Masahiro Fukaya

Kitasato University, Edo, Tōkyō, Japan

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Publications (129)546.4 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Development of correct topographical connections between peripheral receptors and central somatosensory stations requires activity-dependent synapse refinement, in which the NMDA type of glutamate receptors plays a key role. Here we compared functional roles of GluN2B (GluRε2 or NR2B) and GluN2D (GluRε4 or NR2D), two major regulatory subunits of neonatal NMDA receptors, in development of whisker-related patterning at trigeminal relay stations. Compared with control littermates, both the appearance of whisker-related patterning and the termination of the critical period, as assessed by unilateral infraorbital nerve transection, were delayed by nearly a day in the somatosensory cortex of GluN2B(+/-) mice but advanced by nearly a day in GluN2D(-/-) mice. Similar temporal shifts were found at subcortical relay stations in the thalamus and brainstem of GluN2B(+/-) and GluN2D(-/-) mice. In comparison, the magnitude of lesion-induced critical period plasticity in the somatosensory cortex, as assessed following row-C whisker removal, was normal in both mutants. Thus, GluN2B and GluN2D play counteractive roles in temporal development and maturation of somatosensory maps without affecting the magnitude of critical period plasticity. To understand the opposing action, we then examined neuronal and synaptic expressions of the two subunits along the trigeminal pathway. At each trigeminal station, GluN2B was predominant at asymmetrical synapses of non-GABAergic neurons, whereas GluN2D was selective to asymmetrical synapses of GABAergic neurons. Together, our findings suggest that GluN2B expressed at glutamatergic synapses on glutamatergic projection neurons facilitates refinement of ascending pathway synapses directly, whereas GluN2D expressed at glutamatergic synapses on GABAergic interneurons delays it indirectly.
    Journal of Neuroscience 08/2014; 34(35):11534-48. · 6.75 Impact Factor
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    ABSTRACT: Type I phosphatidylinositol 4-phosphate 5 kinase γ (PIP5KIγ) constitutes a major pathway for the generation of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) that regulates a variety of neuronal functions at both presynaptic and postsynaptic compartments. In this study, we examined the expression and localization of PIP5KIγ in the adult mouse retina. RT-PCR analysis revealed that PIP5KIγ_v2 was predominantly expressed in the retina while PIP5KIγ_v3 was also expressed faintly. Immunostaining of the adult mouse retina revealed intense PIP5KIγ-immunoreactivity in the inner and outer plexiform layers in a punctate manner. In the photoreceptor ribbon synapse, PIP5KIγ was highly concentrated at the periactive zone. These findings suggest that PIP5KIγ, especially PIP5KIγ_i2, is localized at the periactive zone, a functionally suitable compartment for the endocytosis of synaptic vesicles in photoreceptor ribbon synapses.
    Brain Research 08/2014; · 2.83 Impact Factor
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    ABSTRACT: Endosomal trafficking mediated by Rab11 and Arf6 small GTPases is essential for various neuronal functions. Family of Rab11-interacting protein 3 (FIP3)/Arfophilin-1, also termed Eferin, is a dual effector for Rab11 and Arf6 and implicated in endosomal trafficking during cytokinesis. To understand the neuronal functions of FIP3, we first showed the widespread neuronal expression of FIP3 mRNA in adult mouse brain by in situ hybridization. Immunohistochemical analysis showed the association of FIP3 with a subpopulation of endosomes labeled with EEA1 and syntaxin 12 in hippocampal neurons. Immunoblot analysis showed the progressive increase of FIP3 with a peak around postnatal day 15 during hippocampal development. Furthermore, knockdown of endogenous FIP3 decreased the total dendritic length of cultured hippocampal neurons with a concomitant increase in the number of short (<40 μm) primary dendrites. Together, FIP3 is suggested to regulate dendritic formation possibly through Rab11- and Arf6-mediated endosomal trafficking.
    Brain research 04/2014; · 2.83 Impact Factor
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    ABSTRACT: The metabotropic glutamate receptor subtype 1 (mGluR1, Grm1) in cerebellar Purkinje cells (PCs) is essential for motor coordination and motor learning. At the synaptic level, mGluR1 has a critical role in long-term synaptic depression (LTD) at parallel fiber (PF)-PC synapses, and in developmental elimination of climbing fiber (CF)-PC synapses. mGluR1a, a predominant splice variant in PCs, has a long carboxyl (C)-terminal domain that interacts with Homer scaffolding proteins. Cerebellar roles of the C-terminal domain at both synaptic and behavior levels remain poorly understood. To address this question, we introduced a short variant, mGluR1b, which lacks this domain into PCs of mGluR1-knock-out (KO) mice (mGluR1b-rescue mice). In mGluR1b-rescue mice, mGluR1b showed dispersed perisynaptic distribution in PC spines. Importantly, mGluR1b-rescue mice exhibited impairments in inositol 1,4,5-trisphosphate receptor (IP3R)-mediated Ca(2+) release, CF synapse elimination, LTD induction, and delay eyeblink conditioning: they showed normal transient receptor potential canonical (TRPC) currents and normal motor coordination. In contrast, PC-specific rescue of mGluR1a restored all cerebellar defects of mGluR1-KO mice. We conclude that the long C-terminal domain of mGluR1a is required for the proper perisynaptic targeting of mGluR1, IP3R-mediated Ca(2+) release, CF synapse elimination, LTD, and motor learning, but not for TRPC currents and motor coordination.
    Journal of Neuroscience 02/2014; 34(7):2702-12. · 6.75 Impact Factor
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    ABSTRACT: The membrane trafficking and actin cytoskeleton remodeling mediated by ADP ribosylation factor 6 (Arf6) are functionally linked to various neuronal processes including neurite formation and maintenance, neurotransmitter release, and receptor internalization. EFA6A is an Arf6-specific guanine nucleotide exchange factor that is abundantly expressed in the brain. In this study, we identified sorting nexin-1 (SNX1), a retromer component that is implicated in endosomal sorting and trafficking, as a novel interacting partner for EFA6A by yeast two-hybrid screening. The interaction was mediated by the C-terminal region of EFA6A and a BAR domain of SNX1, and further confirmed by pull-down assay and immunoprecipitation from mouse brain lysates. In situ hybridization analysis demonstrated the widespread expression of SNX1 in the mouse brain, which overlapped with the expression of EFA6A in the forebrain. Immunofluorescent analysis revealed the partial colocalization of EFA6A and SNX1 in the dendritic fields of the hippocampus. Immunoelectron microscopic analysis revealed the overlapping subcellular localization of EFA6A and SNX1 at the post-synaptic density and endosomes in dendritic spines. In Neuro-2a neuroblastoma cells, expression of either EFA6A or SNX1 induced neurite outgrowth, which was further enhanced by co-expression of EFA6A and SNX1. The present findings suggest a novel mechanism by which EFA6A regulates Arf6-mediated neurite formation through the interaction with SNX1. We identified sorting nexin-1 (SNX1) as a novel binding partner for EFA6A, and demonstrated overlapping ultrastructural localization of EFA6A and SNX1 in spines and dendrites of hippocampal neurons. Furthermore, we showed that this interaction enhanced neurite outgrowth of Neuro-2a cells. The present findings suggest the importance of the interaction between EFA6A and SNX1 in Arf6-mediated neuronal functions in dendritic spines.
    Journal of Neurochemistry 11/2013; · 4.24 Impact Factor
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    ABSTRACT: Syntaxin-1A is a t-SNARE that is involved in vesicle docking and vesicle fusion; it is important in presynaptic exocytosis in neurons because it interacts with many regulatory proteins. Previously, we found 1) that autophosphorylated Ca2+/calmodulin-dependent protein kinase II (CaMKII), an important modulator of neural plasticity, interacts with syntaxin-1A to regulate exocytosis, and 2) that a syntaxin missense mutation [R151G] attenuated this interaction. To more precisely determine the physiological importance of this interaction between CaMKII and syntaxin, we generated mice with a knock-in (KI) syntaxin-1A [R151G] mutation. Complexin is a molecular clamp involved in exocytosis, and in the KI mice, recruitment of complexin to the SNARE complex was reduced because of an abnormal CaMKII-syntaxin interaction. Nevertheless, SNARE complex formation was not inhibited, and, consequently, basal neurotransmission was normal. However, the KI mice did exhibit abnormal presynaptic plasticity, and they had a more pronounced synaptic response than did wild-type littermates; this pronounced response included several behavioral abnormalities. Notably, the R151G phenotypes were generally similar to previously reported CaMKII mutant phenotypes. Additionally, synaptic recycling in these KI mice was delayed, and the density of synaptic vesicles was reduced. Taken together, our results indicated that this single point mutation in syntaxin-1A causes abnormal regulation of neuronal plasticity and vesicle recycling, and that the affected syntaxin-1A-CaMKII interaction is essential to normal brain and synaptic functions in vivo.
    Journal of Biological Chemistry 10/2013; · 4.60 Impact Factor
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    ABSTRACT: Type I phosphatidylinositol 4-phosphate 5-kinase (PIP5KI)γ is one of the phosphoinositide kinases that produce phosphatidylinositol 4,5-bisphosphate, which is a critical regulator of cell adhesion formation, actin dynamics and membrane trafficking. Here, we examined the functional roles of PIP5KIγ in radial neuronal migration during cortical formation. Reverse transcription-polymerase chain reaction analysis revealed that PIP5KIγ_v2/v6 and PIP5KIγ_v3 were expressed throughout cortical development with distinct expression patterns. In situ hybridisation analysis showed that PIP5KIγ mRNA was expressed throughout the cortical layers. Immunohistochemical analysis revealed that PIP5KIγ was localised in a punctate manner in the radial glia and migrating neuroblasts. Knockdown of PIP5KIγ using in utero electroporation disturbed the radial neuronal migration and recruitment of talin and focal adhesion kinase to puncta beneath the plasma membrane. The same inhibitory effect on neuronal migration was observed by overexpression of a catalytically inactive mutant of PIP5KIγ_v2 but not PIP5KIγ_v1 or PIP5KIγ_v3. These findings suggest an essential role of PIP5KIγ, particularly PIP5KIγ_i2, in neuronal migration, possibly through recruitment of adhesion components to the plasma membrane.
    European Journal of Neuroscience 06/2013; · 3.67 Impact Factor
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    ABSTRACT: The adult CNS contains an abundant population of oligodendrocyte precursor cells (NG2(+) cells) that generate oligodendrocytes and repair myelin, but how these ubiquitous progenitors maintain their density is unknown. We generated NG2-mEGFP mice and used in vivo two-photon imaging to study their dynamics in the adult brain. Time-lapse imaging revealed that NG2(+) cells in the cortex were highly dynamic; they surveyed their local environment with motile filopodia, extended growth cones and continuously migrated. They maintained unique territories though self-avoidance, and NG2(+) cell loss though death, differentiation or ablation triggered rapid migration and proliferation of adjacent cells to restore their density. NG2(+) cells recruited to sites of focal CNS injury were similarly replaced by a proliferative burst surrounding the injury site. Thus, homeostatic control of NG2(+) cell density through a balance of active growth and self-repulsion ensures that these progenitors are available to replace oligodendrocytes and participate in tissue repair.
    Nature Neuroscience 04/2013; · 14.98 Impact Factor
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    ABSTRACT: Oligodendrocytes associate with axons to establish myelin and provide metabolic support to neurons. In the spinal cord of amyotrophic lateral sclerosis (ALS) mice, oligodendrocytes downregulate transporters that transfer glycolytic substrates to neurons and oligodendrocyte progenitors (NG2(+) cells) exhibit enhanced proliferation and differentiation, although the cause of these changes in oligodendroglia is unknown. We found extensive degeneration of gray matter oligodendrocytes in the spinal cord of SOD1 (G93A) ALS mice prior to disease onset. Although new oligodendrocytes were formed, they failed to mature, resulting in progressive demyelination. Oligodendrocyte dysfunction was also prevalent in human ALS, as gray matter demyelination and reactive changes in NG2(+) cells were observed in motor cortex and spinal cord of ALS patients. Selective removal of mutant SOD1 from oligodendroglia substantially delayed disease onset and prolonged survival in ALS mice, suggesting that ALS-linked genes enhance the vulnerability of motor neurons and accelerate disease by directly impairing the function of oligodendrocytes.
    Nature Neuroscience 03/2013; · 14.98 Impact Factor
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    ABSTRACT: Pathological examination of dementia with Lewy bodies patients identified the presence of abnormal α-synuclein (αSyn) aggregates in the presynaptic terminals. αSyn is involved in the regulation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex. Importantly, αSyn-transgenic mouse and postmortem examination of patients with Parkinson's disease have demonstrated the abnormal distribution of SNARE protein in presynaptic terminals. In this study, we investigated the effects of SNARE dysfunction on endogenous αSyn using Snap25(S187A/S187A) mutant mice. These mice have homozygous knock-in gene encoding unphosphorylatable S187A-substituted synaptosomal-associated protein of 25 kDa (SNAP-25). The mice displayed a significant age-dependent change in the distribution of αSyn and its Ser(129)-phosphorylated form in abnormally hypertrophied glutamatergic nerve terminals in the striatum. Electron-microscopic analysis revealed the abnormally condensed synaptic vesicles with concomitant mislocalization of αSyn protein to the periactive zone in the glutamatergic nerve terminals. However, the Snap25(S187A/S187A) mutant mouse harbored no abnormalities in the nigrostriatal dopaminergic neurons. Our present results suggest that SNARE dysfunction is the initial trigger of mislocalization and accumulation of αSyn, and probably is an important pathomechanism of α-synucleinopathies.
    Journal of Neuroscience 11/2012; 32(48):17186-96. · 6.75 Impact Factor
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    ABSTRACT: The BRAG/IQSEC is a family of guanine nucleotide exchange factors for ADP ribosylation factors, small GTPases that regulate membrane trafficking and actin cytoskeleton, and comprises three structurally related members (BRAG1-3) generated from different genes. In the mouse retina, BRAG1 (also known as IQSEC2) was previously shown to localize at synaptic ribbons of photoreceptor terminals and to form a protein complex with RIBEYE. In this study, we examined immunohistochemical localization of BRAG2 (IQSEC1) and BRAG3 (IQSEC3) in the adult mouse retina at the light and electron microscopic levels. In the outer plexiform layer, BRAG2 showed a punctate distribution in intimate association with dystrophin and β-dystroglycan. Immunoelectron microscopic analysis revealed that BRAG2 localized at specific subcompartments of photoreceptor terminals in both rod spherules and cone pedicles. In the inner plexiform layer, immunolabeling for both BRAG2 and BRAG3 had a punctate appearance, suggestive of synaptic labeling. Double immunostaining demonstrated that BRAG2 colocalized preferentially with PSD-95 and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate-type glutamate receptors (AMPARs). By contrast, BRAG3 colocalized with gephyrin and a subpopulation of inhibitory synapses expressing glycine receptors or γ-aminobutyric acid type A receptors (GABA(A) Rs). Immunoelectron microscopic analysis revealed that BRAG2 localized to postsynaptic processes at bipolar dyads, whilst BRAG3 localized to postsynaptic components at conventional synapses. These findings suggest that BRAG/IQSEC family members have key roles in the function and organization of distinct excitatory and inhibitory synapses in the retina. J.Comp.Neurol.,2012.©2012 Wiley Periodicals, Inc.
    The Journal of Comparative Neurology 08/2012; · 3.51 Impact Factor
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    ABSTRACT: Protein incorporated later into tight junctions (Pilt), also termed tight junction-associated protein 1 or tight junction protein 4, is a coiled-coil domain-containing protein that was originally identified as a human discs large-interacting protein. In this study, we identified Pilt as an Arf6-binding protein by yeast two-hybrid screening. By immunocytochemical analysis, Pilt was shown to be predominantly localized at the trans-Golgi complex and to exhibit diffuse cytoplasmic distribution in association with endosomes and plasma membrane in NIH3T3 cells. Silencing of endogenous Pilt disrupted the Golgi structure. The present findings suggest the functional involvement of Pilt in the maintenance of the Golgi structure. STRUCTURED SUMMARY OF PROTEIN INTERACTIONS: GM130 and Piltcolocalize by fluorescence microscopy (View interaction) Arf6(Q67L)physically interacts with Pilt by two hybrid (View Interaction: 1, 2) Piltphysically interacts with Arf6(Q67L) by pull down (View interaction).
    FEBS letters 07/2012; 586(19):3064-70. · 3.54 Impact Factor
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    ABSTRACT: Mitochondria divide and fuse continuously, and the balance between these two processes regulates mitochondrial shape. Alterations in mitochondrial dynamics are associated with neurodegenerative diseases. Here we investigate the physiological and cellular functions of mitochondrial division in postmitotic neurons using in vivo and in vitro gene knockout for the mitochondrial division protein Drp1. When mouse Drp1 was deleted in postmitotic Purkinje cells in the cerebellum, mitochondrial tubules elongated due to excess fusion, became large spheres due to oxidative damage, accumulated ubiquitin and mitophagy markers, and lost respiratory function, leading to neurodegeneration. Ubiquitination of mitochondria was independent of the E3 ubiquitin ligase parkin in Purkinje cells lacking Drp1. Treatment with antioxidants rescued mitochondrial swelling and cell death in Drp1KO Purkinje cells. Moreover, hydrogen peroxide converted elongated tubules into large spheres in Drp1KO fibroblasts. Our findings suggest that mitochondrial division serves as a quality control mechanism to suppress oxidative damage and thus promote neuronal survival.
    The Journal of Cell Biology 05/2012; 197(4):535-51. · 9.69 Impact Factor
  • Masahiro Fukaya, Yoshinobu Hara, Hiroyuki Sakagami
    Neuroscience Research 09/2011; 71. · 2.15 Impact Factor
  • Neuroscience Research 09/2011; 71. · 2.15 Impact Factor
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    ABSTRACT: Oligodendrocyte precursor cells (OPCs) express NMDA receptors (NMDARs) and form synapses with glutamatergic neurons throughout the CNS. Although glutamate influences the proliferation and maturation of these progenitors in vitro, the role of NMDAR signaling in oligodendrogenesis and myelination in vivo is not known. Here, we investigated the consequences of genetically deleting the obligatory NMDAR subunit NR1 from OPCs and their oligodendrocyte progeny in the CNS of developing and mature mice. NMDAR-deficient OPCs proliferated normally, achieved appropriate densities in gray and white matter, and differentiated to form major white matter tracts without delay. OPCs also retained their characteristic physiological and morphological properties in the absence of NMDAR signaling and were able to form synapses with glutamatergic axons. However, expression of calcium-permeable AMPA receptors (AMPARs) was enhanced in NMDAR-deficient OPCs. These results suggest that NMDAR signaling is not used to control OPC development but to regulate AMPAR-dependent signaling with surrounding axons, pointing to additional functions for these ubiquitous glial cells.
    Journal of Neuroscience 08/2011; 31(35):12650-62. · 6.75 Impact Factor
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    ABSTRACT: The planar cell polarity (PCP) protein, Prickle (Pk), is conserved in invertebrates and vertebrates, and regulates cellular morphogenesis and movement. Vertebrate Pk consists of at least two family members, Pk1 and Pk2, both of which are expressed in the brain; however, their localization and function at synapses remain elusive. Here, we show that Pk2 is expressed mainly in the adult brain and is tightly associated with the postsynaptic density (PSD) fraction obtained by subcellular fractionation. In primary cultured rat hippocampal neurons, Pk2 is colocalized with PSD-95 and synaptophysin at synapses. Moreover, immunoelectron microcopy shows that Pk2 is localized at the PSD of asymmetric synapses in the hippocampal CA1 region. Biochemical assays identified that Pk2 forms a complex with PSD proteins including PSD-95 and NMDA receptor subunits via the direct binding to the C-terminal guanylate kinase domain of PSD-95. These results indicate that Pk2 is a novel PSD protein that interacts with PSD-95 and NMDA receptors through complex formations in the brain.
    Journal of Biochemistry 02/2011; 149(6):693-700. · 3.07 Impact Factor
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    ABSTRACT: SynArfGEF, also known as BRAG3 or IQSEC3, is a member of the brefeldin A-resistant Arf-GEF/IQSEC family and was originally identified by screening for mRNA species associated with the post-synaptic density fraction. In this study, we demonstrate that synArfGEF activates Arf6, using Arf pull down and transferrin incorporation assays. Immunohistochemical analysis reveals that synArfGEF is present in somata and dendrites as puncta in close association with inhibitory synapses, whereas immunoelectron microscopic analysis reveals that synArfGEF localizes preferentially at post-synaptic specializations of symmetric synapses. Using yeast two-hybrid and pull down assays, we show that synArfGEF is able to bind utrophin/dystrophin and S-SCAM/MAGI-2 scaffolding proteins that localize at inhibitory synapses. Double immunostaining reveals that synArfGEF co-localizes with dystrophin and S-SCAM in cultured hippocampal neurons and cerebellar cortex, respectively. Both β-dystroglycan and S-SCAM were immunoprecipitated from brain lysates using anti-synArfGEF IgG. Taken together, these findings suggest that synArfGEF functions as a novel regulator of Arf6 at inhibitory synapses and associates with the dystrophin-associated glycoprotein complex and S-SCAM.
    Journal of Neurochemistry 12/2010; 116(6):1122-37. · 4.24 Impact Factor
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    ABSTRACT: The mammalian CNS contains a ubiquitous population of glial progenitors known as NG2+ cells that have the ability to develop into oligodendrocytes and undergo dramatic changes in response to injury and demyelination. Although it has been reported that NG2+ cells are multipotent, their fate in health and disease remains controversial. Here, we generated PDGFαR-CreER transgenic mice and followed their fate in vivo in the developing and adult CNS. These studies revealed that NG2+ cells in the postnatal CNS generate myelinating oligodendrocytes, but not astrocytes or neurons. In regions of neurodegeneration in the spinal cord of ALS mice, NG2+ cells exhibited enhanced proliferation and accelerated differentiation into oligodendrocytes but remained committed to the oligodendrocyte lineage. These results indicate that NG2+ cells in the normal CNS are oligodendrocyte precursors with restricted lineage potential and that cell loss and gliosis are not sufficient to alter the lineage potential of these progenitors.
    Neuron 11/2010; 68(4):668-81. · 15.77 Impact Factor

Publication Stats

5k Citations
546.40 Total Impact Points


  • 2010–2014
    • Kitasato University
      • Department of Anatomy
      Edo, Tōkyō, Japan
  • 2003–2014
    • Hokkaido University
      • Department of Medicine II
      Sapporo, Hokkaidō, Japan
  • 2010–2013
    • Johns Hopkins University
      Baltimore, Maryland, United States
  • 2004–2010
    • Niigata University
      • Division of Cellular Neurobiology
      Niahi-niigata, Niigata, Japan
    • Tohoku University
      • Graduate School of Pharmaceutical Sciences
      Sendai-shi, Miyagi-ken, Japan
    • Kanazawa University
      • Graduate School of Medical Sciences
      Kanazawa, Ishikawa, Japan
  • 1999–2010
    • Hokkaido University Hospital
      • Division of Urology
      Sapporo, Hokkaidō, Japan
  • 2009
    • Hyogo College of Medicine
      Nishinomiya, Hyōgo, Japan
  • 1989–2009
    • The University of Tokyo
      • Faculty & Graduate School of Medicine
      Tokyo, Tokyo-to, Japan
  • 2006–2007
    • Osaka University
      • Division of Cellular Neuroscience
      Ibaraki, Osaka-fu, Japan
  • 2000
    • Hirosaki University
      • Department of Neuropathology
      Khirosaki, Aomori Prefecture, Japan
  • 1997
    • Niigata Institute of Technology
      Edo, Tōkyō, Japan