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[
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>
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[
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.
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[
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.
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[
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.
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[
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.
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[
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.
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[
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.
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[
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.
-
[
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.
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[
Neuronal Development, Synaptic Function, and Behavior Meeting,
2006]
We obtained in vivo intracellular recordings from the amphid sensory neuron pair AFD. Nematodes whose AFD neurons are laser ablated fail to track their cultivation temperature (Mori & Ohshima, 1995, Nature 376:344) and Ca2+ imaging has revealed increases in intracellular Ca2+ in AFD in response to warming (Kimura et al., 2004, Current Biology 14:1291), identifying AFD as a likely thermosensor. Consistent with this idea, we find that AFD responds to changes in temperature: warming elicits an inward current and cooling an outward current. These thermoreceptor currents (TRCs) activate rapidly and are sufficient to modulate the cell"s membrane potential by more than 20 mV. Both inward and outward TRCs are retained in the absence of intracellular K+, suggesting that K+ channels do not play a role in generating TRCs. TRCs were not detected in AWA, an amphid sensory neuron pair that synapse onto AFD, indicating that TRCs are a specific property of AFD.
Nematodes migrate down temperature gradients when placed above a threshold temperature. This threshold for thermotaxis is set by the worms" cultivation temperature (Tc) and can be reset within ~2 hours by shifting worms to a new Tc. Paralleling the behavior, TRCs in AFD activate above a threshold temperature defined by recent experience. Interestingly, the sensory neuron adapts far more rapidly than the behavior. We estimate that thresholds for TRC activation can be reset in less than 15 minutes. The behavioral and sensory responses also differ in their dependence on feeding state: whereas animals starved for >3 hours cease to perform thermotaxis, TRC"s are preserved in animals starved for >10 hours. Our results support the hypothesis that AFD is a temperature-sensing neuron, and provide direct insight into how temperature is encoded in the C. elegans nervous system