--- _id: '9756' abstract: - lang: eng text: High-resolution visualization and quantification of membrane proteins contribute to the understanding of their functions and the roles they play in physiological and pathological conditions. Sodium dodecyl sulfate-digested freeze-fracture replica labeling (SDS-FRL) is a powerful electron microscopy method to study quantitatively the two-dimensional distribution of transmembrane proteins and their tightly associated proteins. During treatment with SDS, intracellular organelles and proteins not anchored to the replica are dissolved, whereas integral membrane proteins captured and stabilized by carbon/platinum deposition remain on the replica. Their intra- and extracellular domains become exposed on the surface of the replica, facilitating the accessibility of antibodies and, therefore, providing higher labeling efficiency than those obtained with other immunoelectron microscopy techniques. In this chapter, we describe the protocols of SDS-FRL adapted for mammalian brain samples, and optimization of the SDS treatment to increase the labeling efficiency for quantification of Cav2.1, the alpha subunit of P/Q-type voltage-dependent calcium channels utilizing deep learning algorithms. acknowledgement: This work was supported by the European Union (European Research Council Advanced grant no. 694539 and Human Brain Project Ref. 720270 to R. S.) and the Austrian Academy of Sciences (DOC fellowship to D.K.). alternative_title: - Neuromethods article_processing_charge: No author: - first_name: Walter full_name: Kaufmann, Walter id: 3F99E422-F248-11E8-B48F-1D18A9856A87 last_name: Kaufmann orcid: 0000-0001-9735-5315 - first_name: David full_name: Kleindienst, David id: 42E121A4-F248-11E8-B48F-1D18A9856A87 last_name: Kleindienst - first_name: Harumi full_name: Harada, Harumi id: 2E55CDF2-F248-11E8-B48F-1D18A9856A87 last_name: Harada orcid: 0000-0001-7429-7896 - first_name: Ryuichi full_name: Shigemoto, Ryuichi id: 499F3ABC-F248-11E8-B48F-1D18A9856A87 last_name: Shigemoto orcid: 0000-0001-8761-9444 citation: ama: 'Kaufmann W, Kleindienst D, Harada H, Shigemoto R. High-Resolution localization and quantitation of membrane proteins by SDS-digested freeze-fracture replica labeling (SDS-FRL). In: Receptor and Ion Channel Detection in the Brain. Vol 169. Neuromethods. New York: Humana; 2021:267-283. doi:10.1007/978-1-0716-1522-5_19' apa: 'Kaufmann, W., Kleindienst, D., Harada, H., & Shigemoto, R. (2021). High-Resolution localization and quantitation of membrane proteins by SDS-digested freeze-fracture replica labeling (SDS-FRL). In Receptor and Ion Channel Detection in the Brain (Vol. 169, pp. 267–283). New York: Humana. https://doi.org/10.1007/978-1-0716-1522-5_19' chicago: 'Kaufmann, Walter, David Kleindienst, Harumi Harada, and Ryuichi Shigemoto. “High-Resolution Localization and Quantitation of Membrane Proteins by SDS-Digested Freeze-Fracture Replica Labeling (SDS-FRL).” In Receptor and Ion Channel Detection in the Brain, 169:267–83. Neuromethods. New York: Humana, 2021. https://doi.org/10.1007/978-1-0716-1522-5_19.' ieee: 'W. Kaufmann, D. Kleindienst, H. Harada, and R. Shigemoto, “High-Resolution localization and quantitation of membrane proteins by SDS-digested freeze-fracture replica labeling (SDS-FRL),” in Receptor and Ion Channel Detection in the Brain, vol. 169, New York: Humana, 2021, pp. 267–283.' ista: 'Kaufmann W, Kleindienst D, Harada H, Shigemoto R. 2021.High-Resolution localization and quantitation of membrane proteins by SDS-digested freeze-fracture replica labeling (SDS-FRL). In: Receptor and Ion Channel Detection in the Brain. Neuromethods, vol. 169, 267–283.' mla: Kaufmann, Walter, et al. “High-Resolution Localization and Quantitation of Membrane Proteins by SDS-Digested Freeze-Fracture Replica Labeling (SDS-FRL).” Receptor and Ion Channel Detection in the Brain, vol. 169, Humana, 2021, pp. 267–83, doi:10.1007/978-1-0716-1522-5_19. short: W. Kaufmann, D. Kleindienst, H. Harada, R. Shigemoto, in:, Receptor and Ion Channel Detection in the Brain, Humana, New York, 2021, pp. 267–283. date_created: 2021-07-30T09:34:56Z date_published: 2021-07-27T00:00:00Z date_updated: 2024-03-28T23:30:31Z day: '27' ddc: - '573' department: - _id: RySh - _id: EM-Fac doi: 10.1007/978-1-0716-1522-5_19 ec_funded: 1 has_accepted_license: '1' intvolume: ' 169' keyword: - 'Freeze-fracture replica: Deep learning' - Immunogold labeling - Integral membrane protein - Electron microscopy language: - iso: eng month: '07' oa_version: None page: 267-283 place: New York project: - _id: 25CA28EA-B435-11E9-9278-68D0E5697425 call_identifier: H2020 grant_number: '694539' name: 'In situ analysis of single channel subunit composition in neurons: physiological implication in synaptic plasticity and behaviour' - _id: 25CBA828-B435-11E9-9278-68D0E5697425 call_identifier: H2020 grant_number: '720270' name: Human Brain Project Specific Grant Agreement 1 (HBP SGA 1) publication: ' Receptor and Ion Channel Detection in the Brain' publication_identifier: eisbn: - '9781071615225' isbn: - '9781071615218' publication_status: published publisher: Humana quality_controlled: '1' related_material: record: - id: '9562' relation: dissertation_contains status: public series_title: Neuromethods status: public title: High-Resolution localization and quantitation of membrane proteins by SDS-digested freeze-fracture replica labeling (SDS-FRL) type: book_chapter user_id: D865714E-FA4E-11E9-B85B-F5C5E5697425 volume: 169 year: '2021' ... --- _id: '7391' abstract: - lang: eng text: Electron microscopy (EM) is a technology that enables visualization of single proteins at a nanometer resolution. However, current protein analysis by EM mainly relies on immunolabeling with gold-particle-conjugated antibodies, which is compromised by large size of antibody, precluding precise detection of protein location in biological samples. Here, we develop a specific chemical labeling method for EM detection of proteins at single-molecular level. Rational design of α-helical peptide tag and probe structure provided a complementary reaction pair that enabled specific cysteine conjugation of the tag. The developed chemical labeling with gold-nanoparticle-conjugated probe showed significantly higher labeling efficiency and detectability of high-density clusters of tag-fused G protein-coupled receptors in freeze-fracture replicas compared with immunogold labeling. Furthermore, in ultrathin sections, the spatial resolution of the chemical labeling was significantly higher than that of antibody-mediated labeling. These results demonstrate substantial advantages of the chemical labeling approach for single protein visualization by EM. article_processing_charge: No article_type: original author: - first_name: Shigekazu full_name: Tabata, Shigekazu id: 4427179E-F248-11E8-B48F-1D18A9856A87 last_name: Tabata - first_name: Marijo full_name: Jevtic, Marijo id: 4BE3BC94-F248-11E8-B48F-1D18A9856A87 last_name: Jevtic - first_name: Nobutaka full_name: Kurashige, Nobutaka last_name: Kurashige - first_name: Hirokazu full_name: Fuchida, Hirokazu last_name: Fuchida - first_name: Munetsugu full_name: Kido, Munetsugu last_name: Kido - first_name: Kazushi full_name: Tani, Kazushi last_name: Tani - first_name: Naoki full_name: Zenmyo, Naoki last_name: Zenmyo - first_name: Shohei full_name: Uchinomiya, Shohei last_name: Uchinomiya - first_name: Harumi full_name: Harada, Harumi id: 2E55CDF2-F248-11E8-B48F-1D18A9856A87 last_name: Harada orcid: 0000-0001-7429-7896 - first_name: Makoto full_name: Itakura, Makoto last_name: Itakura - first_name: Itaru full_name: Hamachi, Itaru last_name: Hamachi - first_name: Ryuichi full_name: Shigemoto, Ryuichi id: 499F3ABC-F248-11E8-B48F-1D18A9856A87 last_name: Shigemoto orcid: 0000-0001-8761-9444 - first_name: Akio full_name: Ojida, Akio last_name: Ojida citation: ama: Tabata S, Jevtic M, Kurashige N, et al. Electron microscopic detection of single membrane proteins by a specific chemical labeling. iScience. 2019;22(12):256-268. doi:10.1016/j.isci.2019.11.025 apa: Tabata, S., Jevtic, M., Kurashige, N., Fuchida, H., Kido, M., Tani, K., … Ojida, A. (2019). Electron microscopic detection of single membrane proteins by a specific chemical labeling. IScience. Elsevier. https://doi.org/10.1016/j.isci.2019.11.025 chicago: Tabata, Shigekazu, Marijo Jevtic, Nobutaka Kurashige, Hirokazu Fuchida, Munetsugu Kido, Kazushi Tani, Naoki Zenmyo, et al. “Electron Microscopic Detection of Single Membrane Proteins by a Specific Chemical Labeling.” IScience. Elsevier, 2019. https://doi.org/10.1016/j.isci.2019.11.025. ieee: S. Tabata et al., “Electron microscopic detection of single membrane proteins by a specific chemical labeling,” iScience, vol. 22, no. 12. Elsevier, pp. 256–268, 2019. ista: Tabata S, Jevtic M, Kurashige N, Fuchida H, Kido M, Tani K, Zenmyo N, Uchinomiya S, Harada H, Itakura M, Hamachi I, Shigemoto R, Ojida A. 2019. Electron microscopic detection of single membrane proteins by a specific chemical labeling. iScience. 22(12), 256–268. mla: Tabata, Shigekazu, et al. “Electron Microscopic Detection of Single Membrane Proteins by a Specific Chemical Labeling.” IScience, vol. 22, no. 12, Elsevier, 2019, pp. 256–68, doi:10.1016/j.isci.2019.11.025. short: S. Tabata, M. Jevtic, N. Kurashige, H. Fuchida, M. Kido, K. Tani, N. Zenmyo, S. Uchinomiya, H. Harada, M. Itakura, I. Hamachi, R. Shigemoto, A. Ojida, IScience 22 (2019) 256–268. date_created: 2020-01-29T15:56:56Z date_published: 2019-12-20T00:00:00Z date_updated: 2024-03-28T23:30:12Z day: '20' ddc: - '570' department: - _id: RySh doi: 10.1016/j.isci.2019.11.025 ec_funded: 1 external_id: isi: - :000504652000020 pmid: - '31786521' file: - access_level: open_access checksum: f3e90056a49f09b205b1c4f8c739ffd1 content_type: application/pdf creator: dernst date_created: 2020-02-04T10:48:36Z date_updated: 2020-07-14T12:47:57Z file_id: '7448' file_name: 2019_iScience_Tabata.pdf file_size: 7197776 relation: main_file file_date_updated: 2020-07-14T12:47:57Z has_accepted_license: '1' intvolume: ' 22' issue: '12' language: - iso: eng license: https://creativecommons.org/licenses/by/4.0/ month: '12' oa: 1 oa_version: Published Version page: 256-268 pmid: 1 project: - _id: 25CA28EA-B435-11E9-9278-68D0E5697425 call_identifier: H2020 grant_number: '694539' name: 'In situ analysis of single channel subunit composition in neurons: physiological implication in synaptic plasticity and behaviour' - _id: 25CBA828-B435-11E9-9278-68D0E5697425 call_identifier: H2020 grant_number: '720270' name: Human Brain Project Specific Grant Agreement 1 (HBP SGA 1) publication: iScience publication_identifier: issn: - 2589-0042 publication_status: published publisher: Elsevier quality_controlled: '1' related_material: record: - id: '11393' relation: dissertation_contains status: public scopus_import: '1' status: public title: Electron microscopic detection of single membrane proteins by a specific chemical labeling tmp: image: /images/cc_by.png legal_code_url: https://creativecommons.org/licenses/by/4.0/legalcode name: Creative Commons Attribution 4.0 International Public License (CC-BY 4.0) short: CC BY (4.0) type: journal_article user_id: c635000d-4b10-11ee-a964-aac5a93f6ac1 volume: 22 year: '2019' ... --- _id: '736' abstract: - lang: eng text: The neurotransmitter receptor subtype, number, density, and distribution relative to the location of transmitter release sites are key determinants of signal transmission. AMPA-type ionotropic glutamate receptors (AMPARs) containing GluA3 and GluA4 subunits are prominently expressed in subsets of neurons capable of firing action potentials at high frequencies, such as auditory relay neurons. The auditory nerve (AN) forms glutamatergic synapses on two types of relay neurons, bushy cells (BCs) and fusiform cells (FCs) of the cochlear nucleus. AN-BC and AN-FC synapses have distinct kinetics; thus, we investigated whether the number, density, and localization of GluA3 and GluA4 subunits in these synapses are differentially organized using quantitative freeze-fracture replica immunogold labeling. We identify a positive correlation between the number of AMPARs and the size of AN-BC and AN-FC synapses. Both types of AN synapses have similar numbers of AMPARs; however, the AN-BC have a higher density of AMPARs than AN-FC synapses, because the AN-BC synapses are smaller. A higher number and density of GluA3 subunits are observed at AN-BC synapses, whereas a higher number and density of GluA4 subunits are observed at AN-FC synapses. The intrasynaptic distribution of immunogold labeling revealed that AMPAR subunits, particularly GluA3, are concentrated at the center of the AN-BC synapses. The central distribution of AMPARs is absent in GluA3-knockout mice, and gold particles are evenly distributed along the postsynaptic density. GluA4 gold labeling was homogenously distributed along both synapse types. Thus, GluA3 and GluA4 subunits are distributed at AN synapses in a target-cell-dependent manner. article_processing_charge: No author: - first_name: María full_name: Rubio, María last_name: Rubio - first_name: Ko full_name: Matsui, Ko last_name: Matsui - first_name: Yugo full_name: Fukazawa, Yugo last_name: Fukazawa - first_name: Naomi full_name: Kamasawa, Naomi last_name: Kamasawa - first_name: Harumi full_name: Harada, Harumi id: 2E55CDF2-F248-11E8-B48F-1D18A9856A87 last_name: Harada orcid: 0000-0001-7429-7896 - first_name: Makoto full_name: Itakura, Makoto last_name: Itakura - first_name: Elek full_name: Molnár, Elek last_name: Molnár - first_name: Manabu full_name: Abe, Manabu last_name: Abe - first_name: Kenji full_name: Sakimura, Kenji last_name: Sakimura - first_name: Ryuichi full_name: Shigemoto, Ryuichi id: 499F3ABC-F248-11E8-B48F-1D18A9856A87 last_name: Shigemoto orcid: 0000-0001-8761-9444 citation: ama: Rubio M, Matsui K, Fukazawa Y, et al. The number and distribution of AMPA receptor channels containing fast kinetic GluA3 and GluA4 subunits at auditory nerve synapses depend on the target cells. Brain Structure and Function. 2017;222(8):3375-3393. doi:10.1007/s00429-017-1408-0 apa: Rubio, M., Matsui, K., Fukazawa, Y., Kamasawa, N., Harada, H., Itakura, M., … Shigemoto, R. (2017). The number and distribution of AMPA receptor channels containing fast kinetic GluA3 and GluA4 subunits at auditory nerve synapses depend on the target cells. Brain Structure and Function. Springer. https://doi.org/10.1007/s00429-017-1408-0 chicago: Rubio, María, Ko Matsui, Yugo Fukazawa, Naomi Kamasawa, Harumi Harada, Makoto Itakura, Elek Molnár, Manabu Abe, Kenji Sakimura, and Ryuichi Shigemoto. “The Number and Distribution of AMPA Receptor Channels Containing Fast Kinetic GluA3 and GluA4 Subunits at Auditory Nerve Synapses Depend on the Target Cells.” Brain Structure and Function. Springer, 2017. https://doi.org/10.1007/s00429-017-1408-0. ieee: M. Rubio et al., “The number and distribution of AMPA receptor channels containing fast kinetic GluA3 and GluA4 subunits at auditory nerve synapses depend on the target cells,” Brain Structure and Function, vol. 222, no. 8. Springer, pp. 3375–3393, 2017. ista: Rubio M, Matsui K, Fukazawa Y, Kamasawa N, Harada H, Itakura M, Molnár E, Abe M, Sakimura K, Shigemoto R. 2017. The number and distribution of AMPA receptor channels containing fast kinetic GluA3 and GluA4 subunits at auditory nerve synapses depend on the target cells. Brain Structure and Function. 222(8), 3375–3393. mla: Rubio, María, et al. “The Number and Distribution of AMPA Receptor Channels Containing Fast Kinetic GluA3 and GluA4 Subunits at Auditory Nerve Synapses Depend on the Target Cells.” Brain Structure and Function, vol. 222, no. 8, Springer, 2017, pp. 3375–93, doi:10.1007/s00429-017-1408-0. short: M. Rubio, K. Matsui, Y. Fukazawa, N. Kamasawa, H. Harada, M. Itakura, E. Molnár, M. Abe, K. Sakimura, R. Shigemoto, Brain Structure and Function 222 (2017) 3375–3393. date_created: 2018-12-11T11:48:14Z date_published: 2017-11-01T00:00:00Z date_updated: 2023-09-27T14:14:51Z day: '01' ddc: - '571' department: - _id: RySh doi: 10.1007/s00429-017-1408-0 external_id: isi: - '000414761700002' file: - access_level: open_access checksum: 73787a22507de8fb585bb598e1418ca7 content_type: application/pdf creator: system date_created: 2018-12-12T10:10:20Z date_updated: 2020-07-14T12:47:56Z file_id: '4806' file_name: IST-2017-881-v1+1_s00429-017-1408-0.pdf file_size: 4011126 relation: main_file file_date_updated: 2020-07-14T12:47:56Z has_accepted_license: '1' intvolume: ' 222' isi: 1 issue: '8' language: - iso: eng month: '11' oa: 1 oa_version: Published Version page: 3375 - 3393 publication: Brain Structure and Function publication_identifier: issn: - '18632653' publication_status: published publisher: Springer publist_id: '6932' pubrep_id: '881' quality_controlled: '1' scopus_import: '1' status: public title: The number and distribution of AMPA receptor channels containing fast kinetic GluA3 and GluA4 subunits at auditory nerve synapses depend on the target cells tmp: image: /images/cc_by.png legal_code_url: https://creativecommons.org/licenses/by/4.0/legalcode name: Creative Commons Attribution 4.0 International Public License (CC-BY 4.0) short: CC BY (4.0) type: journal_article user_id: c635000d-4b10-11ee-a964-aac5a93f6ac1 volume: 222 year: '2017' ... --- _id: '1094' abstract: - lang: eng text: Immunogold labeling of freeze-fracture replicas has recently been used for high-resolution visualization of protein localization in electron microscopy. This method has higher labeling efficiency than conventional immunogold methods for membrane molecules allowing precise quantitative measurements. However, one of the limitations of freeze-fracture replica immunolabeling is difficulty in keeping structural orientation and identifying labeled profiles in complex tissues like brain. The difficulty is partly due to fragmentation of freeze-fracture replica preparations during labeling procedures and limited morphological clues on the replica surface. To overcome these issues, we introduce here a grid-glued replica method combined with SEM observation. This method allows histological staining before dissolving the tissue and easy handling of replicas during immunogold labeling, and keeps the whole replica surface intact without fragmentation. The procedure described here is also useful for matched double-replica analysis allowing further identification of labeled profiles in corresponding P-face and E-face. acknowledged_ssus: - _id: EM-Fac acknowledgement: 'We thank Prof. Elek Molnár for providing us a pan-AMPAR anti-body used in Fig.2 and Dr. Ludek Lovicar for technical assistance in scanning electron microscope imaging. This work was supported by the European Union (HBP—Project Ref. 604102). ' alternative_title: - Methods in Molecular Biology article_processing_charge: No author: - first_name: Harumi full_name: Harada, Harumi id: 2E55CDF2-F248-11E8-B48F-1D18A9856A87 last_name: Harada orcid: 0000-0001-7429-7896 - first_name: Ryuichi full_name: Shigemoto, Ryuichi id: 499F3ABC-F248-11E8-B48F-1D18A9856A87 last_name: Shigemoto orcid: 0000-0001-8761-9444 citation: ama: 'Harada H, Shigemoto R. Immunogold protein localization on grid-glued freeze-fracture replicas. In: High-Resolution Imaging of Cellular Proteins. Vol 1474. Springer; 2016:203-216. doi:10.1007/978-1-4939-6352-2_12' apa: Harada, H., & Shigemoto, R. (2016). Immunogold protein localization on grid-glued freeze-fracture replicas. In High-Resolution Imaging of Cellular Proteins (Vol. 1474, pp. 203–216). Springer. https://doi.org/10.1007/978-1-4939-6352-2_12 chicago: Harada, Harumi, and Ryuichi Shigemoto. “Immunogold Protein Localization on Grid-Glued Freeze-Fracture Replicas.” In High-Resolution Imaging of Cellular Proteins, 1474:203–16. Springer, 2016. https://doi.org/10.1007/978-1-4939-6352-2_12. ieee: H. Harada and R. Shigemoto, “Immunogold protein localization on grid-glued freeze-fracture replicas,” in High-Resolution Imaging of Cellular Proteins, vol. 1474, Springer, 2016, pp. 203–216. ista: 'Harada H, Shigemoto R. 2016.Immunogold protein localization on grid-glued freeze-fracture replicas. In: High-Resolution Imaging of Cellular Proteins. Methods in Molecular Biology, vol. 1474, 203–216.' mla: Harada, Harumi, and Ryuichi Shigemoto. “Immunogold Protein Localization on Grid-Glued Freeze-Fracture Replicas.” High-Resolution Imaging of Cellular Proteins, vol. 1474, Springer, 2016, pp. 203–16, doi:10.1007/978-1-4939-6352-2_12. short: H. Harada, R. Shigemoto, in:, High-Resolution Imaging of Cellular Proteins, Springer, 2016, pp. 203–216. date_created: 2018-12-11T11:50:06Z date_published: 2016-08-12T00:00:00Z date_updated: 2023-09-05T14:09:01Z day: '12' department: - _id: RySh doi: 10.1007/978-1-4939-6352-2_12 ec_funded: 1 intvolume: ' 1474' language: - iso: eng month: '08' oa_version: None page: 203 - 216 project: - _id: 25CD3DD2-B435-11E9-9278-68D0E5697425 call_identifier: FP7 grant_number: '604102' name: Localization of ion channels and receptors by two and three-dimensional immunoelectron microscopic approaches publication: High-Resolution Imaging of Cellular Proteins publication_identifier: eissn: - 1611-3349 issn: - 0302-9743 publication_status: published publisher: Springer publist_id: '6281' quality_controlled: '1' status: public title: Immunogold protein localization on grid-glued freeze-fracture replicas type: book_chapter user_id: c635000d-4b10-11ee-a964-aac5a93f6ac1 volume: 1474 year: '2016' ... --- _id: '1546' abstract: - lang: eng text: Synaptic efficacy and precision are influenced by the coupling of voltage-gated Ca2+ channels (VGCCs) to vesicles. But because the topography of VGCCs and their proximity to vesicles is unknown, a quantitative understanding of the determinants of vesicular release at nanometer scale is lacking. To investigate this, we combined freeze-fracture replica immunogold labeling of Cav2.1 channels, local [Ca2+] imaging, and patch pipette perfusion of EGTA at the calyx of Held. Between postnatal day 7 and 21, VGCCs formed variable sized clusters and vesicular release became less sensitive to EGTA, whereas fixed Ca2+ buffer properties remained constant. Experimentally constrained reaction-diffusion simulations suggest that Ca2+ sensors for vesicular release are located at the perimeter of VGCC clusters (<30nm) and predict that VGCC number per cluster determines vesicular release probability without altering release time course. This "perimeter release model" provides a unifying framework accounting for developmental changes in both synaptic efficacy and time course. acknowledgement: This work was supported by the Core Research for Evolutional Science and Technology (CREST) of Japan Science and Technology Agency to T.T. and R.S.; by the funding provided by Okinawa Institute of Science and Technology (OIST) to T.T. and Y.N.; by JSPS Core-to-Core Program, A. Advanced Networks to T.T.; by the Grant-in-Aid for Young Scientists from the Japanese Ministry of Education, Culture, Sports, Science and Technology (#23700474) to Y.N.; by the Centre National de la Recherche Scientifique through the Actions Thematiques et Initatives sur Programme, Fondation Fyssen, Fondation pour la Recherche Medicale, Federation pour la Recherche sur le Cerveau, Agence Nationale de la Recherche (ANR-2007-Neuro-008-01 and ANR-2010-BLAN-1411-01) to D.D. and Y.N.; and by the European Commission Coordination Action ENINET (LSHM-CT-2005-19063) to D.D. and R.A.S. R.A.S. and J.S.R. were funded by Wellcome Trust Senior (064413) and Principal (095667) Research Fellowship and an ERC advance grant (294667) to RAS. author: - first_name: Yukihiro full_name: Nakamura, Yukihiro last_name: Nakamura - first_name: Harumi full_name: Harada, Harumi id: 2E55CDF2-F248-11E8-B48F-1D18A9856A87 last_name: Harada orcid: 0000-0001-7429-7896 - first_name: Naomi full_name: Kamasawa, Naomi last_name: Kamasawa - first_name: Ko full_name: Matsui, Ko last_name: Matsui - first_name: Jason full_name: Rothman, Jason last_name: Rothman - first_name: Ryuichi full_name: Shigemoto, Ryuichi id: 499F3ABC-F248-11E8-B48F-1D18A9856A87 last_name: Shigemoto orcid: 0000-0001-8761-9444 - first_name: R Angus full_name: Silver, R Angus last_name: Silver - first_name: David full_name: Digregorio, David last_name: Digregorio - first_name: Tomoyuki full_name: Takahashi, Tomoyuki last_name: Takahashi citation: ama: Nakamura Y, Harada H, Kamasawa N, et al. Nanoscale distribution of presynaptic Ca2+ channels and its impact on vesicular release during development. Neuron. 2015;85(1):145-158. doi:10.1016/j.neuron.2014.11.019 apa: Nakamura, Y., Harada, H., Kamasawa, N., Matsui, K., Rothman, J., Shigemoto, R., … Takahashi, T. (2015). Nanoscale distribution of presynaptic Ca2+ channels and its impact on vesicular release during development. Neuron. Elsevier. https://doi.org/10.1016/j.neuron.2014.11.019 chicago: Nakamura, Yukihiro, Harumi Harada, Naomi Kamasawa, Ko Matsui, Jason Rothman, Ryuichi Shigemoto, R Angus Silver, David Digregorio, and Tomoyuki Takahashi. “Nanoscale Distribution of Presynaptic Ca2+ Channels and Its Impact on Vesicular Release during Development.” Neuron. Elsevier, 2015. https://doi.org/10.1016/j.neuron.2014.11.019. ieee: Y. Nakamura et al., “Nanoscale distribution of presynaptic Ca2+ channels and its impact on vesicular release during development,” Neuron, vol. 85, no. 1. Elsevier, pp. 145–158, 2015. ista: Nakamura Y, Harada H, Kamasawa N, Matsui K, Rothman J, Shigemoto R, Silver RA, Digregorio D, Takahashi T. 2015. Nanoscale distribution of presynaptic Ca2+ channels and its impact on vesicular release during development. Neuron. 85(1), 145–158. mla: Nakamura, Yukihiro, et al. “Nanoscale Distribution of Presynaptic Ca2+ Channels and Its Impact on Vesicular Release during Development.” Neuron, vol. 85, no. 1, Elsevier, 2015, pp. 145–58, doi:10.1016/j.neuron.2014.11.019. short: Y. Nakamura, H. Harada, N. Kamasawa, K. Matsui, J. Rothman, R. Shigemoto, R.A. Silver, D. Digregorio, T. Takahashi, Neuron 85 (2015) 145–158. date_created: 2018-12-11T11:52:39Z date_published: 2015-01-07T00:00:00Z date_updated: 2021-01-12T06:51:31Z day: '07' ddc: - '570' department: - _id: RySh doi: 10.1016/j.neuron.2014.11.019 file: - access_level: open_access checksum: 725f4d5be2dbb44b283ce722645ef37d content_type: application/pdf creator: system date_created: 2018-12-12T10:15:47Z date_updated: 2020-07-14T12:45:01Z file_id: '5170' file_name: IST-2016-482-v1+1_1-s2.0-S0896627314010472-main.pdf file_size: 3080111 relation: main_file file_date_updated: 2020-07-14T12:45:01Z has_accepted_license: '1' intvolume: ' 85' issue: '1' language: - iso: eng month: '01' oa: 1 oa_version: Published Version page: 145 - 158 publication: Neuron publication_status: published publisher: Elsevier publist_id: '5625' pubrep_id: '482' quality_controlled: '1' scopus_import: 1 status: public title: Nanoscale distribution of presynaptic Ca2+ channels and its impact on vesicular release during development tmp: image: /images/cc_by.png legal_code_url: https://creativecommons.org/licenses/by/4.0/legalcode name: Creative Commons Attribution 4.0 International Public License (CC-BY 4.0) short: CC BY (4.0) type: journal_article user_id: 2DF688A6-F248-11E8-B48F-1D18A9856A87 volume: 85 year: '2015' ... --- _id: '1898' abstract: - lang: eng text: Fast synaptic transmission is important for rapid information processing. To explore the maximal rate of neuronal signaling and to analyze the presynaptic mechanisms, we focused on the input layer of the cerebellar cortex, where exceptionally high action potential (AP) frequencies have been reported invivo. With paired recordings between presynaptic cerebellar mossy fiber boutons and postsynaptic granule cells, we demonstrate reliable neurotransmission upto ~1 kHz. Presynaptic APs are ultrafast, with ~100μs half-duration. Both Kv1 and Kv3 potassium channels mediate the fast repolarization, rapidly inactivating sodium channels ensure metabolic efficiency, and little AP broadening occurs during bursts of up to 1.5 kHz. Presynaptic Cav2.1 (P/Q-type) calcium channels open efficiently during ultrafast APs. Furthermore, a subset of synaptic vesicles is tightly coupled to Ca2+ channels, and vesicles are rapidly recruited to the release site. These data reveal mechanisms of presynaptic AP generation and transmitter release underlying neuronal kHz signaling. author: - first_name: Andreas full_name: Ritzau Jost, Andreas last_name: Ritzau Jost - first_name: Igor full_name: Delvendahl, Igor last_name: Delvendahl - first_name: Annika full_name: Rings, Annika last_name: Rings - first_name: Niklas full_name: Byczkowicz, Niklas last_name: Byczkowicz - first_name: Harumi full_name: Harada, Harumi id: 2E55CDF2-F248-11E8-B48F-1D18A9856A87 last_name: Harada orcid: 0000-0001-7429-7896 - first_name: Ryuichi full_name: Shigemoto, Ryuichi id: 499F3ABC-F248-11E8-B48F-1D18A9856A87 last_name: Shigemoto orcid: 0000-0001-8761-9444 - first_name: Johannes full_name: Hirrlinger, Johannes last_name: Hirrlinger - first_name: Jens full_name: Eilers, Jens last_name: Eilers - first_name: Stefan full_name: Hallermann, Stefan last_name: Hallermann citation: ama: Ritzau Jost A, Delvendahl I, Rings A, et al. Ultrafast action potentials mediate kilohertz signaling at a central synapse. Neuron. 2014;84(1):152-163. doi:10.1016/j.neuron.2014.08.036 apa: Ritzau Jost, A., Delvendahl, I., Rings, A., Byczkowicz, N., Harada, H., Shigemoto, R., … Hallermann, S. (2014). Ultrafast action potentials mediate kilohertz signaling at a central synapse. Neuron. Elsevier. https://doi.org/10.1016/j.neuron.2014.08.036 chicago: Ritzau Jost, Andreas, Igor Delvendahl, Annika Rings, Niklas Byczkowicz, Harumi Harada, Ryuichi Shigemoto, Johannes Hirrlinger, Jens Eilers, and Stefan Hallermann. “Ultrafast Action Potentials Mediate Kilohertz Signaling at a Central Synapse.” Neuron. Elsevier, 2014. https://doi.org/10.1016/j.neuron.2014.08.036. ieee: A. Ritzau Jost et al., “Ultrafast action potentials mediate kilohertz signaling at a central synapse,” Neuron, vol. 84, no. 1. Elsevier, pp. 152–163, 2014. ista: Ritzau Jost A, Delvendahl I, Rings A, Byczkowicz N, Harada H, Shigemoto R, Hirrlinger J, Eilers J, Hallermann S. 2014. Ultrafast action potentials mediate kilohertz signaling at a central synapse. Neuron. 84(1), 152–163. mla: Ritzau Jost, Andreas, et al. “Ultrafast Action Potentials Mediate Kilohertz Signaling at a Central Synapse.” Neuron, vol. 84, no. 1, Elsevier, 2014, pp. 152–63, doi:10.1016/j.neuron.2014.08.036. short: A. Ritzau Jost, I. Delvendahl, A. Rings, N. Byczkowicz, H. Harada, R. Shigemoto, J. Hirrlinger, J. Eilers, S. Hallermann, Neuron 84 (2014) 152–163. date_created: 2018-12-11T11:54:36Z date_published: 2014-10-01T00:00:00Z date_updated: 2021-01-12T06:53:55Z day: '01' department: - _id: RySh doi: 10.1016/j.neuron.2014.08.036 intvolume: ' 84' issue: '1' language: - iso: eng month: '10' oa_version: None page: 152 - 163 publication: Neuron publication_status: published publisher: Elsevier publist_id: '5197' quality_controlled: '1' scopus_import: 1 status: public title: Ultrafast action potentials mediate kilohertz signaling at a central synapse type: journal_article user_id: 4435EBFC-F248-11E8-B48F-1D18A9856A87 volume: 84 year: '2014' ... --- _id: '2478' abstract: - lang: eng text: Despite the pivotal functions of the NMDA receptor (NMDAR) for neural circuit development and synaptic plasticity, the molecular mechanisms underlying the dynamics of NMDAR trafficking are poorly understood. The cell adhesion molecule neuroligin-1 (NL1) modifies NMDAR-dependent synaptic transmission and synaptic plasticity, but it is unclear whether NL1 controls synaptic accumulation or function of the receptors. Here, we provide evidence that NL1 regulates the abundance of NMDARs at postsynaptic sites. This function relies on extracellular, NL1 isoform-specific sequences that facilitate biochemical interactions between NL1 and the NMDAR GluN1 subunit. Our work uncovers NL1 isoform-specific cisinteractions with ionotropic glutamate receptors as a key mechanism for controlling synaptic properties. author: - first_name: Elaine full_name: Budreck, Elaine C last_name: Budreck - first_name: Oh full_name: Kwon, Oh-Bin last_name: Kwon - first_name: Jung full_name: Jung, Jung-Hoon last_name: Jung - first_name: Stéphane full_name: Baudouin, Stéphane J last_name: Baudouin - first_name: Albert full_name: Thommen, Albert last_name: Thommen - first_name: Hye full_name: Kim, Hye-Sun last_name: Kim - first_name: Yugo full_name: Fukazawa, Yugo last_name: Fukazawa - first_name: Harumi full_name: Harumi Harada id: 2E55CDF2-F248-11E8-B48F-1D18A9856A87 last_name: Harada orcid: 0000-0001-7429-7896 - first_name: Katsuhiko full_name: Tabuchi, Katsuhiko last_name: Tabuchi - first_name: Ryuichi full_name: Ryuichi Shigemoto id: 499F3ABC-F248-11E8-B48F-1D18A9856A87 last_name: Shigemoto orcid: 0000-0001-8761-9444 - first_name: Peter full_name: Scheiffele, Peter last_name: Scheiffele - first_name: Joung full_name: Kim, Joung-Hun last_name: Kim citation: ama: Budreck E, Kwon O, Jung J, et al. Neuroligin-1 controls synaptic abundance of NMDA-type glutamate receptors through extracellular coupling. PNAS. 2013;110(2):725-730. doi:10.1073/pnas.1214718110 apa: Budreck, E., Kwon, O., Jung, J., Baudouin, S., Thommen, A., Kim, H., … Kim, J. (2013). Neuroligin-1 controls synaptic abundance of NMDA-type glutamate receptors through extracellular coupling. PNAS. National Academy of Sciences. https://doi.org/10.1073/pnas.1214718110 chicago: Budreck, Elaine, Oh Kwon, Jung Jung, Stéphane Baudouin, Albert Thommen, Hye Kim, Yugo Fukazawa, et al. “Neuroligin-1 Controls Synaptic Abundance of NMDA-Type Glutamate Receptors through Extracellular Coupling.” PNAS. National Academy of Sciences, 2013. https://doi.org/10.1073/pnas.1214718110. ieee: E. Budreck et al., “Neuroligin-1 controls synaptic abundance of NMDA-type glutamate receptors through extracellular coupling,” PNAS, vol. 110, no. 2. National Academy of Sciences, pp. 725–730, 2013. ista: Budreck E, Kwon O, Jung J, Baudouin S, Thommen A, Kim H, Fukazawa Y, Harada H, Tabuchi K, Shigemoto R, Scheiffele P, Kim J. 2013. Neuroligin-1 controls synaptic abundance of NMDA-type glutamate receptors through extracellular coupling. PNAS. 110(2), 725–730. mla: Budreck, Elaine, et al. “Neuroligin-1 Controls Synaptic Abundance of NMDA-Type Glutamate Receptors through Extracellular Coupling.” PNAS, vol. 110, no. 2, National Academy of Sciences, 2013, pp. 725–30, doi:10.1073/pnas.1214718110. short: E. Budreck, O. Kwon, J. Jung, S. Baudouin, A. Thommen, H. Kim, Y. Fukazawa, H. Harada, K. Tabuchi, R. Shigemoto, P. Scheiffele, J. Kim, PNAS 110 (2013) 725–730. date_created: 2018-12-11T11:57:54Z date_published: 2013-01-08T00:00:00Z date_updated: 2021-01-12T06:57:43Z day: '08' doi: 10.1073/pnas.1214718110 extern: 1 intvolume: ' 110' issue: '2' month: '01' page: 725 - 730 publication: PNAS publication_status: published publisher: National Academy of Sciences publist_id: '4423' quality_controlled: 0 status: public title: Neuroligin-1 controls synaptic abundance of NMDA-type glutamate receptors through extracellular coupling type: journal_article volume: 110 year: '2013' ... --- _id: '2690' abstract: - lang: eng text: Establishing the spatiotemporal concentration profile of neurotransmitter following synaptic vesicular release is essential for our understanding of inter-neuronal communication. Such profile is a determinant of synaptic strength, short-term plasticity and inter-synaptic crosstalk. Synaptically released glutamate has been suggested to reach a few millimolar in concentration and last for <1 ms. The synaptic cleft is often conceived as a single concentration compartment, whereas a huge gradient likely exists. Modelling studies have attempted to describe this gradient, but two key parameters, the number of glutamate in a vesicle (NGlu) and its diffusion coefficient (DGlu) in the extracellular space, remained unresolved. To determine this profile, the rat calyx of Held synapse at postnatal day 12-16 was studied where diffusion of glutamate occurs two-dimensionally and where quantification of AMPA receptor distribution on individual postsynaptic specialization on medial nucleus of the trapezoid body principal cells is possible using SDS-digested freeze-fracture replica labelling. To assess the performance of these receptors as glutamate sensors, a kinetic model of the receptors was constructed from outside-out patch recordings. From here, we simulated synaptic responses and compared them with the EPSC recordings. Combinations of NGlu and DGlu with an optimum of 7000 and 0.3 μm2 ms-1 reproduced the data, suggesting slow diffusion. Further simulations showed that a single vesicle does not saturate the synaptic receptors, and that glutamate spillover does not affect the conductance amplitude at this synapse. Using the estimated profile, we also evaluated how the number of multiple vesicle releases at individual active zones affects the amplitude of postsynaptic signals. author: - first_name: Timotheus full_name: Budisantoso, Timotheus last_name: Budisantoso - first_name: Harumi full_name: Harumi Harada id: 2E55CDF2-F248-11E8-B48F-1D18A9856A87 last_name: Harada orcid: 0000-0001-7429-7896 - first_name: Naomi full_name: Kamasawa, Naomi last_name: Kamasawa - first_name: Yugo full_name: Fukazawa, Yugo last_name: Fukazawa - first_name: Ryuichi full_name: Ryuichi Shigemoto id: 499F3ABC-F248-11E8-B48F-1D18A9856A87 last_name: Shigemoto orcid: 0000-0001-8761-9444 - first_name: Ko full_name: Matsui, Ko last_name: Matsui citation: ama: Budisantoso T, Harada H, Kamasawa N, Fukazawa Y, Shigemoto R, Matsui K. Evaluation of glutamate concentration transient in the synaptic cleft of the rat calyx of Held. Journal of Physiology. 2013;591(1):219-239. doi:10.1113/jphysiol.2012.241398 apa: Budisantoso, T., Harada, H., Kamasawa, N., Fukazawa, Y., Shigemoto, R., & Matsui, K. (2013). Evaluation of glutamate concentration transient in the synaptic cleft of the rat calyx of Held. Journal of Physiology. Wiley-Blackwell. https://doi.org/10.1113/jphysiol.2012.241398 chicago: Budisantoso, Timotheus, Harumi Harada, Naomi Kamasawa, Yugo Fukazawa, Ryuichi Shigemoto, and Ko Matsui. “Evaluation of Glutamate Concentration Transient in the Synaptic Cleft of the Rat Calyx of Held.” Journal of Physiology. Wiley-Blackwell, 2013. https://doi.org/10.1113/jphysiol.2012.241398. ieee: T. Budisantoso, H. Harada, N. Kamasawa, Y. Fukazawa, R. Shigemoto, and K. Matsui, “Evaluation of glutamate concentration transient in the synaptic cleft of the rat calyx of Held,” Journal of Physiology, vol. 591, no. 1. Wiley-Blackwell, pp. 219–239, 2013. ista: Budisantoso T, Harada H, Kamasawa N, Fukazawa Y, Shigemoto R, Matsui K. 2013. Evaluation of glutamate concentration transient in the synaptic cleft of the rat calyx of Held. Journal of Physiology. 591(1), 219–239. mla: Budisantoso, Timotheus, et al. “Evaluation of Glutamate Concentration Transient in the Synaptic Cleft of the Rat Calyx of Held.” Journal of Physiology, vol. 591, no. 1, Wiley-Blackwell, 2013, pp. 219–39, doi:10.1113/jphysiol.2012.241398. short: T. Budisantoso, H. Harada, N. Kamasawa, Y. Fukazawa, R. Shigemoto, K. Matsui, Journal of Physiology 591 (2013) 219–239. date_created: 2018-12-11T11:59:05Z date_published: 2013-01-01T00:00:00Z date_updated: 2021-01-12T06:59:04Z day: '01' doi: 10.1113/jphysiol.2012.241398 extern: 1 intvolume: ' 591' issue: '1' month: '01' page: 219 - 239 publication: Journal of Physiology publication_status: published publisher: Wiley-Blackwell publist_id: '4207' quality_controlled: 0 status: public title: Evaluation of glutamate concentration transient in the synaptic cleft of the rat calyx of Held type: journal_article volume: 591 year: '2013' ... --- _id: '2615' abstract: - lang: eng text: Taste-mGluR4, cloned from taste tissues, is a truncated variant of brain-expressed mGluR4a (brain-mGluR4), and is known to be a candidate for the receptor involved in the umami taste sense. Although the expression patterns of taste- and brain-mGluR4 mRNAs have been demonstrated, no mention has so far been made of the expression of these two mGluR4 proteins in taste tissues. The present study examined the expression of taste-mGluR4 and brain-mGluR4 proteins in rat taste tissues by using a specific antibody for mGluR4a which shared a C-terminus of both taste- and brain-mGluR4, for immunoblot analysis and immunohistochemistry. Immunoblot analysis showed that both brain-mGluR4 and taste-mGluR4 were expressed in the taste tissues. Taste-mGluR4 was not detected in the cerebellum. The immunoreactive band for brain-mGluR4 protein was much stronger than that for taste-mGluR4 protein. In the cryosections of fungiform, foliate and circumvallate papillae, the antibody against taste-mGluR4 exhibited intense labeling of the taste pores and taste hairs in all the taste buds of gustatory papillae examined; the immunoreaction to the antibody against brain-mGluR4 was more intense at the same sites of the taste buds. The portions of the taste bud cells below the taste pore and surrounding keratinocytes did not show any immunoreactivities. The results of the present study strongly suggest that, in addition to taste-mGluR4, brain-mGluR4 may function even more importantly than the former as a receptor for glutamate, i.e. the umami taste sensation. article_processing_charge: No article_type: original author: - first_name: Takashi full_name: Toyono, Takashi last_name: Toyono - first_name: Yuji full_name: Seta, Yuji last_name: Seta - first_name: Shinji full_name: Sataoka, Shinji last_name: Sataoka - first_name: Harumi full_name: Harada, Harumi id: 2E55CDF2-F248-11E8-B48F-1D18A9856A87 last_name: Harada orcid: 0000-0001-7429-7896 - first_name: Takahiko full_name: Morotomi, Takahiko last_name: Morotomi - first_name: Shintaro full_name: Kawano, Shintaro last_name: Kawano - first_name: Ryuichi full_name: Shigemoto, Ryuichi id: 499F3ABC-F248-11E8-B48F-1D18A9856A87 last_name: Shigemoto orcid: 0000-0001-8761-9444 - first_name: Kuniaki full_name: Toyoshima, Kuniaki last_name: Toyoshima citation: ama: Toyono T, Seta Y, Sataoka S, et al. Expression of the metabotropic glutamate receptor, mGluR4a, in the taste hairs of taste buds in rat gustatory papillae. Archives of Histology and Cytology. 2002;65(1):91-96. doi:10.1679/aohc.65.91 apa: Toyono, T., Seta, Y., Sataoka, S., Harada, H., Morotomi, T., Kawano, S., … Toyoshima, K. (2002). Expression of the metabotropic glutamate receptor, mGluR4a, in the taste hairs of taste buds in rat gustatory papillae. Archives of Histology and Cytology. Japan Society of Histological Documentation. https://doi.org/10.1679/aohc.65.91 chicago: Toyono, Takashi, Yuji Seta, Shinji Sataoka, Harumi Harada, Takahiko Morotomi, Shintaro Kawano, Ryuichi Shigemoto, and Kuniaki Toyoshima. “Expression of the Metabotropic Glutamate Receptor, MGluR4a, in the Taste Hairs of Taste Buds in Rat Gustatory Papillae.” Archives of Histology and Cytology. Japan Society of Histological Documentation, 2002. https://doi.org/10.1679/aohc.65.91. ieee: T. Toyono et al., “Expression of the metabotropic glutamate receptor, mGluR4a, in the taste hairs of taste buds in rat gustatory papillae,” Archives of Histology and Cytology, vol. 65, no. 1. Japan Society of Histological Documentation, pp. 91–96, 2002. ista: Toyono T, Seta Y, Sataoka S, Harada H, Morotomi T, Kawano S, Shigemoto R, Toyoshima K. 2002. Expression of the metabotropic glutamate receptor, mGluR4a, in the taste hairs of taste buds in rat gustatory papillae. Archives of Histology and Cytology. 65(1), 91–96. mla: Toyono, Takashi, et al. “Expression of the Metabotropic Glutamate Receptor, MGluR4a, in the Taste Hairs of Taste Buds in Rat Gustatory Papillae.” Archives of Histology and Cytology, vol. 65, no. 1, Japan Society of Histological Documentation, 2002, pp. 91–96, doi:10.1679/aohc.65.91. short: T. Toyono, Y. Seta, S. Sataoka, H. Harada, T. Morotomi, S. Kawano, R. Shigemoto, K. Toyoshima, Archives of Histology and Cytology 65 (2002) 91–96. date_created: 2018-12-11T11:58:41Z date_published: 2002-01-01T00:00:00Z date_updated: 2023-07-25T10:00:15Z day: '01' doi: 10.1679/aohc.65.91 extern: '1' external_id: pmid: - '12002614' intvolume: ' 65' issue: '1' language: - iso: eng month: '01' oa_version: None page: 91 - 96 pmid: 1 publication: Archives of Histology and Cytology publication_identifier: issn: - 0914-9465 publication_status: published publisher: Japan Society of Histological Documentation publist_id: '4283' quality_controlled: '1' scopus_import: '1' status: public title: Expression of the metabotropic glutamate receptor, mGluR4a, in the taste hairs of taste buds in rat gustatory papillae type: journal_article user_id: ea97e931-d5af-11eb-85d4-e6957dddbf17 volume: 65 year: '2002' ...