Language：English   Publishing type：Research paper (scientific journal)  

Gastrin-releasing peptide (GRP) and its receptor (GRPR) have been identified as itch mediators in the spinal and trigeminal somatosensory systems in rodents. In primates, there are few reports of GRP/GRPR expression or function in the spinal sensory system and virtually nothing is known in the trigeminal system. The aim of the present study was to characterize GRP and GRPR in the trigeminal and spinal somatosensory system of Japanese macaque monkeys (Macaca fuscata). cDNA encoding GRP was isolated from the macaque dorsal root ganglion (DRG) and exhibited an amino acid sequence that was highly conserved among mammals and especially in primates. Immunohistochemical analysis demonstrated that GRP was expressed mainly in the small-sized trigeminal ganglion and DRG in adult macaque monkeys. Densely stained GRP-immunoreactive (ir) fibers were observed in superficial layers of the spinal trigeminal nucleus caudalis (Sp5C) and the spinal cord. In contrast, GRP-ir fibers were rarely observed in the principal sensory trigeminal nucleus and oral and interpolar divisions of the spinal trigeminal nucleus. cDNA cloning, in situ hybridization, and Western blot revealed substantial expression of GRPR mRNA and GRPR protein in the macaque spinal dorsal horn and Sp5C. Our Western ligand blot and ligand derivative stain for GRPR revealed that GRP directly bound in the macaque Sp5C and spinal dorsal horn as reported in rodents. Finally, GRP-ir fibers were also detected in the human spinal dorsal horn. The spinal and trigeminal itch neural circuits labeled with GRP and GRPR appear to function also in primates.

DOI： 10.1002/cne.25376

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media {LLC}  

<jats:title>Abstract</jats:title><jats:p>Bombesin is a putative antibacterial peptide isolated from the skin of the frog, <jats:italic>Bombina bombina</jats:italic>. Two related (bombesin-like) peptides, gastrin-releasing peptide (GRP) and neuromedin B (NMB) have been found in mammals. The history of GRP/bombesin discovery has caused little attention to be paid to the evolutionary relationship of GRP/bombesin and their receptors in vertebrates. We have classified the peptides and their receptors from the phylogenetic viewpoint using a newly established genetic database and bioinformatics. Here we show, by using a clawed frog (<jats:italic>Xenopus tropicalis</jats:italic>), that GRP is not a mammalian counterpart of bombesin and also that, whereas the GRP system is widely conserved among vertebrates, the NMB/bombesin system has diversified in certain lineages, in particular in frog species. To understand the derivation of GRP system in the ancestor of mammals, we have focused on the GRP system in <jats:italic>Xenopus</jats:italic>. Gene expression analyses combined with immunohistochemistry and Western blotting experiments demonstrated that GRP peptides and their receptors are distributed in the brain and stomach of <jats:italic>Xenopus</jats:italic>. We conclude that GRP peptides and their receptors have evolved from ancestral (GRP-like peptide) homologues to play multiple roles in both the gut and the brain as one of the <jats:italic>‘gut-brain peptide’</jats:italic> systems.</jats:p>

DOI： 10.1038/s41598-021-92528-x

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Other Link： http://www.nature.com/articles/s41598-021-92528-x

Language：English   Publishing type：Research paper (scientific journal)   Publisher：{MDPI} {AG}  

Translational research often requires the testing of experimental therapies in primates, but research in non-human primates is now stringently controlled by law around the world. Tissues fixed in formaldehyde without glutaraldehyde have been thought to be inappropriate for use in electron microscopic analysis, particularly those of the brain. Here we report the immunoelectron microscopic characterization of arginine vasopressin (AVP)-producing neurons in macaque hypothalamo-pituitary axis tissues fixed by perfusion with 4% formaldehyde and stored at -25 °C for several years (4-6 years). The size difference of dense-cored vesicles between magnocellular and parvocellular AVP neurons was detectable in their cell bodies and perivascular nerve endings located, respectively, in the posterior pituitary and median eminence. Furthermore, glutamate and the vesicular glutamate transporter 2 could be colocalized with AVP in perivascular nerve endings of both the posterior pituitary and the external layer of the median eminence, suggesting that both magnocellular and parvocellular AVP neurons are glutamatergic in primates. Both ultrastructure and immunoreactivity can therefore be sufficiently preserved in macaque brain tissues stored long-term, initially for light microscopy. Taken together, these results suggest that this methodology could be applied to the human post-mortem brain and be very useful in translational research.

DOI： 10.3390/ijms22179180

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Authorship：Lead author   Publishing type：Research paper (scientific journal)   Publisher：{MDPI} {AG}  

The spinal ejaculation generator (SEG) is located in the central gray (lamina X) of the rat lumbar spinal cord and plays a pivotal role in the ejaculatory reflex. We recently reported that SEG neurons express the oxytocin receptor and are activated by oxytocin projections from the paraventricular nucleus of hypothalamus (PVH). However, it is unknown whether the SEG responds to oxytocin in vivo. In this study, we analyzed the characteristics of the brain–spinal cord neural circuit that controls male sexual function using a newly developed in vivo electrophysiological technique. Optogenetic stimulation of the PVH of rats expressing channel rhodopsin under the oxytocin receptor promoter increased the spontaneous firing of most lamina X SEG neurons. This is the first demonstration of the in vivo electrical response from the deeper (lamina X) neurons in the spinal cord. Furthermore, we succeeded in the in vivo whole-cell recordings of lamina X neurons. In vivo whole-cell recordings may reveal the features of lamina X SEG neurons, including differences in neurotransmitters and response to stimulation. Taken together, these results suggest that in vivo electrophysiological stimulation can elucidate the neurophysiological response of a variety of spinal neurons during male sexual behavior.

DOI： 10.3390/ijms22073400

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Informa UK Limited  

Oxytocin is produced in the hypothalamus and stimulates uterine contraction and milk ejection. While many people consider oxytocin to be a female hormone, it is reported that, in men, the plasma oxytocin level increases markedly after ejaculation. However, this aspect of oxytocin physiology is poorly understood. The spinal ejaculation generator (SEG), which expresses the neuropeptide, gastrin-releasing peptide (GRP), can trigger ejaculation in rats. Therefore, we focused on systemic effects of oxytocin on the GRP/SEG neuron system in the lumbar spinal cord controlling sexual activity in male rats. We found that systemic administration of oxytocin significantly shortened the latency to the first mount, intromission and ejaculation during male copulatory behavior. In addition, the local oxytocin level in the lumbar cord was significantly higher in males than in females. Histological analysis showed that oxytocin-binding is apparent in spinal GRP/SEG neurons. We therefore conclude that oxytocin influences male sexual activity via the SEG.

DOI： 10.1080/19420889.2021.1902056

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Language：English   Publishing type：Research paper (scientific journal)  

Astrocytes are critical regulators of CNS function and are proposed to be heterogeneous in the developing brain and spinal cord. Here we identify a population of astrocytes located in the superficial laminae of the spinal dorsal horn (SDH) in adults that is genetically defined by Hes5. In vivo imaging revealed that noxious stimulation by intraplantar capsaicin injection activated Hes5+ SDH astrocytes via α1A-adrenoceptors (α1A-ARs) through descending noradrenergic signaling from the locus coeruleus. Intrathecal norepinephrine induced mechanical pain hypersensitivity via α1A-ARs in Hes5+ astrocytes, and chemogenetic stimulation of Hes5+ SDH astrocytes was sufficient to produce the hypersensitivity. Furthermore, capsaicin-induced mechanical hypersensitivity was prevented by the inhibition of descending locus coeruleus-noradrenergic signaling onto Hes5+ astrocytes. Moreover, in a model of chronic pain, α1A-ARs in Hes5+ astrocytes were critical regulators for determining an analgesic effect of duloxetine. Our findings identify a superficial SDH-selective astrocyte population that gates descending noradrenergic control of mechanosensory behavior.

DOI： 10.1038/s41593-020-00713-4

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

DOI： 10.1002/cne.25026

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Other Link： https://onlinelibrary.wiley.com/doi/full-xml/10.1002/cne.25026

Language：English   Publishing type：Research paper (scientific journal)  

Exposure to endocrine-disrupting chemicals may adversely affect animals, particularly during development. Tris(1,3-dichloroisopropyl) phosphate (TDCIPP) is an organophosphate with anti-androgen function in vitro that is present in indoor dust at relatively high concentrations. In male rats, androgens are necessary for the development of reproductive organs, as well as the endocrine and central nervous systems. However, we currently do not know the exact effects of TDCIPP exposure through suckling on subsequent reproductive behavior in males. Here, we show that TDCIPP exposure (25-250 mg kg-1 via oral administration over 28 consecutive days post-birth) suppressed male sexual behavior and reduced testes size. These changes were dose-dependent and appeared first in adults rather than in juveniles. These results demonstrate that TDCIPP exposure led to normal body growth and appearance in juveniles, but disrupted the endocrine system and physiology in adults. Therefore, assays should be performed using adult animals to ensure accuracy, and to confirm the influence of chemical substances given during early mammalian life.

DOI： 10.1002/jat.3569

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Nature Publishing Group  

Study design: Experimental animal study. Objectives: Although a population of gastrin-releasing peptide (GRP) neurons in the lumbar spinal cord has an important role in erection and ejaculation in rats, little information exists on this GRP system in primates. To identify the male-specific GRP system in the primate spinal cord, we studied the lumbosacral cord in macaque monkeys as a non-human primate model. Setting: University laboratory in Japan. Methods: To determine the gene sequence of GRP precursors, the rhesus macaque monkey genomic sequence data were searched, followed by phylogenetic analysis. Subsequently, immunocytochemical analysis for GRP was performed in the monkey spinal cord. Results: We have used bioinformatics to identify the ortholog gene for GRP precursor in macaque monkeys. Phylogenetic analysis suggested that primate prepro-GRP is separated from that of other mammalian species and clustered to an independent branch as primates. Immunocytochemistry for GRP further demonstrated that male-dominant sexual dimorphism was found in the spinal GRP system in monkeys as in rodents. Conclusion: We have demonstrated in macaque monkeys that the GRP system in the lower spinal cord shows male-specific dimorphism and May have an important role in penile functions not only in rodents but also in primates.

DOI： 10.1038/sc.2017.105

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

Several regions of the brain and spinal cord control male reproductive function. We previously demonstrated that the gastrin-releasing peptide (GRP) system, located in the lumbosacral spinal cord of rats, controls spinal centers to promote penile reflexes during male copulatory behavior. However, little information exists on the male-specific spinal GRP system in animals other than rats. The objective of this study was to examine the functional generality of the spinal GRP system in mammals using the Asian house musk shrew (Suncus murinus; suncus named as the laboratory strain), a specialized placental mammal model. Mice are also used for a representative model of small laboratory animals. We first isolated complementary DNA encoding GRP in suncus. Phylogenetic analysis revealed that suncus preproGRP was clustered to an independent branch. Reverse transcription-PCR showed that GRP and its receptor mRNAs were both expressed in the lumbar spinal cord of suncus and mice. Immunohistochemistry for GRP demonstrated that the sexually dimorphic GRP system and male-specific expression/distribution patterns of GRP in the lumbosacral spinal cord in suncus are similar to those of mice. In suncus, we further found that most GRP-expressing neurons in males also express androgen receptors, suggesting that this male-dominant system in suncus is also androgen-dependent. Taken together, these results indicate that the sexually dimorphic spinal GRP system exists not only in mice but also in suncus, suggesting that this system is a conserved property in mammals. J. Comp. Neurol. 525:1586-1598, 2017. (c) 2016 Wiley Periodicals, Inc.

DOI： 10.1002/cne.24138

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPAN ACAD  

A sexually dimorphic spinal gastrin-releasing peptide (GRP) system in the lumbosacral spinal cord, which projects to the lower spinal centers, controls erection and ejaculation in rats. However, little is known about the postnatal development of this system. In this study, we therefore examined the postnatal development of the male-dominant spinal GRP system and its sexual differentiation in rats using immunohistochemistry. Our results show that male-dominant expression of GRP is prominent from the onset of puberty and that sexually dimorphism persists into adulthood. These results suggest that androgen surge during male puberty plays an important role in the development and maintenance of the male-specific GRP function in the rat spinal cord.

DOI： 10.2183/pjab.92.69

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Language：English   Publisher：Tohoku University  

Neural circuits underlying male sexual function comprise several nuclei located in the brain and spinal cord. We have previously demonstrated in rats that the gastrin-releasing peptide (GRP) system influences spinal centers promoting penile reflexes. Moreover, a group of oxytocin (OXT) neurons, situated in the parvocellular part of the paraventricular nucleus of the hypothalamus, project into the spinal cord and control penile reflexes. Therefore, it has been hypothesized that OXT is transported by long descending paraventriculospinal pathways and activates proerectile spinal centers. Consequently, we have shown that in rats, axonal distribution of OXT in the lumbar spinal cord exhibits a male-dominant sexual dimorphism. Furthermore, OXT binding is observed in the spinal GRP neurons. Thus, OXT axons may secrete OXT from spinal axonal terminals and regulate male sexual function via an OXT receptor-mediated mechanism in spinal GRP neurons. Future studies should address the relationship between the hypothalamic OXT and spinal GRP systems. Identification of the male-specific brain-spinal cord neural circuit that regulates male sexual behavior may provide new avenues for therapeutic approaches to masculine reproductive dysfunction, including erectile dysfunction and/or ejaculation disorder.

DOI： 10.4036/iis.2015.B.08

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Other Link： https://jlc.jst.go.jp/DN/JLC/20015114165?from=CiNii

Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Arginine vasopressin (AVP) is a neurohypophysial hormone synthesized as a part of a prepropeptide precursor containing the signal peptide, AVP hormone, AVP-associated neurophysin II and copeptin in the hypothalamic neurosecretory neurons. A transgenic (Tg) rat line expressing the AVP-eGFP fusion gene has been generated. To establish the AVP-eGFP Tg rat as a unique model for an analysis of AVP dynamics invivo, we first examined the invivo molecular dynamics of the AVP-eGFP fusion gene, and then the release of GFP in response to physiological stimuli. Double immunoelectron microscopy demonstrated that GFP was specifically localized in neurosecretory vesicles of AVP neurons in this Tg rat. After stimulation of the posterior pituitary with high potassium we demonstrated the exocytosis of AVP neurosecretory vesicles containing GFP at the ultrastructural level. Biochemical analyses indicated that the AVP-eGFP fusion gene is subjected to invivo post-translational modifications like the native AVP gene, and is packaged into neurosecretory vesicles as a fusion protein: copeptin(1-14)-GFP. Moreover, GFP release into the circulating blood appeared to be augmented after osmotic stimulation, like native AVP. Thus, here we show for the first time the invivo molecular processing of the AVP-eGFP fusion gene and stimulated secretion after osmotic stimulation in rats. Because GFP behaved like native AVP in the hypothalamo-pituitary axis, and in particular was released into the circulation in response to a physiological stimulus, the AVP-eGFP Tg rat model appears to be a powerful tool for analyzing neuroendocrine systems at the organismal level.

DOI： 10.1111/febs.13291

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Gastrin-releasing peptide (GRP) has recently been identified as an itch-specific neuropeptide in the spinal sensory system in mice, but there are no reports of the expression and distribution of GRP in the trigeminal sensory system in mammals. We characterized and compared GRP-immunoreactive (ir) neurons in the trigeminal ganglion (TG) with those in the rat spinal dorsal root ganglion (DRG). GRP immunoreactivity was expressed in 12% of TG and 6% of DRG neurons and was restricted to the small- and medium-sized type cells. In both the TG and DRG, many GRP-ir neurons also expressed substance P and calcitonin gene-related peptide, but not isolectin B-4. The different proportions of GRP and transient receptor potential vanilloid 1 double-positive neurons in the TG and DRG imply that itch sensations via the TG and DRG pathways are transmitted through distinct mechanisms. The distribution of the axon terminals of GRP-ir primary afferents and their synaptic connectivity with the rat trigeminal sensory nuclei and spinal dorsal horn were investigated by using light and electron microscopic histochemistry. Although GRP-ir fibers were rarely observed in the trigeminal sensory nucleus principalis, oralis, and interpolaris, they were predominant in the superficial layers of the trigeminal sensory nucleus caudalis (Vc), similar to the spinal dorsal horn. Ultrastructural analysis revealed that GRP-ir terminals contained clear microvesicles and large dense-cored vesicles, and formed asymmetric synaptic contacts with a few dendrites in the Vc and spinal dorsal horn. These results suggest that GRP-dependent orofacial and spinal pruriceptive inputs are processed mainly in the superficial laminae of the Vc and spinal dorsal horn. J. Comp. Neurol. 522:1858-1873, 2014. (c) 2013 Wiley Periodicals, Inc.

DOI： 10.1002/cne.23506

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

IntroductionThe pelvic striated muscles play an important role in mediating erections and ejaculation, and together these muscles compose a tightly coordinated neuromuscular system that is androgen sensitive and sexually dimorphic.
AimTo identify spinal and brains neurons involved in the control of the levator ani (LA) and bulbospongiosus (BS) in the male adult and preadolescent rat.
MethodsRats were anesthetized, and the transsynaptic retrograde tracer pseudorabies virus (PRV) was injected into the LA muscle of adults or the ventral BS muscle in 30-day-old rats. After 3-5 days rats were sacrificed, and PRV-labeled neurons in the spinal cords and brains were identified using immunohistochemistry. The presence of gastrin-releasing peptide (GRP) in the lumbar spinal neurons was examined.
Main Outcomes MeasuresThe location and number of PRV-labeled neurons in the spinal cord and brain and GRP colocalization in the lumbar spinal cord.
ResultsPRV-labeled spinal interneurons were found distributed throughout T11-S1 of the spinal cord, subsequent to dorsal medial motoneuron infection. The majority of spinal interneurons were found in the lumbosacral spinal cord in the region of the dorsal gray commissure and parasympathetic preganglionic neurons. Preadolescent rats had more PRV-labeled spinal interneurons at L5-S1 where the motoneurons were located but relatively less spread rostrally in the spinal cord compared with adults. Lumbar spinothalmic neurons in medial gray of L3-L4 co-localized PRV and GRP. In the brain consistent labeling was seen in areas known to be involved in male sexual behavior including the ventrolateral medulla, hypothalamic paraventricular nucleus, and medial preoptic area.
ConclusionCommon spinal and brain pathways project to the LA and BS muscles in the rat suggesting that these muscles act together to coordinate male sexual reflexes. Differences may exist in the amount of synaptic connections/neuronal pathways in adolescents compared with adults. Dobberfuhl AD, Oti T, Sakamoto H, and Marson L. Identification of CNS neurons innervating the levator ani and ventral bulbospongiosus muscles in male rats. J Sex Med 2014;11:664-677.

DOI： 10.1111/jsm.12418

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER IRELAND LTD  

Androgens including testosterone, organize the nervous system as well as masculine external and internal genitalia during the perinatal period. Androgen organization involves promotion of masculine body features, usually by acting through androgen receptors (ARs). We have recently demonstrated that the gastrin-releasing peptide (GRP) system in the lumbar spinal cord also mediates spinal centers promoting penile reflexes during male sexual behavior in rats. Testosterone may induce sexual differentiation of this spinal GRP system during development and maintain its activation in adulthood. In the present study, we examined the role of ARs in the nervous system regulating the development of the sexually dimorphic GRP system. For this purpose, we used a conditional mouse line selectively lacking the AR gene in the nervous system. AR foxed males carrying (mutants) or not (controls) the nestin-Cre transgene were castrated in adulthood and supplemented with physiological amounts of testosterone. Loss of AR expression in the nervous system resulted in a significant decrease in the number of GRP neurons compared to control littermates. Consequently, the intensity of GRP axonal projections onto the lower lumbar and upper sacral spinal cord was greater in control males than in mutant males. These results suggest that ARs expressed in the nervous system play a significant role in the development of the GRP system in the male lumbar spinal cord. The AR-deletion mutation may attenuate sexual behavior and activity of mutant males via spinal GRP system-mediated neural mechanisms. (C) 2013 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.neulet.2013.10.068

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Three-dimensional (3-D) analysis of anatomical ultrastructures is important in biological research. However, 3-D image analysis on exact serial sets of ultra-thin sections from biological specimens is very difficult to achieve, and limited information can be obtained by 3-D reconstruction from these sections due to the small area that can be reconstructed. On the other hand, the high-penetration power of electrons by an ultra-high accelerating voltage enables thick sections of biological specimens to be examined. High-voltage electron microscopy (HVEM) is particularly useful for 3-D analysis of the central nervous system because considerably thick sections can be observed at the ultrastructure level. Here, we applied HVEM tomography assisted by light microscopy to a study of the 3-D chemical neuroanatomy of the rat lower spinal cord annotated by double-labeling immunohistochemistry. This powerful methodology is useful for studying molecular and/or chemical neuroanatomy at the 3-D ultrastructural level.

DOI： 10.1007/s00418-012-0976-6

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DOI： 10.4172/2161-038X.1000e101

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J-GLOBAL

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J-GLOBAL

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J-GLOBAL

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J-GLOBAL

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Language：Japanese   Publisher：日本組織細胞化学会  

CiNii Article

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Other Link： http://search.jamas.or.jp/link/ui/2016046963

Language：Japanese   Publisher：日本組織細胞化学会  

CiNii Article

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Other Link： http://search.jamas.or.jp/link/ui/2016046985

Language：Japanese  

J-GLOBAL

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Event date： 2022.9.8 - 2022.9.10

Language：Japanese   Presentation type：Symposium, workshop panel (nominated)  

Venue：東京都  

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Event date： 2022.7.9

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Venue：京都府  

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Event date： 2022.6.30 - 2022.7.3

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Venue：東京都文京区  

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Venue：山梨県南都留郡  

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Venue：東広島市，広島県  

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Venue：東広島市，広島県  

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Venue：Tsukuba, Ibarakishi  

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Venue：文京区，東京都  

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Venue：文京区，東京都  

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Venue：相模原市，神奈川県  

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Venue：相模原市，神奈川県  

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Venue：武蔵野市，東京都  

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Venue：相模原市，神奈川県  

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Venue：富山市，富山県  

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Venue：倉敷市，岡山県  

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Venue：Hiroshima Grand Prince Hotel, Hiroshima  

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Venue：高知市，高知県  

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Venue：高知市，高知県  

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Venue：瀬戸内市，岡山県  

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Venue：瀬戸内市，岡山県  

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Venue：浜松市，静岡県  

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Venue：浜松市、静岡県  

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Venue：熱海市，静岡県  

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Venue：三鷹市，東京都  

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Venue：新潟市，新潟県  

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Venue：仙台市，宮城県  

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Venue：枚方市，大阪府  

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Venue：枚方市，大阪府  

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Venue：神戸市，兵庫県  

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Venue：大阪市，大阪府  

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Venue：松山市，愛媛県  

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Venue：松山市，愛媛県  

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Venue：神戸市，兵庫県  

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Venue：神戸市，兵庫県  

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Venue：神戸市，兵庫県  

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Venue：岡崎市，愛知県  

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Country：Japan

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Award type：Award from Japanese society, conference, symposium, etc.  Country：Japan

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Award type：Award from Japanese society, conference, symposium, etc.  Country：Japan

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