Yamazaki, S. et al. (2017) International Worm Meeting "Learning-dependent behavioral modulation of sensory behavior revealed by machine learning and optophysiological analysis in a virtual environment."

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.

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

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.

Tanimoto, Y. et al. (2017) International Worm Meeting "Calcium dynamics regulates the timing of decision-making in worms."

Iwasaki, Y., Tanimoto, Y., Hashimoto, K., Nakai, J., Miyanishi, Y., Kawazoe, Y., Fujita, K., Kimura, K., Gengyo-Ando, K., Iino, Y., Yamazoe-Umemoto, A., Busch, K. E., Fei, X., Yamazaki, S.

[International Worm Meeting, 2017]

The brain processes sensory information to generate various physiological responses with different timing (i.e., with different response latencies). In decision-making, for example, animals choose one from multiple behavioral options based on environmental sensory information, with a temporal delay associated with the certainty of sensory information. The neural mechanism of timing, however, is largely unclear. We report the cellular and molecular mechanisms underlying the timing of decision-making during olfactory navigation in worms. Based on subtle changes in concentrations of the repulsive odor 2-nonanone, worms efficiently choose the appropriate migratory direction after multiple trials as a form of behavioral decision-making, which is different from the typical biased random walk. From simultaneous monitoring of behavior and neural activity in virtual odor gradients, we found that two pairs of sensory neurons regulate this behavioral response in an opposing manner with different temporal dynamics. ASH nociceptive neurons exhibit a time-differential response to an increase in the 2-nonanone concentration, which leads to an immediate turning response similar to a "reflex." In contrast, AWB olfactory neurons exhibit a time-integral response to a decrease in the odor concentration, which leads to turn suppression with a temporal delay resembling "deliberation." We further found that the AWB response is independent of synaptic connections and is mediated by a gradual calcium influx, mainly via L-type voltage-gated calcium channel (VGCC) EGL-19, whereas the ASH response is mediated by rapid calcium influx via multiple types of calcium channels. Thus, the timing of neuronal responses, such as deliberate decision-making or rapid reflex, is determined by cell type-dependent involvement of calcium channels. Interestingly, such time-integral neural responses have also been observed in decision-making by primates and rodents, and are considered to be mediated by recurrent neural circuits, although intracellular mechanisms have also been proposed. We suggest that a single-cell temporal integrator with L-type VGCCs, such as the AWB neuron, may be the evolutionarily conserved molecular basis for decision-making.

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.

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

Yamazoe, Akiko, Kimura, Kotaro, Iwasaki, Yuishi

[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.

Mongan NP et al. (1997) International C. elegans Meeting "AN EXTENSIVE NICOTINIC ACETYLCHOLINE RECEPTOR GENE FAMILY IN CAENORHABDITIS ELEGANS."

Baylis HA, Mongan NP, Sattelle DB

[International C. elegans Meeting, 1997]

Although the nicotinic acetylcholine receptors (nAChRs) are the best characterized ionotropic neurotransmitter receptors, the extent of the molecular and functional diversity of the nAChR gene family is not known for a single organism. A number of C. elegans nAChR subunit genes have been identified previously using genetic approaches [lev-1(non-a),unc-38(a),unc-29(non-a),deg-3(a)] and cross-species probing of C. elegans cDNA libraries [acr-2(non-a),acr-3(non-a),and Ce-21(a)]. nAChR a-subunits may be distinguished from all other ionotropic receptor subunits by the presence of adjacent cysteines at positions equivalent to 192,193 in the Torpedo a-subunit. We have applied an RT-PCR strategy to confirm the expression of 8 novel putative nAChR a-subunits predicted by GENEFINDER. Analysis of the regions of the a-subunit considered to (a) contribute to the acetylcholine binding site and (b) the channel lining, has revealed a diversity which may underlie distinct functional roles for members of this multi-gene family.

C Huang et al. (1999) International C. elegans Meeting "Each C. elegans Laminin a Subunit Mediates Distinct Aspects of Morphogenesis"

WG Wadsworth, C Huang, PD Yurchenco

[International C. elegans Meeting, 1999]

Laminin is a heterotrimeric glycoprotein composed of a , b , and g subunits. The genome sequencing project reveals two a , one b and one g laminin subunit genes. Based on sequence analyses, major features and known binding sites are conserved. The predicted a chains, laminin a A and laminin a B, are most similar to the vertebrate a 1/ a 2 and a 5 chains respectively, while the b and g resemble the b 1/ b 2 and g 1 chains. Thus, there are two predicted laminin trimers, a A bg and a B bg . Mutations in epi-1 , which encodes laminin a B, have been isolated and characterized (abstract ECWM 98). They cause defects that are consistent with the developmental roles for basement membranes (BMs). We report the expression patterns of laminin a A and a B. By immunostaining, both first appear at gastrulation between germ layers. However, laminin a A is heavily deposited anteriorly, surrounding pharygeal precursors, whereas laminin a B is more posteriorly localized. Laminin a B becomes localized to the BMs associated with pharynx, intestine, gonad, body wall, body wall muscle, and muscles throughout development. In contrast, laminin a A accumulates in pharyngeal BM, intestinal BM and body wall muscle BM during elongation and its level in intestinal BM and body wall muscle BM gradually decreases. In larvae and adults, laminin a A is only sometimes detected weakly in pharyngeal BM and body wall muscle BM. It is primarily localized in the spermatheca. Injection of dsRNA directed to epi-1 produces phenotypes similar to those observed in epi-1 alleles and the injection of dsRNA directed to lam3 , which encodes laminin aB, causes L1 larval arrest with severe pharyngeal defects. Double dsRNA-mediated interference directed to both genes produces embryos that arrest at morphogenesis with phenotypes more severe than those caused by the RNAi of either epi-1 or lam3 alone, indicating that both genes contribute to morphogenesis. Together, our RNAi and immunostaining results indicate that laminin a B has a broad role in maintaining the structural integrity of BMs and for regulating many aspects of morphogenesis, while laminin a A is restricted to specialized membranes and is required for the morphogenesis of specific tissues.

Yanjue Wu et al. (2005) International Worm Meeting "EGb 761 INHIBITS AMYLOID BETA OLIGOMERIZATION AND TOXICITY IN TRANSGENIC CAENORHABDITIS ELEGANS"

Christopher D Link, Yves Christen, Astrid G Zepeda, Yanjue Wu, Zhixin Wu, Yuan Luo

[International Worm Meeting, 2005]

Amyloid peptide aggregation has been postulated to link oxidative stress and neurodegeneration seen in Alzheimer's diesease (AD). We have previously demonstrated that the standardized Ginkgo Biloba extract, EGb 761, inhibits A aggregation in vitro and attenuates the expression of a stress response gene small heat shock protein (hsp 16-2) in Caenorhabditis elegans. In this study, we use EGb 761 as a pharmacological modulator to associate A species with the levels of oxidative free radicals and with A-induced toxicity in an A-expressing transgenic C.elegans model, which can express A in muscle cells after temperature up-shift. We observed that EGb 761 alleviated A-induced toxicity which correlated with its ability to specifically inhibit A oligomerization. Furthermore, EGb 761 reduces intracellular levels of hydrogen peroxide in the transgenic C.elegans. These findings suggest that A oligomers and A-induced oxidative strss are crucial for A toxicity, and EGb 761 has a clear therapeutic potential for prevention and treatment of AD.

Lyashenko, Alexey et al. (2015) International Worm Meeting "Line scanning fluorescent confocal microscope for a 3D multi-neuron imaging of C. elegans."

Polanco, Edward, Lyashenko, Alexey, Arisaka, Katsushi

[International Worm Meeting, 2015]

Conventional confocal microscope uses a physical aperture to reduce the amount of out of focus light to the image sensor. We developed a line scanning confocal microscope that utilizes a software controlled rolling shutter on a CMOS camera for a high-speed 3D volume imaging of dozens of active neurons. The microscope setup allows for a real time worm tracking for freely navigating C. elegans under a localized external stimulation for phototaxis and thermotaxis. An external photo stimulation for optogenetics was also realized.

Paul Maddox et al. (2007) International Worm Meeting "Functional genomics identifies a Myb domain-containing protein family required for assembly of CENP-A chromatin."

Joost Monen, Arshad Desai, Francie Hyndman, Karen Oegema, Paul Maddox

[International Worm Meeting, 2007]

Nucleosomes containing the centromere-specific histone H3 variant centromere protein A (CENP-A) create the chromatin foundation for kinetochore assembly. To understand the mechanisms that selectively target CENP-A to centromeres, we took a functional genomics approach in the nematode Caenorhabditis elegans, in which failure to load CENP-A results in a signature kinetochore-null (KNL) phenotype. We identified a single protein, KNL-2, that is specifically required for CENP-A incorporation into chromatin. KNL-2 and CENP-A localize to centromeres throughout the cell cycle in an interdependent manner and coordinately direct chromosome condensation, kinetochore assembly, and chromosome segregation. The isolation of KNL-2-associated chromatin coenriched CENP-A, indicating their close proximity on DNA. KNL-2 defines a new conserved family of Myb DNA-binding domain-containing proteins. The human homologue of KNL-2 is also specifically required for CENP-A loading and kinetochore assembly but is only transiently present at centromeres after mitotic exit. These results implicate a new protein class in the assembly of centromeric chromatin and suggest that holocentric and monocentric chromosomes share a common mechanism for CENP-A loading.