[{"scopus_import":"1","month":"09","intvolume":" 41","abstract":[{"lang":"eng","text":"Rab-interacting molecule (RIM)-binding protein 2 (BP2) is a multidomain protein of the presynaptic active zone (AZ). By binding to RIM, bassoon (Bsn), and voltage-gated Ca2+ channels (CaV), it is considered to be a central organizer of the topography of CaV and release sites of synaptic vesicles (SVs) at the AZ. Here, we used RIM-BP2 knock-out (KO) mice and their wild-type (WT) littermates of either sex to investigate the role of RIM-BP2 at the endbulb of Held synapse of auditory nerve fibers (ANFs) with bushy cells (BCs) of the cochlear nucleus, a fast relay of the auditory pathway with high release probability. Disruption of RIM-BP2 lowered release probability altering short-term plasticity and reduced evoked EPSCs. Analysis of SV pool dynamics during high-frequency train stimulation indicated a reduction of SVs with high release probability but an overall normal size of the readily releasable SV pool (RRP). The Ca2+-dependent fast component of SV replenishment after RRP depletion was slowed. Ultrastructural analysis by superresolution light and electron microscopy revealed an impaired topography of presynaptic CaV and a reduction of docked and membrane-proximal SVs at the AZ. We conclude that RIM-BP2 organizes the topography of CaV, and promotes SV tethering and docking. This way RIM-BP2 is critical for establishing a high initial release probability as required to reliably signal sound onset information that we found to be degraded in BCs of RIM-BP2-deficient mice in vivo. SIGNIFICANCE STATEMENT: Rab-interacting molecule (RIM)-binding proteins (BPs) are key organizers of the active zone (AZ). Using a multidisciplinary approach to the calyceal endbulb of Held synapse that transmits auditory information at rates of up to hundreds of Hertz with submillisecond precision we demonstrate a requirement for RIM-BP2 for normal auditory signaling. Endbulb synapses lacking RIM-BP2 show a reduced release probability despite normal whole-terminal Ca2+ influx and abundance of the key priming protein Munc13-1, a reduced rate of SV replenishment, as well as an altered topography of voltage-gated (CaV)2.1 Ca2+ channels, and fewer docked and membrane proximal synaptic vesicles (SVs). This hampers transmission of sound onset information likely affecting downstream neural computations such as of sound localization."}],"pmid":1,"oa_version":"Published Version","issue":"37","volume":41,"publication_identifier":{"eissn":["1529-2401"],"issn":["0270-6474"]},"publication_status":"published","file":[{"success":1,"checksum":"769ab627c7355a50ccfd445e43a5f351","file_id":"11423","content_type":"application/pdf","relation":"main_file","access_level":"open_access","file_name":"2021_JourNeuroscience_Butola.pdf","date_created":"2022-05-31T09:10:15Z","file_size":11571961,"date_updated":"2022-05-31T09:10:15Z","creator":"dernst"}],"language":[{"iso":"eng"}],"article_type":"original","type":"journal_article","tmp":{"legal_code_url":"https://creativecommons.org/licenses/by/4.0/legalcode","image":"/images/cc_by.png","name":"Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)","short":"CC BY (4.0)"},"status":"public","_id":"10051","department":[{"_id":"RySh"}],"file_date_updated":"2022-05-31T09:10:15Z","date_updated":"2023-08-14T06:56:30Z","ddc":["570"],"publisher":"Society for Neuroscience","quality_controlled":"1","oa":1,"acknowledgement":"This work was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) through the Collaborative Sensory Research Center 1286 [to C.W. (A4) and T.M. (B5)] and under Germany’s Excellence Strategy Grant EXC 2067/1-390729940. We thank S. Gerke, A.J. Goldak, and C. Senger-Freitag for expert technical assistance; G. Hoch for developing image analysis routines; and S. Chepurwar and N. Strenzke for technical support and discussion regarding in vivo experiments. We also thank Dr. Christian Rosenmund, Dr. Katharina Grauel, and Dr. Stephan Sigrist for providing RIM-BP2 KO mice and Dr. Masahiko Watanabe for providing the anti-neurexin-antibody, and Dr. Toshihisa Ohtsuka for the anti-ELKS-antibody. J. Neef for help with the STED imaging and image analysis; E. Neher and S. Rizzoli for discussion and comments on the manuscript; K. Eguchi for help with the statistical analysis; and C. H. Huang and J. Neef for constant support and scientific discussion.","page":"7742-7767","doi":"10.1523/JNEUROSCI.0586-21.2021","date_published":"2021-09-15T00:00:00Z","date_created":"2021-09-27T14:33:13Z","isi":1,"has_accepted_license":"1","year":"2021","day":"15","publication":"Journal of Neuroscience","author":[{"first_name":"Tanvi","last_name":"Butola","full_name":"Butola, Tanvi"},{"first_name":"Theocharis","last_name":"Alvanos","full_name":"Alvanos, Theocharis"},{"first_name":"Anika","full_name":"Hintze, Anika","last_name":"Hintze"},{"last_name":"Koppensteiner","full_name":"Koppensteiner, Peter","orcid":"0000-0002-3509-1948","first_name":"Peter","id":"3B8B25A8-F248-11E8-B48F-1D18A9856A87"},{"last_name":"Kleindienst","full_name":"Kleindienst, David","id":"42E121A4-F248-11E8-B48F-1D18A9856A87","first_name":"David"},{"id":"499F3ABC-F248-11E8-B48F-1D18A9856A87","first_name":"Ryuichi","last_name":"Shigemoto","full_name":"Shigemoto, Ryuichi","orcid":"0000-0001-8761-9444"},{"full_name":"Wichmann, Carolin","last_name":"Wichmann","first_name":"Carolin"},{"full_name":"Moser, Tobias","last_name":"Moser","first_name":"Tobias"}],"external_id":{"pmid":["34353898"],"isi":["000752287700005"]},"article_processing_charge":"No","title":"RIM-binding protein 2 organizes Ca21 channel topography and regulates release probability and vesicle replenishment at a fast central synapse","citation":{"chicago":"Butola, Tanvi, Theocharis Alvanos, Anika Hintze, Peter Koppensteiner, David Kleindienst, Ryuichi Shigemoto, Carolin Wichmann, and Tobias Moser. “RIM-Binding Protein 2 Organizes Ca21 Channel Topography and Regulates Release Probability and Vesicle Replenishment at a Fast Central Synapse.” Journal of Neuroscience. Society for Neuroscience, 2021. https://doi.org/10.1523/JNEUROSCI.0586-21.2021.","ista":"Butola T, Alvanos T, Hintze A, Koppensteiner P, Kleindienst D, Shigemoto R, Wichmann C, Moser T. 2021. RIM-binding protein 2 organizes Ca21 channel topography and regulates release probability and vesicle replenishment at a fast central synapse. Journal of Neuroscience. 41(37), 7742–7767.","mla":"Butola, Tanvi, et al. “RIM-Binding Protein 2 Organizes Ca21 Channel Topography and Regulates Release Probability and Vesicle Replenishment at a Fast Central Synapse.” Journal of Neuroscience, vol. 41, no. 37, Society for Neuroscience, 2021, pp. 7742–67, doi:10.1523/JNEUROSCI.0586-21.2021.","ieee":"T. Butola et al., “RIM-binding protein 2 organizes Ca21 channel topography and regulates release probability and vesicle replenishment at a fast central synapse,” Journal of Neuroscience, vol. 41, no. 37. Society for Neuroscience, pp. 7742–7767, 2021.","short":"T. Butola, T. Alvanos, A. Hintze, P. Koppensteiner, D. Kleindienst, R. Shigemoto, C. Wichmann, T. Moser, Journal of Neuroscience 41 (2021) 7742–7767.","apa":"Butola, T., Alvanos, T., Hintze, A., Koppensteiner, P., Kleindienst, D., Shigemoto, R., … Moser, T. (2021). RIM-binding protein 2 organizes Ca21 channel topography and regulates release probability and vesicle replenishment at a fast central synapse. Journal of Neuroscience. Society for Neuroscience. https://doi.org/10.1523/JNEUROSCI.0586-21.2021","ama":"Butola T, Alvanos T, Hintze A, et al. RIM-binding protein 2 organizes Ca21 channel topography and regulates release probability and vesicle replenishment at a fast central synapse. Journal of Neuroscience. 2021;41(37):7742-7767. doi:10.1523/JNEUROSCI.0586-21.2021"},"user_id":"4359f0d1-fa6c-11eb-b949-802e58b17ae8"},{"ec_funded":1,"volume":10,"related_material":{"link":[{"url":"https://doi.org/10.1101/2020.04.16.045112","relation":"earlier_version"}],"record":[{"relation":"dissertation_contains","id":"9562","status":"public"}]},"language":[{"iso":"eng"}],"file":[{"relation":"main_file","access_level":"open_access","content_type":"application/pdf","success":1,"checksum":"6ebcb79999f889766f7cd79ee134ad28","file_id":"9440","creator":"cziletti","file_size":8174719,"date_updated":"2021-05-31T09:43:09Z","file_name":"2021_eLife_Bhandari.pdf","date_created":"2021-05-31T09:43:09Z"}],"publication_status":"published","publication_identifier":{"eissn":["2050-084X"]},"intvolume":" 10","month":"04","scopus_import":"1","oa_version":"Published Version","abstract":[{"text":"The synaptic connection from medial habenula (MHb) to interpeduncular nucleus (IPN) is critical for emotion-related behaviors and uniquely expresses R-type Ca2+ channels (Cav2.3) and auxiliary GABAB receptor (GBR) subunits, the K+-channel tetramerization domain-containing proteins (KCTDs). Activation of GBRs facilitates or inhibits transmitter release from MHb terminals depending on the IPN subnucleus, but the role of KCTDs is unknown. We therefore examined the localization and function of Cav2.3, GBRs, and KCTDs in this pathway in mice. We show in heterologous cells that KCTD8 and KCTD12b directly bind to Cav2.3 and that KCTD8 potentiates Cav2.3 currents in the absence of GBRs. In the rostral IPN, KCTD8, KCTD12b, and Cav2.3 co-localize at the presynaptic active zone. Genetic deletion indicated a bidirectional modulation of Cav2.3-mediated release by these KCTDs with a compensatory increase of KCTD8 in the active zone in KCTD12b-deficient mice. The interaction of Cav2.3 with KCTDs therefore scales synaptic strength independent of GBR activation.","lang":"eng"}],"file_date_updated":"2021-05-31T09:43:09Z","department":[{"_id":"RySh"},{"_id":"PeJo"}],"ddc":["570"],"date_updated":"2024-03-27T23:30:30Z","status":"public","tmp":{"legal_code_url":"https://creativecommons.org/licenses/by/4.0/legalcode","image":"/images/cc_by.png","name":"Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)","short":"CC BY (4.0)"},"type":"journal_article","article_type":"original","_id":"9437","date_created":"2021-05-30T22:01:23Z","date_published":"2021-04-29T00:00:00Z","doi":"10.7554/ELIFE.68274","publication":"eLife","day":"29","year":"2021","isi":1,"has_accepted_license":"1","oa":1,"quality_controlled":"1","publisher":"eLife Sciences Publications","acknowledgement":"We are grateful to Akari Hagiwara and Toshihisa Ohtsuka for CAST antibody, and Masahiko Watanabe for neurexin antibody. We thank David Adams for kindly providing the stable Cav2.3 cell line. Cav2.3 KO mice were kindly provided by Tsutomu Tanabe. This project has received funding from the European Research Council (ERC) and European Commission (EC), under the European Union’s Horizon 2020 research and innovation programme (ERC grant agreement no. 694539 to Ryuichi Shigemoto, no. 692692 to Peter Jonas, and the Marie Skłodowska-Curie grant agreement no. 665385 to Cihan Önal), the Swiss National Science Foundation Grant 31003A-172881 to Bernhard Bettler and Deutsche Forschungsgemeinschaft (For 2143) and BIOSS-2 to Akos Kulik.","title":"GABAB receptor auxiliary subunits modulate Cav2.3-mediated release from medial habenula terminals","article_processing_charge":"No","external_id":{"isi":["000651761700001"]},"author":[{"first_name":"Pradeep","id":"45EDD1BC-F248-11E8-B48F-1D18A9856A87","last_name":"Bhandari","full_name":"Bhandari, Pradeep","orcid":"0000-0003-0863-4481"},{"full_name":"Vandael, David H","orcid":"0000-0001-7577-1676","last_name":"Vandael","first_name":"David H","id":"3AE48E0A-F248-11E8-B48F-1D18A9856A87"},{"full_name":"Fernández-Fernández, Diego","last_name":"Fernández-Fernández","first_name":"Diego"},{"last_name":"Fritzius","full_name":"Fritzius, Thorsten","first_name":"Thorsten"},{"id":"42E121A4-F248-11E8-B48F-1D18A9856A87","first_name":"David","last_name":"Kleindienst","full_name":"Kleindienst, David"},{"first_name":"Hüseyin C","id":"4659D740-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0002-2771-2011","full_name":"Önal, Hüseyin C","last_name":"Önal"},{"full_name":"Montanaro-Punzengruber, Jacqueline-Claire","last_name":"Montanaro-Punzengruber","id":"3786AB44-F248-11E8-B48F-1D18A9856A87","first_name":"Jacqueline-Claire"},{"first_name":"Martin","last_name":"Gassmann","full_name":"Gassmann, Martin"},{"orcid":"0000-0001-5001-4804","full_name":"Jonas, Peter M","last_name":"Jonas","id":"353C1B58-F248-11E8-B48F-1D18A9856A87","first_name":"Peter M"},{"last_name":"Kulik","full_name":"Kulik, Akos","first_name":"Akos"},{"last_name":"Bettler","full_name":"Bettler, Bernhard","first_name":"Bernhard"},{"id":"499F3ABC-F248-11E8-B48F-1D18A9856A87","first_name":"Ryuichi","last_name":"Shigemoto","full_name":"Shigemoto, Ryuichi","orcid":"0000-0001-8761-9444"},{"first_name":"Peter","id":"3B8B25A8-F248-11E8-B48F-1D18A9856A87","last_name":"Koppensteiner","full_name":"Koppensteiner, Peter","orcid":"0000-0002-3509-1948"}],"user_id":"4359f0d1-fa6c-11eb-b949-802e58b17ae8","citation":{"short":"P. Bhandari, D.H. Vandael, D. Fernández-Fernández, T. Fritzius, D. Kleindienst, H.C. Önal, J.-C. Montanaro-Punzengruber, M. Gassmann, P.M. Jonas, A. Kulik, B. Bettler, R. Shigemoto, P. Koppensteiner, ELife 10 (2021).","ieee":"P. Bhandari et al., “GABAB receptor auxiliary subunits modulate Cav2.3-mediated release from medial habenula terminals,” eLife, vol. 10. eLife Sciences Publications, 2021.","ama":"Bhandari P, Vandael DH, Fernández-Fernández D, et al. GABAB receptor auxiliary subunits modulate Cav2.3-mediated release from medial habenula terminals. eLife. 2021;10. doi:10.7554/ELIFE.68274","apa":"Bhandari, P., Vandael, D. H., Fernández-Fernández, D., Fritzius, T., Kleindienst, D., Önal, H. C., … Koppensteiner, P. (2021). GABAB receptor auxiliary subunits modulate Cav2.3-mediated release from medial habenula terminals. ELife. eLife Sciences Publications. https://doi.org/10.7554/ELIFE.68274","mla":"Bhandari, Pradeep, et al. “GABAB Receptor Auxiliary Subunits Modulate Cav2.3-Mediated Release from Medial Habenula Terminals.” ELife, vol. 10, e68274, eLife Sciences Publications, 2021, doi:10.7554/ELIFE.68274.","ista":"Bhandari P, Vandael DH, Fernández-Fernández D, Fritzius T, Kleindienst D, Önal HC, Montanaro-Punzengruber J-C, Gassmann M, Jonas PM, Kulik A, Bettler B, Shigemoto R, Koppensteiner P. 2021. GABAB receptor auxiliary subunits modulate Cav2.3-mediated release from medial habenula terminals. eLife. 10, e68274.","chicago":"Bhandari, Pradeep, David H Vandael, Diego Fernández-Fernández, Thorsten Fritzius, David Kleindienst, Hüseyin C Önal, Jacqueline-Claire Montanaro-Punzengruber, et al. “GABAB Receptor Auxiliary Subunits Modulate Cav2.3-Mediated Release from Medial Habenula Terminals.” ELife. eLife Sciences Publications, 2021. https://doi.org/10.7554/ELIFE.68274."},"project":[{"call_identifier":"H2020","_id":"25CA28EA-B435-11E9-9278-68D0E5697425","name":"In situ analysis of single channel subunit composition in neurons: physiological implication in synaptic plasticity and behaviour","grant_number":"694539"},{"name":"Biophysics and circuit function of a giant cortical glumatergic synapse","grant_number":"692692","_id":"25B7EB9E-B435-11E9-9278-68D0E5697425","call_identifier":"H2020"},{"name":"International IST Doctoral Program","grant_number":"665385","call_identifier":"H2020","_id":"2564DBCA-B435-11E9-9278-68D0E5697425"}],"article_number":"e68274"},{"oa":1,"publisher":"Institute of Science and Technology Austria","page":"124","date_created":"2021-06-17T14:10:47Z","date_published":"2021-06-01T00:00:00Z","doi":"10.15479/at:ista:9562","year":"2021","has_accepted_license":"1","day":"01","article_processing_charge":"No","author":[{"full_name":"Kleindienst, David","last_name":"Kleindienst","id":"42E121A4-F248-11E8-B48F-1D18A9856A87","first_name":"David"}],"title":"2B or not 2B: Hippocampal asymmetries mediated by NMDA receptor subunit GluN2B C-terminus and high-throughput image analysis by Deep-Learning","citation":{"chicago":"Kleindienst, David. “2B or Not 2B: Hippocampal Asymmetries Mediated by NMDA Receptor Subunit GluN2B C-Terminus and High-Throughput Image Analysis by Deep-Learning.” Institute of Science and Technology Austria, 2021. https://doi.org/10.15479/at:ista:9562.","ista":"Kleindienst D. 2021. 2B or not 2B: Hippocampal asymmetries mediated by NMDA receptor subunit GluN2B C-terminus and high-throughput image analysis by Deep-Learning. Institute of Science and Technology Austria.","mla":"Kleindienst, David. 2B or Not 2B: Hippocampal Asymmetries Mediated by NMDA Receptor Subunit GluN2B C-Terminus and High-Throughput Image Analysis by Deep-Learning. Institute of Science and Technology Austria, 2021, doi:10.15479/at:ista:9562.","short":"D. Kleindienst, 2B or Not 2B: Hippocampal Asymmetries Mediated by NMDA Receptor Subunit GluN2B C-Terminus and High-Throughput Image Analysis by Deep-Learning, Institute of Science and Technology Austria, 2021.","ieee":"D. Kleindienst, “2B or not 2B: Hippocampal asymmetries mediated by NMDA receptor subunit GluN2B C-terminus and high-throughput image analysis by Deep-Learning,” Institute of Science and Technology Austria, 2021.","ama":"Kleindienst D. 2B or not 2B: Hippocampal asymmetries mediated by NMDA receptor subunit GluN2B C-terminus and high-throughput image analysis by Deep-Learning. 2021. doi:10.15479/at:ista:9562","apa":"Kleindienst, D. (2021). 2B or not 2B: Hippocampal asymmetries mediated by NMDA receptor subunit GluN2B C-terminus and high-throughput image analysis by Deep-Learning. Institute of Science and Technology Austria. https://doi.org/10.15479/at:ista:9562"},"user_id":"c635000d-4b10-11ee-a964-aac5a93f6ac1","alternative_title":["ISTA Thesis"],"month":"06","acknowledged_ssus":[{"_id":"EM-Fac"}],"abstract":[{"text":"Left-right asymmetries can be considered a fundamental organizational principle of the vertebrate central nervous system. The hippocampal CA3-CA1 pyramidal cell synaptic connection shows an input-side dependent asymmetry where the hemispheric location of the presynaptic CA3 neuron determines the synaptic properties. Left-input synapses terminating on apical dendrites in stratum radiatum have a higher density of NMDA receptor subunit GluN2B, a lower density of AMPA receptor subunit GluA1 and smaller areas with less often perforated PSDs. On the other hand, left-input synapses terminating on basal dendrites in stratum oriens have lower GluN2B densities than right-input ones. Apical and basal synapses further employ different signaling pathways involved in LTP. SDS-digested freeze-fracture replica labeling can visualize synaptic membrane proteins with high sensitivity and resolution, and has been used to reveal the asymmetry at the electron microscopic level. However, it requires time-consuming manual demarcation of the synaptic surface for quantitative measurements. To facilitate the analysis of replica labeling, I first developed a software named Darea, which utilizes deep-learning to automatize this demarcation. With Darea I characterized the synaptic distribution of NMDA and AMPA receptors as well as the voltage-gated Ca2+ channels in CA1 stratum radiatum and oriens. Second, I explored the role of GluN2B and its carboxy-terminus in the establishment of input-side dependent hippocampal asymmetry. In conditional knock-out mice lacking GluN2B expression in CA1 and GluN2B-2A swap mice, where GluN2B carboxy-terminus was exchanged to that of GluN2A, no significant asymmetries of GluN2B, GluA1 and PSD area were detected. We further discovered a previously unknown functional asymmetry of GluN2A, which was also lost in the swap mouse. These results demonstrate that GluN2B carboxy-terminus plays a critical role in normal formation of input-side dependent asymmetry.","lang":"eng"}],"oa_version":"Published Version","related_material":{"record":[{"relation":"part_of_dissertation","id":"9756","status":"public"},{"relation":"part_of_dissertation","status":"public","id":"9437"},{"id":"8532","status":"public","relation":"part_of_dissertation"},{"id":"612","status":"public","relation":"part_of_dissertation"}]},"degree_awarded":"PhD","publication_status":"published","publication_identifier":{"issn":["2663-337X"]},"language":[{"iso":"eng"}],"file":[{"file_name":"Thesis.pdf","date_created":"2021-06-17T14:03:14Z","creator":"dkleindienst","file_size":77299142,"date_updated":"2022-07-02T22:30:04Z","embargo":"2022-07-01","file_id":"9563","checksum":"659df5518db495f679cb1df9e9bd1d94","relation":"main_file","access_level":"open_access","content_type":"application/pdf"},{"file_id":"9564","checksum":"3bcf63a2b19e5b6663be051bea332748","content_type":"application/zip","embargo_to":"open_access","access_level":"closed","relation":"source_file","date_created":"2021-06-17T14:04:30Z","file_name":"Thesis_source.zip","date_updated":"2022-07-02T22:30:04Z","file_size":369804895,"creator":"dkleindienst"}],"type":"dissertation","status":"public","_id":"9562","file_date_updated":"2022-07-02T22:30:04Z","department":[{"_id":"GradSch"},{"_id":"RySh"}],"date_updated":"2023-09-11T12:55:53Z","supervisor":[{"last_name":"Shigemoto","full_name":"Shigemoto, Ryuichi","orcid":"0000-0001-8761-9444","first_name":"Ryuichi","id":"499F3ABC-F248-11E8-B48F-1D18A9856A87"}],"ddc":["570"]},{"intvolume":" 169","place":"New York","month":"07","alternative_title":["Neuromethods"],"oa_version":"None","abstract":[{"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.","lang":"eng"}],"ec_funded":1,"related_material":{"record":[{"relation":"dissertation_contains","status":"public","id":"9562"}]},"volume":169,"language":[{"iso":"eng"}],"publication_status":"published","publication_identifier":{"eisbn":["9781071615225"],"isbn":["9781071615218"]},"keyword":["Freeze-fracture replica: Deep learning","Immunogold labeling","Integral membrane protein","Electron microscopy"],"status":"public","type":"book_chapter","_id":"9756","series_title":"Neuromethods","department":[{"_id":"RySh"},{"_id":"EM-Fac"}],"ddc":["573"],"date_updated":"2024-03-27T23:30:30Z","quality_controlled":"1","publisher":"Humana","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.).","date_created":"2021-07-30T09:34:56Z","doi":"10.1007/978-1-0716-1522-5_19","date_published":"2021-07-27T00:00:00Z","page":"267-283","publication":" Receptor and Ion Channel Detection in the Brain","day":"27","year":"2021","has_accepted_license":"1","project":[{"grant_number":"694539","name":"In situ analysis of single channel subunit composition in neurons: physiological implication in synaptic plasticity and behaviour","_id":"25CA28EA-B435-11E9-9278-68D0E5697425","call_identifier":"H2020"},{"_id":"25CBA828-B435-11E9-9278-68D0E5697425","call_identifier":"H2020","name":"Human Brain Project Specific Grant Agreement 1 (HBP SGA 1)","grant_number":"720270"}],"title":"High-Resolution localization and quantitation of membrane proteins by SDS-digested freeze-fracture replica labeling (SDS-FRL)","article_processing_charge":"No","author":[{"first_name":"Walter","id":"3F99E422-F248-11E8-B48F-1D18A9856A87","last_name":"Kaufmann","orcid":"0000-0001-9735-5315","full_name":"Kaufmann, Walter"},{"id":"42E121A4-F248-11E8-B48F-1D18A9856A87","first_name":"David","last_name":"Kleindienst","full_name":"Kleindienst, David"},{"id":"2E55CDF2-F248-11E8-B48F-1D18A9856A87","first_name":"Harumi","last_name":"Harada","full_name":"Harada, Harumi","orcid":"0000-0001-7429-7896"},{"first_name":"Ryuichi","id":"499F3ABC-F248-11E8-B48F-1D18A9856A87","last_name":"Shigemoto","full_name":"Shigemoto, Ryuichi","orcid":"0000-0001-8761-9444"}],"user_id":"D865714E-FA4E-11E9-B85B-F5C5E5697425","citation":{"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.","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.","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.","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.","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."}},{"month":"09","intvolume":" 21","scopus_import":"1","oa_version":"Published Version","abstract":[{"lang":"eng","text":"The molecular anatomy of synapses defines their characteristics in transmission and plasticity. Precise measurements of the number and distribution of synaptic proteins are important for our understanding of synapse heterogeneity within and between brain regions. Freeze–fracture replica immunogold electron microscopy enables us to analyze them quantitatively on a two-dimensional membrane surface. Here, we introduce Darea software, which utilizes deep learning for analysis of replica images and demonstrate its usefulness for quick measurements of the pre- and postsynaptic areas, density and distribution of gold particles at synapses in a reproducible manner. We used Darea for comparing glutamate receptor and calcium channel distributions between hippocampal CA3-CA1 spine synapses on apical and basal dendrites, which differ in signaling pathways involved in synaptic plasticity. We found that apical synapses express a higher density of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and a stronger increase of AMPA receptors with synaptic size, while basal synapses show a larger increase in N-methyl-D-aspartate (NMDA) receptors with size. Interestingly, AMPA and NMDA receptors are segregated within postsynaptic sites and negatively correlated in density among both apical and basal synapses. In the presynaptic sites, Cav2.1 voltage-gated calcium channels show similar densities in apical and basal synapses with distributions consistent with an exclusion zone model of calcium channel-release site topography."}],"volume":21,"related_material":{"record":[{"id":"9562","status":"public","relation":"dissertation_contains"}]},"issue":"18","ec_funded":1,"file":[{"file_size":5748456,"date_updated":"2020-09-21T14:08:58Z","creator":"dernst","file_name":"2020_JournMolecSciences_Kleindienst.pdf","date_created":"2020-09-21T14:08:58Z","content_type":"application/pdf","relation":"main_file","access_level":"open_access","success":1,"file_id":"8551","checksum":"2e4f62f3cfe945b7391fc3070e5a289f"}],"language":[{"iso":"eng"}],"publication_identifier":{"eissn":["14220067"],"issn":["16616596"]},"publication_status":"published","status":"public","type":"journal_article","article_type":"original","tmp":{"legal_code_url":"https://creativecommons.org/licenses/by/4.0/legalcode","image":"/images/cc_by.png","name":"Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)","short":"CC BY (4.0)"},"_id":"8532","department":[{"_id":"RySh"}],"file_date_updated":"2020-09-21T14:08:58Z","ddc":["570"],"date_updated":"2024-03-27T23:30:30Z","publisher":"MDPI","quality_controlled":"1","oa":1,"acknowledgement":"This research was funded by Austrian Academy of Sciences, DOC fellowship to D.K., European Research\r\nCouncil Advanced Grant 694539 and European Union Human Brain Project (HBP) SGA2 785907 to R.S.\r\nWe acknowledge Elena Hollergschwandtner for technical support.","date_published":"2020-09-14T00:00:00Z","doi":"10.3390/ijms21186737","date_created":"2020-09-20T22:01:35Z","day":"14","publication":"International Journal of Molecular Sciences","has_accepted_license":"1","isi":1,"year":"2020","project":[{"grant_number":"694539","name":"In situ analysis of single channel subunit composition in neurons: physiological implication in synaptic plasticity and behaviour","_id":"25CA28EA-B435-11E9-9278-68D0E5697425","call_identifier":"H2020"},{"name":"Mechanism of formation and maintenance of input side-dependent asymmetry in the hippocampus","_id":"25D32BC0-B435-11E9-9278-68D0E5697425"},{"_id":"26436750-B435-11E9-9278-68D0E5697425","call_identifier":"H2020","name":"Human Brain Project Specific Grant Agreement 2 (HBP SGA 2)","grant_number":"785907"}],"article_number":"6737","title":"Deep learning-assisted high-throughput analysis of freeze-fracture replica images applied to glutamate receptors and calcium channels at hippocampal synapses","author":[{"last_name":"Kleindienst","full_name":"Kleindienst, David","id":"42E121A4-F248-11E8-B48F-1D18A9856A87","first_name":"David"},{"full_name":"Montanaro-Punzengruber, Jacqueline-Claire","last_name":"Montanaro-Punzengruber","first_name":"Jacqueline-Claire","id":"3786AB44-F248-11E8-B48F-1D18A9856A87"},{"id":"45EDD1BC-F248-11E8-B48F-1D18A9856A87","first_name":"Pradeep","orcid":"0000-0003-0863-4481","full_name":"Bhandari, Pradeep","last_name":"Bhandari"},{"last_name":"Case","full_name":"Case, Matthew J","id":"44B7CA5A-F248-11E8-B48F-1D18A9856A87","first_name":"Matthew J"},{"first_name":"Yugo","last_name":"Fukazawa","full_name":"Fukazawa, Yugo"},{"full_name":"Shigemoto, Ryuichi","orcid":"0000-0001-8761-9444","last_name":"Shigemoto","id":"499F3ABC-F248-11E8-B48F-1D18A9856A87","first_name":"Ryuichi"}],"article_processing_charge":"No","external_id":{"isi":["000579945300001"]},"user_id":"4359f0d1-fa6c-11eb-b949-802e58b17ae8","citation":{"ista":"Kleindienst D, Montanaro-Punzengruber J-C, Bhandari P, Case MJ, Fukazawa Y, Shigemoto R. 2020. Deep learning-assisted high-throughput analysis of freeze-fracture replica images applied to glutamate receptors and calcium channels at hippocampal synapses. International Journal of Molecular Sciences. 21(18), 6737.","chicago":"Kleindienst, David, Jacqueline-Claire Montanaro-Punzengruber, Pradeep Bhandari, Matthew J Case, Yugo Fukazawa, and Ryuichi Shigemoto. “Deep Learning-Assisted High-Throughput Analysis of Freeze-Fracture Replica Images Applied to Glutamate Receptors and Calcium Channels at Hippocampal Synapses.” International Journal of Molecular Sciences. MDPI, 2020. https://doi.org/10.3390/ijms21186737.","ama":"Kleindienst D, Montanaro-Punzengruber J-C, Bhandari P, Case MJ, Fukazawa Y, Shigemoto R. Deep learning-assisted high-throughput analysis of freeze-fracture replica images applied to glutamate receptors and calcium channels at hippocampal synapses. International Journal of Molecular Sciences. 2020;21(18). doi:10.3390/ijms21186737","apa":"Kleindienst, D., Montanaro-Punzengruber, J.-C., Bhandari, P., Case, M. J., Fukazawa, Y., & Shigemoto, R. (2020). Deep learning-assisted high-throughput analysis of freeze-fracture replica images applied to glutamate receptors and calcium channels at hippocampal synapses. International Journal of Molecular Sciences. MDPI. https://doi.org/10.3390/ijms21186737","short":"D. Kleindienst, J.-C. Montanaro-Punzengruber, P. Bhandari, M.J. Case, Y. Fukazawa, R. Shigemoto, International Journal of Molecular Sciences 21 (2020).","ieee":"D. Kleindienst, J.-C. Montanaro-Punzengruber, P. Bhandari, M. J. Case, Y. Fukazawa, and R. Shigemoto, “Deep learning-assisted high-throughput analysis of freeze-fracture replica images applied to glutamate receptors and calcium channels at hippocampal synapses,” International Journal of Molecular Sciences, vol. 21, no. 18. MDPI, 2020.","mla":"Kleindienst, David, et al. “Deep Learning-Assisted High-Throughput Analysis of Freeze-Fracture Replica Images Applied to Glutamate Receptors and Calcium Channels at Hippocampal Synapses.” International Journal of Molecular Sciences, vol. 21, no. 18, 6737, MDPI, 2020, doi:10.3390/ijms21186737."}},{"publication_status":"published","language":[{"iso":"eng"}],"file":[{"creator":"system","date_updated":"2020-07-14T12:47:20Z","file_size":5542926,"date_created":"2018-12-12T10:15:36Z","file_name":"IST-2018-1013-v1+1_2018_Kleindienst_Differential.pdf","access_level":"open_access","relation":"main_file","content_type":"application/pdf","checksum":"a55b3103476ecb5f4f983d8801807e8b","file_id":"5157"}],"ec_funded":1,"issue":"3","volume":223,"related_material":{"record":[{"id":"9562","status":"public","relation":"dissertation_contains"}]},"abstract":[{"lang":"eng","text":"Metabotropic GABAB receptors mediate slow inhibitory effects presynaptically and postsynaptically through the modulation of different effector signalling pathways. Here, we analysed the distribution of GABAB receptors using highly sensitive SDS-digested freeze-fracture replica labelling in mouse cerebellar Purkinje cells. Immunoreactivity for GABAB1 was observed on presynaptic and, more abundantly, on postsynaptic compartments, showing both scattered and clustered distribution patterns. Quantitative analysis of immunoparticles revealed a somato-dendritic gradient, with the density of immunoparticles increasing 26-fold from somata to dendritic spines. To understand the spatial relationship of GABAB receptors with two key effector ion channels, the G protein-gated inwardly rectifying K+ (GIRK/Kir3) channel and the voltage-dependent Ca2+ channel, biochemical and immunohistochemical approaches were performed. Co-immunoprecipitation analysis demonstrated that GABAB receptors co-assembled with GIRK and CaV2.1 channels in the cerebellum. Using double-labelling immunoelectron microscopic techniques, co-clustering between GABAB1 and GIRK2 was detected in dendritic spines, whereas they were mainly segregated in the dendritic shafts. In contrast, co-clustering of GABAB1 and CaV2.1 was detected in dendritic shafts but not spines. Presynaptically, although no significant co-clustering of GABAB1 and GIRK2 or CaV2.1 channels was detected, inter-cluster distance for GABAB1 and GIRK2 was significantly smaller in the active zone than in the dendritic shafts, and that for GABAB1 and CaV2.1 was significantly smaller in the active zone than in the dendritic shafts and spines. Thus, GABAB receptors are associated with GIRK and CaV2.1 channels in different subcellular compartments. These data provide a better framework for understanding the different roles played by GABAB receptors and their effector ion channels in the cerebellar network."}],"oa_version":"Published Version","scopus_import":"1","intvolume":" 223","month":"04","date_updated":"2024-03-27T23:30:30Z","ddc":["571"],"department":[{"_id":"RySh"}],"file_date_updated":"2020-07-14T12:47:20Z","_id":"612","tmp":{"legal_code_url":"https://creativecommons.org/licenses/by/4.0/legalcode","image":"/images/cc_by.png","name":"Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)","short":"CC BY (4.0)"},"type":"journal_article","article_type":"original","pubrep_id":"1013","status":"public","year":"2018","isi":1,"has_accepted_license":"1","publication":"Brain Structure and Function","day":"01","page":"1565 - 1587","date_created":"2018-12-11T11:47:29Z","date_published":"2018-04-01T00:00:00Z","doi":"10.1007/s00429-017-1568-y","oa":1,"publisher":"Springer","quality_controlled":"1","citation":{"chicago":"Luján, Rafael, Carolina Aguado, Francisco Ciruela, Javier Cózar, David Kleindienst, Luis De La Ossa, Bernhard Bettler, et al. “Differential Association of GABAB Receptors with Their Effector Ion Channels in Purkinje Cells.” Brain Structure and Function. Springer, 2018. https://doi.org/10.1007/s00429-017-1568-y.","ista":"Luján R, Aguado C, Ciruela F, Cózar J, Kleindienst D, De La Ossa L, Bettler B, Wickman K, Watanabe M, Shigemoto R, Fukazawa Y. 2018. Differential association of GABAB receptors with their effector ion channels in Purkinje cells. Brain Structure and Function. 223(3), 1565–1587.","mla":"Luján, Rafael, et al. “Differential Association of GABAB Receptors with Their Effector Ion Channels in Purkinje Cells.” Brain Structure and Function, vol. 223, no. 3, Springer, 2018, pp. 1565–87, doi:10.1007/s00429-017-1568-y.","apa":"Luján, R., Aguado, C., Ciruela, F., Cózar, J., Kleindienst, D., De La Ossa, L., … Fukazawa, Y. (2018). Differential association of GABAB receptors with their effector ion channels in Purkinje cells. Brain Structure and Function. Springer. https://doi.org/10.1007/s00429-017-1568-y","ama":"Luján R, Aguado C, Ciruela F, et al. Differential association of GABAB receptors with their effector ion channels in Purkinje cells. Brain Structure and Function. 2018;223(3):1565-1587. doi:10.1007/s00429-017-1568-y","short":"R. Luján, C. Aguado, F. Ciruela, J. Cózar, D. Kleindienst, L. De La Ossa, B. Bettler, K. Wickman, M. Watanabe, R. Shigemoto, Y. Fukazawa, Brain Structure and Function 223 (2018) 1565–1587.","ieee":"R. Luján et al., “Differential association of GABAB receptors with their effector ion channels in Purkinje cells,” Brain Structure and Function, vol. 223, no. 3. Springer, pp. 1565–1587, 2018."},"user_id":"c635000d-4b10-11ee-a964-aac5a93f6ac1","external_id":{"isi":["000428419500030"]},"article_processing_charge":"No","author":[{"first_name":"Rafael","full_name":"Luján, Rafael","last_name":"Luján"},{"full_name":"Aguado, Carolina","last_name":"Aguado","first_name":"Carolina"},{"last_name":"Ciruela","full_name":"Ciruela, Francisco","first_name":"Francisco"},{"last_name":"Cózar","full_name":"Cózar, Javier","first_name":"Javier"},{"full_name":"Kleindienst, David","last_name":"Kleindienst","first_name":"David","id":"42E121A4-F248-11E8-B48F-1D18A9856A87"},{"full_name":"De La Ossa, Luis","last_name":"De La Ossa","first_name":"Luis"},{"full_name":"Bettler, Bernhard","last_name":"Bettler","first_name":"Bernhard"},{"first_name":"Kevin","full_name":"Wickman, Kevin","last_name":"Wickman"},{"last_name":"Watanabe","full_name":"Watanabe, Masahiko","first_name":"Masahiko"},{"last_name":"Shigemoto","orcid":"0000-0001-8761-9444","full_name":"Shigemoto, Ryuichi","first_name":"Ryuichi","id":"499F3ABC-F248-11E8-B48F-1D18A9856A87"},{"first_name":"Yugo","full_name":"Fukazawa, Yugo","last_name":"Fukazawa"}],"publist_id":"7192","title":"Differential association of GABAB receptors with their effector ion channels in Purkinje cells","project":[{"_id":"25CBA828-B435-11E9-9278-68D0E5697425","call_identifier":"H2020","grant_number":"720270","name":"Human Brain Project Specific Grant Agreement 1 (HBP SGA 1)"},{"call_identifier":"FP7","_id":"25681D80-B435-11E9-9278-68D0E5697425","name":"International IST Postdoc Fellowship Programme","grant_number":"291734"}]}]