Ishihara T (2008) Tanpakushitsu Kakusan Koso "[Mechanisms of behavioral plasticity in C. elegans]."

Ishihara T

[Tanpakushitsu Kakusan Koso, 2008]

Ishihara T et al. (1996) Worm Breeder's Gazette "Metabotropic glutamate receptor in C. elegans."

Ishihara T, Katsura I

[Worm Breeder's Gazette, 1996]

Metabotropic glutamate receptors (mGluRs) are a class of glutamate receptors that contain seven transmembrane domains and that are coupled with G-proteins. In mammals, eight mGluR genes are identified, and those receptor subtypes are divided into three groups according to homology, pharmacology, and downstream signal transduction system. The receptors belonging to one of the groups (mGluR1 and mGluR5) are coupled to IP3/Ca2+ signal transduction systems, while the other six receptors are involved in the inhibition of cAMP formation. The latter are subdivided further into two groups by the response to agonists. By studying knockout mice lacking mGluR genes, mGluRs are shown to be involved in higher order functions in the mammalian brain. Mice lacking mGluR1 gene are deficient in associative learning dependent on hippocampus and cerebellum, in accordance with reduced long-term potentiation in hippocampal CA1 region and with deficit of the cerebellar long-term depression, respectively. Mice lacking mGluR5 gene show deficiency in ON response in visual transmission, while they show normal OFF response. Recently, it was found that mice lacking mGluR1 gene show abnormal neuronal network formation in cerebellum. In C. elegans genome project, a mGluR gene (ZC506.4) has been identified. According to the amino acid sequence homology to mammalian mGluRs, it belongs to the Gi-coupled mGluR groups, but it is difficult to classify it further. To investigate the expression pattern of the C. elegans mGluR gene, we generated transgenic worms carrying a chimeric mGluR-GFP fusion gene, which contains most of the mGluR coding region, under the control of the mGluR gene promoter. The fusion gene is expressed in several kinds of interneurons and motorneurons including RMD, AIA and PVQ. It is also expressed in pharyngeal interneurons. We also examined the expression of the GFP gene that is transcriptionally fused to the 5' regulatory region of the mGluR gene. Its expression seems essentially the same as that of the chimeric gene. To elucidate the functions of the mGluR in C. elegans, we isolated a mutant of the mGluR gene by insertion and imprecise excision of Tc1. The mutation deletes a 1.8kb fragment within the gene that encodes about 300 amino acids of the first extracellular domain. The mutant grows normally but shows a Loopy phenotype. Since laser ablation of the RMD neurons in a wild type animal causes the Loopy phenotype (Elkes and Kaplan, WBG 12 (5) 66), the expression of the mGluR in RMD neurons may be responsible for normal head movement. We also examined other behaviors including chemotaxis , adaptation, and osmotic avoidance. However, as far as we tested, all of them are normal except that thermotaxis seems to be slightly affected. We will further examine behavioral assays to find function of the mGluR gene in interneurons.

Ishihara T (2004) Tanpakushitsu Kakusan Koso "[Molecular genetics on behavioral plasticity in Caenorhabditis elegans: mechanisms for associative learning]"

Ishihara T

[Tanpakushitsu Kakusan Koso, 2004]

Ishihara T et al. (1997) International C. elegans Meeting "METABOTROPIC GLUTAMATE RECEPTORS IN C. ELEGANS"

Francesconi A, Katsura I, Ishihara T, Duvoisin RM

[International C. elegans Meeting, 1997]

Glutamate receptors are classified into ionotropic receptors and metabotropic receptors (mGluRs). mGluRs, which couple with G-proteins, are divided into three subgroups. The receptors in the first subgroup are coupled to Gq and the IP3/Ca2+ signal transduction system, while the other receptors are involved in inhibiting cAMP formation through Gi. By studying the knockout mice lacking the mGluR gene, it was shown that the mGluRs are involved in higher order functions in mammalian nervous systems, as well as in neural network formation. In the C. elegans genome project, two mGluR genes (mgl-1, mgl-2) have been identified. The predicted amino acid sequences show MGL-1 belongs to the Gi coupled mGluR group, while MGL-2 belongs to the Gq coupled group. Indeed, we found that the HEK293 cells expressing the mgl-2 cDNA increase in phosphoinostitol turnover, responding to glutamate. To elucidate the expression pattern of the C. elegans mGluR genes, we generated transgenic worms carrying a chimeric mGluR-GFP fusion gene. The fusion gene of mgl-2 is expressed essentially in interneurons, while that of mgl-1 is expressed in motorneurons and pharyngeal neurons as well as interneurons. To elucidate functions of mGluRs in C. elegans, we isolated mutants of mGluR genes by the Tc1 insertion/ deletion strategy. We examined various behavioral analyses of them. It seems that the mgl-2 mutant shows abnormal head movement and stronger tap reversal reflexes. We are testing whether these abnormalities can be rescued by the wild type mgl-2 gene.

Ishihara T et al. (1995) International C. elegans Meeting "CONSTRUCTION OF FLUORESCENT MARKERS SPECIFIC TO THE NERVOUS SYSTEM OF C. ELEGANS"

Katsura I, Ishihara T

[International C. elegans Meeting, 1995]

To elucidate how the neuronal circuit is constructed, it may be useful to devise an easy way to observe the morphology of the nervous system in living animals. We are developing GFP markers that are expressed in various sets of neurons in C. elegans. The neuron specific markers are prepared by "promoter trapping" and by using promoters of putatively neuronal genes as predicted by the C. elegans genome project. From about 25,000 clones of a genomic library for promoter trapping constructed in lacZ fusion vector, we isolated three positive clones, of which the lacZ gene was exchanged subsequently for the GFP cDNA. One of them gives GFP expression in almost all neurons, while another gives very specific expression in the AFD cells. The C. elegans genome project predicts some neuronal genes by sequence homology. Among those, promoters of a glutamate receptor gene and a neurotransmitter transporter gene lead to expression in some interneurons and in the dopaminergic neurons, respectively. To identify the marked neurons or to observe neurites of these neurons, we use GFP fused with a nuclear localization signal or with a part of the Unc-76 protein as a marker, respectively. Using these markers, we hope to identify mutations causing defects in neural differentiation or connectivity. This method should be applicable even to lethal mutations, which prevent behavioral assay to detect neuronal disorder.

Ishihara T et al. (1993) International C. elegans Meeting "Construction of lacZ markers specific to the nervous system by promoter trapping."

Katsura I, Ishihara T

[International C. elegans Meeting, 1993]

Ishihara T et al. (2002) Cell "HEN-1, a secretory protein with an LDL receptor motif, regulates sensory integration and learning in Caenorhabditis elegans."

Gengyo-Ando K, Iino Y, Mori I, Katsura I, Ishihara T, Mitani S, Mohri A

[Cell, 2002]

Animals sense many environmental stimuli simultaneously and integrate various sensory signals within the nervous system both 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 and in behavioral plasticity by paired stimuli, although their sensation capability seems to be identical to that of the wild-type. The HEN-1 protein is expressed in two pairs of neurons, but expression in other neurons is sufficient for wild-type behavior. In addition, expression of HEN-1 at the adult stage is sufficient. Thus, HEN-1 regulates sensory processing non-cell-autonomously in the mature neuronal circuit.

T Ishihara et al. (1999) International C. elegans Meeting "Analyses of the Interaction of Two Sensory Signals on the Behavior under Well-fed and Starved Conditions"

T Ishihara, I Katsura

[International C. elegans Meeting, 1999]

Among many environmental stimuli, C. elegans seems to respond to only a few stimuli simultaneously. To elucidate how the neural circuit processes many sensory signals to respond properly, we have designed a new assay system to analyze interaction between two responses: chemotaxis to odorants and avoidance from copper ion. In this assay, the behavior of C. elegans depends on the concentration of both two stimuli, which are sensed by different neurons. This observation suggests that these two responses repress each other. We can argue that this mutual interaction takes place in a defined part of the neural circuit, which consists of about 10 pairs of neurons. We have identified several mutants that show abnormality in this interaction. One of the mutants, ut236 prefers to avoid copper ion rather than to go to odorants, while glr-1 mutants show the opposite phenotype. However, dose responses to the odorants or the copper ion of these mutants are indistinguishable from those of the wild type. We mapped the ut236 mutation between stP33 and unc-18 on the chromosome X and have identified a 15 kb DNA fragment of the cosmid C36B7 that can rescue the ut236 phenotype. Further analysis of the predicted genes in this region is underway. When worms are starved for several hours, their behavior in this assay is changed. They prefer to go to odorants rather than to avoid copper ions as compared with worms in well-fed conditions. This behavioral change is due, at least partially, to weaker avoidance behavior from copper ion by starved animals. Furthermore, in the presence of serotonin, this behavioral change by starvation is not observed. We also isolated mutants that show abnormality in this behavioral change. One of the mutants, ut235 does not change the response to copper ion by starvation, although this mutant behaves almost like the wild type in the change of movement phenotype by starvation and by serotonin. In addition, the ut235 ut236 double mutant entirely prefers to avoid copper ions than to go to the odorants even if they are starved. By characterizing these mutants, we hope to clarify the modification mechanism of the neuronal activity.

Arai M et al. (2018) Brain Nerve "[Molecular and Neuronal Mechanisms of Forgetting]."

Arai M, Ishihara T

[Brain Nerve, 2018]

Animals sense several types of information in their environments and some of those information are stored as memories. The retention of these memories must be regulated for the animals to survive continuously changing environments. However, the mechanisms underlying memory forgetting remain to be understood, partly because psychological studies have suggested that forgetting is a passive process. Recent behavioral genetic studies, using model animals, including mice, Drosophila and Caenorhabditis elegans, have revealed that forgetting is actively regulated in neuronal circuits. In particular, "forgetting cells" non-cell-autonomously accelerate forgetting in other neurons. In this review, we describe recent studies of the mechanisms of forgetting in model animals.

Inoue A et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "The P38/JNK MAP kinase pathway regulates forgetting in Caenorhabditis elegans"

Inoue A, Ishihara T

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

An acquired short-term memory is usually disappeared within hours if not consolidated into a stable long-term memory. However, the mechanisms of the forgetting processes remain largely unknown. Since C. elegans possesses a simple nervous system and their memories usually do not persist more than hours, it is suitable to study the mechanisms of forgetting processes at the molecular level. To identify the gene that regulates those processes, we isolated a mutant qj56 whose memories for the olfactory adaptation and the salt chemotaxis learning retained longer than those in wild type animals. In the qj56 mutant, the memory for the olfactory adaptation was extended from a few hours to more than one day, and the memory for the salt chemotaxis learning was also prolonged. Since, strength of adaptation did not affect memory retention time in wild type animals, the prolonged retention of memory in the qj56 mutant may caused by defect in forgetting processes. We found that qj56 is an allele of tir-1, which encodes an ortholog of human SARM, a Toll interleukin 1 receptor (TIR) domain protein. In C. elegans, TIR-1 regulates innate immune responses and asymmetric expression of STR-2 in the AWC neurons via the P38/MAPK pathway. We found that mutants of CaMKII(UNC-43), p38/JNK MAPKKK(NSY-1), MAPKK(SEK-1), and the JNK-1 show defects in forgetting processes. The forgetting defect of tir-1 mutants could be rescued by the expression of the wild type TIR-1 in the AWC sensory neurons. In addition, mutants of CEH-36, in which the functional AWC sensory neurons were lost, also showed prolonged retention of memory for the olfactory adaptation, suggesting that P38/JNK signals in AWC sensory neurons regulate forgetting processes in C. elegans. Although, TIR-1 regulates both forgetting and left-right asymmetry of AWC neurons, JNK-1 mutant did not show asymmetric abnormality in AWC neurons. Additionally, both nsy-5 mutants, which did not express STR-2 in both AWC neurons (2AWCoff), and nsy-5 mutant with olrn-1(OE), which express STR-2 in both AWC neurons (2AWCon), did not show prolonged retention of memory, suggesting that AWCon or AWCoff cells can regulate the forgetting process adequately. These results indicate that the forgetting is the active process regulated by the P38/JNK MAP kinase pathways in the AWC sensory neurons.