Mori, K. et al. (2017) International Worm Meeting "Labeling of active neural circuits by the calcium probe CaMPARI."

Toyoshima, Y., Mori, K., Iino, Y.

[International Worm Meeting, 2017]

To understand the information processing by the nervous system, it is necessary to reveal the functional connectivity of component neurons. In C. elegans the wiring diagram of all its neurons has been reported. However, the functional networks that are used for particular behaviors such as sensory perception or goal-oriented behaviors, have not yet been fully clarified. Thus, we are currently working on mapping the functional circuits to the connectome data. Here, we constructed an experimental system to extract the functional neural circuits by using a new type of genetically encoded Ca2+ indicator called CaMPARI (1) . CaMPARI fluorescence changes from green to red irreversibly only when high calcium concentration and ultraviolet (UV) light are present simultaneously. Since this probe has been used only in other model organisms such as D. melanogaster, zebrafish and mouse, we tested whether this probe works efficiently in the neurons of C. elegans. Several CaMPARI variants which show different dissociation constant (Kd) for Ca2+ have been reported. We tested some of these variants to find which one would be suitable for this new experimental system. We used ASER neuron and NaCl downstep stimulus for the verification of the CaMPARI function in C.elegans. We next made a strain that expresses CaMPARI in all neurons. We localized CaMPARI at nuclei of each neuron so that we can identify each neuron automatically and annotate them (2). By using this strain, we labeled the neurons that are active after the decrease of NaCl concentration. Our laboratory has previously shown that pairing starvation with exposure to NaCl causes salt avoidance learning in C. elegans. We are currently searching for neurons that respond differently before and after the formation of memory by using this system. (1) Fosque et al. Labeling of active neural circuits in vivo with designed calcium integrators Science (2015) (2) Toyoshima et al. Accurate Automatic Detection of Densely Distributed Cell Nuclei in 3D Space PLOS Computational Biology (2016)</em>

Chun-Yi Huang et al. (2007) International Worm Meeting "eif-3.K is a pro-apoptotic gene in C. elegans."

Jia-Yun Chen, Yi-Chun Wu, Chun-Yi Huang

[International Worm Meeting, 2007]

Programmed cell death (or apoptosis) is an important feature of C. elegans development. Previous studies have identified pro-apoptotic genes egl-1, ced-3 and ced-4 and anti-apoptotic genes ced-9 and icd-1 that control programmed cell death.. We have identified and characterized a novel pro-apoptotic gene eif-3.K. Loss-of-function by mutation or RNAi inactivation in eif-3.K resulted in a decrease of cell corpses, whereas heatshock-induced over-expression of eif-3.K weakly but significantly increased cell corpses. Interestingly, the eif-3.K mutation partially suppressed ectopic cell deaths caused by over-expression of egl-1 or ced-4. This result suggests that eif-3.K may act downstream of or in parallel to egl-1 and ced-4 in the programmed cell death pathway. Using a cell-specific promoter to express eif-3.k in touch neurons, we showed that eif-3.K likely promoted cell death in a cell-autonomous manner. To further explore EIF-3.K function, we generated antibodies against bacterially expressed EIF-3.K protein. We found that EIF-3.K was ubiquitously expressed during embryogenesis and localized to the cytoplasm. As human eif-3.K can functionally substitute C. elegans eif-3.K in an eif-3.K mutant, the function of eif-3.K in apoptosis is likely conserved in evolution.

Chun-Yi Huang et al. (2006) East Asia C. elegans Meeting "eif-3.K is a pro-apoptotic gene in C. elegans"

Teng-Wei Huang, Chun-Yi Huang

[East Asia C. elegans Meeting, 2006]

Programmed cell death or apoptosis is an important feature during C. elegans development. The pro-apoptotic genes egl-1, ced-4 and ced-3 are required for the execution of cell death. We have identified and characterized a novel pro-apoptotic gene eif-3.K. A loss-of-function mutation or inactivation by RNA interference in eif-3.K resulted in a reduction of cell corpse number during embryogenesis, whereas heatshock-induced over-expression of eif-3.K weakly but significantly promoted programmed cell death. In addition, eif-3.K mutation partially suppressed ectopic cell deaths caused by over-expression of egl-1 and ced-4. This result suggests that eif-3.K may act downstream of or in parallel to egl-1 and ced-4 in the genetic pathway during programmed cell death. Using a cell-specific promoter we showed that eif-3.K likely promoted cell death in a cell-autonomous manner. We generated antibodies against bacterially expressed EIF-3.K protein. The immunostaining result showed that EIF-3.K was ubiquitously expressed during embryogenesis and localized to the cytoplasm. To better understand the cell-death defect of eif-3.K mutants, we are currently performing a 4D microscopic analysis of the cell death process in wild-type and eif-3.K mutants.

Schwarz EM et al. (1996) East Coast Worm Meeting "Cellular and molecular analysis of thermosensation"

Chalfie M, Schwarz EM

[East Coast Worm Meeting, 1996]

While senses like sight, hearing, and mechanosensation are becoming well understood, thermosensation remains obscure. Work by Hedgecock (1) and Mori (2) has shown that C. elegans is a promising model system for metazoan thermosensation, but neither the cells nor the genes required for thermosensation in C. elegans are fully known. Previous work in this laboratory (3) has shown that at least three new deg mutations cause cells in the head to degenerate while partially crippling thermosensation; none of these are allelic to previously described ttx mutations . We have therefore begun to determine which cells are defective in these deg mutations. We have also begun genetic mapping experiments aimed at positional cloning of the wild-type deg loci. References: 1. Hedgecock, E.M. and Russell, R.L. (1975). Proc. Natl. Acad. Sci. U.S.A. 72, 4061-4065. 2. Mori, I. and Ohshima, Y. (1995). Nature 376, 344-348. 3. Treinin, M. and Chalfie, M. (1993). International C. elegans meeting abstracts, p. 446.

Johnson, Christina K. et al. (2019) International Worm Meeting "Requirement of glial Na+/K+-ATPase in touch sensation highlights ionic and metabolic link between glia and touch neurons in C. elegans."

Johnson, Christina K., Bianchi, Laura, Wang, Ying, Fernandez Abascal, Jesus

[International Worm Meeting, 2019]

Isolated microenvironments, such as the tripartite synapse, where the concentration of ions is regulated independently from the surrounding tissues, exist throughout the nervous system, including in mechanoreceptors. Modulation of the ionic composition of these microenvironments has been suggested to be achieved by glia and other accessory cells. However, the molecular mechanisms of ionic regulation and effects on neuronal output and animal behavior are poorly understood. Using the model organism C. elegans, our lab published that Na+ channels of the DEG/ENaC family expressed in glia control neuronal Ca2+ transients and animal behavior in response to sensory stimuli. DEG/ENaC Na+ channels are known to establish a favorable driving force for K+ excretion, which occurs via inward rectifier K+ channels, in epithelial tissues across species. We hypothesized that a similar mechanism exists in the nervous system. Using molecular, genetic, in vivo imaging, and behavioral approaches, we showed that expression in glia of inward rectifier K+ channels and cationic channels rescues the sensory deficits caused by knock-out of glial DEG/ENaCs without disrupting neuronal morphology, supporting our hypothesis. Based on this model, Na+/K+-ATPases are also needed to maintain ionic concentrations following influx of Na+ and excretion of K+. We show here that, in addition to glial Na+ and K+ channels, two specific glial Na+/K+-ATPases, EAT-6 and CATP-1, are needed for touch sensation and that their requirement can be bypassed by a high glucose diet. The effect of glucose is dependent on ATP binding capability of the pump, translation, transcription, and the activity of CATP-2, a third Na+/K+-ATPase ?-subunit. Taken together, our results support metabolic and ionic cooperation between glia and neurons in C. elegans mechanosensors, a mechanism that is essential to regulating neuronal output and may be conserved across species.

Ikeda, Shingo et al. (2011) International Worm Meeting "Integration of temperature signals in interneurons of C. elegans."

Kimata, Tsubasa, Ikeda, Shingo, Mori, Ikue

[International Worm Meeting, 2011]

Animals show complex behaviors as a consequence of integrating environmental information through neural circuits. In order to reveal the mechanism of integration, we are focusing on a neuronal circuit of C. elegans, composed of three interneurons, AIY, AIZ and RIA, which play an important role in thermotaxis behavior. The RIA neuron receives two upstream signals, one from AIY for thermophilic movement and the other from AIZ for cryophilic movement, and is supposed to integrate them to transmit to downstream motorneurons SMD and RMD, which connect with neck muscles directly (Mori and Ohshima, Nature, 1995; White et al., Phil. Trans. R. Soc. London, 1986). To understand how RIA integrates two opposite signals from AIY and AIZ, thus reflecting in the behavior, we tried to monitor the activity of AIY, AIZ and RIA with Ca2+ imaging using calcium sensor, GCaMP3 (Tian et al., Nat Methods, 2009). We so far observed significant responses of AIY and RIA to thermal stimuli, some of which were temperature-independent. The previous study using Cameleon showed that AIY responded significantly to thermal stimuli, while the response of RIA to thermal stimuli was minimal (Kuhara and Mori, J. Neurosci.,2006). Further, temperature-independent responses of AIY and RIA have not been reported, suggesting that GCaMP3 can detect different concentration range of intracellular Ca2+ compared to Cameleon YC2.12 or YC3.60. To further analyze how the circuit functions in integrating neural signals, we are currently trying to image the activity of AIZ and will perform simultaneous imaging of AIY, AIZ and RIA.

Maelle Jospin et al. (2001) International C. elegans Meeting "Basic electrophysiological properties of body wall muscle from the Nematode C elegans."

Maelle Jospin, Laurent Segalat, Bruno Allard

[International C. elegans Meeting, 2001]

Electrophysiological properties of striated muscle cells were investigated with the patch clamp technique in the Nematode C elegans . Worms were immobilised with cyanoacrylate glue and longitudinally incised using a tungsten rod sharpened by electrolysis. Recording pipettes were sealed on GFP-expressing body wall muscle cells. In the whole cell configuration, under current clamp conditions, in the presence of Ascaris medium in the bath and K-rich solution in the pipette, no action potential could be induced in response to current injection. Under voltage clamp control and in the same ionic conditions, depolarizations above -30 mV from a holding potential of -70 mV gave rise to outward K currents. Outward K currents resulted from two components, one fast inactivating component blocked by 4-aminopyridine, one delayed sustained component blocked by tetraethylammonium. In the presence of both blockers, an inward Ca current was revealed and inhibited by cadmium. Single channel recording using the inside-out configuration revealed the existence of a Ca-activated Cl channel and a Ca-activated K channel. Single channel experiments are currently performed to characterise voltage-gated conductances at the unitary level.

Paola Jurado et al. (2009) European Worm Neurobiology Meeting "Insights into the molecular mechanisms of Caenorhabditis elegans memory"

Paola Jurado, Ikue Mori

[European Worm Neurobiology Meeting, 2009]

Caenorhabditis elegans responds to a variety of environmental signals (volatile and water soluble chemicals, temperature, etc). The animals memorize these cues and are able to associate them with other existent conditions. Thermotaxis is a well characterized behavior in which C. elegans associates its cultivation temperature with food (Escherichia coli). This association drives the worms to seek their memorized temperature even in the absence of bacteria (1,2). This presents an ideal situation to investigate the molecular and cellular bases of sensory integration leading to complex processes such as memory and learning. We are trying to thoroughly investigate how the assimilation of thermo-sensory cues leads to memory, and how this thermal memory is modified over time. We have performed a screen to look for mutants with an abnormal memory or an altered decission making balance over thermal conditioning. We searched for animals conditioned to find E. coli at a certain temperature that take longer to leave it, in the absence of food, than wild type animals. These animals display a longer lasting memory or a slower decission making when thermal memory is confronted with hunger or the absence of food. One of the mutants isolated from this screening has been mapped to chromosome I and is currently under characterization. 1. I. Mori and Y. Ohshima (1995). "Neural regulation of thermotaxis in C. elegans." Nature 376(6538): 344-8. 2. I. Mori, H. Sasakura and A. Kuhara (2008). "Worm thermotaxis: a model system for analyzing thermosensation and neural plasticity." Curr Opin Neubiol. 2007 Dec 17(6):712-9.

Jurado, Paola et al. (2009) International Worm Meeting "Insights into the molecular mechanisms of Caenorhabditis elegans memory."

Jurado, Paola, Goto, Fuyuki, Mori, Ikue

[International Worm Meeting, 2009]

Caenorhabditis elegans responds to a variety of environmental signals (volatile and water soluble chemicals, temperature, etc). The animals memorize these cues and are able to associate them with other existent conditions. Thermotaxis is a well characterized behavior in which C. elegans associates its cultivation temperature with food (Escherichia coli). This association drives the worms to seek their memorized temperature even in the absence of bacteria (1,2). This presents an ideal situation to investigate the molecular and cellular bases of sensory integration leading to complex processes such as memory and learning. We are investigating how the assimilation of thermo-sensory cues leads to memory, and how this thermal memory is modified over time. We have performed two different screens to look for mutants with an abnormal memory or an altered decission making balance over thermal conditioning. On the first screen we searched for animals conditioned to find E. coli at a certain temperature that take longer to leave it, in the absence of food, than wild type animals. These animals display a longer lasting memory or a slower decission making when thermal memory is confronted with hunger or the absence of food. On the second screen we isolated several animals with a shorter memory or a faster decission making by selecting those that leave their memorized temperaure faster than the wild type animals. All these putative mutants are currently under characterization. 1. I. Mori and Y. Ohshima (1995). "Neural regulation of thermotaxis in C. elegans." Nature 376(6538): 344-8. 2. I. Mori, H. Sasakura and A. Kuhara (2008). "Worm thermotaxis: a model system for analyzing thermosensation and neural plasticity." Curr Opin Neubiol. 2007 Dec 17(6):712-9.

Kuhara, Atsushi et al. (2009) International Worm Meeting "Exploring the neural code in the neural circuit for thermotaxis behavior."

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

[International Worm Meeting, 2009]

Behavior is an ultimate consequence of orchestrated neural calculation. Thermotaxis of C. elegans is an ideal system for comprehensively understanding how a neural circuit encodes a behavioral output (1). In thermotaxis neural circuit, temperature is mainly sensed by AFD neuron, and its information is conveyed to AIY interneuron (1, 2). To elucidate neural calculation, we aimed to inactivate neuronal activity by employing light-activated chloride pump halorhodopsin (HR) (3). Millisecond scale pulsed-light system, which allows Hz-controllable deliveries of light, was developed and equipped in both calcium imaging microscope and C. elegans auto-tracking microscope. Calcium imaging analysis demonstrated that pulsed-excitation of HR in AFD partially reduced calcium influx in AFD for thermal stimuli. Since AFD-ablated animals move randomly or migrate to lower temperature than the cultivation temperature on a thermal gradient (1), we speculated that exciting HR in AFD induces cryophilic or athermotactic abnormalities. We unexpectedly found that pulsed-excitation of HR in AFD induced thermophlic abnormality. Similar abnormality was observed in the mutant exhibiting an abnormal glutamate synaptic transmission in AFD due to AFD-specific defect in EAT-4 (vesicular glutamate transporter) (N. O., A. K. and I. M., this meeting). Previous report (4) and our calcium imaging analysis revealed that calcium influx in AFD for thermal stimuli induced calcium influx in AIY. This suggests that temperature information of AFD is conveyed to AIY through "excitatory" connection. We however found that pulsed-excitation of HR in AFD notably enhanced calcium influx of AIY for thermal stimuli, despite AFD activity itself was partially reduced. Similar abnormal thermal responses of both AFD and AIY were observed in thermophilic mutant tax-6 (calcineurin) that is defective in AFD thermosensory signaling (5, 6), implicating "inhibitory" connection from AFD to AIY. Altogether, these physiological and behavioral results are consistent with the notion that temperature signal in AFD dynamically affects AIY activity through both "inhibitory" and "excitatory" transmissions, which as a consequence likely generates opposite thermotactic behaviors, thermophilic and cryophlic migration on a temperature gradient. (1) Mori and Ohshima, Nature, 1995 (2) Kuhara, Okumura, et al., Science, 2008 (3) Zhang et al., Nature, 2007 (4) Biron et al., Nature Neuroscience, 2006 (5) Kuhara et al., Neuron, 2002 (6) Kuhara and Mori, J. Neurosci, 2006.