Teramoto, Takayuki et al. (2013) International Worm Meeting "4-D Ca2+ imaging of the multiple neurons in a local circuit regulating behavioral choice."

Teramoto, Takayuki, Yamamoto, Yuta, Ishihara, Takeshi

[International Worm Meeting, 2013]

Animals receive multiple environmental signals simultaneously, and then they integrate these signals to execute a proper behavior. Although this sensory integration process is important for their behavior, its neural mechanisms remain unclear. In C. elegans, sensory integration between a repellent Cu2+ and an attractive odorant diacetyl is regulated by a local circuit including sensory neurons AWA and ASH, and interneurons AIA and AIB. AIA interneurons receive chemical synapses from ASH, which are activated by Cu2+, and connect with gap-junctions to AWA, which are activated by diacetyl. This diagram suggests that the signals integrated at AIA may regulate the downstream interneurons AIB, of which activation leads to backward movements. This model is consistent with results from our behavioral analyses; however, simultaneous measurements of the multiple neurons have not been succeeded because they are arranged in three-dimensional space. To visualize and measure the activities of these neurons, we designed a 4-D imaging system based on a confocal microscope with a piezo lens positioner. Combining this system and the Ca2+ indicator Yellow Cameleon, we carried out Ca2+ imaging of multiple neurons: AWA, AIA, and AIB. We observed the following: 1) AWA and AIA were simultaneously activated when AWA were stimulated; 2) AIB interneurons, which receive chemical synapses from AIA, exhibit reciprocal responses to AWA/AIA. These imaging results are consistent with the model that we proposed. Therefore this 4-D imaging system enables us to visualize and measure the multi-neuronal activities, and it may contribute to understanding mechanisms not only for the sensory integration but also for other information processing. On the other hand, 4-D imaging of multiple neurons still has technical difficulties such as indistingishability of each neuronal cells and inconsistent expression of Ca2+ indicators. We are now performing 4-D imaging of many neurons using integrated transgenic lines expressing Ca2+ indicators in the nuclei that makes us able to distinguish individual neurons.

Teramoto, Takayuki et al. (2015) International Worm Meeting "Whole-brain imaging at cellular resolution reveals multi-neuronal dynamics under non-stimulus condition."

Yoshida, Ryo, Teramoto, Takayuki, Toyoshima, Yu, Ishihara, Takeshi, Hirose, Osamu, Iino, Yuichi, Tokunaga, Terumasa

[International Worm Meeting, 2015]

One of the fundamental challenges on neurosciences is whole-brain imaging at cellular resolution using a live animal. Simultaneous measurement of the multi-neuronal activities of the whole brain of a live animal will provide a new sight for exploring and understanding neural mechanisms for information processing. To realize this, we used the C. elegans CNS as a simple model brain and we designed a 4-D imaging system that can acquire time-sequential 3-D images at three wavelength. For image processing of acquired data, we developed a line of programs, which perform positional tracking and segmentation of each neuron.Combining this imaging system and a ratio-metric fluorescent Ca2+ indicator and mCherry as a positional marker, we carried out 4-D Ca2+ imaging of the C. elegans CNS, and we succeeded in visualizing and measurement of neuronal activities of most neurons of the CNS without any anesthesia. Analysis of temporal changes in ratio of the individual neurons revealed that multiple neurons responded with positive and negative correlation to stimulation by such as an attractive odor and a repulsive metal ion. This result may suggest that these neurons are components of the neuronal circuit, which are involved in the sensory signaling pathway. On the other hand under non-stimulus condition, we observed synchronized rhythmic activity in multiple neurons, implying that pacemakers or pattern generators may exist in C. elegans.Taken together, our 4-D Ca2+ imaging techniques provide a measurement method of neuronal activities in the CNS of C. elegans and revealed that multi-neuronal dynamics: 1) responding multi-neurons may involve in information transfer or/and processing, 2) rhythmic activity of other group of neurons may confer a pace or a pattern generation that correlates to behavior such as locomotion. We are now trying to reveal relationship between these neuronal dynamics by analysis of these multi-neuronal activities. Moreover, we plan to identify these neurons by combining neuronal specific promoters and fluorescent with distinguishable wavelength to adapt these neurons on the synaptic connectivity map. .

Takayuki Teramoto et al. (2010) East Asia Worm Meeting "Combinatorial regulation of three TRPM channels governs magnesium homeostasis"

Laura A. Sternick, Kouichi Iwasaki, Takayuki Teramoto, Jakub Siembida, Eric J. Lambie, Eriko Kage-Nakadai, Shirine Sajjadi, Shohei MItani

[East Asia Worm Meeting, 2010]

Mg2+ homeostasis is essential for animal health, and it is mainly regulated by the balance between absorption in the intestine and excretion from the kidney. In human, mutations in the TRPM6 gene cause hypomagnesemia. TRPM7 has been shown to be essential for cell growth; loss of TRPM7 results in poor growths of animals cells, which can be recued by excess supply of Mg2+. Our previous studies have demonstrated that the C. elegans TRPM6/7 homologs, GON-2 and GTL-1, mediate Mg2+ uptake by the intestinal cells. These findings indicate that TRPM6/7 channels are essential for Mg2+ absorption in animals. However, no roles for TRPM channels in mediating Mg2+ excretion have previously been described. In this report, we have investigated the physiological function of the remaining member of the C. elegans TRPM6/7 homologs, GTL-2. GTL-2 is expressed on the basal surface of the C. elegans kidney-equivalent, the excretory cell. Whole-cell patch clamp recordings of primary cultured excretory cells showed an outwardly-rectifying current, suggesting that GTL-2 mediate cations on the excretory cell. Mass spectrometry measurements of trace metals in animals indicated that the gtl-2 mutants contain significantly higher levels of Mg2+ than wild-type animals. The gtl-2 mutant animals show strong growth defects in response to Mg2+ in the culture medium, and these defects can be rescued by reducing Mg2+ in the culture medium, or by secondary mutations in GTL-1 and GON-2. These results indicate that GTL-2 mediate Mg2+ on the basal surface of the excretory cell, and that it functions in excretion of excess Mg2+ from the body cavity of the animals. Based on the these results and our previous studies, we suggest that Mg2+ homeostasis in C. elegans are regulated by the balance of the activities of the three TRPM channels, GON-2, GTL-1, and GTL-2.

Takayuki Teramoto et al. (2004) West Coast Worm Meeting "Molecular cloning and physiological characterization of the defecation rhythm controlling gene dec-1"

Kouichi Iwasaki, Takayuki Teramoto

[West Coast Worm Meeting, 2004]

The C. elegans defecation is a rhythmic behavior: with abundant food, a defecation motor program (DMP) is activated every 45 seconds. It was reported that this defecation rhythm is regulated by the inositol 1,4,5-trisphosphate (IP3) receptor in the intestine (Dal Santo et al., 1999). Therefore, the defecation phenotype can be used to monitor activities of an IP3-dependent Ca ++ -signaling pathway. To visualize cytoplasmic Ca ++ dynamics in the C. elegans intestine, we expressed the Ca ++ -indicating protein, cameleon. The first step of the defecation motor program (the posterior body wall muscle contraction, pBoc) coincided with a Ca ++ spike. This Ca ++ spike was initiated at the posterior end of the intestine and propagated through the entire intestine toward the anterior end. To make this imaging system more useful for physiological study, we have developed a culture system of the C. elegans adult intestine. In this system, we can reproduce a variety of phenomena observed in the intact adult intestine, such as execution of a defection motor program step, Ca ++ oscillation, and Ca ++ -wave propagation. We have also adapted Ca ++ imaging using the chemical indicators Fluo-4 and Fura Red for the dissected intestinal preparations. Fluo-4 increases its fluorescence intensity while Fura Red decreases when [Ca ++ ] increases, therefore, the ratiometric measurement using these indicators maximizes the detection sensitivity and is less likely to pick up false signals. To elucidate the regulatory mechanism of this rhythm generation, we are molecularly cloning dec-1 . The dec-1 mutant was originally isolated by J.H. Thomas based on a constipated phenotype and shows a prolonged DMP rhythm. Along with molecular cloning, we are currently investigating if the intestinal Ca ++ dynamics is altered in the dec-1 mutant. Dal Santo et al . (1999) Cell 98: 757-767 Thomas. (1990) Genetics 124: 855-872

Takayuki Teramoto et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "TRPM Channels Regulate Magnesium Homeostasis in C. elegans"

Kouichi Iwasaki, Takayuki Teramoto, Eric Lambie

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

Magnesium (Mg2+) is an important cofactor for many biological processes, such as protein synthesis, nucleic acid stability, or neuromuscular excitability. In spite of the biological importance of Mg2+ homeostasis, little is known about the mechanisms of Mg2+ uptake and its maintenance. Previously, two M-type transient receptor potential channels, TRPM6 and TRPM7, have been implicated in vertebrate Mg2+ homeostasis. In particular, human TRPM6, when mutated, causes a combined defect of intestinal magnesium absorption and renal magnesium transport as observed in primary hypomagnesaemia with secondary hypocalcaemia (HSH). Since C.elegans has GTL-1 and GON-2 that are the homologues of TRPM6 and TRPM7, this system is a good model to study the molecular mechanisms of TRPM-dependent Mg2+ homeostasis in animal cells. In this study, we investigated the function of the TRPM channels of C.elegans using gtl-1 and gon-2 mutant strains. The gon-2;gtl-1-double mutants showed growth defects under low Mg2+ conditions, and these defects can be largely rescued by dietary supplementation with excess Mg2+. GTL-1 and GON-2 are also important for intestinal Ni2+ uptake because gon-2;gtl-1-double mutants showed resistance to Ni2+ cytotoxicity. Using a Ni2+ indicator, we found that the wild-type intestine incorporates Ni2+, but the double-mutant intestine does so to a lesser extent. Further our analysis showed that this TRPM-dependent Ni2+ transport is regulated by Mg2+ levels. Finally, our electrophysiological data showed that the large outwardly-rectifying current observed in the wild type intestinal cells was mainly due to the activity of the GON-2 channel, and that GTL-1 played important roles in the Mg2+-dependent regulation of current generation. Based on these observations, we conclude that differential regulation of two TRPM channels in Mg2+ absorption ensures a constant supply of Mg2+ through GTL-1, while the occasional bursts of GON-2 activity allow rapid return to normal Mg2+ concentrations following physiological perturbations.

Takayuki Teramoto et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "Intestinal calcium waves coordinate a behavioral motor program in C. elegans"

Takayuki Teramoto, Kouichi Iwasaki

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

Intracellular Ca2+ signals that are generated by inositol 1,4,5-trisphosphate (IP3) control a variety of cellular functions ranging from muscle contraction to gene expression. In many cell types, Ca2+ signals are temporally and spatially organized into transient changes in the cytosolic Ca2+ concentration, and can be expressed as intercellular Ca2+ waves. It has been proposed that the intercellular Ca2+ waves function as synchronizing cellular activities in a particular organ. We are interested in dissecting the functions and mechanisms of such intercellular Ca2+ wave signaling in C. elegans. The defecation behavior in C. elegans appears to be regulated by the periodic activation of inositol 1,4,5-trisphosphate receptor (IP3R) in the intestine. This behavioral motor program consists of a simple stereotyped series of muscle contractions with a precise timing: in the presence of abundant food, the defecation motor program is activated every 45~50 seconds. When the IP3 receptor activity is reduced, the resulting animals show prolonged motor program cycles. Conversely, over-expression of itr-1 shortened the cycle. Therefore, the motor program cycle depends on the activity of IP3 receptor, and the phenotype of the defecation behavior can be used to monitor cellular physiology in the C. elegans intestine. To investigate how the intestinal Ca2+ regulates this motor program, we developed an assay system to study Ca2+ dynamics in the C. elegans intestine. Using this system, we found that the timing between the first and second motor steps is coordinated by intercellular Ca2+-wave propagation in the intestine. When the Ca2+-wave propagation was prematurely terminated in the UNC-43 (CaMKII) mutant, the second/third motor steps were not expressed while the first step was normal. Also, when the Ca2+-wave propagation was blocked by the IP3 receptor inhibitor heparin at the mid-intestine in wild type, the second/third motor steps were eliminated. These phenotypes phenocopies ablation of the motor neurons AVL and DVB. Based on these observations, we conclude that an intestinal Ca2+-wave propagation governs the timing of neural activities that controls specific behavioral patterns in C. elegans.

Kuge, Sayuri et al. (2013) International Worm Meeting "Ca2+ dynamics of a whole single neuron."

Teramoto, Takayuki, Ishihara, Takeshi, Kuge, Sayuri

[International Worm Meeting, 2013]

The nematode C.elegans is an excellent model organism for studying how neuronal circuits activity transform sensory signals. Ca2+ imaging of neurons using Ca2+sensitive fluorescent proteins including GCaMP and yellow cameleon has revealed functions of the neuronal circuits. However, the mechanism of circuit function at single-neuron resolution is still unclear. To elucidate the functional characteristics of a single neuron, we analyzed Ca2+ dynamics of a whole single neuron by a 4-D imaging system based on a confocal microscope with a piezo lens positioner. The system enabled us to capture 3-D reconstructed images of a whole single neuron with more than 4 steric images per second. To analyze the Ca2+ dynamics of a whole neuron, we used G-GECO series of Ca2+ sensors, which were developed from G-CAMP. To observe the various basal Ca2+ concentrations, we developed G-GECO derivatives with higher affinity to Ca2+. We analyzed animals expressing Ca2+ indicators in AWCON neuron by str-2 promoter in the olfactory chip. We hope that our 4-D imaging system enables us to observe Ca2+ dynamics at dendrites, a cell body, and an axon independently and to analyze precise mechanisms of sequential rapid neuronal activation and inactivation in a whole single neuron.

Usuyama, Mamoru et al. (2015) International Worm Meeting "Ca2+ dependent negative feedback loop in AWC olfactory neurons: from mathematical viewpoint."

Usuyama, Mamoru, Ishihara, Takeshi, Iwasaki, Yuishi, Kuge, Sayuri, Teramoto, Takayuki

[International Worm Meeting, 2015]

AWC receptor neurons show inhibitory response for odor stimulus and transient excitement for odor removal. This means that AWC neurons have mechanism which suppresses neural activity by external stimulation and activates by removal of the stimulation. AWC neurons change [Ca2+] via cGMP as second messenger. However, how AWC neurons change [cGMP] by stimulation is still unclear. In order to reproduce odor response in AWC neurons, we propose the negative feedback system to control [cGMP] in stimulation. The mathematical model consists of biochemical reactions and ionic transports through ion channels. Each biochemical molecules in the model, such as Ca2+, cGMP and so on, give concentrations and reaction rate constants to describe the intracellular biochemical reactions. The model is designed to easily mimic genetic assay such as gene over expression and its knock out experiments. We consider the two experimental features of odor response: (1) Peak [Ca2+] of off-response was linearly increased with duration of stimulus. (2)[Ca2+] was quickly decreased when neurons receive stimulus. Our previous work suggests that two or more Ca2+ dependent negative feedback loops for controlling cGMP production is necessary to reproduce odor responses in AWC (Usuyama, et. al 2012). In our study, however, further speculations in signaling pathway and parameter search show that only one negative feedback loop is enough to reproduce such experimental features. In addition to the experimental features, we consider the response of AWC neurons to successive stepwise stimulation. And we report "neural response" to odor stimulus in "wild type" and "mutant" in sillco, such as peak value and time constants of [Ca2+] change.

Kuge, Sayuri et al. (2015) International Worm Meeting "A quenching-type of fluorescent Ca2+ indicator for detecting neuronal inhibition."

Nishihara, Tomonobu, Ishihara, Takeshi, Teramoto, Takayuki, Nagai, Takeharu, Matsuda, Tomoki, Kuge, Sayuri, Furuie, Hironobu

[International Worm Meeting, 2015]

Fluorescent Ca2+ indicators have been rapidly improved for the last several decades and now are indispensable tools for cell biology, especially neuronal science, because they enable us to monitor neuronal activities. However, these fluorescent Ca2+ indicators including GCaMPs are ideal only for monitoring the activation of neurons. To understand precise functions of the neuronal network, a fluorescent Ca2+ indicator that is suitable for monitoring the neuronal inhibition would be necessary. We previously reported various "pericam" types of indicators that are based on circularly permuted green fluorescent protein (cpGFP) fused to calmodulin and calmodulin-target peptide, M13. Among these, "inverse-pericam" has unique property; the fluorescence intensity gets 7-fold dimmer upon Ca2+ binding. Although the dynamic range is relatively big among that of the reported Ca2+ indicators, it should be enlarged to effectively monitor the neuronal inhibition. To this end, we first created a large mutant library of "inverse-pericam" by iterative error-prone PCR. The mutant libraries of genetic variants of inverse-pericam indicators were expressed in the E. coli periplasm and were screened by comparing fluorescence intensity between high- and low-Ca2+ conditions. After screening of about 20,000 bacterial colonies, we found a variant, which shows nearly 20-folds dynamic range in vitro. When the inverse-pericam variant, which we named IP2.0 was expressed in HeLa cells, the fluorescence of IP2.0 was greatly quenched responding to histamine stimulation, indicating that IP2.0 is useful to detect change of the intracellular concentration of Ca2+. Then we examined whether the decrease of Ca2+ concentration during the stimulation of isoamyl alcohol, as well as the increase of Ca2+ concentration after removing isoamyl alcohol can be monitored by expressing IP2.0 in AWCON neuron of C. elegans. While the fluorescence of GCaMPs has known to be reduced slightly during the stimulation, we observed the increase of fluorescence of IP2.0 during the stimulation. These results suggest that odor stimuli decreases Ca2+ concentration in AWCON neurons and so IP2.0 will be an indispensable tool for studying neuronal inhibition.

Ashley Taylor et al. (2007) International Worm Meeting "Characterization of a C. elegans defecation mutant: pbo-1(sa7)."

Ashley Taylor, Kiri Ulmschneider, Maureen Peters

[International Worm Meeting, 2007]

The C. elegans intestine functions to control the worms defecation motor program. The motor program consists of three stereotyped, sequential muscle contractions: a contraction of the posterior body wall muscles, followed after a few seconds by contraction of the anterior body wall muscles, and finally by contraction of the enteric muscles. Intestinal cells contain a molecular clock that signals each motor step. Posterior body contraction is directly controlled by the intestine; it does not require neuronal function. In contrast, anterior body contraction and enteric muscle contraction require two neurons. A crucial component of the clock is calcium oscillation mediated by the inositol 1,4,5 - trisphosphate (IP3) receptor1. In wild-type animals, cyclic calcium spikes in the posterior intestinal cells directly precede the initiation of the motor program and these spikes are absent in the IP3 receptor mutant1. The calcium spike in the posterior cells leads to a calcium wave that proceeds to the anterior cells of the intestine2. Genetic and pharmacological disruptions of the calcium wave result in motor program defects suggesting that the calcium wave directly instructs the motor steps2. We are investigating how calcium activated signals instruct the steps of the motor program. We are focusing on the most direct signaling event, initiation of the posterior body contraction (pBoc). The signaling from the intestine to the posterior body wall muscles is direct; it does not require neurons. Our model for this signaling event is as follows: the posterior body contraction signaling molecule(s) should be released in response to calcium rise within the intestine and transduced to the overlying muscles where it elicits contraction; disrupting the generation of the signal or the reception of the signal should alter pBoc specifically. To test our model we are studying the genes in this signaling pathway. We have characterized and fine mapped a previously isolated posterior body contraction mutant, pbo-1(sa7). pbo-1(sa7) worms have pBoc deficits, are developmentally delayed, and exhibit reduced brood size. To fine map the pbo-1(sa7) gene, we performed SNP mapping. After identifying candidate genes, we tested each one using RNA interference experiments. Promising candidates are being sequenced to identify the mutation. Cloning the pbo-1(sa7) gene will uncover a key element of the direct signaling between the intestine and body wall muscles. 1. Dal Santo et al., (1999) Cell, 98, 757-67. 2. Teramoto and Iwasaki, (2006) Cell Calcium, 40(3):319-27.