SDS-digested freeze-fracture replica labeling(SDS-FRL) was innovated as a suitable technique to reveal the fine structure of plasma membrane and quantitative localization of biomolecules over the membrane structure. However, it was difficult to apply this technique to molecules immobilized on protoplasmic faces(P-face) quantitatively. Thus, in this research project, I tried to optimize labeling protocols of SDS-FRL. The optimization was carried out at steps of SDS treatment, pretreatment and blocking of replicated membranes as well as components on each labeling solutions. As a result, although details of labeling protocol differ among experiments, new protocol provided high-sensitive labeling not only for molecules on exoplasmic faces but also for those on P-faces. Localization data obtained by these investigations were published as original papers.

• Project/Area Number: 21500311
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Neuroscience in general
• Research Institution: Nagoya University (2011); National Institute for Physiological Sciences (2009-2010)
• Principal Investigator: FUKAZAWA Yugo 名古屋大学, 大学院・医学系研究科, 准教授 (60343745)
• Project Period (FY): 2009 – 2011
• Project Status: Completed (Fiscal Year 2011)
• Budget Amount: ¥4,550,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥1,050,000); Fiscal Year 2011: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2010: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000); Fiscal Year 2009: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
• Keywords: 凍結割断レプリカ / 電子顕微鏡 / 分子局在 / 細胞膜

Research Abstract

SDS-digested freeze-fracture replica labeling(SDS-FRL) was innovated as a suitable technique to reveal the fine structure of plasma membrane and quantitative localization of biomolecules over the membrane structure. However, it was difficult to apply this technique to molecules immobilized on protoplasmic faces(P-face) quantitatively. Thus, in this research project, I tried to optimize labeling protocols of SDS-FRL. The optimization was carried out at steps of SDS treatment, pretreatment and blocking of replicated membranes as well as components on each labeling solutions. As a result, although details of labeling protocol differ among experiments, new protocol provided high-sensitive labeling not only for molecules on exoplasmic faces but also for those on P-faces. Localization data obtained by these investigations were published as original papers.

Research Products

• [Journal Article] Virus-mediated swapping of zolpidem-insensitive with zolpidem-sensitive GABAA receptors in cortical pyramidal cells (2012)
  • Author(s): Sumegi M, Fukazawa Y, Matsui K, Lorincz A, Eyre MD, Nusser Z
  • Journal Title: J Physiol Volume: (in press)
  • Peer Reviewed
• [Journal Article] Intra-synapse-type and inter-synapse-type relationships between synaptic size and AMPAR expression (2012)
  • Author(s): Fukazawa Y, Shigemoto R.
  • Journal Title: Current Opinion Neurobiology Volume: 22 Pages: 1-7
  • Peer Reviewed
• [Journal Article] Mechanisms underlying signal filtering at a multi-synapse contact (2012)
  • Author(s): Budisantoso T, Matsui K, Kamasawa N, Fukazawa Y, Shigemoto R
  • Journal Title: J Neurosci Volume: (in press)
  • Peer Reviewed
• [Journal Article] Intra-synapse-type and inter-synapse-type relationships between synapticsize and AMPAR expression (2012)
  • Author(s): Fukazawa Y
  • Journal Title: Current Opinion Neurobiology Volume: 22 Issue: 3 Pages: 1-7
  • DOI: 10.1016/j.conb.2012.01.006
  • Peer Reviewed
• [Journal Article] Developmental profile of SK2 channel expression and function in CA1 neurons (2012)
  • Author(s): Ballesteros-Merino C
  • Journal Title: Hippocampus Volume: (印刷中) Issue: 6 Pages: 1467-1480
  • DOI: 10.1002/hipo.20986
  • Peer Reviewed
• [Journal Article] Developmental profile of SK2 channel expression and function in CA1 neurons (2011)
  • Author(s): Ballesteros-Merino C, Lin M, Wu WW, Ferrandiz-Huertas C, Cabanero MJ, Watanabe M, Fukazawa Y, Shigemoto R, Maylie J, Adelman JP, Lujan R
  • Journal Title: Hippocampus Volume: (in press)
  • Peer Reviewed
• [Journal Article] NMDA receptors in hippocampal GABA ergic synapses and their role in nitric oxide signaling (2011)
  • Author(s): Szabadits E, Cserep C, Sz. nyi A, Fukazawa Y, Shigemoto R, Watanabe M, Itohara S, Freund T, Nyiri G
  • Journal Title: J Neurosci Volume: 31 Pages: 5893-5904
  • Peer Reviewed
• [Journal Article] NMDA Receptors in Hippocampal GABAergic Synapses and Their Role in Nitric Oxide Signaling (2011)
  • Author(s): Szabadits, E.; Cserep, C.; Szonyi, A., et al
  • Journal Title: Journal of Neuroscience Volume: 31(16) Issue: 16 Pages: 5893-5904
  • DOI: 10.1523/jneurosci.5938-10.2011
  • Peer Reviewed
• [Journal Article] Quantitative localization of synaptic and extra synaptic GABAA receptor subunits on hippocampal pyramidal cells by freeze-fracture replica immunolabelling (2010)
  • Author(s): Kasugai Y, Swinny JD, Roberts JDB, Dalezios Y, Fukazawa Y, Sieghart W, Shigemoto R, Somogyi P
  • Journal Title: Eu J Neurosci Volume: 32 Pages: 1868-1888
  • Peer Reviewed
• [Journal Article] Differential postsynaptic compartments in the laterocapsular division of the central nucleus of amygdale for afferents from the Para brachial nucleus and the basolateral nucleus in the rat (2010)
  • Author(s): Dong Y-L, Fukazawa Y, Wang W, Kamasawa N, Shigemoto R
  • Journal Title: J Comp Neurol Volume: 518 Pages: 4771-4791
  • Peer Reviewed
• [Journal Article] Subcellular distribution ofα1G subunit of T-type calcium channel in the mouse dorsal lateral geniculate nucleus (2010)
  • Author(s): Parajuli KL, Fukazawa Y, Watanabe M, Shigemoto R
  • Journal Title: J Comp Neurol Volume: 518 Pages: 4362-4374
  • Peer Reviewed
• [Journal Article] Cbln1 is a ligand for an orphan glutamate receptorχ2, bidirectional synapse organizer (2010)
  • Author(s): Matsuda K, Miura E, Miyazaki T, Kakegawa W, Emi K, Narumi S, Fukazawa Y, Ito-Ishida A, Kondo T, Shigemoto R, Watanabe M, Yuzaki M
  • Journal Title: Science Volume: 328 Pages: 363-368
  • Peer Reviewed
• [Journal Article] Cblnl is a ligand for an orphan glutamate receptor δ2, bidirectional synapse organizer. (2010)
  • Author(s): Matsuda K, Miura E, Miyazaki T, Kakegawa W, Emi K, Narumi S, Fukazawa Y, Ito-Ishida A, Kondo T, Shigemoto R, Watanabe M, Yuzaki M
  • Journal Title: Science Volume: 328 Pages: 363-368
  • Peer Reviewed
• [Journal Article] Subcellular distribution of α1G subunit of T-type calcium channel in the mouse dorsal lateral geniculate nucleus. (2010)
  • Author(s): Parajuli KL, Fukazawa Y, Watanabe M, Shigemoto R
  • Journal Title: J Comp Neurol Volume: 518 Pages: 4362-4374
  • Peer Reviewed
• [Journal Article] Differential postsynaptic compartments in the laterocapsular division of the central nucleus of amygdale for afferents from the parabrachial nucleus and the basolateral nucleus in the rat. (2010)
  • Author(s): Dong Y-L, Fukazawa Y, Wang W, Kamasawa N, Shigemoto R
  • Journal Title: J Comp Neurol Volume: 518 Pages: 4771-4791
  • Peer Reviewed
• [Journal Article] Quantitative localization of synaptic and extrasynaptic GABA_A receptor subunits on hippocampal pyramidal cells by freeze-fracture replica immuno labelling. (2010)
  • Author(s): Kasugai Y, Swinny JD, Roberts JDB, Dalezios Y, Fukazawa Y, Sieghart W, Shigemoto R, Somogyi P
  • Journal Title: Eu J Neurosci Volume: 32 Pages: 1868-1888
  • Peer Reviewed
• [Journal Article] Input-specific intrasynaptic arrangements of ionotropic glutamater eceptors and their impact on postsynaptic responses (2009)
  • Author(s): Tarusawa E, Matsui K, Budisantoso T, Molnar E, Watanabe M, Matsui M, Fukazawa Y, Shigemoto R
  • Journal Title: J Neurosci Volume: 29 Pages: 12896-12908
  • Peer Reviewed
• [Journal Article] Crosstalk between GABA_(B) and mGlu1 & receptors reveals new insight into GPCR signal integration (2009)
  • Author(s): Rives ML, Vol C, Fukazawa Y, Tinel N, Trinquet E, Ayoub MA, Shigemoto R, Pin JP, Prezeau L
  • Journal Title: EMBO J Volume: 28 Pages: 2195-2208
  • Peer Reviewed
• [Journal Article] Large-conductance calcium-activated potassium channels in Purkinje cell plasma membranes are clustered at sites of hypolemmal microdomains (2009)
  • Author(s): Kaufmann WA, Ferraguti F, FukazawaY, Kasugai Y, Shigemoto R, Laake P, Sexton JA, Ruth P, Wietzorrek G, KnausH-G, Strom JF, Ottersen OP
  • Journal Title: J Comp Neurol Volume: 515 Pages: 215-230
  • Peer Reviewed
• [Journal Article] Input-specific intrasynaptic arrangements of ionotropic glutamate receptors and their impact on postsynaptic responses (2009)
  • Author(s): Tarusawa E, Matsui K, Budisantoso T, Molnar E, Watanabe M, Matsui M, Fukazawa Y, Shigemoto R
  • Journal Title: J Neurosci 29 Pages: 12896-12908
  • Peer Reviewed
• [Journal Article] Crosstalk between GABA_(B)and mGlulα receptors reveals new insight in to GPCR signal integration (2009)
  • Author(s): Rives ML, Vol C, Fukazawa Y, Tinel N, Trinquet E, Ayoub MA, Shigemoto R, Pin JP, Prezeau L
  • Journal Title: EMBO J 28 Pages: 2195-2208
  • Peer Reviewed
• [Journal Article] Large-conductance calcium-activated potassium channels in Purkinje cell plasma membranes are clustered at sites of hypolemmal microdomains (2009)
  • Author(s): Kaufmann WA, Ferraguchi F, Fukazawa Y, Kasugai Y, Shigemoto R, Laake P, Sexton JA, Ruth P, Wietzorrek G, Knaus H-G, Strom JF, Ottersen OP
  • Journal Title: J Comp Neurol 515 Pages: 215-230
  • Peer Reviewed
• [Presentation] R型電位依存性カルシウムチャネル(Cav2. 3)のマウス脳内微細局在 (2011)
  • Author(s): Parajuli L, Kulik A,重本隆一,深澤有吾
  • Organizer: 第71回日本解剖学会中部支部学術集会
  • Place of Presentation: 名古屋
  • Year and Date: 2011-10-14
• [Presentation] Presynaptic localization of R-type calcium channel in the in terpeduncular nucleus (2011)
  • Author(s): Parajuli LK, Fukazawa Y, Shigemoto R
  • Organizer: 第34回日本神経科学大会
  • Place of Presentation: 横浜
  • Year and Date: 2011-09-17
• [Presentation] Analysis of functional connectivity among cortical layers during opto genetically induced perturbations (2011)
  • Author(s): Kuki T, Matuzaka Y, Fukazawa Y, Yawo H, Muchiake H
  • Organizer: 第34回日本神経科学大会
  • Place of Presentation: 横浜
  • Year and Date: 2011-09-16
• [Presentation] Right-hemispheric dominance of dentate granular cell c-fos expression after spatial exploration in split-brain mice (2011)
  • Author(s): Ahmed H, Kawakami R, Fukazawa Y, Shigemoto R
  • Organizer: 第34回日本神経科学大会
  • Place of Presentation: 横浜
  • Year and Date: 2011-09-15
• [Presentation] Ultrastructural localization analysis of septins in mammalian nervous system (2011)
  • Author(s): Kurita H, Fukazawa Y, Ageta-Ishihara N, Shigemoto R, Kinoshita M
  • Organizer: 第34回日本神経科学大会
  • Place of Presentation: 横浜
  • Year and Date: 2011-09-15
• [Presentation] Right hemisphere dominance of dentate granule cell activation after exploration of novel environment in wild-type and iv mutant mice (2011)
  • Author(s): Shigemoto R, Armed H, Kawakami H, Fukazawa Y
  • Organizer: 8^<th> IBRO world congress of neuroscience
  • Place of Presentation: Florence, Italy
  • Year and Date: 2011-07-16
• [Presentation] Structural correlate of long-term motor memory formation in cerebeller cortex of mice (2011)
  • Author(s): Aziz W, Wang W, Fukazawa Y, Shigemoto R
  • Organizer: 8^<th> IBRO world congress of neuroscience
  • Place of Presentation: Florence, Italy
  • Year and Date: 2011-07-15
• [Presentation] 定量的分子局在解析と計算論的手法によるシナプス構造-機能連関の解析 (2011)
  • Organizer: 日本顕微鏡学会.第67回学術講演会
  • Place of Presentation: 福岡
  • Year and Date: 2011-05-16
• [Presentation] 定量的分子局在解析と計算論的手法によるシナプス構造-機能連関の解析 (2011)
  • Author(s): 深澤有吾
  • Organizer: 日本顕微鏡学会
  • Place of Presentation: 福岡(招待講演)
  • Year and Date: 2011-05-16
• [Presentation] Orexin neurons are directly and indirectly activated by orexin through the orexin 2receptor (2010)
  • Author(s): Tabuchi S, Tsunematsu T, Fukazawa Y, Tominaga M, Yamanaka A
  • Organizer: Neuroscience2010
  • Place of Presentation: SanDiego, USA
  • Year and Date: 2010-11-17
• [Presentation] NMDA receptors in hippocampal GABAergic synapses (2010)
  • Author(s): Szabadits E, Sz. nyi A, Cserep C, FukazawaY, Watanabe M, Shigemoto R, Freund TF, Nyiri G
  • Organizer: Neuroscience2010
  • Place of Presentation: SanDiego, USA
  • Year and Date: 2010-11-16
• [Presentation] Structural changes at parallel fiber-Purkinje cell synapses after long-term adaptation of horizontal optokinetic response in mice (2010)
  • Author(s): Shigemoto R, Aziz W, Fukazawa Y, WangW, Tarusawa E
  • Organizer: Neuroscience2010
  • Place of Presentation: SanDiego, USA
  • Year and Date: 2010-11-16
• [Presentation] GABA(B) receptor activity dinamically regulates expression of Kir3 channels (2010)
  • Author(s): Kulik A, Fukazawa Y, Shigemoto R, Frotscher M, Vida I
  • Organizer: Neuroscience2010
  • Place of Presentation: SanDiego, USA
  • Year and Date: 2010-11-04
• [Presentation] Characterization of DRG neuron subpopulations selectively expressing ChR2 in Thy-1. 2 transgenic rat (2010)
  • Author(s): Ji Z, Ishizuka T, Fukazawa Y, Shigemoto R, Yawo H
  • Organizer: 第33回日本神経科学大会
  • Place of Presentation: 神戸
  • Year and Date: 2010-09-03
• [Presentation] Presynaptic, postsynaptic, and morphological determinants of signal transmission at the retinogeniculate synapse (2010)
  • Author(s): Matsui K, Budisantoso T, Kamasawa N, Fukazawa Y, Shigemoto R
  • Organizer: 第33回日本神経科学大会
  • Place of Presentation: 神戸
  • Year and Date: 2010-09-03
• [Presentation] Regional and subcellular distribution of the αlE subunit of voltage-gated calcium channel in the adult mouse brain. (2010)
  • Author(s): Parajuli KP, Fukazawa Y, Kulik A, Shigemoto R
  • Organizer: 第33回日本神経科学大会
  • Place of Presentation: 神戸コンベンションセンター(兵庫県)
  • Year and Date: 2010-09-03
• [Presentation] Exploring functions of the septin cytoskeleton in the formation and remodeling of neuroglial network (2010)
  • Author(s): Ageta-Ishihara N, Hagiwara A, Kurita H, Morita T, Tominaga T, Fukazawa Y,: Shigemoto R, Kinoshita M
  • Organizer: 第33回日本神経科学大会
  • Place of Presentation: 神戸
  • Year and Date: 2010-09-02
• [Presentation] Regional and subcellular distribution of theα1E subunit of voltage-gated calcium channel in the adult mouse brain (2010)
  • Author(s): Parajuli KP, Fukazawa Y, Kulik A, Shigemoto R
  • Organizer: 第33回日本神経科学大会
  • Place of Presentation: 神戸
  • Year and Date: 2010-09-02
• [Presentation] GABA(B) receptor activity dynamically regulates surface expression of Kir3 channels (2010)
  • Author(s): Kulik A, Fukazawa Y, Shigemoto R, Frotscher M, Vida I.
  • Organizer: FENS2010
  • Place of Presentation: Amsterdam, Netherland
  • Year and Date: 2010-04-07
• [Presentation] Cross-regulation between GABAB and mGlu1a receptors reveals new insight into signal integration between GPCRs (2010)
  • Author(s): Prezeau L, Rives ML, Fukazawa Y, Tinel N, Trinquet E, Ayoub M, Shigemoto R, Pin JP
  • Organizer: FENS2010
  • Place of Presentation: Amsterdam, Netherland
  • Year and Date: 2010-04-07
• [Presentation] 水平性視機性眼球反応適応時の小脳回路変化とそのメカニズム (2010)
  • Organizer: 第115回日本解剖学会全国学術集会
  • Place of Presentation: 盛岡
  • Year and Date: 2010-03-30
• [Presentation] 水平性視機性眼球反応適応時の小脳回路変化とそのメカニズム (2010)
  • Author(s): 深澤有吾
  • Organizer: 第115回日本解剖学会全国学術集会
  • Place of Presentation: 盛岡・岩手県民会館
  • Year and Date: 2010-03-30
• [Presentation] Compartmentalized distribution of a1G subunit of T-type calcium channel in the dorsal lateral geniculate nucleus of mouse brain (2009)
  • Author(s): Parajuli LK, Fukazawa Y, Watanabe M, Shigemoto R
  • Organizer: Neuroscience 2009
  • Place of Presentation: Nagoya, Japan
  • Year and Date: 2009-09-17
• [Presentation] In vivo remodeling of postsynaptic glutamate receptor or ganization revealed by freeze-fracture replica labeling (2009)
  • Author(s): Fukazawa Y, Itakura M, Takahashi T, SaitohY, Inokuchi K, Molnar E, Shigemoto R
  • Organizer: Neuroscience 2009
  • Place of Presentation: Nagoya, Japan
  • Year and Date: 2009-09-17
• [Presentation] Structural changes at parallel fiber to Purkinje cells synapses after long-term adaptation of horizontal opt kinetic response (2009)
  • Author(s): Aziz W, Fukazawa Y, Tarusawa E, Shigemoto R
  • Organizer: Neuroscience 2009
  • Place of Presentation: Nagoya, Japan
  • Year and Date: 2009-09-16
• [Presentation] Orexin 2 receptor positively regulates the activity of orexin neurons (2009)
  • Author(s): Tabuchi S, Tsunematsu T, Tominaga M, Fukazawa Y, Yamanaka A
  • Organizer: 第33回日本神経科学大会
  • Place of Presentation: 神戸
  • Year and Date: 2009-09-02
• [Presentation] Zernike phase contract electron microscopy visualizes detailed structures of vitrified biological specimens (2009)
  • Author(s): Fukuda Y, Fukazawa Y, Danev RS, Shigemoto R, Nagayama K
  • Organizer: IUPS2009
  • Place of Presentation: Kyoto, Japan
  • Year and Date: 2009-08-01
• [Presentation] The enhancement of PSD size and AMPA receptor density in the central amygdala in rat modle of neuropathic pain (2009)
  • Author(s): Dong D, Fukazawa Y, Shigemoto R
  • Organizer: IUPS2009
  • Place of Presentation: Kyoto, Japan
  • Year and Date: 2009-07-31
• [Presentation] Structural changes at parallel fiber-Purkinje cells synapses after long-term adaptation of horizontal optokinetic response in mice (2009)
  • Author(s): Aziz W, Fukazawa Y, Tarusawa E, Shigemoto R
  • Organizer: IUPS2009
  • Place of Presentation: Kyoto, Japan
  • Year and Date: 2009-07-29
• [Presentation] Remodeling of postsynaptic glutamate receptor organization in awake rats revealed by freeze-fracture replica labeling (2009)
  • Author(s): Fukazawa Y, Itakura M, Takahashi T, SaitohY, Inokuchi K, Molnar E, Shigemoto R
  • Organizer: IUPS2009
  • Place of Presentation: Kyoto, Japan
  • Year and Date: 2009-07-28
• [Presentation] Immunohistochemical localization ofα1Gsubunit of T-type calcium channel in thedorsal lateral geniculate nucleus of mouse brain (2009)
  • Author(s): Parajuli LK, Fukazawa Y, Watanabe M, Shigemoto R
  • Organizer: IUPS2009
  • Place of Presentation: Kyoto, Japan
  • Year and Date: 2009-07-28
• [Book] Electron microscopy of biological membrane. Encyclopedia of Biophysics (2012)
  • Author(s): Fujimoto T, Fukazawa Y
  • Publisher: Encyclopedia of Biophysics(in press)
• [Book] Electron microscopy of biological membrane In Encyclopedia of Biophysics (2012)
  • Author(s): Fujimoto T, Fukazawa Y
  • Total Pages: 3
  • Publisher: Academic Press
• [Book] Glutamate receptor organization : ultrastructural insights (2009)
  • Author(s): Fukazawa Y, Shigemoto R
  • Publisher: Encyclopedia of Neuroscience(volume 4)