Yamazoe, Akiko et al. (2013) International Worm Meeting "A neuronal mechanism for navigation along a repulsive odor gradient."

Busch, Karl Emanuel, Tanimoto, Yuki, Miyanishi, Yosuke, Kimura, Kotaro, Yamazoe, Akiko, Iwasaki, Yuishi, Yamazaki, Shuhei, Kawazoe, Yuya, Iino, Yuichi, Hashimoto, Koichi, Gengyo-Ando, Keiko, Fujita, Kosuke, Nakai, Junichi, Fei, Xianfeng

[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.

Yamazoe, Akiko et al. (2011) International Worm Meeting "Worms maintain their response to the repulsive odor 2-nonanone during avoidance behavior."

Yamazoe, Akiko, Iwasaki, Yuishi, Kimura, Kotaro

[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.

Akiko Yamazoe et al. (2010) East Asia Worm Meeting "The worms respond to the gradient direction of the repulsive odor 2-nonanone"

Kotaro Kimura, Akiko Yamazoe

[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.

Kohara Y et al. (1994) Worm Breeder's Gazette "The C. elegans cDNA Project: A Progress Report."

Sugimoto A, Motohashi T, Tabara H, Kohara Y, Watanabe H

[Worm Breeder's Gazette, 1994]

The C.elegans cDNA project: A progress report Yuji Kohara, Tomoko Motohashi, Akiko Sugimoto, Hisako Watanabe and Hiroaki Tabara Gene Library Lab, National Institute of Genetics, Mishima 411, Japan. e-mail: ykohara@lddbj.nig.ac.jp

Yamazaki, Shuhei et al. (2015) International Worm Meeting "Identifying neural signaling that cooperates with dopamine signaling in repulsive odor learning."

Yamazoe, Akiko, Yamazaki, Shuhei, Kimura, Kotaro

[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.

Tanimoto, Yuki et al. (2013) International Worm Meeting "A virtual reality running machine for worms-a highly integrated microscope system for olfactory behavior."

Kimura, Kotaro, Fei, Xianfeng, Nakai, Junichi, Yamazaki, Shuhei, Busch, Karl Emanuel, Kawazoe, Yuya, Gengyo-Ando, Keiko, Miyanishi, Yosuke, Hashimoto, Koichi, Tanimoto, Yuki, Fujita, Kosuke

[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.

Tanimoto, Yuki et al. (2015) International Worm Meeting "Neuronal mechanisms for C. elegans olfactory navigation revealed by a highly integrated microscope system."

Nakai, Junichi, Yamazoe, Akiko, Hashimoto, Koichi, Tanimoto, Yuki, Kimura, Kotaro, Iwasaki, Yuishi, Fei, Xianfeng, Gengyo-Ando, Keiko, Fujita, Kosuke, Miyanishi, Yosuke, Yamazaki, Shuhei, Kawazoe, Yuya

[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.

Miyanishi, Yosuke et al. (2011) International Worm Meeting "Functional imaging of neuronal activity for 2-nonanone stimulation."

Nakai, Junichi, Kimura, Kotaro, Miyanishi, Yosuke

[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.

A Asano et al. (1999) International C. elegans Meeting "Homologues of claudin, integral protein of mammalian tight junction, are present and important in C. elegans"

S Tsukita, K Asano, A Asano, H Sasaki, M Furuse

[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.

Kohara Y et al. (1995) Worm Breeder's Gazette "The C. elegans cDNA Project: A Progress Report."

Motohashi T, Sugimoto A, Watanabe H, Tabara H, Kohara Y

[Worm Breeder's Gazette, 1995]

The C.elegans cDNA project: A progress report Yuji Kohara, Tomoko Motohashi, Akiko Sugimoto, Hisako Watanabe and Hiroaki Tabara Gene Library Lab, National Institute of Genetics, Mishima 411, Japan e-mail: ykohara/*ddbj.nig.ac.jp Tag sequencing is now on the third set of cDNA dones. After analysis of the first set of cDNA clones (some 4,400 clones), each 10,000 clones were picked up randomly from 3 different cDNA libraries (an embryonic cDNA library and libraries of >2kb cDNA and unfractionated cDNA made from mixed stage population). The total 30,000 clones were gridded and probed with the cDNA clones belonging to the species which had been represented by more than 4 clones in the analysis of the first set. A set of some 4,800 cDNA clones (the second set) were selected out of the unhybridized clones (from rare or not analyzed cDNA species) and has been subjected to the tag sequencing. This analysis produced 3,667 clean 3'-tags which gave 1,532 more unique cDNA species (see Fig.). As the next step, the grids were further screened with the cDNA probes the groups containing more than 4 clones at the point. A set of some 4,000 cDNA clones (the third set) was selected out of the unhybridized clones and tag sequencing has been continued on this set. The current status of our progress is that we have identified 3,324 unique cDNA species out of 7,647 clones (clean 3'-tags). The unique cDNA species were assigned serial numbers from CELK00001 to CELK03324. These analyses have also detected many pairs of clones which appeared to be generated by alternative splicing. In some cases, two groups were turned out that they were derived from the same gene but had different 3'-end sequences due to alternative splicing or differential poly-A addition. We are going to make a list of such differential splicings. BLASTX search showed that 653 groups out of the 1,816 groups identified through the analysis of the second and the third sets gave significant similarities (blastx score > 100), which are listed below. (Note; "-" in the column of "Frame" means BLASTX search was made using only 3'-tag sequences so far.) Mapping and in situ analysis are in progress.