Sato, Hirofumi et al. (2015) International Worm Meeting "A gustatory neural circuit for experience-dependent salt chemotaxis in C. elegans."

Hashimoto, Koichi, Iino, Yuichi, Fei, Xianfeng, Kunitomo, Hirofumi, Sato, Hirofumi

[International Worm Meeting, 2015]

Caenorhabditis elegans shows experience-dependent behaviors to many environmental cues. For sodium chloride, worms are known to memorize a particular salt concentration and approach the memorized concentration. In this study, we therefore searched for the neural circuit required for the memory of salt concentration. First, we conditioned worms in different salt concentrations, and monitored the activity of the salt-sensing chemosensory neuron ASER and three downstream interneurons; AIA, AIB, and AIY. We found that ASER, AIB, and AIY changed the responses depending on the previously exposed salt concentrations. We investigated the response of ASER in more detail, and found that the basal calcium level of ASER might change depending on cultivation concentration, and the plasticity of ASER response seemed to be independent of inputs from other neurons. Next, to assess the contribution of the three interneurons to the behavior, we ablated them individually, and compared behavioral responses of those worms with wild type. As a result, the reversal frequency of cell-ablated worms was different from that of the wild type. However, cell-ablated worms showed normal salt chemotaxis under the tested conditions, indicating that there are redundancies in the neural circuit that processes the salt perception signal. Furthermore, we investigated the relationship between the neural response and locomotion of worms. We used a tracking-imaging system with microfluidic arena that allowed worms to crawl in a controlled liquid environment (Albrecht et al., 2011), and recorded locomotion of worms and neural responses simultaneously. The result showed that the speed of worms decreased only when salt concentration was decreased below cultivation concentration. However, ASER always showed an off-response to salt, indicating that there is an experience-dependent plasticity in the process that links the ASER response to moving velocity.

Zheng, Ying Grace et al. (2015) International Worm Meeting "Optophysiological dissection of individual dopaminergic neuron pairs during food-dependent behavioral modulation."

Kimura, Kotaro, Zheng, Ying Grace, Tanimoto, Yuki, Fei, Xianfeng, Hashimoto, Koichi

[International Worm Meeting, 2015]

Dopamine (DA) functions as an important neuromodulator responsible for regulating various behaviors in animals. While DA signaling has been shown to play a significant role in behavioral regulation, the mechanism by which DA-releasing (DAergic) neurons are activated in response to environmental stimuli remains unclear. In C. elegans, DA functions as a "presence of food" signal responsible for modulating various food-dependent behaviors (Chase and Koelle, Wormbook, 2007). Four pairs of DAergic neurons, CEPD, CEPV, ADE, and PDE neurons, work redundantly for behavioral modulation in response to a bacterial lawn, which is sensed as a mechanical stimulus (Sawin et al., Neuron, 2000). A previous calcium imaging study revealed that one class of DAergic neurons, the CEP neurons, are indeed activated by mechanical stimuli (Kindt et al., Neuron, 2007). However, the timing and manner of activation of specific DAergic neuron pairs in response to the bacterial lawn have not yet been revealed since functional dissection of DAergic neurons by calcium imaging and/or optogenetics is difficult. All the known DAergic neuron-specific promoters express genes in all DAergic neurons-not in just one or a subset of them. Therefore, sophisticated machine vision techniques are necessary to target one of the many DAergic neurons in a worm migrating upon an agar surface to enter a bacterial lawn. In an attempt to understand how individual DAergic neuron pairs are activated by food stimuli and how this activation affects worm behavior, we conducted optophysiological analyses using our original auto-tracking integrated microscope system (Tanimoto et al., this meeting). First, we monitored the activation patterns of each DA neuron pair in freely moving worms by calcium imaging; we found that CEPD, CEPV, and PDE pairs were differently activated upon food-entry (The ADE pair was not analyzed because of dim fluorescence from the cells). At present, we are optogenetically stimulating individual DAergic neuron pairs in freely moving worms to test whether these differences in activation patterns reflect functional differences in the neurons. Our analysis will shed light on activities and functions of each of the genetically indistinguishable neurons in this small model organism.

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

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.

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

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.

Sandrine Fraboulet et al. (2009) European Worm Neurobiology Meeting "Chemical rescue of paralysis in a transgenic model of Caenorhabditis elegans overexpressing human Beta-amyloid"

David I C Scopes, Andrew K Jones, David B Sattelle, Aileen Moloney, Christopher D Link, Steven D Buckingham, Mark J Treherne, Hozefa Amijee, Sandrine Fraboulet

[European Worm Neurobiology Meeting, 2009]

Authors acknowledge support from MRC. Numerous studies support a role for aggregated Ab as a mediator of the toxicity that underlies Alzheimer.s disease (AD) and other diseases such as Inclusion Body myositis (IBM), an acquired muscle disease affecting people over 50 years old. In this pathology, muscle weakness and degeneration is accompanied by chronic muscle inflammation. Remarkably, despite the inflammation component of the disease, IBM patients are only poorly responsive to anti-inflammatory drugs suggesting that inflammation per se may not be the primary cause of the pathology. We have used a C. elegans transgenic line over-expressing human amyloid 1-42 peptide in the muscles as a model with which to test the efficiency of new compounds on in vivo amyloid toxicity. This C. elegans line becomes paralyzed shortly after the induction of amyloid expression in the muscles, which makes it an easily-recordable phenotype useful for exploring candidate treatments to alleviate the paralysis. We report on the actions of two novel chemicals, which inhibit amyloid aggregation and partially rescue the amyloid-induced phenotype. References: Wu Y, W.Z., Butko P, Christen Y, Lambert MP, Klein WL, Link CD, Luo Y. (2006) J. Neurosci., 26, 13102-13. Link CD, T.A., Kapulkin V, Duke K, Kim S, Fei Q, Wood DE, Sahagan BG. (2003) Neurobiol Aging., 24, 397-413. Jones, A.K., Buckingham, S.D. and Sattelle, D.B. (2005). Nat Rev Drug Discov, 4, 321-30.

Gobel V et al. (1994) Worm Breeder's Gazette "Neurofibromatosis 2 Tumor Suppressor Gene Homologs."

Fleming JT, Winge P, Gobel V, Friend S, FitzGerald M, Moshiach S

[Worm Breeder's Gazette, 1994]

Neurofibromatosis 2 tumor suppressor gene homologs Verena Gobel*+, Per Winge*, Michael FitzGerald*, Simon Moshiach*, Stephen Friend*, and John Fleming*+. MGH *Cancer Center and +Dept. of Pediatrics, Boston, MA. The loss of function of the human Neurofibromatosis 2 (NF2) gene predisposes for nervous system tumors. NF2 has structural similarity to the moezin-ezrin-radixin family of cytoskeletal membrane linker proteins which are thought to connect membrane receptors to the cytoskeleton. NF2 thus belongs to a new class of tumor suppressor genes directly involving members of the cytoskeleton. We are interested in studying how the cytoskeleton may contribute to growth regulation. Our hypothesis is that members of the above mentioned class of proteins are necessary to transduce negative growth regulating signals induced by cell-to-cell contact known as "contact inhibition". Using a combination of degenerate PCR and low homology screening based on conserved regions in the human NF2-moezin- ezrin-radixin gene family we have isolated and sequenced full length c-DNA clones of the C. elegans homologs of human NF2 and radixin. In spite of extensive screening we have not obtained other family members and we thus assume that these genes represent the complete gene family in C. elegans. For radixin we have also isolated genomic clones and are currently in the process of finishing the genomic sequence. The NF2 genomic sequence w2, kindly provided by LaDeana Hillier/Richard Wilson for the genome sequencing project. The general structure of the C. elegans proteins preserves the structure of their human counterparts with a highly conserved N-terminal half (70 to 75% homology between human/human, human/C.elegans and C.elegans/C.elegans members) and a non conserved C-terminal half. The N terminus is predicted to be directed towards the membrane and to provide the link to transmembrane proteins whereas the C-terminal half provides the connection,to actin and/or other cytoskeletal proteins. Humradixin 208 EYLKIAQDLEMYG VNY FE/ KNKK 229 Ceradixin EYLKIA QDLEMYG VNY FEI RNKK CeNF2 EYLRVAQDLEMYG I L Y YP/ QNKK HumNF2 EYLKIA QDLEMYGVNY FA/ RNKK The genomic structure of NF2 preserves most of the intron/exon junctions of the human NF2 gene, but is quite distinct from that of radixin (the human genomic structure of radixin is not known). Using a peptide antibody raised against a conserved part of the human gene family and also conserved in the C. elegans homologs (kindly provided by Frank Solomon, MIT), we have obtained staining not seen with control antibodies. We are in the process of confirming these preliminary data using different fixation- and staining protocols (thanks to Ralf Baumeister and Bob Waterston). We hope that by using confocal microscopy we may also be able to get some information on the subcellular localization. Additionally, we are generating promotor-GFP constructs to obtain more expression data and to be able to differentiate between the anitbody staining patterns of the two proteins. The map position of radixin (fingerprinting by Alan Coulson, Cambridge) is close to spe- 11 on chromosome I, NF2 is located in proximity to daf-4 on chromosome III. Ann Rose has performed an extensive lethal screen around the location of radixin (about 120 different lethals are consistent with its location). Hoping (?!) that a radixin loss-of-function mutant will be lethal (vertebrate radixin has been shown to cap the barbed ends of actin), Colin Thacker has started to try to obtain rescue of the lethal strains generated from this area with cosmids that cover the gene. Based on the detailled protocols and generous help of Joel Rothman, Susan Mango and Ed Maryon we have generated a TCl transposon library with the mutator strain MT 3126 and have isolated transposon insertions in NF2 as well as in radixin. We are in the process of sib selection to obtain pure strains in order to generate loss-of-function mutants. Finally, we have also constructed antisense vectors with both genes under heat shock promotor control to be able to differentially delete one or both genes at different stages of development. There is some evidence in mammalian tissue culture systems that NF may suppress ras induced transformation (Maruta, pers. comm.). To test this observation in C. elegans we plan to inject NF2 under control of the let-60 promotor to see if we can suppress the let-60 gain-of-function Muv phenotype.