Takeshi Ishihara et al. (2003) International Worm Meeting "HEN-1 specifically binds to a subpopulation of C.elegans primary culture cells."

Isao Katsura, Takeshi Ishihara

[International Worm Meeting, 2003]

Integration of two sensory signals is one of the most fundamental step in the informational processing in the neural circuit. To elucidate its molecular mechanisms, we study mutants defective in the interaction of two sensory signals. Among those, hen-1 mutant animals prefer avoidance of Cu2+ ion to chemotaxis to diacetyl, although they show normal response to Cu2+ ion or to diacetyl. In addition, hen-1 shows defects in behavioral plasticity after conditioned with NaCl and starvation, and with temperature and starvation. The positional cloning reveals that the hen-1 gene encodes a secretory protein with a LDL receptor motif. HEN-1 protein is localized in the axons of ASE and AIY neurons. The expression of HEN-1 by cell type specific promoters and a heatshock promoter in the hen-1 mutant reveals that HEN-1 protein functions cell-non-autonomously in the mature nervous system. These results suggests that HEN-1 is a neuromodulator for regulating the sensory integration and neuronal plasticity. To elucidate how HEN-1 regulates neuronal modulation, we are searching a binding protein for HEN-1 protein. The HEN-1-alkaline phosphatase fusion protein (HEN-1-AP) expressed in HEK293 cells was secreted to the medium. To identify the receptor for the HEN-1, we analyzed whether HEN-1-AP can bind to C.elegans cells. HEN-1-AP bound a subpopulation of cell surface of the C. elegans primary culture cells, but alkaline phosphatase did not. This result indicates that a specific receptor for HEN-1 exists in C.elegans. Therefore, we are screening an expression cDNA library of the C. elegans primary culture cells by using this binding assay system.

Takeshi Ishihara et al. (2004) East Asia Worm Meeting "Receptor tyrosine kinase, SCD-2, regulates sensory integration and behavioral plasticity"

Isao Katsura, Takeshi Ishihara, Yuichi Iino

[East Asia Worm Meeting, 2004]

Animals sense many environmental stimuli,and process the sensory information in the nervous system. This sensoryprocessing includes selection of appropriate information, proper integration,and modification by experience or memory. To elucidate molecular and neuronalmechanisms for such sensory processing, we are studying mutants defective insensory integration and learning. hen-1 mutants show defects inintegration of two sensory signals: Cu 2+ and attractive odorants. hen-1 mutant animals show weakertendency to cross the Cu 2+ barrier during migration toward diacetyl, although they appear notto exhibit defects in chemotaxis to diacetyl nor avoidance of Cu 2+ . Furthermore, hen-1 mutantanimals show abnormality in behavioral plasticities induced by pairedpresentation of NaCl and starvation and of temperature and starvation. The hen-1 gene encodes a secretory protein with an LDLa motif, which is most homologus tothe Drosophila signaling molecule Jeb. HEN-1 is expressed in ASE and AIYneurons and functions cell-non-autonomously in the mature nervous system. Recently, Jeb in Drosophila was reported to regulate development of mesodermal cellsthrough the receptor tyrosine kinase DAlk, which is a homologue of human proto-oncogeneAlk. Since the LDLa domain in Jeb is similar to that in HEN-1, we started toanalyze the scd-2 gene, which encodes a receptor tyrosine kinase similarto DAlk. Expression analyses by using an scd-2 promoter-GFP construct suggestedthat SCD-2 is expressed in several sensory neurons and interneurons. scd-2 mutants showed the same behavioral defects as hen-1 mutants in paradigmsfor sensory integration and learning. The expression of SCD-2 in scd-2 mutants driven by neuron specific promoters and a heatshock promoter suggestedthat SCD-2 functions in the mature nervous system, like HEN-1. These resultssuggest that SCD-2 may be a receptor for HEN-1. Determination of the cellswhere SCD-2 functions for sensory integration and learning may reveal the centerof informational processing in C.elegans.

Takeshi Ishihara et al. (2002) Japanese Worm Meeting "Searching for a protein interacting with HEN-1, which regulates sensory integration and learning in Caenorhabditis elegans"

Isao Katsura, Ikue Mori, Yuichi Iino, Akiko Mohri, Keiko Gengyo-Ando, Takeshi Ishihara, Shohei MItani

[Japanese Worm Meeting, 2002]

Animals sense many environmental stimuli simultaneously, and integrate various sensory signals within the nervous system to generate proper behavioral responses and also to form relevant memories. HEN-1, a secretory protein with an LDL receptor motif, regulates such processes in Caenorhabditis elegans. The hen-1 mutants show defects in the integration of two sensory signals: Cu2+ ion and diacetyl, although they show normal response to Cu2+ ion or diacetyl. Furthermore, they show defects in two types of behavioral plasticity induced by paired stimuli: NaCl and starvation or temperature and starvation, while they show normal responses to NaCl, temperature, or starvation. The HEN-1 protein is expressed in two pairs of neurons, ASE and AIY, but expression in other neurons is sufficient for wild-type behavior. In addition, the HEN-1 protein functions in the mature neural circuit. These results indicate that the HEN-1 protein regulates sensory integration and learning. These features of HEN-1 suggest various possible mechanisms for its function. For example, it functions as a neuromodulator or an antagonist of a neurotransmitter. Another possibility is that it is a component of an extracellular matrix essential for proper transmission of neuromodulators. To elucidate molecular mechanisms of the sensory processing involved in the HEN-1 protein, we are searching a protein interacting with HEN-1. To date, we have found several candidates by the yeast 2-hybrid screening. We analyzed their expression by GFP fusion genes and found that one of them is expressed in the nervous system. This gene encodes a novel protein with weak similarity to ZYG-11. In addition, we isolated mutants of this gene by screening pools of mutagenized worms using PCR. We are analyzing their phenotypes by the interaction assay.

Takeshi Ishihara et al. (2005) International Worm Meeting "Receptor tyrosine kinase, SCD-2, regulates sensory integration and learning in C. elegans"

Yuichi Iino, Daisuke D Ikeda, Isao Katsura, Takashi Tabata, Takeshi Ishihara

[International Worm Meeting, 2005]

Animals sense many environmental stimuli, and process the sensory information in the nervous system. This sensory processing includes selection of appropriate information, proper integration, and modification by experience or memory. To elucidate molecular and neuronal mechanisms for such sensory processing, we are studying mutants defective in sensory integration and learning in C. elegans. hen-1 mutants show defects in integration of two sensory signals and in behavioral plasticities induced by paired presentation of two sensory signals. The hen-1 gene encodes a secretory protein with an LDLa motif, which is most homologous to the Drosophila signaling molecule Jeb. HEN-1 is expressed in AIY and ASE and functions cell-non-autonomously in the mature nervous system. These results suggest that HEN-1 signals may regulate sensory integration to form proper memory in the neuronal circuit. Recently, Jeb in Drosophila was reported to regulate development of mesodermal cells through the receptor tyrosine kinase DAlk, which is a homologue of human proto-oncogene Alk. Since the LDLa domain in Jeb is similar to that in HEN-1, we started to analyze the scd-2 gene, which encodes a receptor tyrosine kinase similar to DAlk. scd-2 mutants showed the same behavioral defects as hen-1 mutants in paradigms for sensory integration and learning, suggesting that SCD-2 is a candidate for a HEN-1 receptor. Expression studies using a functional scd-2::GFP fusion gene suggest that SCD-2 is expressed in sensory neurons and interneurons in head and tail regions and localized in plasma memblanes. Expression of SCD-2 driven by various promoters in scd-2 mutants suggests that SCD-2 functions in a few pairs of amphid interneurons at adult stage to regulate these behaviors. These results suggest that HEN-1 regulates neuronal functions of interneurons through SCD-2 receptor for adequate sensory integration and learning.

Takeshi Ishihara et al. (2001) International C. elegans Meeting "A novel secretory protein, HEN-1, regulates integration of sensory signals and behavioral plasticity."

Takeshi Ishihara, Ikue Mori, Akiko Mohri, Isao Katsura, Yuichi Iino

[International C. elegans Meeting, 2001]

Integration of sensory signals and neuronal plasticity are essential steps for informational processing in the nervousl system. Animals sense many environmental stimuli simultaneously, and these sensory signals are modulated depending on their experiences and environment to generate a proper response. To elucidate its molecular mechanisms, we study a hen-1(ut236) mutant, which was originally identified as a mutant defective in interaction of two sensory signals. C. elegans move toward attractive odorants and avoid aversive Cu 2+ ion. In our paradigm, worms must cross aversive Cu 2+ ion barrier to reach attractive odorants, and their behavior varies with the concentration of both stimuli. In this assay, hen-1 prefers to avoid Cu 2+ ions rather than to move toward the attractive odorants, although the dose responses to odorants and to Cu 2+ are indistinguishable from those of wild type. This phenotype suggests that hen-1 has defects in the integration of two sensory signals but not in the chemosensation. Positional cloning reveals that the hen-1 gene encodes a novel secretory protein with an LDL-ligand binding motif and is expressed in ASE and AIY neurons. Immunohistochemical studies showed that HEN-1 is localized in their processes at the nerve ring and their cell bodies in wild type animals, but it is not transported to the processes and mislocalized to cell bodies in the unc-104 mutant in which synaptic vesicles are not transported. By using cell type specific promoters, we found that the HEN-1 functions cell nonautonomously in the nervous system. In addition, the expression of the HEN-1 in embryonic stage is not sufficient for the rescue, but expression in the late larval or adult stage is. This result suggests that it is necessary for functions in the mature neuronal circuit, but not for development of the nervous system. The hen-1 mutant has also defects in two types of behavioral plasticity induced by paired sensory stimuli. Wild type animals conditioned with NaCl and starvation change their behavior to avoid NaCl, but hen-1 shows weak behavioral change after conditioning. In another paradigm, wild type animals cultivated with food migrate to the cultivated temperature, but, after starvation, they change their behavior to avoid the temperature (see abstract by Mohri et al.). The hen-1 mutant shows normal thermotaxis in well-fed condition, but, unlike wild type animals, hen-1 conditioned with starvation at a cultivated temperature does not change their behavior to avoid the temperature (Aho phenotype). These defects in learning can be rescued by the wild type hen-1 transgene. Although, in these paradigms, worms are conditioned with starvation and other stimuli, we cannot find any abnormality in behavioral change induced by sole starvation. These results suggest that the HEN-1 controls neuromodulation of sensory signals and, as a result, it affects integration of these signals and learning induced by paired stimuli.

Kitazono, Tomohiro et al. (2013) International Worm Meeting "Identification of regulatory factors for forgetting in C. elegans."

Ishihara, Takeshi, Inoue, Akitoshi, Kitazono, Tomohiro

[International Worm Meeting, 2013]

To properly respond to continuously changing environment, animals have to forget past memories that are inconsistent with present circumstances. To elucidate the regulatory mechanisms of forgetting, we use the olfactory adaptation in C. elegans as a simple model. By forward genetic analyses, we found that TIR-1, a p38 MAPK adaptor protein, and its downstream signaling pathway (TIR-1/JNK-1 pathway) mutants exhibit prolonged retention of the olfactory adaptation to diacetyl and isoamylalchol, which are the odorants sensed by AWA and AWC, respectively. We also demonstrated that the TIR-1/JNK-1 pathway accelerates forgetting by releasing forgetting signals from AWC neurons to other neurons. Our study suggested that the neuronal circuit including AWC neurons regulates forgetting. However, the molecular mechanisms underlying this forgetting remain largely unknown. Therefore, to identify downstream components, we carried out a suppressor screening of the gain of function mutants of tir-1, which shows a weak adaptation phenotype probably because forgetting signals are always released. By the screening, we isolated 71 independent lines, which showed the prolonged retention of olfactory adaptation to diacetyl. These mutants are classified into 2 classes. One class shows the prolonged retention of olfactory adaptation to both diacetyl and isoamylalcohol. The other class shows the prolonged retention of olfactory adaptation to diacetyl, but not to isoamylalcohol. This result suggests that the distinct downstream signaling pathways regulate forgetting of olfactory adaptation in the distinct neurons. To identify downstream components of TIR-1/JNK-1 pathway, we analyzed the isolated mutants with the whole genome sequencing and genetic mapping. Among those mutants, we determined the complete DNA sequences of 20 mutants, which showed strong phenotypes. We identified a gene responsible for one of these mutants, qj143 as maco-1, which is conserved across the species and is orthologous to human macoilin. This study enables us to provide new insights on the regulatory mechanisms of forgetting.

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.

Sato, Noriko et al. (2021) International Worm Meeting "Simultaneous measurements of membrane voltage and intracellular Ca2+ of AWA neurons by a gene encoded voltage indicator and GCaMP."

Tokunaga, Terumasa, Sato, Noriko, Ishihara, Takeshi

[International Worm Meeting, 2021]

Measurement of neuronal activities in non-invasive and unanesthetized condition is important for understanding neuronal function in intact animals. Ca2+ imaging by fluorescent gene encoded calcium indicators (GECI) are a powerful way to measure neuronal activities in C. elegans. Although Ca2+ imaging revealed important aspects in neuronal functions, the measurement of neuronal membrane voltage is important to understand the neuronal functions. Furthermore, the relations of change of membrane voltages and changes of Ca2+ has not been fully understood. Recently, several types of gene encoded voltage indicators (GEVI) that are derived from 7TM proteins used for optogenetics has been developed to measure changes of membrane voltage in living animals. Even though the fluorescence of these GEVIs is dim, they showed fast time constants and relatively high fluorescent change depend on voltages. Among those GEVIs, we use paQuasAr3 for the voltage measurement, because it shows relatively higher fluorescence with other superior characteristics. Since AWA, one of the olfactory sensory neurons, which is responsible for diacetyl sensation, was reported to show all-or-none action potentials (Liu et al. 2018), we firstly analyzed AWA voltage changes induced by diacetyl. We found that fluorescence of paQuasAr3 expressed in AWA cell body is changed in response to diacetyl stimulation with high reproducibility. At the beginning of the stimulation, the transient increase and decrease of fluorescence intensity was observed, whereas the relatively higher fluorescence intensity was sustained during the stimulation. To elucidate relations between the Ca2+ responses and the voltage responses, we made wild-type animals expressing paQuasAr3 and GCaMP6f in AWA neurons, and measured both fluorescence at a cell body simultaneously. We found that the changes of paQuasAr3 started faster than the changes of GCaMP. These analyses will give insights on the neuronal functions in informational processing.

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.

Teo, J.Hooi Min. et al. (2019) International Worm Meeting "Cellular analysis in forgetting of an olfactory memory in Caenorhabditis elegans."

Teo, J.Hooi Min., Ishihara, Takeshi, Kitazono, Tomohiro

[International Worm Meeting, 2019]

Learning and forgetting information acquired from surrounding is a crucial process for the animals to strive for survival in changing environments. However, working mechanisms in learning and forgetting remain unclear. C. elegans shows a weak chemoattraction after prolonged exposure to an attractive odorant in the absence of food. Adapted wild-type animals are able to recover their chemoattraction toward the conditioned odorant after 4 hours of normal cultivation on food. We regard this behavioral recovery as forgetting. Previous study showed that TIR-1/JNK-1 pathway in AWC neurons plays a role to regulate forgetting the adaptation memory retained in AWA neurons. Still, the mechanism and neuronal network between AWC and AWA underlying forgetting are not known. To study how AWC regulate forgetting in AWA, we investigated several interneurons which work downstream of AWC and AWA neurons in the olfactory circuit. We discovered that animals lacking functional AIA interneurons showed normal adaptation but displayed a defect in forgetting of olfactory adaptation to both diacetyl and isoamylalcohol. This suggests that AIA play a role in forgetting of two distinct types of olfactory adaptation. Furthermore, acute silencing of AIA during recovery using Drosophila histamine-gated chloride channel (HisCl1) was sufficient to cause a forgetting defect in olfactory adaptation, suggests that the activity of AIA during recovery is required to actively regulate forgetting. To elucidate the forgetting mechanisms in which AIA are associated, we inspected the interaction of AIA interneurons with TIR-1 pathway by examining the effect of AIA interneurons in tir-1(gf) animal. The strain not only suppressed a weak adaptation phenotype in tir-1(gf), but also showed prolonged retention of adaptation, suggesting that AIA work downstream of TIR-1 pathway in regulate forgetting. We also found that mutation in egl-21 gene, a gene which encoded carboxypeptidase E (CPE) protein required for neuropeptides processing, caused abnormality in forgetting in olfactory memory, indicates that neuropeptide might play a certain role in regulation of forgetting. However, our results suggested that AIA did not regulate forgetting via neuropeptide release. To examine how AIA engage in active forgetting, we are currently studying the effect of AIA on AWA calcium responses to diacetyl.