Tsukada, Yuki et al. (2009) International Worm Meeting "Quantitative behavioral analysis of freely moving C. elegans."

Miyara, Akiko, Shimowada, Tomoyasu, Ohnishi, Noriyuki, Kuhara, Atsushi, Tsukada, Yuki, Mori, Ikue

[International Worm Meeting, 2009]

C. elegans is an ideal system to study information processing mechanism by neural network because the patterns of connectivity for all 302 neurons had been mapped out. In addition, input stimuli and output behavioral responses are robust for qualitative measurement. Although previous studies have identified several neurons and molecules related to particular types of information processing, little is known about the mechanism of information processing in C. elegans. This is because the lack of dynamic information for stimuli and behaviors as input and output functions, respectively. We are approaching this problem by quantitatively measuring input stimulus and output behavior. To begin our analysis, we took advantage of our live imaging and tracking system that pictured freely moving single animals; the time-lapse images were recorded up to several hours at video rate (about 30 frames/sec). Then, curvature maps of worm body along head-to-tail axis were constructed by using homemade matlab programs, and thus locomotory states were represented by the patterns in the map. The map identified bending frequencies at each position along the body and speed of curvature wave from head to tail. As previously reported (Croll NA, 1975), curvature wave along head-to-tail axis showed various patterns. For example, reverse wave (tail-to-head) of curvature was observed when the animals moved backward. We compared wild-type (N2) animals with ttx-8 (nj34) mutants that showed defects in thermotaxis by circular thermal gradient assay and calcium imaging of thermosensory neurons (Miyara et al. unpublished). Our quantitative behavioral map analysis distinguished behavioral patterns of wild-type animals and the mutants. Since the behavioral patterns of the ttx-8 mutant include normal migratory patterns, we assume that the mutants are normal in motor control but sensory and/or information processing of the neural network is defective. More speculatively, we suppose that judgmental process and/or maintenance of action planning in the mutants should be defective. We are currently working on the quantitative analysis for temperature input stimuli and attempting to establish mathematical model for information processing during thermotaxis.

Tsukada, Yuki et al. (2011) International Worm Meeting "Long term calcium imaging of AFD thermosensory neuron revealed behavioral strategy for exploratory movements during thermotaxis."

Tsukada, Yuki, Mori, Ikue, Ohnishi, Noriyuki, Kuhara, Atsushi, Shimowada, Tomoyasu

[International Worm Meeting, 2011]

Despite the identification of neurons, their connectivity, and molecules related to the specific mechanisms of neural circuits, little is known about the mechanism of information processing in Caenorhabditis elegans. An essential obstacle is the lack of methods that can monitor time course of information flow during information processing tasks. To address this issue, we focus on thermotaxis behavior of C. elegans because the information flow is relatively simple: the essential neural circuit related to thermotaxis is thought to consist of only several neurons. We quantitatively monitored information input (temperature) and output (behavior) by using thermography and our live imaging/tracking system that pictures freely moving single animals; the time-lapse images of a single animal were recorded up to several hours at about 30 frames/sec video rate with migratory trajectory. We simultaneously monitored activity of AFD thermosensory neuron and behavior during thermotaxis by combining the tracking system with the calcium imaging system with genetically encoded calcium indicator, Cameleon YC 3.60. Thus we quantified exact temperature, AFD activity, and behavioral state of freely moving animal during thermotaxis. Wild-type N2 animals on an agar plate with thermal gradient showed thermotaxis behavior depending on their cultivated temperature or food availability. Long-term monitoring of AFD activity showed spike-like increments of calcium concentration despite the continuous, slow temperature change of about 0.1 deg C/min. By comparing AFD activity and the behavioral states, we found that the peak of AFD activity corresponded to the timing of turn, although the AFD activity and turning behavior was not one-to-one coupling and one activity peak seemed to correspond to a few turns. This relationship changed when the animals were conditioned with off-food plate, by experiencing the animal off-food for two hours before the thermotaxis assay. Off-food conditioned animals tended to show one AFD activity peak for one turn or AFD activity peaks without any turn. According to these results, we suggest that AFD thermosensory neurons recognize thermal environment as discrete system even in shallow continuous thermal environment and that the animals change behavioral strategy during thermotaxis by adjusting relationship between AFD activity input and turning output. These hypotheses brush up biased random walk model of exploratory behavior of C. elegans and shed light on a specific type of information processing systems which utilize random signal.

Ochiai, Yuta et al. (2011) International Worm Meeting "Quantitative analysis of exploratory patterns during thermotaxis."

Tsukada, Yuki, Mori, Ikue, Ochiai, Yuta

[International Worm Meeting, 2011]

Exploratory behavior is essential for animals to obtain food, partners and other resources. When transferred from the plate with food to the plate without food, Caenorhabditis elegans shows several behavioral patterns, and the neural circuit that controls those patterns is identified (Gray et al., 2005). While exploratory behaviors generally utilize various sensory cues, little is known about the exploratory behavioral patterns when specific sensory cues are available. Thermotaxis of C. elegans is a model of the environmental information-based exploratory behaviors because the neural circuit critical for thermotaxis is identified (Mori and Ohshima 1995; Kuhara et al., 2008), and input and output of temperature information flow can be quantitatively measured in our system. Here, we aimed to assess the exploratory patterns during thermotaxis and clarify the neuronal mechanism regulating the exploratory patterns in accordance with sensory cues. We videotaped the freely moving individual animals on agar plate for up to 60 minutes using a computer-driven tracking system that keeps the animal in the microscopic field. Timings of reorientation turns were detected by analyzing the movies with image processing program. Then, we calculated the turn frequency for every ten minutes. We also analyzed the relationship between number of turns and temperatures. Wild-type N2 animals were cultivated at 17 deg C, 20 deg C and 23 deg C with food, OP50, and then were placed on the uniform 20 deg C 6x9cm agar plate without food. The turn frequency rapidly decreased within the initial 20 minutes and continued to gradually decrease until 30 minutes in 17 deg C, 20 deg C or 23 deg C cultivated animals. After 30 minutes, turn frequency of 20 deg C cultivated animals showed a constant turn frequency for another 30 minutes, but the turn frequency of 17 deg C or 23 deg C cultivated animals changed irregularly. By contrast, when the animals were placed on the plate with linear thermal gradient ranging from 15 deg C to 25 deg C, and with the temperature steepness of 1 deg C per 1cm, the turn frequency did not decrease rapidly in the initial 20 minutes. We classified the exploratory patterns on the thermal gradient into four classes according to the number of turns in relation to the temperatures. Mutants for cGMP-gated channel (tax-4) and AFD neuron-specific guanylyl cyclases (gcy-23, gcy-8, and gcy-18 triple mutant) showed lower turn frequency on thermal gradient than wild type animals, thus showing different exploratory patterns during thermotaxis. Our results indicate that the change in turn frequency during exploratory behavior is modulated by temperature information. Our further analysis with thermotaxis mutants would be a key for clarifying this exploratory behavior mechanism.

Kano, Amane et al. (2019) International Worm Meeting "Toward understanding neural computations in an interneuron AIY through optogenetical manipulation of its presynaptic sensory neurons AFD and AWC."

Kano, Amane, Tsukada, Yuki, Mori, Ikue

[International Worm Meeting, 2019]

How an interneuron integrates environmental cues transformed by sensory neurons is an important problem in neuroscience. The simplest model to elucidate this problem is a neural circuit consisting of two sensory neurons and their postsynaptic interneuron. One such model in C. elegans is a circuit consisting of thermosensory neurons AFD and AWC, and their postsynaptic interneuron AIY, which has been identified as a part of the circuit for thermotaxis (Mori & Ohshima, Nature, 1995; Kuhara, Okumura et al., Science, 2008). Previously, one of our studies shows that AFD either excited or inhibited AIY activity depending on thermal stimuli (Kuhara et al., Nat. Commun., 2011). Similarly, other study reports that activation of AWC suppressed AIY in response to thermal stimulus (Kuhara, Okumura et al., Science, 2008). However, how AIY responses depend on states of both AFD and AWC activities remains unclear. In this study, we investigated how AIY activity is affected by optogenetically controlling states of AFD and AWC activities. We first introduced GCaMP6f and Chrimson in either AFD or AWC, and confirmed that AFD and AWC activity could be excited by Chrimson. We next quantified AIY activity under controlling AFD, AWC, or both AFD and AWC was evoked by Chrimson by scoring spike-like AIY activity peaks to define as the frequency of AIY activity. We found that when AFD was excited by Chrimson, the frequency of AIY activity also increased. By contrast, when AWC was excited by Chrimson, the frequency of AIY activity was unchanged. However, when both AFD and AWC were simultaneously evoked, we observed two states, increment or decrement, of the frequency of AIY activity. Our results suggest that AIY responses are indeed dependent on the state of both AFD and AWC. Based on this result on simultaneous evoking of AFD and AWC activity, we are currently seeking the critical time window between excitation of AFD and AWC, which may affect AIY responses. To reveal critical time window, we use projection mapping system that enables illumination of pinpointed areas. We so far established our projection mapping system to illuminate AFD soma and AWC soma, both of which are about 10 m close to each other. We are also improving the software aiming to detect the increment of calcium signal and feedback it to excitation timing and illumination areas. By using this feedback software, we hope to reveal how states of AFD and AWC dynamically affect AIY activity, thereby contributing to clarify how sensory signals integrate within an interneuron.

Miyara, Akiko et al. (2009) International Worm Meeting "Studies on neuronal function of a novel and conserved protein TTX-8."

Tsukada, Yuki, Miyara, Akiko, Ohta, Akane, Okochi, Yoshifumi, Mori, Ikue, Kuhara, Atsushi

[International Worm Meeting, 2009]

Animals can sense and respond properly to variable signals in surrounding environment. From receiving signals to behavioral outputs, many neurons contribute to transfer signal information. In principle, a particular stimulus is perceived by sensory neurons and then transmitted to downstream interneurons, and finally behavioral output is induced as a result of neural processing. During this processing, those neurons are needed to function accrately as might be expected. Here, we report that a novel protein TTX-8 plays a role in assisting basal neuronal function. TTX-8 is predicted to have transmembrane region in N-terminus and coiled coil region in C-terminus. The ttx-8 mutants showed several abnormal behaviors including thermotaxis, chemotaxis to NaCl and odorants, and locomotion (Tsukada et. al., this meeting). We found that TTX-8 is expressed and functions in neurons. A human homologue of TTX-8 weakly but effectively rescued abnormal thermotaxis phenotype of the ttx-8 mutant, suggesting that TTX-8 is conserved across species. When ttx-8 cDNA was expressed simultaneously in major thermosensory neuron AFD, and downstream interneuron AIY and AIZ of ttx-8 mutants, about 50% of transgenic animals showed normal thermotaxis phenotype. This result suggests that TTX-8 functions in AFD, AIY and AIZ neurons for thermotaxis. Consistently, calcium imaging analysis revealed that calcium influx in both AFD and AIY neurons of ttx-8 mutant were notably decreased in response to thermal stimuli, indicating that TTX-8 is required for proper activation of AFD and AIY neurons. To determine which region of TTX-8 is necessary for its accurate function, we constructed two truncated TTX-8 proteins, one lacks transmembrane region and the other lacks coiled coil region, and did rescue experiments using each truncated protein. Both truncated forms could not rescue abnormal thermotaxis of ttx-8, suggesting that both regions are important to proper TTX-8 function. RIC-3, which resembles TTX-8 in protein structure, has been reported to be required for the maturation of acetylcoline receptor (Halevi et. al., 2002). In ttx-8 mutant, a presynaptic marker SNB-1::GFP was mislocalized in motorneurons. Taken together, TTX-8 might be involved in the maturation of synaptic proteins. Consistent with this possibility, immunostaining experiments using HeLa cell showed that TTX-8 was localized to peri-nuclei region of cytoplasm, perhaps to ER and/or Golgi-body. Our data suggest that TTX-8 plays a role in supporting neuronal function probably through regulation of neural activation or maturation of synaptic proteins.

Giles, Andrew C. et al. (2013) International Worm Meeting "Toward the identification of behavioral strategies underlying C. elegans thermotaxis using the Multi-Worm Tracker."

Tsukada, Yuki, Giles, Andrew C., Mori, Ikue, Nakano, Shunji

[International Worm Meeting, 2013]

C. elegans navigate thermal gradients to find their cultivation temperature. Studying mechanisms of thermotaxis will provide insight into neural functions including thermosensation, memory and the computations that neurons use to integrate information and decide on motor output. Assays that measure the accumulation of worms on thermal gradients rapidly quantify thermotaxis but are not capable of determining the behavioral strategies that worms utilize while navigating. Analyses of locomotion on thermal gradients and in response to temperature ramps have revealed some of the underlying strategies, such as biased random walk during cryophilic movement, but have not yet identified components needed for other aspects of the behavior, including thermophilic movement. To develop a high-throughput assay that captures components of locomotion in environments where thermophilic movement has been observed, we adapted the Multi-Worm Tracker (MWT), a software-hardware package that measures locomotion-based behavior for dozens of worms simultaneously, to work with our accumulation assay. There were two main obstacles: lighting and the size of the field of view. We found that using black anodized aluminum under the assay plate to transmit the thermal gradient in combination with low angle lighting above provided good contrast between worms and background. To image a 13.5 x 10 cm thermotaxis assay plate, we found the Dalsa Falcon 4 MP camera previously used by the MWT did not provide enough resolution to adequately visualize body shape, which impaired automated identification of certain components of locomotion. We found the Toshiba-Teli CleverDragon 12 MP camera was compatible and improved the imaging of body shape and locomotion while maintaining adequate temporal resolution (~13 Hz). We conclude that this setup can be used to track locomotion components during multi-worm thermotaxis assays and can likely be applied to analyze other behaviors that require a large assay area, such as chemotaxis. We hope that high-throughput analysis with this system will reveal more of the behavioral strategies underlying C. elegans thermotaxis behavior.

Huang, Tzu-Ting et al. (2019) International Worm Meeting "Molecular and Functional Characterization of Age-Dependent Changes in C. elegans Thermosensory behaviors."

Mori, Ikue, Nakano, Shunji, Tsukada, Yuki, Huang, Tzu-Ting, Matsuyama, Hironori J.

[International Worm Meeting, 2019]

Progressive decline in sensory, behavioral and cognitive functions during aging is mostly attributed to deterioration in synaptic structures or activities of the nervous system. It remains elusive however how the primary sensory neurons, which directly sense and gate environmental information, respond to stimuli in the course of senescence and shape associative learning during aging. Here we characterize the molecular and functional aging of primary sensory neurons in the thermotaxis and isothermal tracking behaviors, both of which are circuitry-tractable learning models in Caenorhabditis elegans. As we reported previously, theprimary thermosensory neuron AFD that drives food-associative thermotaxis behavior displays a response threshold and an operating range of temperature-evoked calcium dynamics shaped by its temperature experiences. In this study, we found that AFD neurons in aged animals show a prolonged operating range of temperature-evoked calcium dynamics, while similar to young AFD neurons, capable to set their thermosensory experience-based response threshold. Consistent with this observation, we found at the behavioral level that aged animals carry imprecise thermotactic and isothermal tracking behaviors while retaining the behavioral plasticity to reshape their behaviors. In an attempt to understand a molecular mechanism in functional senescence, we screened mutants for age-related behavioral decline and revealed that an animal carrying a mutation in G protein coupled receptor kinase demonstrates aging-related behavioral phenotypes at early adult stage. Downstream signaling pathways and the suppressors for improving neural functions in senescent nervous systems are under characterization. Our molecular and functional analysis of AFD suggests that age-dependent changes in the primary sensory neurons contribute to behavioral decline associated with senescence, and highlights the importance of sensory pathways as part of age-dependent remodeling in behavioral functions.

Huang, Tzu-Ting et al. (2017) International Worm Meeting "Morphological and Functional Characterization of Age-Dependent Changes in A C. elegans Thermosensory Neuron."

Tamada, Takashi, Huang, Tzu-Ting, Mori, Ikue, Pan, Chun-Liang, Tsukada, Yuki

[International Worm Meeting, 2017]

Widespread age-dependent changes could be found at multiple levels of neural architecture in C. elegans, including the neuronal soma, axon, dendrite or synapses. Whether age-related changes occur to C. elegans ciliated endings, microtubule-based signaling compartments in the sensory neurons, has not been characterized. Here we report age-dependent deterioration of sensory ending architecture in the AFD thermosensory neuron, including reduced microvilli and engorged cilia. Age-dependent changes of the AFD sensory ending were modified by daf-16 or daf-2 mutations in a way that was consistent with alteration of life span by these mutations. This suggests that insulin signaling, one of the most conserved regulatory mechanisms of life span, modulates age-dependent decline of ciliated endings. Age-dependent changes could also be found in ciliated endings of multiple amphid neuronal classes, indicating a global deterioration of primary sensory neurons during aging. To understand whether functions of the AFD neurons deteriorate during aging, we performed calcium imaging and thermotaxis assays. We found that compared to young neurons, aged AFD neurons showed precocious activity at temperature lower than the cultivation temperature (TC), and they continued to respond at temperature higher than the TC. Interestingly, mid-age wild-type animals were cryophilic in thermotaxis assay, consistent with our observations that high AFD activity induced turning behaviors. Our work provides a detailed account of the morphology, function and related behaviors of a C. elegans thermosensory neuron during aging and lays the foundation for future exploration of molecular underpinnings of such age-dependent changes in the sensory system. (supported by National Health Research Institutes, NHRI-EX106-10529NI and the Ministry of Science and Technology, MOST 104-2320-B-002-058-MY3)

Fatima, Nida ul et al. (2017) International Worm Meeting "A mitochondrial isocitrate dehydrogenase prevents direct reprogramming of germ cells to neurons in C. elegans."

Kolundzic, Ena, Fatima, Nida ul, Reid, Anna, Tursun, Baris

[International Worm Meeting, 2017]

Direct reprogramming makes use of transcription factors (TFs) that induce the identity of specific cell types. These TFs often act in a context dependent manner, and are restricted in most cell types by inhibitory mechanisms that maintain cell fates (Graf & Enver, 2009; Zhau & Melton, 2008). In order to identify these barriers, we study the model organism Caenorhabditis elegans (C. elegans) using the zinc-finger TF CHE-1 that is required to induce the glutamatergic ASE neuron fate. Upon ectopic expression of CHE-1 and removal of barrier genes by RNAi, induction of the ASE neuronal fate marker gcy-5prom::gfp can be seen in a variety of cell types including germ cells3, the epidermis and the intestine. We identified a candidate barrier gene for reprogramming germ cells into neurons (we call this the GeCo phenotype), which is the NAD+-dependent mitochondrial isocitrate dehydrogenase idha-1. We observed that upon RNAi knockdown of idha-1 and ectopic expression of CHE-1, cells in the germline acquire neuron-like morphology and express a number of neuronal fate markers. Furthermore, upon ectopic expression of the Pitx family of homeodomain-containing TF UNC-30, which specifies the fate of GABAergic motor neurons, germ cells express a reporter for the GABAergic neuron fate upon knockdown of idha-1. Recent studies on mitochondria in the context of reprogramming and iPSCs, show that mitochondrial dynamics change during the process of differentiation and cells show distinct mitochondrial and metabolic signatures during proliferation and differentiation (Bukowiecki et al. 2014). This suggests that disturbing mitochondrial function may feed back to chromatin thereby altering gene expression and allowing reprogramming. However, this process seems to be more specific since the depletion of most other genes that are required for mitochondrial function do not result in the GeCo phenotype. Interestingly, the idha-1 depletion-mediated reprogramming of germ cells to neurons is partially repressed in animals that lack the hypoxia-induced factor HIF-1. HIF-1 has been implicated in regulating iPSC reprogramming, a process that is triggered by changes in the level of the metabolite alpha-ketoglutarate (DeBerardinis et al., 2008; Xu et al., 2010). Importantly, alpha-ketoglutarate levels are regulated by isocitrate dehydrogenases such as IDHA-1 (Zeng et al., 2014; Zhang et al., 2015). Moreover, alpha-ketoglutarate is known to act as a co-factor of histone demethylases (Tsukada et al., 2006) and we are currently studying the underlying signaling mechanisms as well as the chromatin regulation involved during this cellular conversion.