Suzuki, Michiyo et al. (2017) International Worm Meeting "Region specific irradiation of Caenorhabditis elegans with heavy ion microbeam."

Suzuki, Michiyo, Yokota, Yuichiro, Funayama, Tomoo

[International Worm Meeting, 2017]

Animals including humans are exposed to radiation from natural and artificial sources, cosmic rays, and nuclear accidents. Radiation may affect vital functions such as locomotion, feeding, learning. To develop effective methods to investigate the effects of radiation exposure at tissue level as well as whole body is needed. In the present study, we therefore aimed to establish a novel method for radiation-targeting of specific regions of Caenorhabditis elegans using a collimating microbeam system, thus allowing vital functions, especially locomotion, to be observed immediately after irradiation. We immobilized individual adult C. elegans in straight microfluidic channels (60 m in width) in a polydimethylsiloxane chip and subjected them to irradiation targeted to the nerve ring, mid, or tail region, respectively, using carbon ions. The ions were delivered from the AVF cyclotron at TIARA of QST-Takasaki and then were collimated using a f20 m beam exit (micro-aperture) of a collimating microbeam system. Furthermore, for wider area of the body, a f60 m micro-aperture was used. In the ventral half-body irradiation, we targeted to the f60 m semicircle area in order from head to tail and irradiated at fifteen or more times. For comparison, whole body was irradiated using a scan beam. Immediately after irradiation, we added a drop of buffer solution on a microfluidic chip and collected animals using a picker. Each animal was transferred to a NGM plate without food and the locomotion was evaluated. The effects of whole-body irradiation tended to be more effective than those of region-specific microbeam irradiation at the same dose (500 Gy). We believe that microbeam irradiation method is powerful tool for research into the cellular and molecular effects of radiation, and that the novel techniques developed will be of use in future studies.

Suzuki, Michiyo et al. (2011) International Worm Meeting "Bold TextEffects of ionizing radiation on pharyngeal pumping in Caenorhabditis elegans."

Tsuji, Toshio, Suzuki, Michiyo, Kobayashi, Yasuhiko, Hattori, Yuya, Sakashita, Tetsuya

[International Worm Meeting, 2011]

Caenorhabditis elegans is a good in vivo model system to examine radiobiological effects. Recently, we found that locomotion caused by body-wall muscles was reduced in a dose-dependent manner after gamma-ray irradiation, and that the locomotion was eventually restored (1-2). However, it is not known whether the same effects are observed in other types of movements in C. elegans. The combination of muscles and motor neurons used for locomotion is different from that used for other movements such as chewing and swallowing (pumping motion). Therefore, a study of radiation effects on different types of movements might help to identify those regions among muscles and the nervous system that are strongly related to radiation responses. Here, we examine the radiation effects on pumping by the pharyngeal muscles in particular. In the experiments, 50 or more well-fed adult C. elegans were placed on an agar dish with a bacterial lawn (food) and irradiated with a graded dose of <sup>60</SUP>Co gamma rays. Pharyngeal pumping in 5 or more animals was recorded using a high-speed camera every 2 h from 0 h to 8 h post-irradiation. The frequency of pharyngeal pumping was counted using 60 continuous recording images of 3 s duration. We found that irradiated animals could be classified into 2 groups. One group stopped pumping immediately after irradiation and the other showed normal pumping activity. This tendency of the 2 groups was distinctly different from that of locomotion using body-wall muscles, wherein the motility of the irradiated animals decreased uniformly in a normal distribution wholly in a dose-dependent manner. In addition, the pumping activity was completely restored up to the level of the non-irradiated animals within 2 h, and the restoration level was higher than that in locomotion. Our findings indicate that whole body irradiation reduced the pumping and locomotion and both movements were restored within several hours, although there was an obvious difference in the aspect of the reduction between pumping and locomotion. This difference might depend on the difference of the number and/or type of neurons controlling the pharyngeal muscles for pumping and body-wall muscles for locomotion. Further study on the mechanism underlying the irradiation-induced reduction and restoration in each movement will be required. (1) Sakashita, T. et al. (2008) J. Radiat. Res. 49, 285. (2) Suzuki, M. et al. (2009) J. Radiat. Res. 50, 119.

Suzuki, Michiyo et al. (2013) International Worm Meeting "Quantitative analysis of IR-induced effects on locomotion in Caenorhabditis elegans."

Hattori, Yuya, Sakashita, Tetsuya, Tsuji, Toshio, Suzuki, Michiyo, Kobayashi, Yasuhiko

[International Worm Meeting, 2013]

Background and purpose: C. elegans is a good in vivo model system to examine radiobiological effects. We have previously examined the effects of ionizing radiation (IR) on locomotion in C. elegans using 'body bends' (the number of bends counted in the anterior body region at 20-s intervals) [1] and reported an IR-induced reduction in motility [2],[3]. However, the degree of motility in each region of the body has not been established. In the present study, we employed a video-based analysis and investigated the IR-induced effects on locomotion in more detail. Irradiation and video-based analysis: Young adult wild-type C. elegans were placed on a NGM plate with a bacterial lawn (food) and irradiated with graded single doses (0-1.5 kGy) of 60Co g-rays. Immediately after irradiation, animals were transferred to a NGM plate, either with or without food. The movements of five or more animals placed on each plate were video-recorded. The video images were analyzed off-line based on a previously published method [4]. Briefly, after binarization and denoising, the body line was skeletonized and evenly divided into 12 segments; X- and Y-coordinates of each point on the body were subsequently acquired. To evaluate the motility of each point on the body, the moving distance of each of 13 points over a 5 s period was calculated using the X- and Y-coordinates. In addition, we introduced a novel standard, namely the straight distance from head to tail, to evaluate the body form. Results: Under the -food condition, the moving distance of irradiated animals was reduced in a dose-dependent manner at each point on the body, and there was no difference between the effects on each region of the body. The dose-dependent reduction in locomotion was also observed in animals under the +food condition. Furthermore, we evaluated body form and found that IR-induced quantitative changes in body form. References: [1] Sawin, E.R., et al. (2000) Neuron 26, [2] Sakashita, T., et al. (2008) J. Radiat. Res. 49, [3] Suzuki, M., et al. (2009) J. Radiat. Res. 50, [4] Hattori, T., et al. (2012) Neural Comput. 24.

Michiyo Suzuki et al. (2004) East Asia Worm Meeting "Motion Simulation with a C. elegans Model for Touch Sutimuli"

Michiyo Suzuki, Noboru Takiguchi, Toshio Tsuji, Hisao Ohtake

[East Asia Worm Meeting, 2004]

In recent years, a new approach for analyzing functional mechanism of a living organism has been proposed, in which computer simulation of a mathematical model is fully utilized ['98 H.Ohtake, '01 H.Kitano]. In this analysis using a virtual model instead of the corresponding actual organism, it is possible to change environmental conditions easily and to analyze their behavior repeatedly under the same conditions. This is not only useful to the area of biology, but also possible to be applied to the area of engineering such as establishment of a new brain-like machine based on the mechanism of living organisms. In the approach using a virtual model, analysis of simple' organisms is necessary to understand systems of higher organisms. Therefore, our group has developed computer models of two kinds of unicellular organisms, colibacilli and paramecium, based on the knowledge of both biology and engineering ['02 T.Tsuji et al. , '04 A.Hirano et al. ]. This study deals with multicellular organisms as the next step of the above-mentioned approach. Among multicellular organisms, we focus on Caenorhabditis elegans ( C. elegans ), and aim to develop a computer model of this organism based on the previous studies at the nervous level. So far, many studies of the C. elegans model have been reported [for example, '99 T.C.Ferree et al. , '01 K.Kawamura et al. ]. However, since they focused on only sensing and processing external stimuli, the locomotion which is appeared was extremely simplified. In modeling C. elegans , the motor control system with respect to locomotory responses have to be considered as well as the internal processing system. Consequently, we propose a computer model of C. elegans , which includes the nervous circuit model for processing external stimuli and the kinematic 12-link body model for locomotion control. Although the C. elegans processes many kinds of stimuli, we focus on gentle touch stimuli. In this presentation, we will explain in detail both of a nervous circuit model for touch stimuli and a kinematic model of the body. Also, some properties of our model, particularly those related to the taxis for touch stimuli, will be discussed through the simulation results.

Michiyo Suzuki et al. (2010) East Asia Worm Meeting "Effects of ionizing radiation on pharyngeal pumping in Caenorhabditis elegans"

Toshio Tsuji, Yuya Hattori, Tetsuya Sakashita, Michiyo Suzuki, Yasuhiko Kobayashi

[East Asia Worm Meeting, 2010]

To investigate the effects of ionizing radiation (IR) on vital functions such as learning/memory and volitional/nonvolitional movement is important for understanding the risk of manned space flight or the side-effects of radiation therapy. Caenorhabditis elegans is a good in vivo model system to examine radiobiological effects. Recently, we found that locomotion caused by body-wall muscles was reduced in a dose-dependent manner after gamma-ray irradiation and that the locomotion was eventually restored. However, it is not known whether the same effects are observed in other types of movements in C. elegans. The combination of muscles and motoneurons used for locomotion is different from that used for the other movements such as chewing and swallowing (pumping motion). Therefore, to examine radiation effects on different types of movements might help to clarify the IR-affected regions among muscles and the nervous system. Here, we examine the radiation effects on pumping of pharyngeal muscles.In the experiments, 50 or more well-fed adult animals were placed on an agar dish with a bacterial lawn (food) and irradiated with a graded dose (300, 500, and 1000 Gy) of 60Co gamma rays. Pharyngeal pumping in 4 or more animals was recorded using a high speed camera at every 2 h from 0 h to 8 h after irradiation. The number of times of fast pharyngeal pumping was counted using 60 continuous recording images of 1 s.Interestingly, irradiated animals were classified into 2 groups. One group stopped pumping immediately after irradiation and the other showed normal pumping activity. This tendency of the 2 groups was distinctly different from that of locomotion using body-wall muscles, wherein the motility of the irradiated animals reduced in the normal distribution wholly in a dose-dependent manner. In addition, the pumping activity was completely restored within 2 h and the restoration rate was higher than that of locomotion. These results indicate that the whole body irradiation reduced the pumping and locomotion in a different way, but both movements were restored within several hours. Our findings suggest the existence of multiple action mechanisms of radiation effects on C. elegans's motility.

Michiyo Suzuki et al. (2006) East Asia C. elegans Meeting "Computer simulation of chemotactic neuronal networks in C. elegans: Does food-NaCl associative learning lead to changes in the networks?"

Michiyo Suzuki, Toshio Tsuji, Yasuhiko Kobayashi, Tetsuya Sakashita

[East Asia C. elegans Meeting, 2006]

Under normal conditions, C. elegans produces motion in response to chemical stimuli, e.g., moving toward attractive chemicals and avoiding the noxious ones (chemotaxis). Additionally, C. elegans shows associative learning ability: despite the fact that NaCl is typically a soluble attractant to this nematode, after exposure to NaCl in the absence of food, the chemotaxis toward NaCl changes such that it avoids NaCl [Saeki, S. et al. 2001]. However, this behavior is not observed in the case of benzaldehyde (a volatile attractant) [Eric, L. et al. 2004], although neuronal networks of chemotaxis toward NaCl and benzaldehyde show a partial overlap. We focused on the change in chemotaxis toward NaCl caused by the food-NaCl associative learning and conducted a computer simulation to understand this change at the neuronal network level. Soluble and volatile chemicals are perceived by different sensory neurons. However, since several interneurons connect with multiple sensory neurons, neuronal networks of chemotaxis toward NaCl and benzaldehyde show a partial overlap. Therefore, the change in chemotaxis caused by the food-NaCl associative learning is hypothesized to be induced mainly in some parts of the neuronal networks. In this study, to identify these parts of the neuronal networks in which the change is induced, we modeled the chemotactic neuronal networks based on connections of actual C. elegans [White, J.G. et al. 1986]. The computer simulation of the responses of this model before and after food-NaCl associative learning revealed that this model can be used to replicate the actual responses toward some kinds of chemicals. The preliminary results showed that the signal transduction around the AIY interneuron after the food-NaCl associative learning greatly differed from that before the food-NaCl associative learning. This result is in agreement with the experimental findings of a previous study [Ishihara, T. et al. 2002], which suggested that AIY is an important neuron for food-NaCl associative learning. Based on this, we discuss the partial change in the neuronal networks caused by associative learning.

Yamasaki, Akira et al. (2017) International Worm Meeting "Effects of Region-Specific Irradiation on Locomotion and Autophagy in C. elegans."

Zhang-Akiyama, Qiu-Mei, Suzuki, Michiyo, Kobayashi, Yasuhiko, Yamasaki, Akira, Funayama, Tomoo

[International Worm Meeting, 2017]

Ionizing radiation generates reactive oxygen species, and causes damage to cell components including DNA and protein. In C.elegans, radiation sensitivity is different from germline-cell and somatic cell. When adult C.elegans are irradiated with ten-fold higher dose than the dose that leads to germline-cell death, worms are still alive1. Locomotion using motor neurons and body-wall muscles was reduced immediately after irradiation with 0.5 kGy2. However the mechanism of reduction is not fully understood. In the present study, to investigate a tissue that is responsible for reduction of locomotion, we used region-specific microbeam irradiation. We used energetic carbon ions delivered from the AVF cyclotron at Takasaki Ion accelerators for Advanced Radiation Application facility of QST-Takasaki. After irradiation, a worm was replaced on NGM plate, and the locomotion was video-recorded and then the trajectory for 5-sec duration was derived by image processing of the movie. As a result, the same effects as whole-body irradiation were not observed after region-specific microbeam irradiation to pharynx or anterior half-body (the nerve ring, pharynx, intestine, and gonad) or posterior or posterior half-body (vulva, intestine, gonad, and tail). This suggests that the radiation effects on locomotion depend on the size of irradiation area. To detect the dose whether the reduction of locomotion is restored or leads to individual death, and we investigated alternation of locomotion after irradiation. We found the dose that locomotion of worms were completely stopped immediately after irradiation, and had been stopped at least twenty-four hours. Less than the dose, locomotion of the worms was reduced in a dose dependent manner, and was partially restored after twenty-four hours. Protein damage generated by irradiation may be involved in this reduction and restoration of locomotion, so we investigated whether autophagy is induced after irradiation. Using GFP reporter assay of lgg-1, one of the autophagosome genes, the increased level of GFP::LGG-1 was detected seven hours after irradiation in somatic cells. This suggests that autophagy may be one of the reasons of restoration of locomotion after irradiation. 1. Ishii,N., Suzuki, K., Int. J. Radiat. Biol., 58:827-833. (1990) 2. Suzuki, M., et al., J. Radiat. Res. 50, 119-125. (2009)

Hattori, Yuya et al. (2013) International Worm Meeting "Cell-level modeling and simulation of the pharyngeal pumping in Caenorhabditis elegans."

Suzuki, Michiyo, Hattori, Yuya, Tsuji, Toshio, Kobayashi, Yasuhiko

[International Worm Meeting, 2013]

The pharyngeal pumping in Caenorhabditis elegans is generated and controlled by the pharyngeal neurons and muscular cells. In this study, we proposed a simulation-based approach to estimate the mechanisms of oscillation generation in pharynx at cell level. To conduct the simulations, we previously developed a pharyngeal muscle model including 20 muscular cell models and 9 marginal cell models (Hattori, Y., et al, Artif. Life Robotics, 17, 2012). Output in each cell model was the membrane potential based on FitzHugh-Nagumo equations. These cell models were connected by gap junctions based on the actual connection structure of pharyngeal muscle in C. elegans. The gap junctions transmitted the outputs between cell models. The electropharyngeogram (EPG), which displays the summed activity of the electrophysiological responses of pharyngeal muscle cells and neurons, was used to measure the biological signals from pharyngeal pumping in C. elegans. In our simulation, we obtained the EPG using the outputs of individual cell models.

Soh, Zu et al. (2013) International Worm Meeting "A method of estimating environmental friction based on a body dynamics model of Caenorhabditis elegans."

Tsuji, Toshio, Hattori, Yuya, Masaoka, Kazuya, Soh, Zu, Suzuki, Michiyo

[International Worm Meeting, 2013]

Information on the forces generated by the C. elegans is important in exploring the mechanism of its locomotion. This study was performed to investigate a method for estimating environmental friction based on the use of a dynamics model and image analysis. The model is described using Newton-Euler equations of N rigid links serially coupled by rotational joints. The friction forces acting between the environment and the body are modeled using dynamic and viscous friction. Although the motion equation can be solved for friction coefficients as a function of the worm's motion, this straightforward solution is an ill-posed problem because the friction model contains four unknown coefficients, while a worm crawling on an x-y plane has only two degrees of freedom. To solve this problem, the proposed method involves simultaneous analysis of two different worm motions in the same environment for which identical friction coefficients are assumed. In addition, as the images used in analysis to determine a worm's motion inevitably contain noise, a coefficient-optimization approach was adopted to minimize the error between trajectories of an actual worm and the corresponding dynamics model in terms of the four friction coefficients. To verify the proposed method, undulational motion of the dynamics model was artificially generated and simulated with randomly preset friction coefficients. With this configuration, the method was applied to estimate the preset coefficients. The results indicated that the percentage error between the preset and estimated coefficients was within 4%. The results of the method's subsequent application to actual worms showed that the dynamics model could trace their trajectories within a percentage error of 2% of the body length, and that the torque generated from the model was in a reasonable range as measured in previous studies (Ghanbari et al. 2008; Johari et al,, 2013) in a microstructured environment.

Soh, Zu et al. (2019) International Worm Meeting "Computer simulation of connectome-based neural network model of Caenorhabditis elegans to generate muscle activity patterns for locomotion."

Soh, Zu, Iino, Yuichi, Sakamoto, Kazuma, Suzuki, Michiyo, Tsuji, Toshio

[International Worm Meeting, 2019]

Caenorhabditis elegans can generate coordinated movements using only 58 motor neurons. Calcium imaging studies and connectome-based mathematical models have revealed the roles of command neurons and the importance of proprioceptive feedback in generating undulatory movements. However, the current mathematical model is limited to the simulation of forward locomotion. In this study, we constructed a connectome-based model including 58 motor neurons, command neurons, and 96 muscle cells to generate muscle activity patterns for both forward and backward movements. In the previous study, we modeled the command neurons as units that output on/off signals corresponding to forward and backward movements. The motor neurons and muscle cells were modeled using the integrate-and-fire model and connected by synaptic and electric connections based on the connectome provided by WormAtlas. The strengths of the synaptic and electric connections were adjusted using backpropagation through a time algorithm so that the muscle activity patterns for generating the forward and backward locomotion could be switched by the output of the command neurons. In this paper, we performed simulation on muscle activity generation and analyzed the effects of synaptic connection strengths. The simulation results confirmed that the command neurons can switch the propagation direction of the muscle activity when the muscle activities were fed back to the motor neurons. In addition, we found that the mean activation levels of the B-class motor neurons were higher than those of A-class motor neurons when generating a forward wave and vice versa for generating a backward wave. This observation is consistent with the experimental study by Kawano et al. (2011). Furthermore, when we uniformly reduced the synaptic strengths, both the amplitudes and frequencies of the muscle activity decreased simultaneously. The model also predicts that the local synaptic strengths affect both the amplitudes and frequencies of the whole-body motions.