Researcher Profile and Settings

Master

Affiliation (Master)

• Faculty of Medicine　Physiological Science　Anatomy

Affiliation (Master)

• Faculty of Medicine　Physiological Science　Anatomy

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Profile and Settings

Profile and Settings

• Name (Japanese)

  kohtarou

• Name (Kana)

  konno

• Name

  201101030502015357

Achievement

Research Areas

• Life sciences / Functional biochemistry

Research Experience

• 2010/08 - Today 北海道大学医学研究科 解剖発生学分野 助教
• 2010/04 - 2010/07 北海道大学医学研究科 解剖発生学分野 博士研究員
• 2006/04 - 2010/03 北海道大学医学研究科 神経薬理学分野 大学院博士課程

Awards

• 2024/03 北海道大学医学研究院・医学院・医学部医学科 優秀論文賞
  
• 2023 北海道大学医学研究科・医学部医学科 エクセレント・ティーチャー最優秀賞
  
• 2022 北海道大学医学研究科・医学部医学科 エクセレント・ティーチャー最優秀賞
  
• 2022 北海道大学医学研究科・医学部医学科 エクセレント・ティーチャー優秀賞
  
• 2021 北海道大学医学研究科・医学部医学科 エクセレント・ティーチャー優秀賞
  
• 2020 北海道大学医学研究科・医学部医学科 エクセレント・ティーチャー優秀賞
  
• 2018 次世代脳プロジェクト冬のシンポジウム若手優秀発表賞
  
• 2017 次世代脳プロジェクト冬のシンポジウム若手優秀発表賞
  
• 2017 北海道大学医学研究科・医学部医学科 最優秀賞
  
• 2016 北海道大学医学研究科・医学部医学科 連続最優秀賞
  
• 2016 北海道大学医学研究科・医学部医学科 エクセレント・ティーチャー優秀賞
  
• 2016 北海道大学教育総長賞 奨励賞
  
• 2015 包括脳ネットワーク 若手優秀発表賞
  
• 2015 北海道大学医学研究科・医学部医学科 最優秀教員賞
  
• 2014 包括脳ネットワーク 若手優秀発表賞
  
• 2014 北海道大学教育総長賞 奨励賞
  
• 2014 北海道大学医学研究科・医学部医学科 最優秀教員賞
  
• 2013 北海道大学医学研究科・医学部医学科 優秀教員賞
  

Published Papers

• Direct and indirect pathways for heterosynaptic interaction underlying developmental synapse elimination in the mouse cerebellum.

  Hisako Nakayama, Taisuke Miyazaki, Manabu Abe, Maya Yamazaki, Yoshinobu Kawamura, Myeongjeong Choo, Kohtarou Konno, Shinya Kawata, Naofumi Uesaka, Kouichi Hashimoto, Mariko Miyata, Kenji Sakimura, Masahiko Watanabe, Masanobu Kano

  Communications biology 7 (1) 806 - 806 2024/07/03 

  Developmental synapse elimination is crucial for shaping mature neural circuits. In the neonatal mouse cerebellum, Purkinje cells (PCs) receive excitatory synaptic inputs from multiple climbing fibers (CFs) and synapses from all but one CF are eliminated by around postnatal day 20. Heterosynaptic interaction between CFs and parallel fibers (PFs), the axons of cerebellar granule cells (GCs) forming excitatory synapses onto PCs and molecular layer interneurons (MLIs), is crucial for CF synapse elimination. However, mechanisms for this heterosynaptic interaction are largely unknown. Here we show that deletion of AMPA-type glutamate receptor functions in GCs impairs CF synapse elimination mediated by metabotropic glutamate receptor 1 (mGlu1) signaling in PCs. Furthermore, CF synapse elimination is impaired by deleting NMDA-type glutamate receptors from MLIs. We propose that PF activity is crucial for CF synapse elimination by directly activating mGlu1 in PCs and indirectly enhancing the inhibition of PCs through activating NMDA receptors in MLIs.
• Behavioral analysis of kainate receptor KO mice and the role of GluK3 subunit in anxiety.

  Izumi Iida, Kohtarou Konno, Rie Natsume, Manabu Abe, Masahiko Watanabe, Kenji Sakimura, Miho Terunuma

  Scientific reports 14 (1) 4521 - 4521 2024/02/24 

  Kainate receptors (KARs) are one of the ionotropic glutamate receptors in the central nervous system (CNS) comprised of five subunits, GluK1-GluK5. There is a growing interest in the association between KARs and psychiatric disorders, and there have been several studies investigating the behavioral phenotypes of KAR deficient mice, however, the difference in the genetic background has been found to affect phenotype in multiple mouse models of human diseases. Here, we examined GluK1-5 single KO mice in a pure C57BL/6N background and identified that GluK3 KO mice specifically express anxiolytic-like behavior with an alteration in dopamine D2 receptor (D2R)-induced anxiety, and reduced D2R expression in the striatum. Biochemical studies in the mouse cortex confirmed that GluK3 subunits do not assemble with GluK4 and GluK5 subunits, that can be activated by lower concentration of agonists. Overall, we found that GluK3-containing KARs function to express anxiety, which may represent promising anti-anxiety medication targets.
• Behavioral analysis of kainate receptor KO mice and the role of GluK3 subunit in anxiety

  Izumi Iida, Kohtarou Konno, Rie Natsume, Manabu Abe, Masahiko Watanabe, Kenji Sakimura, Miho Terunuma

  Scientific Reports 14 (1) 2024/02/24 

  Abstract Kainate receptors (KARs) are one of the ionotropic glutamate receptors in the central nervous system (CNS) comprised of five subunits, GluK1-GluK5. There is a growing interest in the association between KARs and psychiatric disorders, and there have been several studies investigating the behavioral phenotypes of KAR deficient mice, however, the difference in the genetic background has been found to affect phenotype in multiple mouse models of human diseases. Here, we examined GluK1-5 single KO mice in a pure C57BL/6N background and identified that GluK3 KO mice specifically express anxiolytic-like behavior with an alteration in dopamine D2 receptor (D2R)-induced anxiety, and reduced D2R expression in the striatum. Biochemical studies in the mouse cortex confirmed that GluK3 subunits do not assemble with GluK4 and GluK5 subunits, that can be activated by lower concentration of agonists. Overall, we found that GluK3-containing KARs function to express anxiety, which may represent promising anti-anxiety medication targets.
• Brain-enriched guanylate kinase-associated protein, a component of the post-synaptic density protein complexes, contributes to learning and memory.

  Tayo Katano, Kohtarou Konno, Keizo Takao, Manabu Abe, Akari Yoshikawa, Tsuyoshi Miyakawa, Kenji Sakimura, Masahiko Watanabe, Seiji Ito, Takuya Kobayashi

  Scientific reports 13 (1) 22027 - 22027 2023/12/12 

  Brain-enriched guanylate kinase-associated protein (BEGAIN) is highly enriched in the post-synaptic density (PSD) fraction and was identified in our previous study as a protein associated with neuropathic pain in the spinal dorsal horn. PSD protein complexes containing N-methyl-D-aspartate receptors are known to be involved in neuropathic pain. Since these PSD proteins also participate in learning and memory, BEGAIN is also expected to play a crucial role in this behavior. To verify this, we first examined the distribution of BEGAIN in the brain. We found that BEGAIN was widely distributed in the brain and highly expressed in the dendritic regions of the hippocampus. Moreover, we found that BEGAIN was concentrated in the PSD fraction of the hippocampus. Furthermore, immunoelectron microscopy confirmed that BEGAIN was localized at the asymmetric synapses. Behavioral tests were performed using BEGAIN-knockout (KO) mice to determine the contribution of BEGAIN toward learning and memory. Spatial reference memory and reversal learning in the Barns circular maze test along with contextual fear and cued fear memory in the contextual and cued fear conditioning test were significantly impaired in BEGAIN-KO mice compared to with those in wild-type mice. Thus, this study reveals that BEGAIN is a component of the post-synaptic compartment of excitatory synapses involved in learning and memory.
• Glyoxal fixation: An approach to solve immunohistochemical problem in neuroscience research.

  Kohtarou Konno, Miwako Yamasaki, Taisuke Miyazaki, Masahiko Watanabe

  Science advances 9 (28) eadf7084  2023/07/14 

  The gold-standard fixative for immunohistochemistry is 4% formaldehyde; however, it limits antibody access to target molecules that are buried within specialized neuronal components, such as ionotropic receptors at the postsynapse and voltage-gated ion channels at the axon initial segment, often requiring additional antigen-exposing techniques to detect their authentic signals. To solve this problem, we used glyoxal, a two-carbon atom di-aldehyde. We found that glyoxal fixation greatly improved antibody penetration and immunoreactivity, uncovering signals for buried molecules by conventional immunohistochemical procedures at light and electron microscopic levels. It also enhanced immunosignals of most other molecules, which are known to be detectable in formaldehyde-fixed sections. Furthermore, we unearthed several specific primary antibodies that were once judged to be unusable in formaldehyde-fixed tissues, allowing us to successfully localize so far controversial synaptic adhesion molecule Neuroligin 1. Thus, glyoxal is a highly effective fixative for immunostaining, and a side-by-side comparison of glyoxal and formaldehyde fixation is recommended for routine immunostaining in neuroscience research.
• Neurexins in serotonergic neurons regulate neuronal survival, serotonin transmission, and complex mouse behaviors.

  Amy Cheung, Kotaro Konno, Yuka Imamura, Aya Matsui, Manabu Abe, Kenji Sakimura, Toshikuni Sasaoka, Takeshi Uemura, Masahiko Watanabe, Kensuke Futai

  eLife 12 2023/01/25 

  Extensive serotonin (5-hydroxytryptamine, 5-HT) innervation throughout the brain corroborates 5-HT's modulatory role in numerous cognitive activities. Volume transmission is the major mode for 5-HT transmission but mechanisms underlying 5-HT signaling are still largely unknown. Abnormal brain 5-HT levels and function have been implicated in autism spectrum disorder (ASD). Neurexin (Nrxn) genes encode presynaptic cell adhesion molecules important for the regulation of synaptic neurotransmitter release, notably glutamatergic and GABAergic transmission. Mutations in Nrxn genes are associated with neurodevelopmental disorders including ASD. However, the role of Nrxn genes in the 5-HT system is poorly understood. Here, we generated a mouse model with all three Nrxn genes disrupted specifically in 5-HT neurons to study how Nrxns affect 5-HT transmission. Loss of Nrxns in 5-HT neurons reduced the number of serotonin neurons in the early postnatal stage, impaired 5-HT release, and decreased 5-HT release sites and serotonin transporter expression. Furthermore, 5-HT neuron-specific Nrxn knockout reduced sociability and increased depressive-like behavior. Our results highlight functional roles for Nrxns in 5-HT neurotransmission, 5-HT neuron survival, and the execution of complex behaviors.
• Tmem45b is essential for inflammation- and tissue injury-induced mechanical pain hypersensitivity.

  Tadashi Tanioku, Masayuki Nishibata, Yasuyuki Tokinaga, Kohtaro Konno, Masahiko Watanabe, Hiroaki Hemmi, Yuri Fukuda-Ohta, Tsuneyasu Kaisho, Hidemasa Furue, Tomoyuki Kawamata

  Proceedings of the National Academy of Sciences of the United States of America 119 (45) e2121989119  2022/11/08 

  Persistent mechanical pain hypersensitivity associated with peripheral inflammation, surgery, trauma, and nerve injury impairs patients' quality of life and daily activity. However, the molecular mechanism and treatment are not yet fully understood. Herein, we show that chemical ablation of isolectin B4-binding (IB4+) afferents by IB4-saporin injection into sciatic nerves completely and selectively inhibited inflammation- and tissue injury-induced mechanical pain hypersensitivity while thermal and mechanical pain hypersensitivities were normal following nerve injury. To determine the molecular mechanism involving the specific types of mechanical pain hypersensitivity, we compared gene expression profiles between IB4+ neuron-ablated and control dorsal root ganglion (DRG) neurons. We identified Tmem45b as one of 12 candidate genes that were specific to somatosensory ganglia and down-regulated by IB4+ neuronal ablation. Indeed, Tmem45b was expressed predominantly in IB4+ DRG neurons, where it was selectively localized in the trans Golgi apparatus of DRG neurons but not detectable in the peripheral and central branches of DRG axons. Tmem45b expression was barely detected in the spinal cord and brain. Although Tmem45b-knockout mice showed normal responses to noxious heat and noxious mechanical stimuli under normal conditions, mechanical pain hypersensitivity was selectively impaired after inflammation and tissue incision, reproducing the pain phenotype of IB4+ sensory neuron-ablated mice. Furthermore, acute knockdown by intrathecal injection of Tmem45b small interfering RNA, either before or after inflammation induction, successfully reduced mechanical pain hypersensitivity. Thus, our study demonstrates that Tmem45b is essential for inflammation- and tissue injury-induced mechanical pain hypersensitivity and highlights Tmem45b as a therapeutic target for future treatment.
• Nna1, Essential for Purkinje Cell Survival, Is also Associated with Emotion and Memory

  Li Zhou, Kohtarou Konno, Maya Yamazaki, Manabu Abe, Rie Natsume, Masahiko Watanabe, Hirohide Takebayashi, Kenji Sakimura

  International Journal of Molecular Sciences 23 (21) 12961 - 12961 2022/10/26 [Refereed]
   

  Nna1/CCP1 is generally known as a causative gene for a spontaneous autosomal recessive mouse mutation, Purkinje cell degeneration (pcd). There is enough evidence that the cytosolic function of the zinc carboxypeptidase (CP) domain at the C-terminus of the Nna1 protein is associated with cell death. On the other hand, this molecule’s two nuclear localization signals (NLSs) suggest some other functions exist. We generated exon 3-deficient mice (Nna1N KO), which encode a portion of the N-terminal NLS. Despite the frameshift occurring in these mice, there was an expression of the Nna1 protein lacking the N-terminal side. Surprisingly, the pcd phenotype did not occur in the Nna1N KO mouse. Behavioral analysis revealed that they were less anxious when assessed by the elevated plus maze and the light/dark box tests compared to the control. Furthermore, they showed impairments in context-dependent and sound stimulus-dependent learning. Biochemical analysis of Nna1N KO mice revealed a reduced level of the AMPA-type glutamine receptor GluA2 in the hippocampal synaptosomal fraction. In addition, the motor protein kinesin-1, which transports GluA2 to dendrites, was also decreased. These results indicate that Nna1 is also involved in emotion and memory learning, presumably through the trafficking and expression of synaptic signaling molecules, besides a known role in cell survival.
• Nna1, Essential for Purkinje Cell Survival, Is also Associated with Emotion and Memory. 2022 Oct 26;23(21):12961. doi: 10.3390/ijms232112961. PMID: 36361749.

  Zhou L, Konno K, Yamazaki M, Abe M, Natsume R, Watanabe M, Takebayashi H, Sakimura K

  Int J Mol Sci. 23(21) (12961) 1 - 16 2022/10 [Refereed]
  
• Neutrophils delay repair process in Wallerian degeneration by releasing NETs outside the parenchyma.

  Yasuhiro Yamamoto, Ken Kadoya, Mohamad Alaa Terkawi, Takeshi Endo, Kohtarou Konno, Masahiko Watanabe, Satoshi Ichihara, Akira Hara, Kazuo Kaneko, Norimasa Iwasaki, Muneaki Ishijima

  Life science alliance 5 (10) 2022/10 [Refereed]
   

  Although inflammation is indispensable for the repair process in Wallerian degeneration (WD), the role of neutrophils in the WD repair process remains unclear. After peripheral nerve injury, neutrophils accumulate at the epineurium but not the parenchyma in the WD region because of the blood-nerve barrier. An increase or decrease in the number of neutrophils delayed or promoted macrophage infiltration from the epineurium into the parenchyma and the repair process in WD. Abundant neutrophil extracellular traps (NETs) were formed around neutrophils, and its inhibition dramatically increased macrophage infiltration into the parenchyma. Furthermore, inhibition of either MIF or its receptor, CXCR4, in neutrophils decreased NET formation, resulting in enhanced macrophage infiltration into the parenchyma. Moreover, inhibiting MIF for just 2 h after peripheral nerve injury promoted the repair process. These findings indicate that neutrophils delay the repair process in WD from outside the parenchyma by inhibiting macrophage infiltration via NET formation and that neutrophils, NETs, MIF, and CXCR4 are therapeutic targets for peripheral nerve regeneration.
• Fluorochromized tyramide-glucose oxidase as a multiplex fluorescent tyramide signal amplification system for histochemical analysis.

  Kenta Yamauchi, Shinichiro Okamoto, Yoko Ishida, Kohtarou Konno, Kisara Hoshino, Takahiro Furuta, Megumu Takahashi, Masato Koike, Kaoru Isa, Masahiko Watanabe, Tadashi Isa, Hiroyuki Hioki

  Scientific reports 12 (1) 14807 - 14807 2022/09/12 

  Tyramide signal amplification (TSA) is a highly sensitive method for histochemical analysis. Previously, we reported a TSA system, biotinyl tyramine-glucose oxidase (BT-GO), for bright-filed imaging. Here, we develop fluorochromized tyramide-glucose oxidase (FT-GO) as a multiplex fluorescent TSA system. FT-GO involves peroxidase-catalyzed deposition of fluorochromized tyramide (FT) with hydrogen peroxide produced by enzymatic reaction between glucose and glucose oxidase. We showed that FT-GO enhanced immunofluorescence signals while maintaining low background signals. Compared with indirect immunofluorescence detections, FT-GO demonstrated a more widespread distribution of monoaminergic projection systems in mouse and marmoset brains. For multiplex labeling with FT-GO, we quenched antibody-conjugated peroxidase using sodium azide. We applied FT-GO to multiplex fluorescent in situ hybridization, and succeeded in labeling neocortical interneuron subtypes by coupling with immunofluorescence. FT-GO immunofluorescence further increased the detectability of an adeno-associated virus tracer. Given its simplicity and a staining with a high signal-to-noise ratio, FT-GO would provide a versatile platform for histochemical analysis.
• Targeted proteoform mapping uncovers specific Neurexin-3 variants required for dendritic inhibition.

  David Hauser, Katharina Behr, Kohtarou Konno, Dietmar Schreiner, Alexander Schmidt, Masahiko Watanabe, Josef Bischofberger, Peter Scheiffele

  Neuron 110 (13) 2094 - 2109 2022/07/06 

  The diversification of cell adhesion molecules by alternative splicing is proposed to underlie molecular codes for neuronal wiring. Transcriptomic approaches mapped detailed cell-type-specific mRNA splicing programs. However, it has been hard to probe the synapse-specific localization and function of the resulting protein splice isoforms, or "proteoforms," in vivo. We here apply a proteoform-centric workflow in mice to test the synapse-specific functions of the splice isoforms of the synaptic adhesion molecule Neurexin-3 (NRXN3). We uncover a major proteoform, NRXN3 AS5, that is highly expressed in GABAergic interneurons and at dendrite-targeting GABAergic terminals. NRXN3 AS5 abundance significantly diverges from Nrxn3 mRNA distribution and is gated by translation-repressive elements. Nrxn3 AS5 isoform deletion results in a selective impairment of dendrite-targeting interneuron synapses in the dentate gyrus without affecting somatic inhibition or glutamatergic perforant-path synapses. This work establishes cell- and synapse-specific functions of a specific neurexin proteoform and highlights the importance of alternative splicing regulation for synapse specification.
• Ubiquitin-Specific Protease 2 in the Ventromedial Hypothalamus Modifies Blood Glucose Levels by Controlling Sympathetic Nervous Activation.

  Mayuko Hashimoto, Masaki Fujimoto, Kohtarou Konno, Ming-Liang Lee, Yui Yamada, Koya Yamashita, Chitoku Toda, Michio Tomura, Masahiko Watanabe, Osamu Inanami, Hiroshi Kitamura

  The Journal of neuroscience : the official journal of the Society for Neuroscience 42 (23) 4607 - 4618 2022/06/08 

  Ubiquitin-specific protease 2 (USP2) participates in glucose metabolism in peripheral tissues such as the liver and skeletal muscles. However, the glucoregulatory role of USP2 in the CNS is not well known. In this study, we focus on USP2 in the ventromedial hypothalamus (VMH), which has dominant control over systemic glucose homeostasis. ISH, using a Usp2-specific probe, showed that Usp2 mRNA is present in VMH neurons, as well as other glucoregulatory nuclei, in the hypothalamus of male mice. Administration of a USP2-selective inhibitor ML364 (20 ng/head), into the VMH elicited a rapid increase in the circulating glucose level in male mice, suggesting USP2 has a suppressive role on glucose mobilization. ML364 treatment also increased serum norepinephrine concentration, whereas it negligibly affected serum levels of insulin and corticosterone. ML364 perturbated mitochondrial oxidative phosphorylation in neural SH-SY5Y cells and subsequently promoted the phosphorylation of AMP-activated protein kinase (AMPK). Consistent with these findings, hypothalamic ML364 treatment stimulated AMPKα phosphorylation in the VMH. Inhibition of hypothalamic AMPK prevented ML364 from increasing serum norepinephrine and blood glucose. Removal of ROS restored the ML364-evoked mitochondrial dysfunction in SH-SY5Y cells and impeded the ML364-induced hypothalamic AMPKα phosphorylation as well as prevented the elevation of serum norepinephrine and blood glucose levels in male mice. These results indicate hypothalamic USP2 attenuates perturbations in blood glucose levels by modifying the ROS-AMPK-sympathetic nerve axis.SIGNIFICANCE STATEMENT Under normal conditions (excluding hyperglycemia or hypoglycemia), blood glucose levels are maintained at a constant level. In this study, we used a mouse model to identify a hypothalamic protease controlling blood glucose levels. Pharmacological inhibition of USP2 in the VMH caused a deviation in blood glucose levels under a nonstressed condition, indicating that USP2 determines the set point of the blood glucose level. Modification of sympathetic nervous activity accounts for the USP2-mediated glucoregulation. Mechanistically, USP2 mitigates the accumulation of ROS in the VMH, resulting in attenuation of the phosphorylation of AMPK. Based on these findings, we uncovered a novel glucoregulatory axis consisting of hypothalamic USP2, ROS, AMPK, and the sympathetic nervous system.
• Insight into the function of a unique voltage-sensor protein (TMEM266) and its short form in mouse cerebellum.

  Takafumi Kawai, Hirotaka Narita, Kohtarou Konno, Sharmin Akter, Rizki Tsari Andriani, Hirohide Iwasaki, Shoji Nishikawa, Norihiko Yokoi, Yuko Fukata, Masaki Fukata, Pattama Wiriyasermkul, Pornparn Kongpracha, Shushi Nagamori, Keizo Takao, Tsuyoshi Miyakawa, Manabu Abe, Kenji Sakimura, Masahiko Watanabe, Atsushi Nakagawa, Yasushi Okamura

  The Biochemical journal 2022/05/16 

  Voltage-sensing proteins generally consist of voltage-sensor domains and pore-gate domains, forming the voltage-gated ion channels. However, there are several unconventional voltage-sensor proteins that lack pore-gate domains, conferring them unique voltage-sensing machinery. TMEM266, which is expressed in cerebellum granule cells, is one of the interesting voltage-sensing proteins that has a putative intracellular coiled-coil and a functionally unidentified cytosolic region instead of a pore-gate domain. Here, we approached the molecular function of TMEM266 by performing co-immunoprecipitation experiments. We unexpectedly discovered that TMEM266 proteins natively interact with the novel short form splice variants that only have voltage-sensor domains and putative cytosolic coiled-coil region in cerebellum. The crystal structure of coiled-coil region of TMEM266 suggested that these coiled-coil regions play significant roles in forming homodimers. In vitro expression experiments supported the idea that short form TMEM266 (sTMEM266) or full length TMEM266 (fTMEM266) form homodimers. We also performed proximity labeling mass spectrometry analysis for fTMEM266 and sTMEM266 using Neuro-2A, neuroblastoma cells, and fTMEM266 showed more interacting molecules than sTMEM266, suggesting that the C-terminal cytosolic region in fTMEM266 binds to various targets. Finally, TMEM266-deficient animals showed the moderate abnormality in open-field test. The present study provides clues about the novel voltage-sensing mechanism mediated by TMEM266.
• Histochemical characterization of the dorsal raphe-periaqueductal grey dopamine transporter neurons projecting to the extended amygdala.

  Qin Zhao, Tetsufumi Ito, Chika Soko, Yoshie Hori, Takafumi Furuyama, Hiroyuki Hioki, Kohtarou Konno, Miwako Yamasaki, Masahiko Watanabe, Satoshi Ohtsuka, Munenori Ono, Nobuo Kato, Ryo Yamamoto

  eNeuro 2022/05/13 

  The dorsal raphe (DR) nucleus contains many tyrosine hydroxylase (TH) positive neurons which are regarded as dopaminergic (DA) neurons. These DA neurons in the DR and periaqueductal grey (PAG) region (DADR-PAG neurons) are a subgroup of the A10 cluster, which is known to be heterogeneous. This DA population projects to the central nucleus of the amygdala (CeA) and the bed nucleus of the stria terminalis (BNST) and has been reported to modulate various affective behaviors. To characterize, the histochemical features of DADR-PAG neurons projecting to the CeA and BNST in mice, the current study combined retrograde labeling with fluoro-gold (FG) and histological techniques, focusing on TH, dopamine transporter (DAT), vasoactive intestinal peptide (VIP), and vesicular glutamate transporter 2 (VGlut2). To identify putative DA neurons, DAT-Cre::Ai14 mice were used. It was observed that DATDR-PAG neurons consisted of the following two subpopulations: TH+/VIP- and TH-/VIP+ neurons. The DAT+/TH-/VIP+ subpopulation would be non-DA non-canonical DAT neurons. Anterograde labeling of DATDR-PAG neurons with AAV in DAT-Cre mice revealed that the fibers exclusively innervated the lateral part of the CeA and the oval nucleus of the BNST. Retrograde labeling with FG injections into the CeA or BNST revealed that the two subpopulations similarly innervated these regions. Furthermore, using VGlut2-Cre::Ai14 mice, it was turned out that the TH-/VIP+ subpopulations innervating both CeA and BNST were VGlut2-positive neurons. These two subpopulations of DATDR-PAG neurons, TH+/VIP- and TH-/VIP+, might differentially interfere with the extended amygdala, thereby modulating affective behaviors.Significance StatementDopaminergic (DA) neurons in the dorsal raphe (DR) and periaqueductal grey (PAG) regions have projections to the extended amygdala and have been reported to modulate various affective behaviors. These DA neurons are a subgroup of the A10 cluster, which is known to be heterogeneous. However, it remains unknown how heterogeneous subpopulations innervate the extended amygdala. We used the DA transporter as a DA neuron marker and found that the DAT DR-PAG neurons are composed of at least two subpopulations, DAT+/tyrosine hydroxylase (TH)+/vasoactive intestinal peptide (VIP)- putative DA neurons and DAT+/TH-/VIP+ putative non-DA glutamatergic neurons, innervating the extended amygdala similarly. These results indicate that the two subpopulations might differently modulate the affective behaviors controlled by the extended amygdala.
• SIPA1L1/SPAR1 interacts with the neurabin family of proteins and is involved in GPCR signaling.

  Ken Matsuura, Shizuka Kobayashi, Kohtarou Konno, Miwako Yamasaki, Takahiro Horiuchi, Takao Senda, Tomoatsu Hayashi, Kiyotoshi Satoh, Fumiko Arima-Yoshida, Kei Iwasaki, Lumi Negishi, Naomi Yasui-Shimizu, Kazuyoshi Kohu, Shigenori Kawahara, Yutaka Kirino, Tsutomu Nakamura, Masahiko Watanabe, Tadashi Yamamoto, Toshiya Manabe, Tetsu Akiyama

  The Journal of neuroscience : the official journal of the Society for Neuroscience 42 (12) 2448 - 2473 2022/02/04 

  SIPA1L1 (also known as SPAR1) has been proposed to regulate synaptic functions that are important in maintaining normal neuronal activities, such as regulating spine growth and synaptic scaling, as a component of the PSD-95/NMDA-R-complex. However, its physiological role remains poorly understood. Here, we performed expression analyses using super-resolution microscopy in mouse brain and demonstrated that SIPA1L1 is mainly localized to general submembranous regions in neurons, but surprisingly, not to PSD. Our screening for physiological interactors of SIPA1L1 in mouse brain identified spinophilin and neurabin-1, regulators of GPCR signaling, but rejected PSD-95/NMDA-R-complex components. Furthermore, Sipa1l1 -/- mice showed normal spine size distribution and NMDA-R-dependent synaptic plasticity. Nevertheless, Sipa1l1 -/- mice showed aberrant responses to α2-adrenergic receptor (a spinophilin target) or adenosine A1 receptor (a neurabin-1 target) agonist stimulation, and striking behavioral anomalies, such as hyperactivity, enhanced anxiety, learning impairments, social interaction deficits, and enhanced epileptic seizure susceptibility. Male mice were used for all experiments. Our findings revealed unexpected properties of SIPA1L1, suggesting a possible association of SIPA1L1 deficiency with neuropsychiatric disorders related to dysregulated GPCR signaling, such as epilepsy, attention deficit hyperactivity disorder (ADHD), autism, or fragile X syndrome.SIGNIFICANCE STATEMENTSIPA1L1 is thought to regulate essential synaptic functions as a component of the PSD-95/NMDA-R-complex. In our screening for physiological SIPA1L1-interactors, we identified GPCR-signaling regulators. Moreover, SIPA1L1 KO mice showed striking behavioral anomalies, which may be relevant to GPCR signaling. Our findings revealed an unexpected role of SIPA1L1, which may open new avenues for research on neuropsychiatric disorders that involve dysregulated GPCR signaling. Another important aspect of this paper is that we showed effective methods for checking PSD association and identifying native protein interactors that are difficult to solubilize. These results may serve as a caution for future claims about interacting proteins and PSD proteins, which could eventually save time and resources for researchers and avoid confusion in the field.
• Intravenous transplantation of amnion-derived mesenchymal stem cells promotes functional recovery and alleviates intestinal dysfunction after spinal cord injury.

  Soichiro Takamiya, Masahito Kawabori, Kazuyoshi Yamazaki, Sho Yamaguchi, Aki Tanimori, Koji Yamamoto, Shunsuke Ohnishi, Toshitaka Seki, Kotaro Konno, Khin Khin Tha, Daigo Hashimoto, Masahiko Watanabe, Kiyohiro Houkin, Miki Fujimura

  PloS one 17 (7) e0270606  2022 

  Spinal cord injury (SCI) is often accompanied by gastrointestinal dysfunction due to the disconnection of the spinal autonomic nervous system. Gastrointestinal dysfunction reportedly upregulates intestinal permeability, leading to bacterial translocation of the gut microbiome to the systemic circulation, which further activates systemic inflammation, exacerbating neuronal damage. Mesenchymal stem cells (MSC) reportedly ameliorate SCI. Here, we aimed to investigate their effect on the associated gastrointestinal dysfunction. Human amnion-derived MSC (AMSCs) were intravenously transplanted one day after a rat model of midthoracic SCI. Biodistribution of transplanted cells, behavioral assessment, and histological evaluations of the spinal cord and intestine were conducted to elucidate the therapeutic effect of AMSCs. Bacterial translocation of the gut microbiome was examined by in situ hybridization and bacterial culture of the liver. Systemic inflammations were examined by blood cytokines, infiltrating immune cells in the spinal cord, and the size of the peripheral immune tissue. AMSCs released various neurotrophic factors and were mainly distributed in the liver and lung after transplantation. AMSC-transplanted animals showed smaller spinal damage and better neurological recovery with preserved neuronal tract. AMSCs transplantation ameliorated intestinal dysfunction both morphologically and functionally, which prevented translocation of the gut microbiome to the systemic circulation. Systemic inflammations were decreased in animals receiving AMSCs in the chronic phase. Intravenous AMSC administration during the acute phase of SCI rescues both spinal damage and intestinal dysfunction. Reducing bacterial translocation may contribute to decreasing systemic inflammation.
• 末梢神経損傷後早期免疫反応の詳細 好中球由来MIFによる修復機転の阻害

  山本 康弘, 角家 健, 市原 理司, 原 章, Terkawi Alaa, 今野 幸太郎, 渡辺 雅彦, 岩崎 倫政, 金子 和夫, 石島 旨章

  末梢神経 日本末梢神経学会 32 (2) 268 - 268 0917-6772 2021/12 [Not refereed]
  
• Excitatory and inhibitory receptors utilize distinct post- and trans-synaptic mechanisms in vivo.

  Taisuke Miyazaki, Megumi Morimoto-Tomita, Coralie Berthoux, Kotaro Konno, Yoav Noam, Tokiwa Yamasaki, Matthijs Verhage, Pablo E Castillo, Masahiko Watanabe, Susumu Tomita

  eLife 10 2021/10/18 

  Ionotropic neurotransmitter receptors at postsynapses mediate fast synaptic transmission upon binding of the neurotransmitter. Post- and trans-synaptic mechanisms through cytosolic, membrane, and secreted proteins have been proposed to localize neurotransmitter receptors at postsynapses. However, it remains unknown which mechanism is crucial to maintain neurotransmitter receptors at postsynapses. In this study, we ablated excitatory or inhibitory neurons in adult mouse brains in a cell-autonomous manner. Unexpectedly, we found that excitatory AMPA receptors remain at the postsynaptic density upon ablation of excitatory presynaptic terminals. In contrast, inhibitory GABAA receptors required inhibitory presynaptic terminals for their postsynaptic localization. Consistent with this finding, ectopic expression at excitatory presynapses of neurexin-3 alpha, a putative trans-synaptic interactor with the native GABAA receptor complex, could recruit GABAA receptors to contacted postsynaptic sites. These results establish distinct mechanisms for the maintenance of excitatory and inhibitory postsynaptic receptors in the mature mammalian brain.
• A comparative analysis of kainate receptor GluK2 and GluK5 knockout mice in a pure genetic background.

  Izumi Iida, Kohtarou Konno, Rie Natsume, Manabu Abe, Masahiko Watanabe, Kenji Sakimura, Miho Terunuma

  Behavioural brain research 405 113194 - 113194 2021/05/07 

  Kainate receptors (KARs) are members of the glutamate receptor family that regulate synaptic function in the brain. Although they are known to be associated with psychiatric disorders, how they are involved in these disorders remains unclear. KARs are tetrameric channels assembled from a combination of GluK1-5 subunits. Among these, GluK2 and GluK5 subunits are the major heteromeric subunits in the brain. To determine the functional similarities and differences between GluK2 and GluK5 subunits, we generated GluK2 KO and GluK5 KO mice on a C57BL/6N background, a well-characterized inbred strain, and compared their behavioral phenotypes. We found that GluK2 KO and GluK5 KO mice exhibited the same phenotypes in many tests, such as reduced locomotor activity, impaired motor function, and enhanced depressive-like behavior. No change was observed in motor learning, anxiety-like behavior, or sociability. Additionally, we identified subunit-specific phenotypes, such as reduced motivation toward their environment in GluK2 KO mice and an enhancement in the contextual memory in GluK5 KO mice. These results revealed that GluK2 and GluK5 subunits not only function in a coordinated manner but also have a subunit-specific role in regulating behavior. To summarize, we demonstrated subunit-specific and common behavioral effects of GluK2 and GluK5 subunits for the first time. Moreover, to the best of our knowledge, this is the first evidence of the involvement of the GluK5 subunit in the expression of depressive-like behavior and contextual memory, which strongly indicates its role in psychiatric disorders.
• Immunohistochemistry for Ion Channels and Their Interacting Molecules: Tips for Improving Antibody Accessibility

  Kohtarou Konno, Masahiko Watanabe

  Neuromethods 169 191 - 199 1940-6045 2021 

  Elucidating the molecular organization at synapses is essential for understanding brain function and plasticity. Immunohistochemistry is widely used as a sensitive and specific method in morphological studies. There are specific antibodies directed against receptors, ion channels, and their interacting molecules however, it is sometimes difficult and ineffective to visualize synaptic proteins by conventional immunocytochemistry. This is mainly owing to the fact that the cross-linking of proteins by chemical fixation hampers the accessibility of antibodies to antigen molecules. This is particularly true for receptors and ion channels condensed in the synaptic cleft, postsynaptic density, or trigger zone of action potentials. To overcome this problem, researchers have devised methods to improve immunohistochemical detection of proteins that are hidden or prone to be hidden in condensed molecular matrices. Of these methods, mild chemical fixation by low paraformaldehyde concentrations or fresh frozen sections is often effective in detecting such hidden proteins. Moreover, pretreatment of sections with proteases such as pepsin is a prerequisite to detect proteins embedded in the core of the postsynaptic density, for example, NMDA-type glutamate receptors and their interacting PSD-95 protein family. In this chapter, we introduce these improving techniques for light microscopic immunohistochemistry.
• Mesenchymal Stem Cell Sheet Promotes Functional Recovery and Palliates Neuropathic Pain in a Subacute Spinal Cord Injury Model.

  Kazuyoshi Yamazaki, Masahito Kawabori, Toshitaka Seki, Soichiro Takamiya, Kotaro Konno, Masahiko Watanabe, Kiyohiro Houkin, Miki Fujimura

  Stem cells international 2021 9964877 - 9964877 2021 

  Stem cell therapy has been shown to reverse the sequelae of spinal cord injury (SCI). Although the ideal treatment route remains unknown, providing a large number of stem cells to the injured site using less invasive techniques is critical to achieving maximal recovery. This study was conducted to determine whether administration of bone marrow stem cell (BMSC) sheet made on its own without a scaffold is superior to intramedullary cell transplantation in a rat subacute SCI model. Adult female Sprague-Dawley rats were subjected to SCI by 30 g clip compression at the level of Th6 and Th7 and were administered BMSC cell sheet (7 × 104 cells, subdural), cell suspension (7 × 104 cells, intramedullary), or control seven days after the injury. Motor and sensory assessments, as well as histological evaluation, were performed to determine the efficacy of the different cell transplantation procedures. While both the cell sheet and cell intramedullary injection groups showed significant motor recovery compared to the control group, the cell sheet group showed better results. Furthermore, the cell sheet group displayed a significant sensory recovery compared to the other groups. A histological evaluation revealed that the cell sheet group showed smaller injury lesion volume, less inflammation, and gliosis compared to other groups. Sensory-related fibers of μ-opioid receptors (MOR, interneuron) and hydroxytryptamine transporters (HTT, descending pain inhibitory pathway), located around the dorsal horn of the spinal cord at the caudal side of the SCI, were preserved only in the cell sheet group. Stem cells could also be found inside the peri-injured spinal cord in the cell sheet group. BMSC cell sheets were able to promote functional recovery and palliate neuropathic pain more effectively than intramedullary injections, thus serving as a good treatment option for SCI.
• Specific Neuroligin3-αNeurexin1 signaling regulates GABAergic synaptic function in mouse hippocampus.

  Motokazu Uchigashima, Kohtarou Konno, Emily Demchak, Amy Cheung, Takuya Watanabe, David G Keener, Manabu Abe, Timmy Le, Kenji Sakimura, Toshikuni Sasaoka, Takeshi Uemura, Yuka Imamura Kawasawa, Masahiko Watanabe, Kensuke Futai

  eLife 9 2020/12/23 

  Synapse formation and regulation require signaling interactions between pre- and postsynaptic proteins, notably cell adhesion molecules (CAMs). It has been proposed that the functions of neuroligins (Nlgns), postsynaptic CAMs, rely on the formation of trans-synaptic complexes with neurexins (Nrxns), presynaptic CAMs. Nlgn3 is a unique Nlgn isoform that localizes at both excitatory and inhibitory synapses. However, Nlgn3 function mediated via Nrxn interactions is unknown. Here we demonstrate that Nlgn3 localizes at postsynaptic sites apposing vesicular glutamate transporter 3-expressing (VGT3+) inhibitory terminals and regulates VGT3+ inhibitory interneuron-mediated synaptic transmission in mouse organotypic slice cultures. Gene expression analysis of interneurons revealed that the αNrxn1+AS4 splice isoform is highly expressed in VGT3+ interneurons as compared with other interneurons. Most importantly, postsynaptic Nlgn3 requires presynaptic αNrxn1+AS4 expressed in VGT3+ interneurons to regulate inhibitory synaptic transmission. Our results indicate that specific Nlgn-Nrxn signaling generates distinct functional properties at synapses.
• Autism spectrum disorder-like behavior caused by reduced excitatory synaptic transmission in pyramidal neurons of mouse prefrontal cortex

  Hiroaki Sacai, Kazuto Sakoori, Kohtarou Konno, Kenichiro Nagahama, Honoka Suzuki, Takaki Watanabe, Masahiko Watanabe, Naofumi Uesaka, Masanobu Kano

  Nature Communications 11 (1) 2020/12 

  Autism spectrum disorder (ASD) is thought to result from deviation from normal development of neural circuits and synaptic function. Many genes with mutation in ASD patients have been identified. Here we report that two molecules associated with ASD susceptibility, contactin associated protein-like 2 (CNTNAP2) and Abelson helper integration site-1 (AHI1), are required for synaptic function and ASD-related behavior in mice. Knockdown of CNTNAP2 or AHI1 in layer 2/3 pyramidal neurons of the developing mouse prefrontal cortex (PFC) reduced excitatory synaptic transmission, impaired social interaction and induced mild vocalization abnormality. Although the causes of reduced excitatory transmission were different, pharmacological enhancement of AMPA receptor function effectively restored impaired social behavior in both CNTNAP2- and AHI1-knockdown mice. We conclude that reduced excitatory synaptic transmission in layer 2/3 pyramidal neurons of the PFC leads to impaired social interaction and mild vocalization abnormality in mice.
• Rats deficient in the GAD65 isoform exhibit epilepsy and premature lethality

  Toshikazu Kakizaki, Tomokazu Ohshiro, Makoto Itakura, Kohtarou Konno, Masahiko Watanabe, Hajime Mushiake, Yuchio Yanagawa

  The FASEB Journal 35 (2) 0892-6638 2020/11/25
• FTY720 attenuates neuropathic pain after spinal cord injury by decreasing systemic and local inflammation in a rat spinal cord compression model.

  Kazuyoshi Yamazaki, Masahito Kawabori, Toshitaka Seki, Soichiro Takamiya, Takahiro Tateno, Kotaro Konno, Masahiko Watanabe, Kiyohiro Houkin

  Journal of neurotrauma 2020/03/26 [Refereed][Not invited]
   

  Neuropathic pain severely impairs rehabilitation and quality of life after spinal cord injury (SCI). The sphingosine-1-phosphate receptor agonist FTY720 plays an important protective role in neuronal injury. This study aims to examine the effects of FTY720 in a rat acute SCI model, focusing on neuropathic pain. Female rats with SCI induced by 1-minute clip compression were administered vehicle or 1.5 mg/kg of FTY720 24 hours after the injury. Using the mechanical nociceptive threshold test, we monitored neuropathic pain and performed histological analysis of the pain pathway, including the  opioid receptor (MOR), hydroxytryptamine transporter (HTT), and calcitonin gene-related peptide (CGRP). The motor score, SCI lesion volume, residual motor axons, inflammatory response, glial scar, and microvascular endothelial dysfunction were also compared between the two groups. FTY720 treatment resulted in significant attenuation of post-traumatic neuropathic pain. It also decreased systemic and local inflammation, thereby reducing the damaged areas and astrogliosis and resulting in motor functional recovery. While there was no difference in the CGRP expression between the two groups, FTY720 significantly preserved the MOR in both the caudal and rostral areas of the spinal dorsal horn. While HTT was preserved in the FTY720 group, it was significantly increased in the rostral side and decreased in the caudal side of the injury in the vehicle group. These results suggest that FTY720 ameliorates post-traumatic allodynia through regulation of neuro-inflammation, maintenance of the blood brain barrier, and inhibition of glial scar formation, thereby preserving the connectivity of the descending inhibitory pathway and reducing neuropathic pain.
• PSA-NCAM colocalized with cholecystokinin-expressing cells in the hippocampus is involved in mediating antidepressant efficacy.

  Yamada J, Sato C, Konno K, Watanabe M, Jinno S

  J Neurosci. 2019/12 [Refereed][Not invited]
  
• Expression mapping, quantification, and complex formation of GluD1 and GluD2 glutamate receptors in adult mouse brain.

  Nakamoto C, Konno K, Miyazaki T, Nakatsukasa E, Natsume R, Abe M, Kawamura M, Fukazawa Y, Shigemto R, Yamasaki M, Sakimura K, Watanabe M

  J Comp Neurol. 2019/10 [Refereed][Not invited]
  
• Heterogeneous localization of muscarinic cholinoceptor M1 in the salivary ducts of adult mice.

  Atsara Rawangwong, Suthankamon Khrongyut, Surang Chomphoo, Kohtaro Konno, Miwako Yamasaki, Masahiko Watanabe, Hisatake Kondo, Wiphawi Hipkaeo

  Archives of oral biology 100 14 - 22 2019/04 [Refereed][Not invited]
   

  We hypothesize variation in expression and localization, along the course of the glandular tubule, of muscarinic cholinergic receptor M1 which plays as a distinct contribution, though minor in comparison with M3 receptor, in saliva secretion. Localization of the M1 receptor was examined using immunohistochemistry in three major salivary glands. Although all glandular cells were more or less M1-immunoreactive, acinar cells were weakly immunoreactive, while ductal cells exhibited substantial M1-immunoreactivity. Many ductal cells exhibited clear polarity with higher immunoreactivity in their apical/supra-nuclear domain. However, some exhibited indistinct polarity because of additional higher immunoreactivity in their basal/infra-nuclear domain. A small group of cells with intense immunoreactivity was found, mostly located in the intercalated ducts or in portions of the striated ducts close to the intercalated ducts. In immuno-electron microscopy, the immunoreactive materials were mainly in the cytoplasm including various vesicles and vacuoles. Unexpectedly, distinct immunoreactivity on apical and basal plasma membranes was infrequent in most ductal cells. The heterogeneous localization of M1-immunoreactivity along the gland tubular system is discussed in view of possible modulatory roles of the M1 receptor in saliva secretion.
• Selective distribution of GLUT3-expressing nerve fibers in the lamina terminalis among the circumventricular organs of mice.

  Toshihiko Iwanaga, Kohtarou Konno, Masahiko Watanabe

  Biomedical research (Tokyo, Japan) 40 (5) 207 - 214 2019 [Refereed][Not invited]
   

  Sensory circumventricular organs contain the subfornical organ, organum vasculosum of the lamina terminalis (OVLT), and area postrema. Here, immunostaining for GLUT3 in the murine brain selectively labeled the sobfornical organ and OVLT. The immunoreactive neural tract of the subfornical organ formed into thin bundles and extended ventro-rostrally over the anterior commissure. After turning over the commissure, the neural tract passed through the median preoptic nucleus (MnPO) and reached the OVLT; thus, a continuous neural tract expressing GLUT3 connected the subfornical organ, MnPO, and OVLT in the lamina terminalis. In the OVLT, GLUT3-immunoreactive fibers gathered in both the dorsal cap and lateral periventricular zone. Electron microscopically, the immunoreactive structures in the subfornical organ corresponded to nerve fibers or nerve terminals containing many small clear vesicles. The area postrema, another sensory organ, was immunonegative for GLUT3. This study not only presented a useful marker tracing the neural tract in the sensory sites of the lamina terminalis but also suggested a unique system for sensing and determining the metabolism of circulating glucose in the circumventricular organs.
• Deletion of exons encoding carboxypeptidase domain of Nna1 results in Purkinje cell degeneration (pcd) phenotype.

  Li Zhou, M Ibrahim Hossain, Maya Yamazaki, Manabu Abe, Rie Natsume, Kohtaro Konno, Shun Kageyama, Masaaki Komatsu, Masahiko Watanabe, Kenji Sakimura, Hirohide Takebayashi

  Journal of neurochemistry 147 (4) 557 - 572 2018/11 [Refereed][Not invited]
   

  Purkinje cell degeneration (pcd) was first identified in a spontaneous mouse mutant showing cerebellar ataxia. In addition to cerebellar Purkinje cells (PCs), retinal photoreceptors, mitral cells in the olfactory bulb, and a discrete subpopulation of thalamic neurons also degenerate in the mutant brains. The gene responsible for the pcd mutant is Nna1, also known as ATP/GTP binding protein 1 or cytosolic carboxypeptidase-like 1, which encodes a zinc carboxypeptidase protein. To investigate pathogenesis of the pcd mutation in detail, we generated a conditional Nna1 allele targeting the carboxypeptidase domain at C-terminus. After Cre recombination and heterozygous crossing, we generated Nna1 knockout (KO) mice and found that the Nna1 KO mice began to show cerebellar ataxia at postnatal day 20 (P20). Most PCs degenerated until 4-week-old, except lobule X. Activated microglia and astrocytes were also observed in the Nna1 KO cerebellum. In the mutant brain, the Nna1 mRNA level was dramatically reduced, suggesting that nonsense-mediated mRNA decay occurs in it. Since the Nna1 protein acts as a de-glutamatase on the C-terminus of α-tubulin and β-tubulin, increased polyglutamylated tubulin was detected in the Nna1 KO cerebellum. In addition, the endoplasmic reticulum stress marker, C/EBP homologous protein (CHOP), was up-regulated in the mutant PCs. We report the generation of a functional Nna1 conditional allele and possible mechanisms of PC death in the Nna1 KO in the cerebellum. OPEN PRACTICES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.
• CDC42EP4, a perisynaptic scaffold protein in Bergmann glia, is required for glutamatergic tripartite synapse configuration.

  Natsumi Ageta-Ishihara, Kohtarou Konno, Maya Yamazaki, Manabu Abe, Kenji Sakimura, Masahiko Watanabe, Makoto Kinoshita

  Neurochemistry international 119 190 - 198 2018/10 [Refereed][Not invited]
   

  Configuration of tripartite synapses, comprising the pre-, post-, and peri-synaptic components (axon terminal or bouton, dendritic spine, and astroglial terminal process), is a critical determinant of neurotransmitter kinetics and hence synaptic transmission. However, little is known about molecular basis for the regulation of tripartite synapse morphology. Previous studies showed that CDC42EP4, an effector protein of a cell morphogenesis regulator CDC42, is expressed exclusively in Bergmann glia in the cerebellar cortex, that it forms tight complex with the septin heterooligomer, and that it interacts indirectly with the glutamate transporter GLAST and MYH10/nonmuscle myosin ΙΙB. Scrutiny of Cdc42ep4-/- mice had revealed that the CDC42EP4-septins-GLAST interaction facilitates glutamate clearance, while the role for CDC42EP4-septins-MYH10 interaction has remained unsolved. Here, we find anomalous configuration of the tripartite synapses comprising the parallel fiber boutons, dendritic spines of Purkinje cells, and Bergmann glial processes in Cdc42ep4-/- mice. The complex anomalies include 1) recession of Bergmann glial membranes from the nearest active zones, and 2) extension of nonactive synaptic contact around active zone. In line with the recession of Bergmann glial membranes by the loss of CDC42EP4, overexpression of CDC42EP4 in heterologous cells promotes cell spreading and partitioning of MYH10 to insoluble (i.e., active) fraction. Paradoxically, however, Cdc42ep4-/- cerebellum contained significantly more MYH10 and N-cadherin, which is attributed to secondary neuronal response mainly in Purkinje cells. Given cooperative actions of N-cadherin and MYH10 for adhesion between neurons, we speculate that their augmentation may reflect the extension of nonactive synaptic contacts in Cdc42ep4-/- cerebellum. Transcellular mechanism that links the absence of CDC42EP4 in Bergmann glia to the augmentation of N-cadherin and MYH10 in neurons is currently unknown, but the phenotypic similarity to GLAST-null mice indicates involvement of the glutamate intolerance. Together, the unique phenotype of Cdc42ep4-/- mice provides a clue to novel molecular network underlying tripartite synapse configuration.
• ASCT1 (Slc1a4) transporter is a physiologic regulator of brain d-serine and neurodevelopment.

  Kaplan E, Zubedat S, Radzishevsky I, Valenta AC, Rechnitz O, Sason H, Sajrawi C, Bodner O, Konno K, Esaki K, Derdikman D, Yoshikawa T, Watanabe M, Kennedy RT, Billard JM, Avital A, Wolosker H

  Proc Natl Acad Sci U S A. 115 (38) 9628 - 9633 2018/09 [Refereed][Not invited]
   

  d-serine is a physiologic coagonist of NMDA receptors, but little is known about the regulation of its synthesis and synaptic turnover. The amino acid exchangers ASCT1 (Slc1a4) and ASCT2 (Slc1a5) are candidates for regulating d-serine levels. Using ASCT1 and ASCT2 KO mice, we report that ASCT1, rather than ASCT2, is a physiologic regulator of d-serine metabolism. ASCT1 is a major d-serine uptake system in astrocytes and can also export l-serine via heteroexchange, supplying neurons with the substrate for d-serine synthesis. ASCT1-KO mice display lower levels of brain d-serine along with higher levels of l-alanine, l-threonine, and glycine. Deletion of ASCT1 was associated with neurodevelopmental alterations including lower hippocampal and striatal volumes and changes in the expression of neurodevelopmental-relevant genes. Furthermore, ASCT1-KO mice exhibited deficits in motor function, spatial learning, and affective behavior, along with changes in the relative contributions of d-serine vs. glycine in mediating NMDA receptor activity. In vivo microdialysis demonstrated lower levels of extracellular d-serine in ASCT1-KO mice, confirming altered d-serine metabolism. These alterations are reminiscent of some of the neurodevelopmental phenotypes exhibited by patients with ASCT1 mutations. ASCT1-KO mice provide a useful model for potential therapeutic interventions aimed at correcting the metabolic impairments in patients with ASCT1 mutations.
• Retrograde Signaling from Progranulin to Sort1 Counteracts Synapse Elimination in the Developing Cerebellum.

  Naofumi Uesaka, Manabu Abe, Kohtarou Konno, Maya Yamazaki, Kazuto Sakoori, Takaki Watanabe, Tzu-Huei Kao, Takayasu Mikuni, Masahiko Watanabe, Kenji Sakimura, Masanobu Kano

  Neuron 97 (4) 796 - 805 2018/02/21 [Refereed][Not invited]
   

  Elimination of redundant synapses formed early in development and strengthening of necessary connections are crucial for shaping functional neural circuits. Purkinje cells (PCs) in the neonatal cerebellum are innervated by multiple climbing fibers (CFs) with similar strengths. A single CF is strengthened whereas the other CFs are eliminated in each PC during postnatal development. The underlying mechanisms, particularly for the strengthening of single CFs, are poorly understood. Here we report that progranulin, a multi-functional growth factor implicated in the pathogenesis of frontotemporal dementia, strengthens developing CF synaptic inputs and counteracts their elimination from postnatal day 11 to 16. Progranulin derived from PCs acts retrogradely onto its putative receptor Sort1 on CFs. This effect is independent of semaphorin 3A, another retrograde signaling molecule that counteracts CF synapse elimination. We propose that progranulin-Sort1 signaling strengthens and maintains developing CF inputs, and may contribute to selection of single "winner" CFs that survive synapse elimination.
• Repeated fluvoxamine treatment recovers early postnatal stress-induced hypersociability-like behavior in adult rats

  Haruko Kumamoto, Taku Yamaguchi, Kohtarou Konno, Takeshi Izumi, Takayuki Yoshida, Yu Ohmura, Masahiko Watanabe, Mitsuhiro Yoshioka

  Journal of Pharmacological Sciences 136 (1) 1 - 8 1347-8648 2018/01/01 [Refereed][Not invited]
   

  Childhood maltreatment is associated with impaired adult brain function, particularly in the hippocampus, and is not only a major risk factor for some psychiatric diseases but also affects early social development and social adaptation in later life. The aims of this study were to determine whether early postnatal stress affects social behavior and whether repeated fluvoxamine treatment reverses these changes. Rat pups were exposed to footshock stress during postnatal days 21–25 (at 3 weeks old: 3wFS). During the post-adolescent period (10–14 weeks postnatal), the social interaction test and Golgi-cox staining of dorsal hippocampal pyramidal neurons were performed. Following exposure to footshock stress, 3wFS rats showed an increase in social interaction time, which might be practically synonymous with hypersociability, and a decrease in spine density in the CA3 hippocampal subregion, but not in CA1. These behavioral and morphological changes were both recovered by repeated oral administration of fluvoxamine at a dose of 10 mg/kg/day for 14 days. These findings suggest that the vulnerability of the hippocampal CA3 region is closely related to social impairments induced by physical stress during the juvenile period and shed some light on therapeutic alternatives for early postnatal stress-induced emotional dysfunction.
• Intrarenal signaling mediated by CCK plays a role in salt intake-induced natriuresis

  Hiromi Takahashi-Iwanaga, Shunsuke Kimura, Kohtarou Konno, Masahiko Watanabe, Toshihiko Iwanaga

  AMERICAN JOURNAL OF PHYSIOLOGY-RENAL PHYSIOLOGY 313 (1) F20 - F29 1931-857X 2017/07 [Refereed][Not invited]
   

  The natriuretic hormone CCK exhibits its gene transcripts in total kidney extracts. To test the possibility of CCK acting as an intrarenal mediator of sodium excretion, we examined mouse kidneys by 1) an in situ hybridization technique for CCK mRNA in animals fed a normal- or a high-sodium diet; 2) immuno-electron microscopy for the CCK peptide, 3) an in situ hybridization method and immunohistochemistry for the CCK-specific receptor CCKAR; 4) confocal image analysis of receptor-mediated Ca2+ responses in isolated renal tubules; and 5) metabolic cage experiments for the measurement of urinary sodium excretion in high-salt-fed mice either treated or untreated with the CCKAR antagonist lorglumide. Results showed the CCK gene to be expressed intensely in the inner medulla and moderately in the inner stripe of the outer medulla, with the expression in the latter being enhanced by high sodium intake. Immunoreactivity for the CCK peptide was localized to the rough endoplasmic reticulum of the medullary interstitial cells in corresponding renal regions, confirming it to be a secretory protein. Gene transcripts, protein products, and the functional activity for CCKAR were consistently localized to the late proximal tubule segments (S2 and S3) in the medullary rays, and the outer stripe of the outer medulla. Lorglumide significantly diminished natriuretic responses of mice to a dietary sodium load without altering the glomerular filtration rate. These findings suggest that the medullary interstitial cells respond to body fluid expansion by CCK release for feedback regulation of the late proximal tubular reabsorption.
• Distribution of corticotropin-releasing factor neurons in the mouse brain: a study using corticotropin-releasing factor-modified yellow fluorescent protein knock-in mouse

  Junko Kono, Kohtarou Konno, Ashraf Hossain Talukder, Toshimitsu Fuse, Manabu Abe, Katsuya Uchida, Shuhei Horio, Kenji Sakimura, Masahiko Watanabe, Keiichi Itoi

  BRAIN STRUCTURE & FUNCTION 222 (4) 1705 - 1732 1863-2653 2017/05 [Refereed][Not invited]
   

  We examined the morphological features of corticotropin-releasing factor (CRF) neurons in a mouse line in which modified yellow fluorescent protein (Venus) was expressed under the CRF promoter. We previously generated the CRF-Venus knock-in mouse, in which Venus is inserted into the CRF gene locus by homologous recombination. In the present study, the neomycin phosphotransferase gene (Neo), driven by the pgk-1 promoter, was deleted from the CRF-Venus mouse genome, and a CRF-Venusa dagger Neo mouse was generated. Venus expression is much more prominent in the CRF-Venusa dagger Neo mouse when compared to the CRF-Venus mouse. In addition, most Venus-expressing neurons co-express CRF mRNA. Venus-expressing neurons constitute a discrete population of neuroendocrine neurons in the paraventricular nucleus of the hypothalamus (PVH) that project to the median eminence. Venus-expressing neurons were also found in brain regions outside the neuroendocrine PVH, including the olfactory bulb, the piriform cortex (Pir), the extended amygdala, the hippocampus, the neocortices, Barrington's nucleus, the midbrain/pontine dorsal tegmentum, the periaqueductal gray, and the inferior olivary nucleus (IO). Venus-expressing perikarya co-expressing CRF mRNA could be observed clearly even in regions where CRF-immunoreactive perikarya could hardly be identified. We demonstrated that the CRF neurons contain glutamate in the Pir and IO, while they contain gamma-aminobutyric acid in the neocortex, the bed nucleus of the stria terminalis, the hippocampus, and the amygdala. A population of CRF neurons was demonstrated to be cholinergic in the midbrain tegmentum. The CRF-Venusa dagger Neo mouse may be useful for studying the structural and functional properties of CRF neurons in the mouse brain.
• Late postnatal shifts of parvalbumin and nitric oxide synthase expression within the GABAergic and glutamatergic phenotypes of inferior colliculus neurons

  Hisataka Fujimoto, Kotaro Konno, Masahiko Watanabe, Shozo Jinno

  JOURNAL OF COMPARATIVE NEUROLOGY 525 (4) 868 - 884 0021-9967 2017/03 [Refereed][Not invited]
   

  The inferior colliculus (IC) is partitioned into three subdivisions: the dorsal and lateral cortices (DC and LC) and the central nucleus (ICC), and serves as an integration center of auditory information. Recent studies indicate that a certain population of IC neurons may represent the non-GABAergic phenotype, while they express well-established cortical/hippocampal GABAergic neuron markers. In this study we used the optical disector to investigate the phenotype of IC neurons expressing parvalbumin (PV) and/or nitric oxide synthase (NOS) in C57BL/6J mice during the late postnatal period. Four major types of IC neurons were defined by the presence (+) or absence (-) of PV, NOS, and glutamic acid decarboxylase 67 (GAD67): PV+/NOS-/GAD67(+), PV+/NOS+/GAD67(+), PV+/NOS-/GAD67(-), and PV-/NOS+/GAD67(-). Fluorescent in situ hybridization for vesicular glutamate transporter 2 mRNA indicated that almost all GAD67(-) IC neurons represented the glutamatergic phenotype. The numerical densities (NDs) of total GAD67(+) IC neurons remained unchanged in all subdivisions. The NDs of PV+/NOS-/GAD67(+) neurons and PV-/NOS+/GAD67(-) neurons were reduced with age in the ICC, while they remained unchanged in the DC and LC. By contrast, the NDs of PV+/NOS+/GAD67(+) neurons and PV+/NOS-/GAD67(-) neurons were increased with age in the ICC, although there were no changes in the DC and LC. The cell body size of GAD67(+) IC neurons did not vary according to the expression of PV with or without NOS. The present findings indicate that the expression of PV and NOS may shift with age within the GABAergic and glutamatergic phenotypes of IC neurons during the late postnatal period. J. Comp. Neurol. 525:868-884, 2017. (c) 2016 Wiley Periodicals, Inc.
• Roles of Cbln1 in Non-Motor Functions of Mice

  Shintaro Otsuka, Kohtarou Konno, Manabu Abe, Junko Motohashi, Kazuhisa Kohda, Kenji Sakimura, Masahiko Watanabe, Michisuke Yuzaki

  JOURNAL OF NEUROSCIENCE 36 (46) 11801 - 11816 0270-6474 2016/11 [Refereed][Not invited]
   

  The cerebellum is thought to be involved in cognitive functions in addition to its well established role in motor coordination and motor learning in humans. Cerebellin 1 (Cbln1) is predominantly expressed in cerebellar granule cells and plays a crucial role in the formation and function of parallel fiber-Purkinje cell synapses. Although genes encoding Cbln1 and its postsynaptic receptor, the delta2 glutamate receptor (GluD2), are suggested to be associated with autistic-like traits and many psychiatric disorders, whether such cognitive impairments are caused by cerebellar dysfunction remains unclear. In the present study, we investigated whether and how Cbln1 signaling is involved in non-motor functions in adult mice. We show that acquisition and retention/retrieval of cued and contextual fear memory were impaired in Cbln1-null mice. In situ hybridization and immunohistochemical analyses revealed that Cbln1 is expressed in various extracerebellar regions, including the retrosplenial granular cortex and the hippocampus. In the hippocampus, Cbln1 immunoreactivity was present at the molecular layer of the dentate gyrus and the stratum lacunosum-moleculare without overt mRNA expression, suggesting that Cbln1 is provided by perforant path fibers. Retention/retrieval, but not acquisition, of cued and contextual fear memory was impaired in forebrain-predominant Cbln1-null mice. Spatial learning in the radial arm water maze was also abrogated. In contrast, acquisition of fear memory was affected in cerebellum-predominant Cbln1-null mice. These results indicate that Cbln1 in the forebrain and cerebellum mediates specific aspects of fear conditioning and spatial memory differentially and that Cbln1 signaling likely regulates motor and non-motor functions in multiple brain regions.
• Neural FFA3 activation inversely regulates anion secretion evoked by nicotinic ACh receptor activation in rat proximal colon

  Izumi Kaji, Yasutada Akiba, Kohtarou Konno, Masahiko Watanabe, Shunsuke Kimura, Toshihiko Iwanaga, Ayaka Kuri, Ken-ichi Iwamoto, Atsukazu Kuwahara, Jonathan D. Kaunitz

  JOURNAL OF PHYSIOLOGY-LONDON 594 (12) 3339 - 3352 0022-3751 2016/06 [Refereed][Not invited]
   

  The proximal colonic mucosa is constantly exposed to high concentrations of microbially-produced short-chain fatty acids (SCFAs). Although luminal SCFAs evoke electrogenic anion secretion and smooth muscle contractility via neural and non-neural cholinergic pathways in the colon, the involvement of the SCFA receptor free fatty acid receptor (FFA)3, one of the free fatty acid receptor family members, has not been clarified. We investigated the contribution of FFA3 to cholinergic-mediated secretory responses in rat proximal colon. FFA3 was immunolocalized to enteroendocrine cells and to the enteric neural plexuses. Most FFA3-immunoreactive nerve fibres and nerve endings were cholinergic, colocalized with protein gene product (PGP)9.5, the vesicular ACh transporter, and the high-affinity choline transporter CHT1. In Ussing chambered mucosa-submucosa preparations (including the submucosal plexus) of rat proximal colon, carbachol (CCh)-induced Cl- secretion was decreased by TTX, hexamethonium, and the serosal FFA3 agonists acetate or propionate, although not by an inactive analogue 3-chloropropionate. Serosal application of a selective FFA3 agonist (N-[2-methylphenyl]-[4-furan-3-yl]-2-methyl-5-oxo-1,4,5,6,7,8-hexahydro-quinoline-3-carboxamide; MQC) dose-dependently suppressed the response to CCh but not to forskolin, with an IC50 of 13m. Pretreatment with MQC inhibited nicotine-evoked but not bethanechol-evoked secretion. The inhibitory effect of MQC was reversed by pretreatment with pertussis toxin, indicating that FFA3 acts via the G(i/o) pathway. Luminal propionate induced Cl- secretion via the cholinergic pathway, which was reduced by MQC, as well as by TTX, hexamethonium or removal of the submucosal plexus. These results suggest that the SCFA-FFA3 pathway has a novel anti-secretory function in that it inhibits cholinergic neural reflexes in the enteric nervous system.
• Serotonin 5-HT7 Receptor in the Ventral Hippocampus Modulates the Retrieval of Fear Memory and Stress-Induced Defecation

  Yu Ohmura, Takayuki Yoshida, Kohtarou Konno, Masabumi Minami, Masahiko Watanabe, Mitsuhiro Yoshioka

  INTERNATIONAL JOURNAL OF NEUROPSYCHOPHARMACOLOGY 19 (6) 1461-1457 2016/06 [Refereed][Not invited]
   

  Background: Patients with posttraumatic stress disorder or panic disorder are often troubled by inappropriate retrieval of fear memory. Moreover, these disorders are often comorbid with irritable bowel syndrome. The main aim of the present study is to elucidate the involvement of hippocampal serotonergic systems in fear memory retrieval and stress-induced defecation. Methods and Results: Microinjection of serotonin 7 receptor antagonist, but not other serotonin receptor antagonists (serotonin (1A), (2A), (2C), (3), (4), and (6)), into the rat ventral hippocampus significantly suppressed the expression of freezing behavior, an index of fear memory retrieval, and decreased the amount of feces, an index of stress-induced defecation, in the contextual fear conditioning test. Electrophysiological data indicated that the serotonin 7 receptor agonist increased the frequency of action potentials in the ventral hippocampal CA3 pyramidal neuron via the activation of the hyperpolarization-activated nonselective cation current I-h. Moreover, in situ hybridization demonstrated that Htr7 mRNA was abundantly expressed in the CA3 compared with other subregions of the hippocampus and that these Htr7 mRNA-positive cells coexpressed hyperpolarization-activated cyclic nucleotide-gated channel 2 and 4 mRNAs, which are components of the I-h channel. Conclusions: These results indicated that the released serotonin activates the serotonin 7 receptor in the CA3 ventral hippocampus subregion, enhances the sensitivity to inputs via hyperpolarization-activated cyclic nucleotide 2 and 4 channels, and thereby facilitates fear memory retrieval. The serotonin 7 receptor might be a target of drug development for the treatment of mental disorders involving fear memory and gastrointestinal problems.
• Transsynaptic Modulation of Kainate Receptor Functions by C1q-like Proteins

  Keiko Matsuda, Timotheus Budisantoso, Nikolaos Mitakidis, Yuki Sugaya, Eriko Miura, Wataru Kakegawa, Miwako Yamasaki, Kohtarou Konno, Motokazu Uchigashima, Manabu Abe, Izumi Watanabe, Masanobu Kano, Masahiko Watanabe, Kenji Sakimura, A. Radu Aricescu, Michisuke Yuzaki

  NEURON 90 (4) 752 - 767 0896-6273 2016/05 [Refereed][Not invited]
   

  Postsynaptic kainate-type glutamate receptors (KARs) regulate synaptic network activity through their slow channel kinetics, most prominently at mossy fiber (MF)-CA3 synapses in the hippocampus. Nevertheless, how KARs cluster and function at these synapses has been unclear. Here, we show that C1q-like proteins C1ql2 and C1ql3, produced by MFs, serve as extracellular organizers to recruit functional postsynaptic KAR complexes to the CA3 pyramidal neurons. C1ql2 and C1ql3 specifically bound the amino-terminal domains of postsynaptic GluK2 and GluK4 KAR subunits and the presynaptic neurexin 3 containing a specific sequence in vitro. In C1ql2/3 double-null mice, CA3 synaptic responses lost the slow, KAR-mediated components. Furthermore, despite induction of MF sprouting in a temporal lobe epilepsy model, KARs were not recruited to postsynaptic sites in C1ql2/3 double-null mice, leading to reduced recurrent circuit activities. C1q family proteins, broadly expressed, are likely to modulate KAR function throughout the brain and represent promising antiepileptic targets.
• Emerging roles of ARHGAP33 in intracellular trafficking of TrkB and pathophysiology of neuropsychiatric disorders (vol 7, pg 10594, 2016)

  Takanobu Nakazawa, Ryota Hashimoto, Kazuto Sakoori, Yuki Sugaya, Asami Tanimura, Yuki Hashimotodani, Kazutaka Ohi, Hidenaga Yamamori, Yuka Yasuda, Satomi Umeda-Yano, Yuji Kiyama, Kohtarou Konno, Takeshi Inoue, Kazumasa Yokoyama, Takafumi Inoue, Shusuke Numata, Tohru Ohnuma, Nakao Iwata, Norio Ozaki, Hitoshi Hashimoto, Masahiko Watanabe, Toshiya Manabe, Tadashi Yamamoto, Masatoshi Takeda, Masanobu Kano

  NATURE COMMUNICATIONS 7 11466  2041-1723 2016/04 [Refereed][Not invited]
  
• Involvement of hormones in olfactory imprinting and homing in chum salmon

  Hiroshi Ueda, Shingo Nakamura, Taro Nakamura, Kaoru Inada, Takashi Okubo, Naohiro Furukawa, Reiichi Murakami, Shigeo Tsuchida, Yonathan Zohar, Kotaro Konno, Masahiko Watanabe

  SCIENTIFIC REPORTS 6 2045-2322 2016/02 [Refereed][Not invited]
   

  The olfactory hypothesis for salmon imprinting and homing to their natal stream is well known, but the endocrine hormonal control mechanisms of olfactory memory formation in juveniles and retrieval in adults remain unclear. In brains of hatchery-reared underyearling juvenile chum salmon (Oncorhynchus keta), thyrotropin-releasing hormone gene expression increased immediately after release from a hatchery into the natal stream, and the expression of the essential NR1 subunit of the N-methyl-D-aspartate receptor increased during downstream migration. Gene expression of salmon gonadotropin-releasing hormone (sGnRH) and NR1 increased in the adult chum salmon brain during homing from the Bering Sea to the natal hatchery. Thyroid hormone treatment in juveniles enhanced NR1 gene activation, and GnRHa treatment in adults improved stream odour discrimination. Olfactory memory formation during juvenile downstream migration and retrieval during adult homing migration of chum salmon might be controlled by endocrine hormones and could be clarified using NR1 as a molecular marker.
• Emerging roles of ARHGAP33 in intracellular trafficking of TrkB and pathophysiology of neuropsychiatric disorders

  Nakazawa Takanobu, Hashimoto Ryota, Sakoori Kazuto, Sugaya Yuki, Tanimura Asami, Hashimotodani Yuki, Ohi Kazutaka, Yamamori Hidenaga, Yasuda Yuka, Umeda-Yano Satomi, Kiyama Yuji, Konno Kohtarou, Inoue Takeshi, Yokoyama Kazumasa, Inoue Takafumi, Numata Shusuke, Ohnuma Tohru, Iwata Nakao, Ozaki Norio, Hashimoto Hitoshi, Watanabe Masahiko, Manabe Toshiya, Yamamoto Tadashi, Takeda Masatoshi, Kano Masanobu

  Nature Communications 7 2016 [Refereed][Not invited]
  
• Determination of kainate receptor subunit ratios in mouse brain using novel chimeric protein standards

  Izumi Watanabe-Iida, Kohtarou Konno, Kaori Akashi, Manabu Abe, Rie Natsume, Masahiko Watanabe, Kenji Sakimura

  JOURNAL OF NEUROCHEMISTRY 136 (2) 295 - 305 0022-3042 2016/01 [Refereed][Not invited]
   

  Kainate-type glutamate receptors (KARs) are tetrameric channels assembled from GluK1-5. GluK1-3 are low-affinity subunits that form homomeric and heteromeric KARs, while GluK4 and GluK5 are high-affinity subunits that require co-assembly with GluK1-3 for functional expression. Although the subunit composition is thought to be highly heterogeneous in the brain, the distribution of KAR subunits at the protein level and their relative abundance in given regions of the brain remain largely unknown. In the present study, we titrated C-terminal antibodies to each KAR subunit using chimeric GluA2-GluK fusion proteins, and measured their relative abundance in the P2 and post-synaptic density (PSD) fractions of the adult mouse hippocampus and cerebellum. Analytical western blots showed that GluK2 and GluK3 were the major KAR subunits, with additional expression of GluK5 in the hippocampus and cerebellum. In both regions, GluK4 was very low and GluK1 was below the detection threshold. The relative amount of low-affinity subunits (GluK2 plus GluK3) was several times higher than that of high-affinity subunits (GluK4 plus GluK5) in both regions. Of note, the highest ratio of high-affinity subunits to low-affinity subunits was found in the hippocampal PSD fraction (0.32), suggesting that heteromeric receptors consisting of high- and low-affinity subunits highly accumulate at hippocampal synapses. In comparison, this ratio was decreased to 0.15 in the cerebellar PSD fraction, suggesting that KARs consisting of low-affinity subunits are more prevalent in the cerebellum. Therefore, low-affinity KARsubunits are predominant in the brain, with distinct subunitcombinations between the hippocampus and cerebellum.
• A CDC42EP4/septin-based perisynaptic glial scaffold facilitates glutamate clearance

  Natsumi Ageta-Ishihara, Maya Yamazaki, Kohtarou Konno, Hisako Nakayama, Manabu Abe, Kenji Hashimoto, Tomoki Nishioka, Kozo Kaibuchi, Satoko Hattori, Tsuyoshi Miyakawa, Kohichi Tanaka, Fathul Huda, Hirokazu Hirai, Kouichi Hashimoto, Masahiko Watanabe, Kenji Sakimura, Makoto Kinoshita

  NATURE COMMUNICATIONS 6 2041-1723 2015/12 [Refereed][Not invited]
   

  The small GTPase-effector proteins CDC42EP1-5/BORG1-5 interact reciprocally with CDC42 or the septin cytoskeleton. Here we show that, in the cerebellum, CDC42EP4 is exclusively expressed in Bergmann glia and localizes beneath specific membrane domains enwrapping dendritic spines of Purkinje cells. CDC42EP4 forms complexes with septin hetero-oligomers, which interact with a subset of glutamate transporter GLAST/EAAT1. In Cdc42ep4(-/-) mice, GLAST is dissociated from septins and is delocalized away from the parallel fibre-Purkinje cell synapses. The excitatory postsynaptic current exhibits a protracted decay time constant, reduced sensitivity to a competitive inhibitor of the AMPA-type glutamate receptors (gamma DGG) and excessive baseline inward current in response to a subthreshold dose of a nonselective inhibitor of the glutamate transporters/EAAT1-5 (DL-TBOA). Insufficient glutamate-buffering/clearance capacity in these mice manifests as motor coordination/learning defects, which are aggravated with subthreshold DL-TBOA. We propose that the CDC42EP4/septin-based glial scaffold facilitates perisynaptic localization of GLAST and optimizes the efficiency of glutamate-buffering and clearance.
• Repeated fluvoxamine treatment recovers juvenile stress-induced morphological changes and depressive-like behavior in rats

  Kerise Lyttle, Yu Ohmura, Kohtarou Konno, Takayuki Yoshida, Takeshi Izumi, Masahiko Watanabe, Mitsuhiro Yoshioka

  BRAIN RESEARCH 1616 88 - 100 0006-8993 2015/08 [Refereed][Not invited]
   

  Human studies have suggested that early life stress such as child abuse could enhance susceptibility to depressive disorders. Moreover, the abnormalities of the prefrontal cortex have been associated with depression. Although clinical studies have implied the negative effects of early life stress on brain development, the causality and the detailed morphogenetic changes has not been clearly elucidated. In the present study, we determined the effect of juvenile stress exposure on the presentation of depressive-like behavior and the neural mechanisms involved using a rodent model. Rat pups were exposed to footshock stress during postnatal days 21-25 followed by repeated oral administration of fluvoxamine (0 or 10 mg/kg/d x 14 days), which is a selective serotonin reuptake inhibitor. At the postadolescent stage forced swim test assessment of depressive-like behavior and Golgi Cox staining of medial prefrontal cortex pyramidal neurons followed by morphological analyses were carried out. Post-adolescent behavioral and morphological studies identified the presentation of increased depressive-like behaviors and reduced spine densities and dendritic lengths of layer II/III pyramidal neuron in the infralimbic cortex, but not in the prelimbic cortex of rats exposed to juvenile stress. Repeated fluvoxamine treatment recovered the increased depressive-like behavior and reduced spine densities/dendritic lengths observed in rats exposed to footshock stress. Cortical thicknesses in the infralimbic cortex and prelimbic cortex were also reduced by juvenile stress, but these reductions were not recovered by fluvoxamine treatment. The results demonstrate cortical sensitivities to stress exposures during the juvenile stage which mediate behavioral impairments, and provide a clue to find therapeutics for early life stress-induced emotional dysfunctions. (C) 2015 Elsevier B.V. All rights reserved.
• VGluT3-Expressing CCK-Positive Basket Cells Construct Invaginating Synapses Enriched with Endocannabinoid Signaling Proteins in Particular Cortical and Cortex-Like Amygdaloid Regions of Mouse Brains

  Yuki Omiya, Motokazu Uchigashima, Kohtarou Konno, Miwako Yamasaki, Taisuke Miyazaki, Takayuki Yoshida, Ichiro Kusumi, Masahiko Watanabe

  JOURNAL OF NEUROSCIENCE 35 (10) 4215 - 4228 0270-6474 2015/03 [Refereed][Not invited]
   

  Invaginating synapses in the basal amygdala are a unique type of GABAergic synapses equipped with molecular-anatomical organization specialized for 2-arachidonoylglycerol (2-AG)-mediated endocannabinoid signaling. Cholecystokinin (CCK)-positive basket cell terminals protrude into pyramidal cell somata and form invaginating synapses, where apposing presynaptic and postsynaptic elements are highly loaded with cannabinoid receptor CB1 or 2-AG synthetic enzyme diacylglycerol lipase-alpha (DGL alpha), respectively. The present study scrutinized their neurochemical and neuroanatomical phenotypes in adult mouse telencephalon. In the basal amygdala, vesicular glutamate transporter-3 (VGluT3) was transcribed in one-fourth of CB1-expressing GABAergic interneurons. The majority of VGluT3-positive CB1-expressing basket cell terminals apposed DGL alpha clusters, whereas the majority of VGluT3-negative ones did not. Importantly, VGluT3-positive basket cell terminals selectively constructed invaginating synapses. GABA(A) receptors accumulated on the postsynaptic membrane of invaginating synapses, whereas metabotropic glutamate receptor-5 (mGluR5) was widely distributed on the somatodendritic surface of pyramidal cells. Moreover, CCK2 receptor (CCK2R) was highly transcribed in pyramidal cells. In cortical regions, pyramidal cells equipped with such VGluT3/CB1/DGL alpha-accumulated invaginating synapses were found at variable frequencies depending on the subregions. Therefore, in addition to extreme proximity of CB1- and DGL alpha-loaded presynaptic and postsynaptic elements, tripartite transmitter phenotype of GABA/glutamate/CCK is the common neurochemical feature of invaginating synapses, suggesting that glutamate, CCK, or both can promote 2-AG synthesis through activating G alpha(q/11) protein-coupled mGluR5 and CCK2R. These molecular configurations led us to hypothesize that invaginating synapses might be evolved to provide some specific mechanisms of induction, regulation, and cooperativity for 2-AG-mediated retrograde signaling in particular cortical and cortex-like amygdaloid regions.
• Cellular and subcellular localization of cholecystokinin (CCK)-1 receptors in the pancreas, gallbladder, and stomach of mice

  Kohtarou Konno, Hiromi Takahashi-Iwanaga, Motokazu Uchigashima, Kyoko Miyasaka, Akihiro Funakoshi, Masahiko Watanabe, Toshihiko Iwanaga

  HISTOCHEMISTRY AND CELL BIOLOGY 143 (3) 301 - 312 0948-6143 2015/03 [Refereed][Not invited]
   

  Information concerning the cellular localization of cholecystokinin (CCK)-1 receptors has been discrepant and remained scanty at ultrastructural levels. The present immunohistochemical study at light and electron microscopic levels revealed the distinct localization of CCK1 receptors in visceral organs. Immunohistochemistry by use of a purified antibody against mouse CCK1 receptor was applied to fixed tissue sections of the pancreas, gallbladder, stomach, and intestine of mice. A silver-intensified immunogold method revealed the subcellular localization under electron microscope. The immunoreactivity for CCK1 receptors was selectively found in the basolateral membrane of pancreatic acinar cells and gastric chief cells but was absent in pancreatic islets and gastric D cells. Another intense expression in the gut was seen in the myenteric nerve plexus of the antro-duodenal region and some populations of c-Kit-expressing pacemaker cells in the duodenal musculature. The gallbladder contained smooth muscle fibers with an intense immunoreactivity of CCK1 receptors on cell surfaces. The restricted localization of CCK1 receptors on the basolateral membrane of pancreatic acinar cells and gastric chief cells, along with their absence in the islets of Langerhans and gastric D cells, provides definitive information concerning the regulatory mechanism by circulating CCK. Especially, the subcellular localization in the acinar cells completes the investigation for the detection of circulating CCK by the basolateral membrane.
• Is D-aspartate produced by glutamic-oxaloacetic transaminase-1 like 1 (Got1l1): a putative aspartate racemase?

  Ayumi Tanaka-Hayashi, Shuuhei Hayashi, Ran Inoue, Tomokazu Ito, Kohtarou Konno, Tomoyuki Yoshida, Masahiko Watanabe, Tohru Yoshimura, Hisashi Mori

  AMINO ACIDS 47 (1) 79 - 86 0939-4451 2015/01 [Refereed][Not invited]
   

  d-Aspartate is an endogenous free amino acid in the brain, endocrine tissues, and exocrine tissues in mammals, and it plays several physiological roles. In the testis, d-aspartate is detected in elongate spermatids, Leydig cells, and Sertoli cells, and implicated in the synthesis and release of testosterone. In the hippocampus, d-aspartate strongly enhances N-methyl-d-aspartate receptor-dependent long-term potentiation and is involved in learning and memory. The existence of aspartate racemase, a candidate enzyme for d-aspartate production, has been suggested. Recently, mouse glutamic-oxaloacetic transaminase 1-like 1 (Got1l1) has been reported to synthesize substantially d-aspartate from l-aspartate and to be involved in adult neurogenesis. In this study, we investigated the function of Got1l1 in vivo by generating and analyzing Got1l1 knockout (KO) mice. We also examined the enzymatic activity of recombinant Got1l1 in vitro. We found that Got1l1 mRNA is highly expressed in the testis, but it is not detected in the brain and submandibular gland, where d-aspartate is abundant. The d-aspartate contents of wild-type and Got1l1 KO mice were not significantly different in the testis and hippocampus. The recombinant Got1l1 expressed in mammalian cells showed l-aspartate aminotransferase activity, but lacked aspartate racemase activity. These findings suggest that Got1l1 is not the major aspartate racemase and there might be an as yet unknown d-aspartate-synthesizing enzyme.
• Neuron type- and input pathway-dependent expression of Slc4a10 in adult mouse brains

  Xiaohong Song, Miwako Yamasaki, Taisuke Miyazaki, Kohtarou Konno, Motokazu Uchigashima, Masahiko Watanabe

  EUROPEAN JOURNAL OF NEUROSCIENCE 40 (5) 2797 - 2810 0953-816X 2014/09 [Refereed][Not invited]
   

  Slc4a10 was originally identified as a Na+-driven Cl-/HCO3- exchanger NCBE that transports extracellular Na+ and HCO3- in exchange for intracellular Cl-, whereas other studies argue against a Cl--dependence for Na+-HCO3- transport, and thus named it the electroneutral Na+/HCO3- cotransporter NBCn2. Here we investigated Slc4a10 expression in adult mouse brains by in situ hybridization and immunohistochemistry. Slc4a10 mRNA was widely expressed, with higher levels in pyramidal cells in the hippocampus and cerebral cortex, parvalbumin-positive interneurons in the hippocampus, and Purkinje cells (PCs) in the cerebellum. Immunohistochemistry revealed an uneven distribution of Slc4a10 within the somatodendritic compartment of cerebellar neurons. In the cerebellar molecular layer, stellate cells and their innervation targets (i.e. PC dendrites in the superficial molecular layer) showed significantly higher labeling than basket cells and their targets (PC dendrites in the basal molecular layer and PC somata). Moreover, the distal dendritic trees of PCs (i.e. parallel fiber-targeted dendrites) had significantly greater labeling than the proximal dendrites (climbing fiber-targeted dendrites). These observations suggest that Slc4a10 expression is regulated in neuron type- and input pathway-dependent manners. Because such an elaborate regulation is also found for K+-Cl- cotransporter KCC2, a major neuronal Cl- extruder, we compared their expression. Slc4a10 and KCC2 overlapped in most somatodendritic elements. However, relative abundance was largely complementary in the cerebellar cortex, with particular enrichments of Slc4a10 in PC dendrites and KCC2 in molecular layer interneurons, granule cells and PC somata. These properties might reflect functional redundancy and distinction of these transporters, and their differential requirements by individual neurons and respective input domains.
• GABAergic neurons in the ventral tegmental area receive dual GABA/enkephalin-mediated inhibitory inputs from the bed nucleus of the stria terminalis

  Takehiro Kudo, Kohtarou Konno, Motokazu Uchigashima, Yuchio Yanagawa, Ichiro Sora, Masabumi Minami, Masahiko Watanabe

  EUROPEAN JOURNAL OF NEUROSCIENCE 39 (11) 1796 - 1809 0953-816X 2014/06 [Refereed][Not invited]
   

  Activation of mu-opioid receptor (MOR) disinhibits dopaminergic neurons in the ventral tegmental area (VTA) through inhibition of -aminobutyric acid (GABA)ergic neurons. This mechanism is thought to play a pivotal role in mediating reward behaviors. Here, we characterised VTA-projecting enkephalinergic neurons in the anterior division of the bed nucleus of the stria terminalis (BST) and investigated their targets by examining MOR expression in the VTA. In the BST, neurons expressing preproenkephalin mRNA were exclusively GABAergic, and constituted 37.2% of the total GABAergic neurons. Using retrograde tracer injected into the VTA, 21.6% of VTA-projecting BST neurons were shown to express preproenkephalin mRNA. Enkephalinergic projections from the BST exclusively formed symmetrical synapses onto the dendrites of VTA neurons. In the VTA, 74.1% of MOR mRNA-expressing neurons were GABAergic, with the rest being glutamatergic neurons expressing type-2 vesicular glutamate transporter mRNA. However, MOR mRNA was below the detection threshold in dopaminergic neurons. By immunohistochemistry, MOR was highly expressed on the extrasynaptic membranes of dendrites in GABAergic VTA neurons, including dendrites innervated by BST-VTA projection terminals. MOR was also expressed weakly on GABAergic and glutamatergic terminals in the VTA. Given that GABAA1 is expressed at GABAergic BST-VTA synapses on dendrites of GABAergic neurons [T. Kudo etal. (2012) J. Neurosci., 32, 18035-18046], our results collectively indicate that the BST sends dual inhibitory outputs targeting GABAergic VTA neurons; GABAergic inhibition via wired' transmission, and enkephalinergic inhibition via volume' transmission. This dual inhibitory system provides the neural substrate underlying the potent disinhibitory control over dopaminergic VTA neurons exerted by the BST.
• Enriched Expression of GluD1 in Higher Brain Regions and Its Involvement in Parallel Fiber-Interneuron Synapse Formation in the Cerebellum

  Kohtarou Konno, Keiko Matsuda, Chihiro Nakamoto, Motokazu Uchigashima, Taisuke Miyazaki, Miwako Yamasaki, Kenji Sakimura, Michisuke Yuzaki, Masahiko Watanabe

  JOURNAL OF NEUROSCIENCE 34 (22) 7412 - 7424 0270-6474 2014/05 [Refereed][Not invited]
   

  Of the two members of the delta subfamily of ionotropic glutamate receptors, GluD2 is exclusively expressed at parallel fiber-Purkinje cell (PF-PC) synapses in the cerebellum and regulates their structural and functional connectivity. However, little is known to date regarding cellular and synaptic expression of GluD1 and its role in synaptic circuit formation. In the present study, we investigated this issue by producing specific and sensitive histochemical probes for GluD1 and analyzing cerebellar synaptic circuits in GluD1-knock-out mice. GluD1 was widely expressed in the adult mouse brain, with high levels in higher brain regions, including the cerebral cortex, striatum, limbic regions (hippocampus, nucleus accumbens, lateral septum, bed nucleus stria terminalis, lateral habenula, and central nucleus of the amygdala), and cerebellar cortex. In the cerebellar cortex, GluD1 mRNA was expressed at the highest level in molecular layer interneurons and its immunoreactivity was concentrated at PF synapses on interneuron somata. In GluD1-knock-out mice, the density of PF synapses on interneuron somata was significantly reduced and the size and number of interneurons were significantly diminished. Therefore, GluD1 is common to GluD2 in expression at PF synapses, but distinct from GluD2 in neuronal expression in the cerebellar cortex; that is, GluD1 in interneurons and GluD2 in PCs. Furthermore, GluD1 regulates the connectivity of PF-interneuron synapses and promotes the differentiation and/or survival of molecular layer interneurons. These results suggest that GluD1 works in concert with GluD2 for the construction of cerebellar synaptic wiring through distinct neuronal and synaptic expressions and also their shared synapse-connecting function.
• Deletions in GRID2 lead to a recessive syndrome of cerebellar ataxia and tonic upgaze in humans

  L. Benjamin Hills, Amira Masri, Kotaro Konno, Wataru Kakegawa, Anh-Thu N. Lam, Elizabeth Lim-Melia, Nandini Chandy, R. Sean Hill, Jennifer N. Partlow, Muna Al-Saffar, Ramzi Nasir, Joan M. Stoler, A. James Barkovich, Masahiko Watanabe, Michisuke Yuzaki, Ganeshwaran H. Mochida

  NEUROLOGY 81 (16) 1378 - 1386 0028-3878 2013/10 [Refereed][Not invited]
   

  Objective: To identify the genetic cause of a syndrome causing cerebellar ataxia and eye movement abnormalities. Methods: We identified 2 families with cerebellar ataxia, eye movement abnormalities, and global developmental delay. We performed genetic analyses including single nucleotide polymorphism genotyping, linkage analysis, array comparative genomic hybridization, quantitative PCR, and Sanger sequencing. We obtained eye movement recordings of mutant mice deficient for the ortholog of the identified candidate gene, and performed immunohistochemistry using human and mouse brain specimens. Results: All affected individuals had ataxia, eye movement abnormalities, most notably tonic upgaze, and delayed speech and cognitive development. Homozygosity mapping identified the disease locus on chromosome 4q. Within this region, a homozygous deletion of GRID2 exon 4 in the index family and compound heterozygous deletions involving GRID2 exon 2 in the second family were identified. Grid2-deficient mice showed larger spontaneous and random eye movements compared to wild-type mice. In developing mouse and human cerebella, GRID2 localized to the Purkinje cell dendritic spines. Brain MRI in 2 affected children showed progressive cerebellar atrophy, which was more severe than that of Grid2-deficient mice. Conclusions: Biallelic deletions of GRID2 lead to a syndrome of cerebellar ataxia and tonic upgaze in humans. The phenotypic resemblance and similarity in protein expression pattern between humans and mice suggest a conserved role for GRID2 in the synapse organization between parallel fibers and Purkinje cells. However, the progressive and severe cerebellar atrophy seen in the affected individuals could indicate an evolutionarily unique role for GRID2 in the human cerebellum.
• Chronic Alterations in Monoaminergic Cells in the Locus Coeruleus in Orexin Neuron-Ablated Narcoleptic Mice

  Natsuko Tsujino, Tomomi Tsunematsu, Motokazu Uchigashima, Kohtarou Konno, Akihiro Yamanaka, Kazuto Kobayashi, Masahiko Watanabe, Yoshimasa Koyama, Takeshi Sakurai

  PLOS ONE 8 (7) 1932-6203 2013/07 [Refereed][Not invited]
   

  Narcolepsy patients often suffer from insomnia in addition to excessive daytime sleepiness. Narcoleptic animals also show behavioral instability characterized by frequent transitions between all vigilance states, exhibiting very short bouts of NREM sleep as well as wakefulness. The instability of wakefulness states in narcolepsy is thought to be due to deficiency of orexins, neuropeptides produced in the lateral hypothalamic neurons, which play a highly important role in maintaining wakefulness. However, the mechanism responsible for sleep instability in this disorder remains to be elucidated. Because firing of orexin neurons ceases during sleep in healthy animals, deficiency of orexins does not explain the abnormality of sleep. We hypothesized that chronic compensatory changes in the neurophysiologica activity of the locus coeruleus (LC) and dorsal raphe (DR) nucleus in response to the progressive loss of endogenous orexin tone underlie the pathological regulation of sleep/wake states. To evaluate this hypothesis, we examined firing patterns of serotonergic (5-HT) neurons and noradrenergic (NA) neurons in the brain stem, two important neuronal populations in the regulation of sleep/wakefulness states. We recorded single-unit activities of 5-HT neurons and NA neurons in the DR nucleus and LC of orexin neuron-ablated narcoleptic mice. We found that while the firing pattern of 5-HT neurons in narcoleptic mice was similar to that in wildtype mice, that of NA neurons was significantly different from that in wildtype mice. In narcoleptic mice, NA neurons showed a higher firing frequency during both wakefulness and NREM sleep as compared with wildtype mice. In vitro patch-clamp study of NA neurons of narcoleptic mice suggested a functional decrease of GABAergic input to these neurons. These alterations might play roles in the sleep abnormality in narcolepsy.
• Unique neural characteristics of GAD67-containing 5-HTergic neurons in the rat dorsal raphe nucleus

  Shikanai Hiroki, Yoshida Takayuki, Konno Kohtarou, Yamasaki Miwako, Izumi Takeshi, Ohmura Yu, Shimamura Keiichi, Watanabe Masahiko, Yoshioka Mitsuhiro

  JOURNAL OF PHARMACOLOGICAL SCIENCES 121 65P  1347-8613 2013 [Refereed][Not invited]
  
• Type 2K+Cl cotransporter is preferentially recruited to climbing fiber synapses during development and the stellate cell-targeting dendritic zone at adulthood in cerebellar Purkinje cells

  Kawakita, I, Uchigashima, M, Konno, K, Miyazaki, T, Yamasaki, M, Watanabe, M

  European Journal of Neuroscience 37 (4) 532 - 543 0953-816X 2013 [Refereed][Not invited]
   

  Postnatal expression of the type 2K+Cl cotransporter (KCC2) in neurons lowers the Cl equilibrium potential to values that are more negative than the resting potential, thereby converting the action of Cl-permeable GABAA and glycine receptors from excitatory to inhibitory. In the present study, we investigated the spatiotemporal expression of KCC2 in mouse cerebella, particularly focusing on Purkinje cells (PCs). First, we confirmed the fundamental expression profiles of KCC2 in the cerebellum, i.e. neuron-specific expression, somatodendritic distribution, and postnatal upregulation. We also found preferential recruitment to climbing fiber (CF) synapses during the second and third postnatal weeks, when perisomatic innervation in PCs switches from CFs to basket cell axons (BAs) and also when single winner CFs translocate from somata to dendrites. In parallel with this synaptic recruitment, the intracellular distribution shifted from a diffuse cytoplasmic to a predominantly cell surface pattern. In adult PCs, CF synapse-associated accumulation was obscured. Instead, significantly high expression was noted on the surface of PC dendrites in the superficial two-thirds of the molecular layer, in which stellate cells reside and project axons to innervate PC dendrites. Thus, the somatodendritic distribution in PCs is regulated in relation to particular inputs or input zones. During development, timed recruitment of KCC2 to CF synapses will augment inhibitory GABAergic actions by incoming BAs, promoting the CF-to-BA switchover in perisomatic PC innervation. In adulthood, enriched KCC2 expression at the stellate cell-targeting territory of PC dendrites might help in maintaining intracellular Cl homeostasis and the polarity of GABAA receptor-mediated responses upon sustained activity of this interneuron.
• Three Types of Neurochemical Projection from the Bed Nucleus of the Stria Terminalis to the Ventral Tegmental Area in Adult Mice

  Takehiro Kudo, Motokazu Uchigashima, Taisuke Miyazaki, Kohtarou Konno, Miwako Yamasaki, Yuchio Yanagawa, Masabumi Minami, Masahiko Watanabe

  JOURNAL OF NEUROSCIENCE 32 (50) 18035 - 18046 0270-6474 2012/12 [Refereed][Not invited]
   

  Dopaminergic (DAergic) neurons in the ventral tegmental area (VTA) play crucial roles in motivational control of behaviors, and their activity is regulated directly or indirectly via GABAergic neurons by extrinsic afferents from various sources, including the bed nucleus of the stria terminalis ( BST). Here, the neurochemical composition of VTA-projecting BST neurons and their outputs to the VTA were studied in adult mouse brains. By combining retrograde tracing with fluorescence in situ hybridization for 67 kDa glutamate decarboxylase (GAD67) and vesicular glutamate transporters (VGluTs), VTA-targeting BST neurons were classified into GAD67-positive (GAD67(+))/VGluT3-negative (VGluT3(-)), GAD67(+)/VGluT3(+), and VGluT2(+) neurons, of which GAD67(+)/VGluT3(-)neurons constituted the majority (similar to 90%) of VTA-projecting BST neurons. GABAergic efferents from the BST formed symmetrical synapses on VTA neurons, which were mostly GABAergic neurons, and expressed GABA(A) receptor alpha 1 subunit on their synaptic and extrasynaptic membranes. In the VTA, VGluT3 was detected in terminals expressing vesicular inhibitory amino acid transporter ( VIAAT), plasmalemmal serotonin transporter, or neither. Of these, VIAAT(+)/VGluT3(+) terminals, which should include those from GAD67(+)/VGluT3(+) BST neurons, formed symmetrical synapses. When single axons from VGluT3(+) BST neurons were examined, almost all terminals were labeled for VIAAT, whereas VGluT3 was often absent from terminals with high VIAAT loads. VGluT2(+) terminals in the VTA exclusively formed asymmetrical synapses, which expressed AMPA receptors on postsynaptic membrane. Therefore, the major mode of the BST-VTA projection is GABAergic, and its activation is predicted to disinhibit VTA DAergic neurons. VGluT2(+) and VGluT3(+) BST neurons further supply additional projections, which may principally convey excitatory or inhibitory inputs, respectively, to the VTA.
• Distinct Neurochemical and Functional Properties of GAD67-Containing 5-HT Neurons in the Rat Dorsal Raphe Nucleus

  Hiroki Shikanai, Takayuki Yoshida, Kohtarou Konno, Miwako Yamasaki, Takeshi Izumi, Yu Ohmura, Masahiko Watanabe, Mitsuhiro Yoshioka

  JOURNAL OF NEUROSCIENCE 32 (41) 14415 - 14426 0270-6474 2012/10 [Refereed][Not invited]
   

  The serotonergic (5-HTergic) system arising from the dorsal raphe nucleus (DRN) is implicated in various physiological and behavioral processes, including stress responses. The DRN is comprised of several subnuclei, serving specific functions with distinct afferent and efferent connections. Furthermore, subsets of 5-HTergic neurons are known to coexpress other transmitters, including GABA, glutamate, or neuropeptides, thereby generating further heterogeneity. However, despite the growing evidence for functional variations among DRN subnuclei, relatively little is known about how they map onto neurochemical diversity of 5-HTergic neurons. In the present study, we characterized functional properties of GAD67-expressing 5-HTergic neurons (5-HT/GAD67 neurons) in the rat DRN, and compared with those of neurons expressing 5-HTergic molecules (5-HT neurons) or GAD67 alone. While 5-HT/GAD67 neurons were absent in the dorsomedial (DRD) or ventromedial (DRV) parts of the DRN, they were selectively distributed in the lateral wing of the DRN (DRL), constituting 12% of the total DRL neurons. They expressed plasmalemmal GABA transporter 1, but lacked vesicular inhibitory amino acid transporter. By using whole-cell patch-clamp recording, we found that 5-HT/GAD67 neurons had lower input resistance and firing frequency than 5-HT neurons. As revealed by c-Fos immunohistochemistry, neurons in the DRL, particularly 5-HT/GAD67 neurons, showed higher responsiveness to exposure to an open field arena than those in the DRD and DRV. By contrast, exposure to contextual fear conditioning stress showed no such regional differences. These findings indicate that 5-HT/GAD67 neurons constitute a unique neuronal population with distinctive neurochemical and electrophysiological properties and high responsiveness to innocuous stressor.
• Developmental Switching of Perisomatic Innervation from Climbing Fibers to Basket Cell Fibers in Cerebellar Purkinje Cells

  Ryoichi Ichikawa, Miwako Yamasaki, Taisuke Miyazaki, Kohtarou Konno, Kouichi Hashimoto, Haruyuki Tatsumi, Yoshiro Inoue, Masanobu Kano, Masahiko Watanabe

  JOURNAL OF NEUROSCIENCE 31 (47) 16916 - 16927 0270-6474 2011/11 [Refereed][Not invited]
   

  In early postnatal development, perisomatic innervation of cerebellar Purkinje cells (PCs) switches from glutamatergic climbing fibers (CFs) to GABAergic basket cell fibers (BFs). Here we examined the switching process in C57BL/6 mice. At postnatal day 7 (P7), most perisomatic synapses were formed by CFs on to somatic spines. The density of CF-spine synapses peaked at P9, when pericellular nest around PCs by CFs was most developed, and CF-spine synapses constituted 88% of the total perisomatic synapses. Thereafter, CF-spine synapses dropped to 63% at P12, 6% at P15, and <1% at P20, whereas BF synapses increased reciprocally. During the switching period, a substantial number of BF synapses existed as BF-spine synapses (37% of the total perisomatic synapses at P15), and free spines surrounded by BFs or Bergmann glia also emerged. By P20, BF-spine synapses and free spines virtually disappeared, and BF-soma synapses became predominant (88%), thus attaining the adult pattern of perisomatic innervation. Parallel with the presynaptic switching, postsynaptic receptor phenotype also switched from glutamatergic to GABAergic. In the active switching period, particularly at P12, fragmental clusters of AMPA-type glutamate receptor were juxtaposed with those of GABA(A) receptor. When examined with serial ultrathin sections, immunogold labeling for glutamate and GABA(A) receptors was often clustered beneath single BF terminals. These results suggest that a considerable fraction of somatic spines is succeeded from CFs to BFs and Bergmann glia in the early postnatal period, and that the switching of postsynaptic receptor phenotypes mainly proceeds under the coverage of BF terminals.
• Retrieval of Conditioned Fear Activates the Basolateral and Intercalated Nucleus of Amygdala

  Takeshi Izumi, Shuken Boku, Weng Shinmin, Takeshi Inoue, Kotaro Konno, Taku Yamaguchi, Takayuki Yoshida, Machiko Matsumoto, Masahiko Watanabe, Tsukasa Koyama, Mitsuhiro Yoshioka

  JOURNAL OF NEUROSCIENCE RESEARCH 89 (5) 773 - 790 0360-4012 2011/05 [Refereed][Not invited]
   

  The amygdala is one of the crucial brain structures for conditioned fear, in which conditioned stimuli are received by the basolateral nucleus of the amygdala (BLA), inducing a fear reaction via the central nucleus of the amygdala (CeA). Whereas BLA sends glutamatergic projections into CeA, the intercalated nucleus of the amygdala (ITC) sends GABAergic projections into CeA, which is doubly regulated by BLA and ITC. In the present study, we investigated the characteristics of the neural cells activated by retrieval of conditioned fear in BLA and ITC using immunohistochemistry, in situ hybridization, and Western blot analysis of transcription factors and neural cell markers. Because most conditioned fear-induced c-Fos-positive cells in BLA were glutaminase positive and 67-kDa isomer of glutamic acid decarboxylase (GAD67) negative, these cells are speculated to be glutamatergic. Seventy-eight percent of the phosphorylated CREB (pCREB)positive cells were glutaminase double positive and 13% of the pCREB-positive cells were GAD67 double positive, indicating that many of the conditioned fear-induced pCREB-positive cells in BLA were glutamatergic, but at least some of the pCREB-positive cells were GABAergic. These results suggested that CREB phosphorylation was increased both in glutamatergic and in GABAergic neurons, but c-Fos expression was increased mainly in glutamatergic neurons in BLA. CREB phosphorylation but not c-Fos expression in ITC was specifically increased by retrieval of conditioned fear. It is therefore speculated that ITC GABAergic neurons were activated by retrieval of conditioned fear and that transcription factors other than c-Fos were relevant to the activation. (C) 2011 Wiley-Liss, Inc.
• Cytochemical and cytological properties of perineuronal oligodendrocytes in the mouse cortex

  Chihiro Takasaki, Miwako Yamasaki, Motokazu Uchigashima, Kohtarou Konno, Yuchio Yanagawa, Masahiko Watanabe

  EUROPEAN JOURNAL OF NEUROSCIENCE 32 (8) 1326 - 1336 0953-816X 2010/10 [Refereed][Not invited]
   

  Neuronal cell bodies are associated with glial cells collectively referred to as perineuronal satellite cells. One such satellite cell is the perineuronal oligodendrocyte, which is unmyelinating oligodendrocytes attaching to large neurons in various neural regions. However, little is known about their cellular characteristics and function. In this study, we identified perineuronal oligodendrocytes as 2',3'-cyclic nucleotide 3'-phosphodiesterase-positive cells attaching to neuronal perikarya immunostained for microtubule-associated protein 2, and examined their cytochemical and cytological properties in the mouse cerebral cortex. 2',3'-Cyclic nucleotide 3'-phosphodiesterase-positive perineuronal oligodendrocytes were immunonegative to representative glial markers for astrocytes (brain-type lipid binding protein and glial fibrillary acidic protein), microglia (Iba-1) and NG2+ glia. However, almost all perineuronal oligodendrocytes expressed glia-specific or glia-enriched metabolic enzymes, i.e. the creatine synthetic enzyme S-adenosylmethionine:guanidinoacetate N-methyltransferase and l-serine biosynthetic enzyme 3-phosphoglycerate dehydrogenase. As to molecules participating in the glutamate-glutamine cycle, none of the perineuronal oligodendrocytes expressed the plasmalemmal glutamate transporters GLAST and GLT-1, although nearly half of the perineuronal oligodendrocytes were immunopositive for glutamine synthetase. Cytologically, perineuronal oligodendrocytes were mainly distributed in deep cortical layers (layers IV-VI), and attached directly and tightly to neuronal cell bodies, making a long concave impression to the contacting neurons. Interestingly, they attached more to glutamatergic principal neurons than to GABAergic interneurons, and this became evident at postnatal day 14, when the cerebral cortex develops and maturates. These cytochemical and cytological properties suggest that perineuronal oligodendrocytes are so differentiated as to fulfill metabolic support to the associating principal cortical neurons, rather than to regulate their synaptic transmission.
• Early postnatal stress alters the extinction of context-dependent conditioned fear in adult rats

  Machiko Matsumoto, Hiroko Togashi, Kohtaro Konno, Hiroyo Koseki, Riki Hirata, Takeshi Izumi, Taku Yamaguchi, Mitsuhiro Yoshioka

  PHARMACOLOGY BIOCHEMISTRY AND BEHAVIOR 89 (3) 247 - 252 0091-3057 2008/05 [Refereed][Not invited]
   

  Fear extinction is hypothesized to be a learning process based on a new inhibitory memory. The present study was conducted to elucidate the effect of early postnatal stress on the extinction of context-dependent fear memory in adult rats, with a focus on the serotonergic system. Extinction was estimated by the expression of freezing behavior during repeated extinction trials (i.e., repeated exposure to contextual fear conditioning) on consecutive days. The decrease in fear expression was attenuated in adult rats that had been subjected to footshock (FS) at the third postnatal week (3wFS), but not in those exposed to footshock at the second postnatal week (2wFS). The decreased attenuation of freezing behavior observed in 3wFS was abolished by repeated treatment with the partial N-methyl-D-aspartate receptor agonist D-Cycloserine (15 mg/kg, i.p., for 4 days), which has been shown to facilitate cue-dependent extinction. Repeated treatment with the serotonin 5-hydroxytryptamine-1A (5-HT1A) receptor agonist tandospirone (I mg/kg, i.p., for 4 days) prevented the expression of freezing behavior in 3wFS, whereas diazepam treatment (I mg/kg, i.p., for 4 days) in 3wFS did not. These results suggest that exposure to early postnatal stress at the third week is responsible for attenuating extinction of contextual fear conditioning and is mediated by a serotonergic 5-HT1A receptor mechanism. In other words, exposure to traumatic events during the early postnatal period might precipitate long-lasting alterations in synaptic function that underlie extinction processes of context-dependent fear memory. (c) 2007 Elsevier Inc. All rights reserved.
• Early postnatal stress affects the serotonergic function in the median raphe nuclei of adult rats

  Kohtarou Konno, Machiko Matsumoto, Hiroko Togashi, Taku Yamaguchi, Takeshi Izumi, Masahiko Watanabe, Toshihiko Iwanaga, Mitsuhiro Yoshioka

  BRAIN RESEARCH 1172 60 - 66 0006-8993 2007/10 [Refereed][Not invited]
   

  Recent studies have focused on the serotonergic mechanism mediated via serotonin (5-HT) receptors underlying regulation of emotional stress during the developmental period. The present study was undertaken to elucidate whether early postnatal stress affects rat brain development and influences the serotonergic function in the midbrain median raphe nuclei (MRN) and dorsal raphe nuclei (DRN) in the adult, focusing on the response to unconditioned fear stress. Rats received aversive foot shock (FS) stimuli at the third week of the postnatal period (3wFS), but not those at the second week (2wFS), had increased percentage of time spent on open arms, estimated by the elevated plus maze test, at the postadolescent period (10-12 weeks old). The anxiolytic behavior observed in 3wFS was similar to that in rats having electrolytic lesion of the MRN, but not the DRN. In addition, the number of MRN 5-HT-immunoreactive cells in 3wFS remarkably was reduced compared to the non-FS control and 2wFS groups. These data suggest that aversive stress at the third week is attributable to the serotonergic function in the MRN underlying regulation of unconditioned fear stress. In other words, the "critical period" appears to be the time of neural circuit development of the MRN serotonergic system, which may be implicated in lifelong susceptibility to emotional stress. Published by Elsevier B.V.

MISC

• 前頭前野における興奮性シナプスに着目した注意欠如/多動性障害の神経基盤解析

  鹿内浩樹, 鹿内浩樹, 千里内啓行, 平出幸子, 今野幸太郎, 木村真一, 吉岡充弘, 渡邊雅彦, 富樫廣子, 島村佳一  日本神経精神薬理学会プログラム・抄録集  45th-  2015
• 注意欠如/多動性障害モデルにおける皮質前頭前野興奮性シナプスの機能形態学的異常

  鹿内浩樹, 鹿内浩樹, 千里内啓行, 平出幸子, 今野幸太郎, 木村真一, 大橋敦子, 吉岡充弘, 渡邊雅彦, 富樫廣子, 飯塚健治, 島村佳一  日本薬理学会北部会プログラム・抄録集  66th-  58  2015  [Not refereed][Not invited]
  
• Brain-enriched Sorting Nexin Family Proteins Regulate Spine Morphogenesis and Are Associated with Risk for Schizophrenia

  Takanobu Nakazawa, Ryota Hashimoto, Asami Tanimura, Kazutaka Ohi, Hidenaga Yamamori, Yuka Yasuda, Satomi Umeda-Yano, Yuji Kiyama, Kohtarou Konno, Takafumi Inoue, Shusuke Numata, Tohru Ohnuma, Nakao Iwata, Norio Ozaki, Hitoshi Hashimoto, Masahiko Watanabe, Toshiya Manabe, Tadashi Yamamoto, Masatoshi Takeda, Masanobu Kano  NEUROPSYCHOPHARMACOLOGY  39-  S306  -S307  2014/12  [Not refereed][Not invited]
  
• Distinct neurochemical and functional properties of GAD67-containing 5-HT neurons in the rat dorsal raphe nucleus

  鹿内 浩樹, 吉田 隆行, 今野 幸太郎, 山崎 美和子, 泉 剛, 大村 優, 渡邊 雅彦, 吉岡 充弘  北海道醫學雜誌 = Acta medica Hokkaidonensia  87-  (6)  288  -288  2012/11/01  [Not refereed][Not invited]
  
• Developmental switching of perisomatic innervation from climbing fibers to basket cell fibers in cerebellar Purkinje cells

  市川 量一, 山崎 美和子, 宮崎 太輔, 今野 幸太郎, 橋本 浩一, 辰巳 治之, 井上 芳郎, 狩野 正伸, 渡辺 雅彦  北海道醫學雜誌 = Acta medica Hokkaidonensia  87-  (2)  73  -73  2012/04/01
• 背側縫線核におけるGABA合成酵素GAD67含有5-HT作動性ニューロンの細胞特性

  鹿内浩樹, 吉田隆行, 今野幸太郎, 泉剛, 大村優, 渡邊雅彦, 吉岡充弘  日本薬理学会北部会プログラム・抄録集  62nd-  2011
• 背側縫線核におけるGABA合成酵素GAD67含有5-HTニューロンの細胞特性

  鹿内浩樹, 吉田隆行, 今野幸太郎, 泉剛, 大村優, 渡邊雅彦, 吉岡充弘  日本臨床精神神経薬理学会・日本神経精神薬理学会合同年会プログラム・抄録集  21st-41st-  2011
• 縫線核5-HT/GAD67共発現ニューロンの細胞学的特性

  鹿内浩樹, 吉田隆行, 今野幸太郎, 泉剛, 山口拓, 吉岡充弘  日本薬理学会北部会プログラム・抄録集  61st-  2010
• Early postnatal stress affects the 5-HTergic and GABAergic function of the median raphe nucleus in adult rats

  Kohtarou Konno, Machiko Matsumoto, Takeshi Izumi, Hiroko Togashi, Takayuki Yoshida, Taku Yamaguchi, Hiroyuki Hasegawa, Masahiko Watanabe, Mitsuhiro Yoshioka  NEUROSCIENCE RESEARCH  68-  E396  -E396  2010  [Not refereed][Not invited]
  
• 条件付け恐怖で活性化される扁桃体基底外側核および間在核神経細胞の性質

  泉剛, 井上猛, 今野幸太郎, 山口拓, 吉田隆行, 渡邊雅彦, 小山司, 吉岡充弘  日本臨床精神神経薬理学会・日本神経精神薬理学会合同年会プログラム・抄録集  19th-39th-  2009
• Characterization and functional meaning of the conditioned fear-related neural cells in the basolateral amygdala

  Takeshi Izumi, Takeshi Inoue, Kotaro Konno, Hirokazu Matsuzaki, Takayuki Yoshida, Taku Yamaguchi, Masahiko Watanabe, Tsukasa Koyama, Mitsuhiro Yoshioka  JOURNAL OF PHARMACOLOGICAL SCIENCES  109-  57P  -57P  2009  [Not refereed][Not invited]
  
• SSRIの扁桃体および内側前頭前野を介した抗不安作用発現

  泉剛, 松本真知子, 井上猛, 小関裕代, 松崎広和, 今野幸太郎, 鹿内浩樹, 富樫廣子, 山口拓, 吉田隆行, 吉岡充弘  分子精神医学  9-  (1)  95  -97  2009  [Not refereed][Invited]
  
• 幼若期のストレス負荷が成熟後の脳機能に及ぼす影響

  泉剛, 松本眞知子, 小関裕代, 松崎広和, 今野幸太郎, 富樫広子, 山口拓, 吉田隆行, 吉岡充弘  脳21  11-  (3)  2008
• 幼若期ストレスによるSocial interaction行動障害

  山口拓, 杉尾英昭, 松崎広和, 今野幸太郎, 泉剛, 松本眞知子, 吉田隆行, 富樫廣子, 吉岡充弘  日本薬理学会北部会プログラム・抄録集  59th-  2008
• 幼若期ストレスによる成熟後の情動行動障害に対するfluvoxamine連続投与の効果

  今野幸太郎, 松本真知子, 山口拓, 富樫廣子, 泉剛, 渡辺雅彦, 岩永敏彦, 吉岡充弘  日本薬理学雑誌  131-  (1)  2008
• Possible involvement of monoaminergic neural systems in ADHD-like behaviors of ADHD

  Nozomu Kojima, Ken-Ichi Ueno, Taku Yamaguchi, Machiko Matsumoto, Mutsuko Terado, Kohtarou Konno, Mitsuhiro Yoshioka, Hiroko Togashi  JOURNAL OF PHARMACOLOGICAL SCIENCES  106-  50P  -50P  2008  [Not refereed][Not invited]
  
• 幼若期ストレス負荷ラットの不安関連行動における性差

  松崎広和, 松崎広和, 山口拓, 今野幸太郎, 松本真知子, 樋口かさね, 樋口かさね, 寺戸睦子, 大野孝正, 白川哲夫, 岩永敏彦, 吉岡充弘, 富樫廣子, 富樫廣子  日本薬理学雑誌  129-  (1)  2007
• 幼若期ストレス負荷による成熟ラット行動異常に対するfluvoxamineによる改善作用

  今野幸太郎, 松本真知子, 山口拓, 富樫廣子, 泉剛, 渡辺雅彦, 岩永敏彦, 吉岡充弘  日本生物学的精神医学会プログラム・講演抄録  29th-  2007
• 幼若期ストレス負荷ラットの発達過程における不安関連行動の変化

  杉尾英昭, 山口拓, 富樫廣子, 富樫廣子, 今野幸太郎, 松本真知子, 松本真知子, 泉剛, 吉岡充弘  日本生物学的精神医学会プログラム・講演抄録  29th-  2007
• Anxiety-related behaviors in adolescent and postadolescent rats experienced with early postnatal stress

  Hideaki Sugio, Taku Yamaguchi, Hiroko Togashi, Kohtarou Konno, Machiko Matsumoto, Takeshi Izumi, Mitsuhiro Yoshioka  JOURNAL OF PHARMACOLOGICAL SCIENCES  103-  234P  -234P  2007  [Not refereed][Not invited]
  
• Chronic treatment with fluvoxamine reverses the early postnatal stress-induced behavioral changes at postadolescent periods of rats.

  Kohtarou Konno, Machiko Matsumoto, Taku Yamaguchi, Hiroko Togashi, Takeshi Izumi, Masahiko Watanabe, Toshihiko Iwanaga, Mitsuhiro Yoshioka  JOURNAL OF PHARMACOLOGICAL SCIENCES  103-  234P  -234P  2007  [Not refereed][Not invited]
  
• Involvement of serotonergic system in early postnatal stress-induced emotional susceptibility at postadolescent period of rats

  Kohtarou Konno, Machiko Matsumoto, Hiroko Togashi, Taku Yamaguchi, Mitsuhiro Yoshioka  JOURNAL OF PHARMACOLOGICAL SCIENCES  101-  140  -140  2006  [Not refereed][Not invited]
  
• 幼若期ストレス負荷による行動ならびに海馬シナプス応答性の変化

  松本真知子, 小関祐代, 富樫広子, 今野幸太郎, 山口拓, 菅野学, 掛端仁, 吉岡充弘  日本生物学的精神医学会プログラム・講演抄録  27th-  2005

Research Projects

• コレシストキニン受容体CCK1Rの脳腸機能相関における伝達基盤解明

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2021/04 -2024/03 

  Author : 今野 幸太郎

   

  コレシストキニン（CCK）は上部小腸から分泌され、食後の胃酸、膵液、胆汁の分泌を制御する消化管ホルモンである。CCK受容体は末梢組織に豊富なCCK1Rと中枢神経系に豊富なCCK2Rが存在し末梢型のCCK1Rは標的となる消化腺や胆嚢に発現するだけでなく、脳にも一部発現することがこれまで報告されているが、その詳細は不明である。はじめにCCK1Rの脳内発現分布を蛍光抗体法を用いて検討した。CCK1Rの特異的な免疫反応は弧束核内側部、最後野で強く、弧束核外側部や迷走神経背側運動核ではまばらに認められた。次にCCK1Rが豊富に認められる孤束核内側領域および最後野においてCCK1Rの詳細な局在を蛍光抗体法および包埋前免疫電顕法を用いて検討した結果、CCK1Rは小胞型グルタミン酸トランスポーター2（VGluT2）陽性グルタミン酸作動性神経終末の細胞膜近傍に豊富に局在することが明らかとなった。最後に孤束核内側領域および最後野に中枢性投射線維を送る迷走神経下神経節においてCCK1R mRNAおよびタンパクの発現分布をin situ hybridization法および蛍光抗体法を用いて検討した結果、VGluT2陽性グルタミン酸作動性神経細胞にCCK1R mRNAの発現が認められ、VGluT2陽性細胞の細胞体表面にCCK1Rタンパクの強い局在が認められた。以上の結果から孤束核内側領域および最後野のCCK1Rは迷走神経下神経節由来の神経終末に豊富に局在することが示唆される。
• localization and function of glutamate receptor GluD2 in the retrosplenial granular cortex

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2018/04 -2021/03 

  Author : Konno Kotaro

   

  Little is known to date as to synaptic expression and function of glutamate receptor GluD2 outside the cerebellum. To address this issue, we examined the retrosplenial granular cortex (RSG), a cortical area that is involved in spatial memory and enriched with GluD2. By light and electron microscopy, GluD2 was concentrated at asymmetrical synapses in RSG neurons. Using anterograde and retrograde tracers, we addressed that afferents contacting GluD2-expressing synapses originated from the anteroventral thalamic nucleus (AV). Intriguingly, most RSG-targeting AV neurons expressed mRNA for Cbln1, a ligand for GluD1. Indeed, Cbln1 was clustered on RSG neurons and tightly apposed to GluD2 clusters. The density of AV afferents terminating on RSG neurons was significantly reduced in both GluD2-KO and Cbln1-KO mice. These results provide the molecular-anatomical evidence that GluD2 and Cbln1 interact to promote the formation of specific wiring between the AV and RSG.
• Analysis for GluD1-Cbln1 signaling in synaptic transmission in thalamic neurons of the ventral posteromedial nucleus

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2016/04 -2018/03 

  Author : Konno Kotaro

   

  We selected the ventral posterior nucleus (VP), a brain region with high level expression of GluD1. GluD1 mRNA was expressed highly in relay neurons, and its immunoreactivity was selectively concentrated at specific synapses formed between glutamatergic neurons in the trigeminal nucleus and relay neurons in the VP. In addition, most VP-targeting neurons expressed Cbln1 mRNA, a ligand for GluD1. To pursue a role of the interaction of GluD1 and Cbln1 in the VP, we analyzed changes in terminals of trigeminal nucleus in wild-type, GluD1-KO, and Cbln1-KO mice by immunofluorescence. In GluD1-knockout and Cbln1-knockout mice, the density of glutamatergic terminals were strikingly increased. These results suggest that GluD1 and Cbln1 interaction plays a inhibitory role in synaptic connectivity between glutamatergic neurons in the trigeminal nucleus and relay neurons in the VP.
• Analyses of an orphan metabotropic receptor which plays a role in the long term retention of memory, and a secretory protein encoded by its splice variant

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2014/04 -2017/03 

  Author : KUBO Yoshihiro, YAMAMOTO Izumi, WATANABE Masahiro, Chen I-Shan, Yamamoto Tomomi, TATEYAMA Michihiro, TAKAO Keizo, MIYAKAWA Tsuyoshi, HATTORI Satoko, FUKATA Masaki, FUKATA Yuko, YOKOI Norihiko, SAKIMURA Kenji, SYUU Rei, NATSUME Rie

   

  Prrt3 is a putative G protein coupled receptor of family C, but its function is totally unknown. We aimed at the elucidation of the molecular function and physiological role of Prrt3, and observed the followings. (1) Prrt3 is partly cleaved at the end of N-terminal extracellular domain by Furin. (2) It binds to glutamate transporters EAAT1, 2 and Gi/o G protein. (3) It is expressed chiefly at the presynaptic terminal of excitatory neurons. (4) Conditional Prrt3 gene knock out homozygous mice in forebrain excitatory neurons showed impairment of long term retention of fear conditioning memory. (5) Prtt3 is activated by Oxo-M, a muscarinic Ach receptor agonist, but not by ACh.
• Molecular-anatomical research on multi-modal regulation of synaptic transmission in higher brain regions

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2012/05 -2017/03 

  Author : WATANABE MASAHIKO, Yamasaki Miwako, Miyazaki Taisuke, Konno Kohtarou, Uchigashima Motokazu, Kano Masanobu, Shigemoto Ryuichi, Kobayashi Kazuto

   

  state-dependent manners. We found that TARP and GluD families play important roles in input-, target cell-, activity-dependent regulations in the cerebellum and hippocampus. In the cortex and cortex-like amygdala, CCK-positive interneurons expressing VGluT3 constructed unique invaginating synapses, which were also characterized by intense expression of endocannabinoid signaling molecules to powerfully drive activity- and state-dependent disinhibition. Furthermore, so-called ‘dopamine synapses’ are important for state-dependent controls of motor and cognitive functions. We address that dopamine synapses were neuroligin-2-mediated heterologous contacts formed between dopaminergic presynapse and GABAergic postsynapse, from which we propose the third mode of neural transmission, anchored transmission, in addition to classical modes of wired and volume transmissions.
• Role of glutamate receptor GluD1 in synapse formation and emotion.

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2013/04 -2015/03 

  Author : KONNO kohtarou

   

  Of the two members of the δ subfamily of ionotropic glutamate receptors, GluD2 is exclusively expressed in Purkinje cell and regulates their structural connectivity. However, little is known to date regarding cellular and synaptic expression of GluD1 and its role in synaptic circuit formation. GluD1 was widely expressed in the adult mouse brain. In the cerebellar cortex, GluD1 was concentrated at parallel fiber (PF) synapses on interneuron somata. In GluD1-knock-out mice, the density of PF synapses on interneuron somata was significantly reduced and the size and number of interneurons were significantly diminised. Therefore, GluD1 regulates the connectivity of PF-interneuron synapses and promotes the differentiation and/or survival of molecular layer interneurons. These results suggest that GluD1 works in concert with GluD2 for the construction of cerebellar synaptic wiring through distinct neuronal and synaptic expressions and also their shared synapse-connecting function.