral responses to a chemical signal, we are studying the avoidance behavior of C. elegans to the repulsive odor 2-nonanone as a model. We have found that the avoidance behavior of the animals to 2-nonanone is enhanced rather than reduced after pre-exposure to the odor: The preexposed animals migrate farther away from the odor source than do the control animals, and this plasticity is acquired as a type of non-associative learning (see abstract by Fujita and Kimura). Here, we present evidence to support that the animal's 2-nonanone avoidance appear to depend on the bearing angle - the angle between the direction of their locomotion and of a putative spatial gradient of 2-nonanone. A bearing angle of 0 deg indicates that the movement is directly down the gradient, and a bearing angle of 180 deg indicates that the movement is directly up the gradient. For a quantitative behavioral analysis, the animals' movements during the 2-nonanone avoidance were divided into periods of straight movements (runs) and of frequent turnings (pirouettes), as previously reported in salt chemotaxis (Pierce-Shimomura et al., J. Neurosci., 1999). When an animal's bearing was within ~60 deg during movement down the gradient, pirouette initiation rates were low and constant. By contrast, when an animal's bearing was greater than ~60 deg, pirouette initiation rate increased. Interestingly, only when an animal's bearing during a run was within ~60 deg, the preexposed animals exhibited much lower pirouette initiation rates and longer run durations than did the control animals; this difference may reflect the memory of pre-exposure to cause the enhancement of 2-nonanone avoidance. Consistent with this sensitive response to bearing, the animals appeared to exhibit a more accurate course correction after pirouetting during 2-nonanone avoidance than during the salt chemotaxis. We are currently attempting to measure the actual changes in the concentration of 2-nonanone during the assay by using a sensitive gas chromatography and planning to confirm our model by a computer simulation. We thank Drs. J. Pierce-Shimomura, M. Fujiwara, and N. Masuda for providing their suggestions on our project.
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[
International Worm Meeting,
2015]
Dopamine in association with other neural signals plays crucial roles in various brain functions such as locomotory regulation, reward, emotion, learning and memory. However, the mechanism by which multiple neural signals cooperatively regulate brain functions is not well understood because of neural circuit complexity. To address this issue, we are studying repulsive odor learning regulated by dopamine signaling in C. elegans (Kimura et al., 2010, J. Neurosci.). Upon preexposure to 2-nonanone, the animals exhibit enhanced avoidance behavior to this odorant as a type of non-associative learning. This enhancement is regulated by dopamine signaling via the D2-like dopamine receptor, DOP-3, in a pair of RIC interneurons. Currently, we are working towards identifying genes that genetically interact with dopamine signaling for repulsive odor learning. We have identified several mutant strains that exhibit behavioral defects similar to those seen in dopamine mutants. We first plan to identify the site-of-action of identified genes by cell-specific rescue experiments. The physiological role of the gene will then be analyzed by our integrated microscope system to quantify the relationship between odor stimuli, neural responses, and behavior (Tanimoto et al., this meeting). These analyses will help us understand the interaction between the newly identified neural signaling and dopamine signaling in modulation of learning.
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Nakai, Junichi, Fei, Xianfeng, Kawazoe, Yuya, Miyanishi, Yosuke, Fujita, Kosuke, Hashimoto, Koichi, Tanimoto, Yuki, Yamazaki, Shuhei, Kimura, Kotaro, Gengyo-Ando, Keiko, Busch, Karl Emanuel
[
International Worm Meeting,
2013]
A major function of the nervous system is to transform sensory information into an appropriate behavioral response. The neural mechanisms that mediate sensorimotor transformation are commonly studied by quantifying the behavioral and neural responses to a controlled sensory stimulus. Presenting a controlled chemical stimulus to freely behaving animals under a high-power microscope, however, is challenging. Here, we present a novel integrated microscope system that stimulates a freely moving worm with a virtual odor gradient, tracks its behavioral response, and optically monitors or manipulates neural activity in the worm during this olfactory behavior. In this system, an unrestricted worm is maintained in the center of a bright field by an auto-tracking motorized stage that is regulated by a pattern-matching algorithm at 200 Hz [1]. In addition, the worm is stimulated continuously by an odor flowing from a tube, the concentration of which can be temporally controlled. The odor concentration used in this system is based on the concentration used in the traditional plate assay paradigm (Yamazoe et al., CeNeuro 2012), and can be monitored with a semiconductor sensor connected to the end of the tube when necessary. Using this system, we investigated the neural basis of behavioral responses to a repulsive odor 2-nonanone in worms. We monitored and modulated sensory neuron activity in behaving worms by using calcium imaging and optogenetics, respectively, and found that the avoidance behavior to 2-nonanone is achieved by two counteracting sensory pathways that respond to changes in temporal odor concentration as small as ~10 nM/s (Yamazoe et al., this meeting). Our integrated microscope system, therefore, will allow us to achieve a new level of understanding for sensorimotor transformation during chemosensory behaviors. [1] Maru et al., IEEE/SICE Int. Symp. Sys. Integr. Proc., 2011.
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Miyanishi, Yosuke, Yamazoe, Akiko, Fei, Xianfeng, Iwasaki, Yuishi, Hashimoto, Koichi, Kawazoe, Yuya, Kimura, Kotaro, Fujita, Kosuke, Nakai, Junichi, Gengyo-Ando, Keiko, Yamazaki, Shuhei, Tanimoto, Yuki
[
International Worm Meeting,
2015]
The nervous system of animals transforms dynamically changing sensory information from the environment into appropriate behavioral responses. In particular, olfactory information plays critical roles in adaptive behaviors in the form of long- and short-range chemical cues that encode spatiotemporal information and chemical identity. To elucidate the neuronal mechanisms underlying olfactory behavior, it is desirable to quantify behaviors and neural circuit activities under realistic olfactory stimulus. However, reproducing realistic spatiotemporal patterns in odor concentrations is challenging due to diffusion, turbulent flow, and measurability of odor signals. We have developed an integrated microscope system that produces a virtual odor environment to quantify behaviors and neural circuit activities of the nematode C. elegans. In this system, C. elegans is maintained in the view field of a calcium imaging microscope by an auto-tracking stage using a pattern-matching algorithm. Simultaneously, odor stimulus is controlled with sub-second and sub-muM precision to reproduce realistic temporal patterns. Using this system, we have found that two types of sensory neurons play significant roles to choose a proper migratory direction for navigation in a gradient of the repulsive odor 2-nonanone. Calcium imaging and optogenetic analysis revealed that temporal increments of repulsive odor trigger turns that randomize the migratory direction, while temporal decrements of the odor suppress turning for migration down the gradient. Further mathematical analysis indicated that these sensory neurons are not only antagonizing, but also responding to odor concentration changes at different time scales for the efficient migration. Using this method will lead to comprehensive understanding of cellular mechanisms of decision making in a simple neural circuit.
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[
International Worm Meeting,
2011]
To better understand the neural basis that regulates a worm's sensory behavior and its modulation by learning, we are studying avoidance behavioral responses to 2-nonanone. We previously reported that the avoidance behavior to 2-nonanone is enhanced, rather than reduced, after preexposure to the odor, and this enhancement is acquired as a non-associative dopamine-dependent learning (Kimura et al., J. Neurosci., 2010; Fujita and Kimura, this abstract). In addition, we observed that worms respond to a spatial gradient of 2-nonanone (Yamazoe and Kimura, CE Neuro, 2010), which cannot be simply explained by the pirouette or weathervane strategies.
2-nonanone is mainly sensed by the AWB neurons, which have been shown to exhibit odor-OFF response in aqueous step stimulation with 2-nonanone (Troemel et al., Cell 1997; Ha et al., Neuron 2010). To understand how the neuronal circuits of worms regulate the characteristic 2-nonanone behavioral response, we are monitoring calcium changes in the AWB and downstream neurons using G-CaMP 4 (Shindo et al., PLoS ONE, 2010). We thank Drs. S. Oda, K. Yoshida, and Y. Iino (U. Tokyo) for suggestions on microfluidics; M. Hendricks and Y. Zhang (Harvard) for aqueous 2-nonanone stimulation; and E. Busch and M. de Bono (MRC) for gaseous microfluidic stimulation.
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[
International C. elegans Meeting,
1999]
Recently, new integral proteins of tight junction were discovered in mouse and human (Furuse et al., J. Cell Biol., 141, 1539, 1998; Morita et al., PNAS, 96, 511, 1999). These claudin family proteins are members of tight junction strands. Although presence of tight junctions in C. elegans is not reported, septate junctions and septate-like junctions seem to play similar functions instead. We searched the gene database of C. elegans , and found two homologues of claudin family proteins (claudin-CE1 and -CE2) with four-transmembrane domains, conserved two Cys in the first loop, and similar molecular weight. Interestingly, a protein (claudinD) was also found that has molecular weight about twice of claudin-CE1, and other characteristic structures are likely to have two claudin molecules tandemly repeated. These 3 proteins are coded from nearby sites on chromosome X. Claudin-CE1::GFP with 1.2kb upstream promoter region was expressed in spermatheca which is known to have septate junctions, and gut. Expression of claudin-CE2::GFP was much less, but tissue distribution was similar. RNAi experiments using dsRNA mixture of claudin-CE1, claudin-CE2 and Exon1-4 fragment of claudinD were performed. About 40% of F1 of the injected worms have decreased F2 production (in average 48% decrease), whereas 22% of F1 have almost normal numbers of F2's. Thus, these proteins seem to be important for reproduction of the worms. When expressed in MDCK-II epithelial cells, Claudin-CE1::GFP was localized at cell-cell junctions. Electron microscopic studies are under way. We are grateful to Miss. Akiko Kamamoto whose technical assistance make this work possible.
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Ikejiri, Y., Hiramatsu, F., Yamazaki, S., Fujita, K., Kimura, K., Tanimoto, Y., Hashimoto, K., Maekawa, T., Yamazoe-Umemoto, A.
[
International Worm Meeting,
2017]
Animals modify their behavior based on experiences as learning, although identifying component(s) of behavior modulated by learning has been difficult. In contrast to neural activities, which can be monitored in large numbers of cells simultaneously recently, behavior in general is still analyzed in classic ways and insufficiently studied using simple measures, such as velocity, migratory distance, and/or the probability of selecting a particular goal. Comprehensive classifications of animals' behavior by using machine vision and machine learning methods have been achieved (Gomez-Marin et al., Nat Neurosci, 2014; Brown et al., PNAS, 2013). However, these methods have not been applied to animals' sensory behavior because of technical limitations in measuring sensory signals that animals receive during the behavior. To overcome this problem and effectively identify behavioral components modulated by learning, we used machine learning aiming to detect changes in navigation of worms in a measured odor gradient. We have previously reported that, after experiencing the repulsive odor 2-nonanone for 1 h, worm's odor avoidance behavior is enhanced, and that they move away from the odor source more efficiently (Kimura et al., J Neurosci, 2010). We have also quantified the dynamic changes in the odor concentration during odor avoidance behavior (Yamazoe-Umemoto et al., Neurosci Res, 2015). In the present study, we used decision tree, a machine learning algorithm, to extract features of the animal's sensorimotor response during navigation modified by odor learning. During the migration down the odor gradient, naive worms responded to slight increases in the repulsive odor concentration by stopping forward movements and initiating turns. In contrast, the probability of response was lowered after learning, suggesting that the learned worms ignore "a yellow light". Consistently, by calcium imaging of ASH neurons, whose activation causes turns under a virtual odor gradient (Tanimoto et al., this meeting), we found that the ASH response to a small increase in the odor concentration was reduced after learning. Furthermore, by applying the decision tree analysis, multiple mutant strains were categorized into several groups based on behavioral features. Thus, the integrative machine learning analysis of sensory information and behavioral response is a powerful tool to obtain comprehensive understanding of dynamic activities of neural circuits and its modulation by learning.
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[
International C. elegans Meeting,
2001]
Homologues of claudin, major integral protein of mammalian tight junction with four span transmembrane structure, were found in the genomic data base of C. elegans . Among them, CLC-1(C09F12.1) was discovered by a blast search with mouse claudin-6, it was found later that homology is also present with mouse claudin-7 and human claudin-14. It has in its first loop two Cys residues that are conserved among mammalian claudins. CLC-2 (T05A10.2) was found by a blast search with CLC-1, and has 33% identity (70% homology) with CLC-1, although no significant homology was found with vertebrate claudins so far known. CLC-3 (ZK563.4) has homology with mouse claudin-10 and cattle claudin-16. CLC-4 was found with a blast search with mouse claudin-6, but it was thought to be an eight span transmembrane protein at that time. Recently, it was found that this gene (C01C10.1) encodes two separate proteins, and the down-stream operon (C01C10.1b) was reported to encode a Gas3/PMP-22 homlogue (Agostoni E.et al., Gene, 234, 267-274, 1999). Product of the other operon (C01C10.1a) was not characterized well. Homology with claudin-6, -7, and -9 was found with this product, therefore named as CLC-4. These four claudin homologues have either PMP-22/EMP/MP20 motif or transmembrane four signature or both, and most of them have srg integral membrane protein motif. Furthermore, two conserved Cys residues are present in all C. elegans claudins. Interestingly, many of vertebrate claudins have one or two of these motifs or signature. Although most of vertebrate claudins has CLAUDIN3 signature, no such signature was found in the nematode claudins. All of coding sequences of the nematode claudins were isolated from cDNA libraries, therefore they are all expressed in the worm. Some ESTs were reported for CLC-1 and -3, previously. Expression of these claudins was studied with GFP-tagged molecules. All of claudins is expressed in spermatheca, intestine and hypodermis. CLC-1 and -4 were expressed strongly in pharynx, and sometimes localized at cell-cell junctions. They are also seems to be expressed in excretory-secretory system. CLC-1::GFP is also expressed at cell-cell junction of vulva. Localization of CLC-1 is under study with HA-tagged molecule. Preparation of antibodies against the nematode claudins was very difficult so far. But, affinity purified antibodies raised against loop 1 of CLC-1 seem to be useful for CLC-1 detection in the worm. Results with these antibodies were very similar to those obtaind with GFP-tagged CLC-1. To see if these claudins function as mammalian claudins do, i.e. barrier function, penetration of TRITC-dextran (MW=10,000) was checked after injection of dsRNA's (RNAi). Experiments with full length CLC-1 dsRNA showed that barriers for the high molecular weight dye were damaged by RNAi, in other words, penetration of the dye to pharynx and some other tissues were observed. Similar experiments with other claudins did not detect any barrier damage. This is because the dye only goes into entrance of intestine under normal condition, therefore, we tried weak osmotic shock to deliver the dye to entire intestinal lumen and excretory-secretory duct system. Under this condition, control injection of dsGFP did not result in penetration of the dye to other area of the body. On the other hand, RNAi with the combination of CLC-1 and -4 RNA's resulted in penetration of the dye to 72% of the worm from pharynx, intestine and vulva (or from excertory-secretory system) to the body, whereas barrier was damaged only 40% of worm with a combination of CLC-3 and -4. RNAi effects with other combination of CLC's will be reported, and effects of RNAi to the retention of the sperm in spermatheca will be studied. Accordingly, claudins of C. elegans seems to function as barrier at least partly. Other functions, if any, will be surveyed. We would like to thank excellent technical assistance of Miss. Akiko Kamamoto, without her help this project could not be completed.