Ryuzo Shingai et al. (2007) International Worm Meeting "Chemotaxis of worms having genetically ablated ASK neurons."

Lin Lin, Tokumitsu Wakabayashi, Ryuzo Shingai

[International Worm Meeting, 2007]

ASK sensory neurons may be considered to have a subsidiary position to chemosensory informational pathways in C. elegans, because ASK neurons do not send synaptic outputs to AIY interneuron that receives numerous synaptic inputs from major chemosensory neurons (ASE, AWA, AWC) and thermosensory neurons (AFD). However, laser ablations of ASK neurons produce longer forward movement duration (i.e. lower frequency of backward movement) than in intact worms, and so it is intriguing what roles of ASK neurons are in neural signal pathways of chemotaxis. ASK neurons were genetically ablated by expressing mouse caspase1 from sra-9::Casp1 transgene. Cell ablations were confirmed by staining these integrated transgenic strains with DiD. Since lysine is sensed by ASK and ASE neurons in intact worms, and lysine should be sensed mainly only by ASE neurons in the ASK-ablated worms. The ASK-ablated worms were not attracted by lysine. The response to lysine was affected by the presence of isoamyl alcohol background. Chemotaxis for isoamyl alcohol of ASK-ablated worms was weaker than that of the intact worms. These indicate that ASK neurons are necessary for the chemotaxis behaviors, and chemosensory information via ASK neurons interacts with information via AWC neurons that sense isoamyl alcohol.

Ryuzo Shingai et al. (2010) East Asia Worm Meeting "Computer simulation for neural network of C. elegans"

Ryuzo Shingai, Kazumi Sakata, Yuishi Iwasaki, Tokumitsu Wakabayashi, Hisanori Takahashi, Tarou Ogurusu

[East Asia Worm Meeting, 2010]

All neural connections of C. elegans are known anatomically. To estimate functions of the neural network in behavioral states, we constructed a partial neural network model that regulates chemotaxis and thermotaxis, and conducted computer simulation. Synaptic excitatory/inhibitory properties were assigned to be consistent to those predicted in published articles, and the excitatory/inhibitory properties of other synapses were given at random. Changes in membrane potential of each neuron were observed when thermal/chemical stimuli were given to the sensory neurons. Possible synaptic properties were predicted by evaluating potential changes of the command interneurons that determine forward/backward movement. Some interneurons and motor neurons in the obtained network showed specific patterns of excitatory or inhibitory responses to single or simultaneous different stimuli.

Shingai, Ryuzo et al. (2009) International Worm Meeting "Integration of thermosensation and chemosensation during simultaneous presentation assay in C. elegans."

Adachi, Ryota, Wakabayashi, Tokumitsu, Shingai, Ryuzo

[International Worm Meeting, 2009]

Multi-sensory integration of neural function is important for organisms to discriminate environmental stimuli and to decide behavior. To address this function, we studied C. elegans behavior during simultaneous presentation of cultivation temperature and chemoattractant. C. elegans shows thermotaxis behavior that attracts to their cultivation temperature on a thermal gradient. Thermotaxis and chemotaxis behaviors are important for C. elegans to search food. It is possible that the integration of thermal and chemical information is occurred in animal''s nervous system. At first, we examined thermotaxis behavior in the radial temperature gradient, and chemotaxis behavior toward sodium chloride or isoamyl alcohol. Then, assays for simultaneous presentation of 15 deg C (colder temperature than 20 deg C room temperature) and chemoattractant were performed using 15 deg C-cultivated wild-type worms because of evident thermotaxis behavior in these worms. Simultaneous presentation resulted in a biased migration to cold regions in the first 10 min for the assay, and sodium chloride-regions in the last 40 min. However, when sodium chloride was replaced with isoamyl alcohol in the simultaneous presentation, the behavioral index was very similar to the sum of separate single presentation indices. We then recorded tracks of single worms and analyzed their behavior. Migration toward 15 deg C in simultaneous presentation was faster than in 15 deg C-single presentation, while migration speed toward the chemoattractant was not affected by the presence of 15 deg C spot. For behavior toward sodium chloride, frequencies of forward and backward movements in simultaneous presentation were significantly different from those in single presentation. Angle formed between a worm, the sodium chloride spot, and 15 deg C spot in simultaneous presentation was larger than that for sodium chloride in single presentation . These results suggest that multi-sensory integration occurred between cultivation temperature and sodium chloride in the C. elegans nervous system.

Lin Lin et al. (2006) East Asia C. elegans Meeting "Mutants Having Altered Preference of Chemotaxis in Simultaneous Presentation of Two Attractants"

Tarou OGURUSU, Ryuzo SHINGAI, Tsutomu SATO, Tokumitsu WAKABAYASHI, Lin Lin, Tomohiro OIKAWA

[East Asia C. elegans Meeting, 2006]

Mutants Having AlteredPreference of Chemotaxis in Simultaneous Presentation of Two Attractants Lin Lin, <SUP>a</SUP> Tokumitsu WAKABAYASHI, <SUP>b</SUP> Tomohiro OIKAWA,<SUP>a</SUP>Tsutomu SATO,<SUP>b</SUP> Tarou OGURUSU <SUP>a, b</SUP> and Ryuzo SHINGAI<SUP>a,b,</SUP> <SUP>a</SUP>Department of Computer and Information Science, Graduate School of Engineering, Iwate University, 4-3-5 Ueda, Morioka, 020-8551 Japan <SUP>b</SUP>Laboratory of Bioscience, Faculty of Engineering, Iwate University, 4-3-5 Ueda, Morioka, 020-8551 Japan Upon presentation of two distinct chemoattractants such as sodium acetate and diacetyl simultaneously, the worms were preferentially attracted by one of these chemoattractants. We isolated two mutants having altered preference of chemotaxis behavior toward simultaneous presentation of sodium acetate and diacetyl. chep-1(qr1) (for chemosensory preference) mutant preferred sodium acetate to diacetyl, while chep-2(qr2) mutant preferred diacetyl to sodium acetate in simultaneous presentation of these chemoattractants. chep-1(qr1) showed decreased preference toward diacetyl in the simultaneous two-spot presentation in all conditions tested. The decreased preference toward diacetyl could be explained by the sole effect of the defective chemotaxis toward diacetyl. The chemotaxis behavior of chep-2(qr2) mutant in simultaneous presentation suggested the function of chep-2 gene products within the chemosensory informational integration pathway as well as in the chemosensory pathway. Sodium acetate is detected by the ASEL chemosensory neuron and the neuron sends its synaptic output onto AIA, AIB, AIY interneurons and several other sensory neurons. A low concentration of diacetyl is detected by a bilateral pair of AWA sensory neurons and send their synaptic outputs onto AIY and AIZ neurons and several sensory neurons including ASE. Multiple information detected separately by ASEL and AWA neurons could be integrated directly within the ASEL neuron and within the AIY interneurons. The integration within the AIA, AIB and/or AIZ interneurons were also possible. chep-2 gene product may function in one to several of these neurons to regulate the informational integration pathway.

Tokumitsu Wakabayashi et al. (2005) International Worm Meeting "Neurons Regulating the Duration of Forward Locomotion in Caenorhabditis elegans"

Tokumitsu Wakabayashi, Ryuzo Shingai

[International Worm Meeting, 2005]

The locomotory behavior of Caenorhabditis elegans consists of four simple events, forward and backward movements, omega-shaped turns and rests. The wide variety of behaviors of this worm is achieved through a combination of these simple locomotions. To gain insight into the neuronal mechanisms regulating this locomotion, we analyzed the locomotory behavior of C. elegans over a long time period. In this study, we revealed the existence of at least two distinct behavioral states - pivoting and traveling - in the forward locomotion of C. elegans in the absence of food. Pivoting is characterized by pronounced directional switching and resulting short-duration forward movement (13 1 s), whereas in the traveling state, forward movement is of longer duration (180 25 s). Pivoting has occurred when we transferred a well-fed worm to unseeded plate, and then transition to traveling has taken place, successively. This behavioral alteration was partly dependent on their chemosensory functions because the mutants defective for chemosensing showed aberrant duration of forward movement in our experimental condition. We examined the function of chemosensory neurons by laser-ablation. Duration of forward movement was shortened by the loss of any one of the six chemosensory neurons (AWA, ASE, ADF, ASH, ASI, and ADL). On the other hand, loss of AWC, ASK or AFD chemosensory neurons lead extension of duration of forward movement. All of these chemosensory neurons send their anatomical synaptic output to one to several of the interneurons, AIA, AIB, AIY, and AIZ. Similar to the case for chemosensory neurons, ablation of AIA, AIY and AIB, AIZ interneuron resulted in short- and long- duration forward movement, respectively. These results suggested that C. elegans regulate their locomotion using multiple chemosensory and interneurons having opposed functions (i.e. neurons that promote forward movement and those that suppress forward movement). We are currently conducting genetic screening for mutants defective in the pivoting behavior.

Tokumitsu Wakabayashi et al. (2007) International Worm Meeting "Caenorhabditis elegans mutants having altered preference of chemotaxis behavior during simultaneous presentation of two chemoattractants."

Jun-ya Ishizeki, Ryuzo Shingai, Tokumitsu Wakabayashi

[International Worm Meeting, 2007]

Upon presentation of two distinct chemoattractants such as sodium acetate and diacetyl simultaneously, the nematode C. elegans was preferentially attracted by one of these chemoattractants even when using a particular concentration of attractants inducing the same strength of chemotaxis behavior in separate single presentation experiments. We referred this behavior as a result of the chemosensory informational integration within the C. elegans nervous system. We isolated mutants having altered preference of chemotaxis behavior toward simultaneous presentation of sodium acetate and diacetyl chep-1(qs1) (for chemosensory preference) mutant preferred sodium acetate to diacetyl while chep-2(qs2) mutant preferred diacetyl to sodium acetate in simultaneous presentation of 1/1,000 dilution of diacetyl and various concentrations of sodium acetate. The altered preference of chemotaxis behavior of chep-1(qs1) mutant in simultaneous presentation could be explained by the defect of the mutant in single presentation. In contrast, the altered preference of chemotaxis behavior of chep-2(qs2) mutant in simultaneous presentation could not be explained by the defect in single presentation, suggesting the function of chep-2 gene products within the chemosensory informational integration pathway as well as in the chemosensory pathway. We also isolated another set of mutants with altered preference of chemotaxis behavior toward multiple chemoattractants by using Mos1-mediated mutagenesis (1). Under the condition in which wild-type C. elegans was attracted more preferentially by sodium acetate (simultaneous two-spot presentation of 0.7M sodium acetate and 1/10,000 dilution of diacetyl), chep(qs3) and chep(qs4) mutants were attracted by either of these chemoattractant evenly. Identification of gene responsible for these mutations is currently in progress. 1. Bessareau, J. L. et al. Nature 413, 70-74. (2001).

Masahiro Kuramochi et al. (2010) East Asia Worm Meeting "Neuronal model including Ca2+ dynamics in C. elegans"

Ryuzo Shingai, Masahiro Kuramochi, Yuishi Iwasaki, Kazumi Sakata

[East Asia Worm Meeting, 2010]

Toward quantitative understanding of nervous system of C. elegans, we propose a neuronal model including Ca2+ dynamics. Since no evidence of Na+ current has been found in electrophysiological recordings, Ca2+ current is a key issue to the nervous system of C. elegans. In recent years, calcium imaging techniques have been developed to understand spatial-temporal dynamics of the neuronal activity. In addition, there are several electrophysiological studies of membrane potential. Here, we should pay attention to the observed data in these experiments. Quantity to be measured in the calcium imaging is not Ca2+ concentration itself but fluorescence intensity. Since a fluorescent probe interacts with intracellular proteins and changes its fluorescence properties, it is difficult to calculate the exact intracellular Ca2+ concentration from the fluorescence intensity. In electrophysiological experiment, a worm is pressed with a cover glass and can not move freely. Therefore, there is no guarantee that patch-clamping data reflects the neuronal activity generated in ""natural behavior"". Keeping the above mentioned issues in our mind, we numerically study the nervous system of C. elegans. Our neuronal model includes variables of membrane potential, Ca2+ concentration and fluorescence intensity. Dynamics of these variables are able to compare with the corresponding experimental data quantitatively. In this study, we apply the neuronal model to chemotaxis in C. elegans. To reduce the degree of freedom in neural modeling, we focus on a small neural circuit which consists of ASE chemosensory neurons and a few interneurons. ASE chemosensory neurons show the left/right asymmetric activity (Suzuki et al., 2008). ASEL is stimulated by increases in NaCl concentration. On the other hand, ASER is stimulated by decreases in NaCl concentration. In our simulation, the asymmetric stimulations are taken into account as external current injections to ASEL and ASER, respectively. The electrophysiological properties of membrane potential in ASE neurons (Goodman et al., 1998) are also considered. The model parameters are determined to satisfy the voltage responses to current injection in patch-clamp recordings.

Kazumi Sakata et al. (2002) Japanese Worm Meeting "A Computer Simulation of the Nervous System of C. elegans"

Tokumitsu Wakabayashi, Ryuzo Shingai, Taro Ogurusu, Kazumi Sakata, Katsunori Hoshi

[Japanese Worm Meeting, 2002]

Computer simulation of the neural system of Caenorhabditis elegans was performed. The model neuron had Hodgkin-Huxley type voltage sensitive channels. Parameters of the channels were obtained by curve fitting to the electrophysiological experimental data (Pierce-Shimomura JT, et al. Nature. 410 , 694 (2001)). Chemical synapses were located within neurites and their transmission properties were set to be graded as those of Ascaris suum (Davis RE, Stretton AO. J Neurosci. 9 , 415 (1989)). Gap junction was reconstructed as the connection of neurite membranes between pre- and post-synaptic neurons. The following conditions were necessary to reconstruct realistic changes of membrane potentials; (1) the conductance of gap junction was lower than that of the mammal, (2) the time constants of the channels at chemical synapse was about 3 msec. When the synaptic delay, which included the whole time course from activation of presynaptic soma membrane to that of postsynaptic cell, was set to 500 msec, the whole network system showed periodic perturbation of membrane potential. However, this synaptic delay is very long and not realistic. Therefore, there should be some factors which make the practical delay long as 500 msec. Additionally, we discuss possibility of other factors to generate periodic activation of membrane potential, e.g., pacemaker-like neuron as observed in Ascaris (Angstadt JD, Stretton AOW. J Comp Physiol A. 166 , 165 (1989)).

Yusuke Hokii et al. (2007) International Worm Meeting "C/D or H/ACA snoRNP proteins do not influence the nucleolar localization of C. elegans CeR-2/CeN21 RNA."

Ryuzo Shingai, Chisato Ushida, Hiroshi Sakamoto, Yumi Sasano, Akito Taneda, Yusuke Hokii, Akira Muto, Kazumi Sakata, Masayoshi Shimoyama, Toshinobu Fujiwara

[International Worm Meeting, 2007]

CeR-2/CeN21 RNA is one of the novel ncRNAs isolated from C. elegans. This is 126 nt in length and have a cap at 5 termini. The sequence and the secondary structure have no similarity to those of other ncRNAs. This RNA was expressed in most cells of the worm from the early embryonic stage to adult. In cells, CeR-2/CeN21 RNA concentrates in the nucleolus. Many small RNAs in the nucleolus are classified into a C/D snoRNA family or an H/ACA snoRNA family according to their structures. To know whether the protein components of C/D snoRNPs or H/ACA snoRNPs have any relationship to CeR-2/CeN21 RNA, fluorescence in situ hybridization was performed against the worms of which snoRNP proteins were knocked down by RNAi. When fib-1 (fibrillalin) or Ce-nop56 (K07C5.4), a C/D snoRNP component, was targeted by RNAi, U18 snoRNA, a C/D snoRNA, diffused into the nucleoplasm and cytoplasm. A part of U18 snoRNAs accumulated in an unknown subnuclear component, which was different from the nucleoli. U17/snR30 snoRNA, an H/ACA snoRNA, stayed in the nucleolus. When Ce-cbf5 (K01G5.5) or Ce-nop10 (C25A1.6), an H/ACA snoRNP protein, was targeted, most U18 snoRNAs were observed in the nucleolus but U17/snR30 snoRNA dispersed into the cytoplasm. In all cases, the localization of CeR-2/CeN21 RNA was not influenced. These results suggested that the nucleolar localization system of CeR-2/CeN21 RNA is different from those of C/D snoRNAs and H/ACA snoRNAs.

Yusuke Hokii et al. (2010) East Asia Worm Meeting "A small nucleolar RNA functions in rRNA processing in C. elegans."

Shigenori Oka, Kazumi Sakata, Toshinobu Fujiwara, Yumi Sasano, Akira Muto, Hyouta Himeno, Mayu Sato, Hiroshi Sakamoto, Akito Taneda, Ryuzo Shingai, Yusuke Hokii, Chisato Ushida

[East Asia Worm Meeting, 2010]

CeR-2 RNA, which is also known as CeN21 or Ce9 RNA, is one of the C. elegans non-protein-coding RNAs (ncRNAs) of unknown function. The characterization of this RNA by RNomic studies has failed to classify it into any known ncRNA family. Here, we examined the spatiotemporal expression patterns and found that this is expressed in most cells from the early embryo to adult stages. The subcellular localization is analogous to that of fibrillarin, a major nucleolar protein. Knockdown of nop56, a component of C/D small nucleolar ribonucleoproteins, but not of H/ACA snoRNP proteins, resulted in the aberrant nucleolar localization of CeR-2 RNA. A mutant worm with a reduced amount of cellular CeR-2 RNA showed changes in its pre-rRNA processing pattern compared with that of the wild-type strain N2. These results suggest that CeR-2 RNA is a C/D snoRNA involved in the processing of rRNAs.