[{"oa_version":"Published Version","pmid":1,"acknowledged_ssus":[{"_id":"M-Shop"},{"_id":"PreCl"},{"_id":"EM-Fac"}],"abstract":[{"lang":"eng","text":"GABAB receptor (GBR) activation inhibits neurotransmitter release in axon terminals in the brain, except in medial habenula (MHb) terminals, which show robust potentiation. However, mechanisms underlying this enigmatic potentiation remain elusive. Here, we report that GBR activation on MHb terminals induces an activity-dependent transition from a facilitating, tonic to a depressing, phasic neurotransmitter release mode. This transition is accompanied by a 4.1-fold increase in readily releasable vesicle pool (RRP) size and a 3.5-fold increase of docked synaptic vesicles (SVs) at the presynaptic active zone (AZ). Strikingly, the depressing phasic release exhibits looser coupling distance than the tonic release. Furthermore, the tonic and phasic release are selectively affected by deletion of synaptoporin (SPO) and Ca\r\n 2+\r\n -dependent activator protein for secretion 2 (CAPS2), respectively. SPO modulates augmentation, the short-term plasticity associated with tonic release, and CAPS2 retains the increased RRP for initial responses in phasic response trains. The cytosolic protein CAPS2 showed a SV-associated distribution similar to the vesicular transmembrane protein SPO, and they were colocalized in the same terminals. We developed the “Flash and Freeze-fracture” method, and revealed the release of SPO-associated vesicles in both tonic and phasic modes and activity-dependent recruitment of CAPS2 to the AZ during phasic release, which lasted several minutes. Overall, these results indicate that GBR activation translocates CAPS2 to the AZ along with the fusion of CAPS2-associated SVs, contributing to persistency of the RRP increase. Thus, we identified structural and molecular mechanisms underlying tonic and phasic neurotransmitter release and their transition by GBR activation in MHb terminals."}],"intvolume":" 121","month":"02","language":[{"iso":"eng"}],"file":[{"file_name":"2024_PNAS_Koppensteiner.pdf","date_created":"2024-03-12T13:42:42Z","file_size":13648221,"date_updated":"2024-03-12T13:42:42Z","creator":"dernst","success":1,"file_id":"15110","checksum":"b25b2a057c266ff317a48b0d54d6fc8a","content_type":"application/pdf","relation":"main_file","access_level":"open_access"}],"publication_status":"published","publication_identifier":{"issn":["0027-8424"],"eissn":["1091-6490"]},"ec_funded":1,"license":"https://creativecommons.org/licenses/by-nc-nd/4.0/","volume":121,"issue":"8","related_material":{"link":[{"description":"News on ISTA Website","relation":"press_release","url":"https://ista.ac.at/en/news/neuronal-insights-flash-and-freeze-fracture/"}],"record":[{"id":"13173","status":"public","relation":"research_data"}]},"_id":"15084","status":"public","tmp":{"short":"CC BY-NC-ND (4.0)","name":"Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0)","legal_code_url":"https://creativecommons.org/licenses/by-nc-nd/4.0/legalcode","image":"/images/cc_by_nc_nd.png"},"article_type":"original","type":"journal_article","ddc":["570"],"date_updated":"2024-03-12T13:44:18Z","department":[{"_id":"RySh"},{"_id":"PeJo"}],"file_date_updated":"2024-03-12T13:42:42Z","acknowledgement":"We thank Erwin Neher and Ipe Ninan for critical comments on the manuscript. This project has received funding from the European Research Council (ERC) and European Commission, under the European Union’s Horizon 2020 research and innovation program (ERC grant agreement no. 694539 to R.S. and the Marie Skłodowska-Curie grant agreement no. 665385 to C.Ö.). This study was supported by the Cooperative Study Program of Center for Animal Resources and Collaborative Study of NINS. We thank Kohgaku Eguchi for statistical analysis, Yu Kasugai for additional EM imaging, Robert Beattie for the design of the slice recovery chamber for Flash and Freeze experiments, Todor Asenov from the ISTA machine shop for custom part preparations for high-pressure freezing, the ISTA preclinical facility for animal caretaking, and the ISTA EM facilities for technical support.","oa":1,"quality_controlled":"1","publisher":"Proceedings of the National Academy of Sciences","publication":"Proceedings of the National Academy of Sciences","day":"20","year":"2024","has_accepted_license":"1","date_created":"2024-03-05T09:23:55Z","doi":"10.1073/pnas.2301449121","date_published":"2024-02-20T00:00:00Z","article_number":"e2301449121","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"},{"name":"International IST Doctoral Program","grant_number":"665385","call_identifier":"H2020","_id":"2564DBCA-B435-11E9-9278-68D0E5697425"}],"user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87","citation":{"ista":"Koppensteiner P, Bhandari P, Önal C, Borges Merjane C, Le Monnier E, Roy U, Nakamura Y, Sadakata T, Sanbo M, Hirabayashi M, Rhee J, Brose N, Jonas PM, Shigemoto R. 2024. GABAB receptors induce phasic release from medial habenula terminals through activity-dependent recruitment of release-ready vesicles. Proceedings of the National Academy of Sciences. 121(8), e2301449121.","chicago":"Koppensteiner, Peter, Pradeep Bhandari, Cihan Önal, Carolina Borges Merjane, Elodie Le Monnier, Utsa Roy, Yukihiro Nakamura, et al. “GABAB Receptors Induce Phasic Release from Medial Habenula Terminals through Activity-Dependent Recruitment of Release-Ready Vesicles.” Proceedings of the National Academy of Sciences. Proceedings of the National Academy of Sciences, 2024. https://doi.org/10.1073/pnas.2301449121.","apa":"Koppensteiner, P., Bhandari, P., Önal, C., Borges Merjane, C., Le Monnier, E., Roy, U., … Shigemoto, R. (2024). GABAB receptors induce phasic release from medial habenula terminals through activity-dependent recruitment of release-ready vesicles. Proceedings of the National Academy of Sciences. Proceedings of the National Academy of Sciences. https://doi.org/10.1073/pnas.2301449121","ama":"Koppensteiner P, Bhandari P, Önal C, et al. GABAB receptors induce phasic release from medial habenula terminals through activity-dependent recruitment of release-ready vesicles. Proceedings of the National Academy of Sciences. 2024;121(8). doi:10.1073/pnas.2301449121","short":"P. Koppensteiner, P. Bhandari, C. Önal, C. Borges Merjane, E. Le Monnier, U. Roy, Y. Nakamura, T. Sadakata, M. Sanbo, M. Hirabayashi, J. Rhee, N. Brose, P.M. Jonas, R. Shigemoto, Proceedings of the National Academy of Sciences 121 (2024).","ieee":"P. Koppensteiner et al., “GABAB receptors induce phasic release from medial habenula terminals through activity-dependent recruitment of release-ready vesicles,” Proceedings of the National Academy of Sciences, vol. 121, no. 8. Proceedings of the National Academy of Sciences, 2024.","mla":"Koppensteiner, Peter, et al. “GABAB Receptors Induce Phasic Release from Medial Habenula Terminals through Activity-Dependent Recruitment of Release-Ready Vesicles.” Proceedings of the National Academy of Sciences, vol. 121, no. 8, e2301449121, Proceedings of the National Academy of Sciences, 2024, doi:10.1073/pnas.2301449121."},"title":"GABAB receptors induce phasic release from medial habenula terminals through activity-dependent recruitment of release-ready vesicles","article_processing_charge":"Yes (in subscription journal)","external_id":{"pmid":["38346189"]},"author":[{"last_name":"Koppensteiner","orcid":"0000-0002-3509-1948","full_name":"Koppensteiner, Peter","id":"3B8B25A8-F248-11E8-B48F-1D18A9856A87","first_name":"Peter"},{"full_name":"Bhandari, Pradeep","orcid":"0000-0003-0863-4481","last_name":"Bhandari","first_name":"Pradeep","id":"45EDD1BC-F248-11E8-B48F-1D18A9856A87"},{"orcid":"0000-0002-2771-2011","full_name":"Önal, Hüseyin C","last_name":"Önal","first_name":"Hüseyin C","id":"4659D740-F248-11E8-B48F-1D18A9856A87"},{"full_name":"Borges Merjane, Carolina","orcid":"0000-0003-0005-401X","last_name":"Borges Merjane","first_name":"Carolina","id":"4305C450-F248-11E8-B48F-1D18A9856A87"},{"full_name":"Le Monnier, Elodie","last_name":"Le Monnier","first_name":"Elodie","id":"3B59276A-F248-11E8-B48F-1D18A9856A87"},{"last_name":"Roy","full_name":"Roy, Utsa","first_name":"Utsa","id":"4d26cf11-5355-11ee-ae5a-eb05e255b9b2"},{"first_name":"Yukihiro","last_name":"Nakamura","full_name":"Nakamura, Yukihiro"},{"first_name":"Tetsushi","full_name":"Sadakata, Tetsushi","last_name":"Sadakata"},{"full_name":"Sanbo, Makoto","last_name":"Sanbo","first_name":"Makoto"},{"last_name":"Hirabayashi","full_name":"Hirabayashi, Masumi","first_name":"Masumi"},{"full_name":"Rhee, JeongSeop","last_name":"Rhee","first_name":"JeongSeop"},{"last_name":"Brose","full_name":"Brose, Nils","first_name":"Nils"},{"last_name":"Jonas","full_name":"Jonas, Peter M","orcid":"0000-0001-5001-4804","first_name":"Peter M","id":"353C1B58-F248-11E8-B48F-1D18A9856A87"},{"orcid":"0000-0001-8761-9444","full_name":"Shigemoto, Ryuichi","last_name":"Shigemoto","first_name":"Ryuichi","id":"499F3ABC-F248-11E8-B48F-1D18A9856A87"}]},{"publisher":"Elsevier","quality_controlled":"1","acknowledgement":"We thank Drs. David DiGregorio and Erwin Neher for critically reading an earlier version of the manuscript, Ralf Schneggenburger for helpful discussions, Benjamin Suter and Katharina Lichter for support with image analysis, Chris Wojtan for advice on numerical solution of partial differential equations, Maria Reva for help with Ripley analysis, Alois Schlögl for programming, and Akari Hagiwara and Toshihisa Ohtsuka for anti-ELKS antibody. We are grateful to Florian Marr, Christina Altmutter, and Vanessa Zheden for excellent technical assistance and to Eleftheria Kralli-Beller for manuscript editing. This research was supported by the Scientific Services Units (SSUs) of ISTA (Electron Microscopy Facility, Preclinical Facility, and Machine Shop). The project received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement no. 692692), the Fonds zur Förderung der Wissenschaftlichen Forschung (Z 312-B27, Wittgenstein award; P 36232-B), all to P.J., and a DOC fellowship of the Austrian Academy of Sciences to J.-J.C.","date_published":"2024-01-11T00:00:00Z","doi":"10.1016/j.neuron.2023.12.002","date_created":"2024-01-21T23:00:56Z","year":"2024","day":"11","publication":"Neuron","project":[{"_id":"25B7EB9E-B435-11E9-9278-68D0E5697425","call_identifier":"H2020","name":"Biophysics and circuit function of a giant cortical glumatergic synapse","grant_number":"692692"},{"call_identifier":"FWF","_id":"25C5A090-B435-11E9-9278-68D0E5697425","name":"The Wittgenstein Prize","grant_number":"Z00312"},{"name":"Mechanisms of GABA release in hippocampal circuits","grant_number":"P36232","_id":"bd88be38-d553-11ed-ba76-81d5a70a6ef5"},{"_id":"26B66A3E-B435-11E9-9278-68D0E5697425","name":"Development of nanodomain coupling between Ca2+ channels and release sensors at a central inhibitory synapse","grant_number":"25383"}],"author":[{"last_name":"Chen","full_name":"Chen, JingJing","id":"2C4E65C8-F248-11E8-B48F-1D18A9856A87","first_name":"JingJing"},{"orcid":"0000-0001-9735-5315","full_name":"Kaufmann, Walter","last_name":"Kaufmann","first_name":"Walter","id":"3F99E422-F248-11E8-B48F-1D18A9856A87"},{"last_name":"Chen","full_name":"Chen, Chong","first_name":"Chong","id":"3DFD581A-F248-11E8-B48F-1D18A9856A87"},{"last_name":"Arai","full_name":"Arai, Itaru","id":"32A73F6C-F248-11E8-B48F-1D18A9856A87","first_name":"Itaru"},{"first_name":"Olena","id":"3F8ABDDA-F248-11E8-B48F-1D18A9856A87","last_name":"Kim","full_name":"Kim, Olena"},{"id":"499F3ABC-F248-11E8-B48F-1D18A9856A87","first_name":"Ryuichi","orcid":"0000-0001-8761-9444","full_name":"Shigemoto, Ryuichi","last_name":"Shigemoto"},{"id":"353C1B58-F248-11E8-B48F-1D18A9856A87","first_name":"Peter M","last_name":"Jonas","orcid":"0000-0001-5001-4804","full_name":"Jonas, Peter M"}],"article_processing_charge":"No","external_id":{"pmid":["38215739"]},"title":"Developmental transformation of Ca2+ channel-vesicle nanotopography at a central GABAergic synapse","citation":{"chicago":"Chen, JingJing, Walter Kaufmann, Chong Chen, itaru Arai, Olena Kim, Ryuichi Shigemoto, and Peter M Jonas. “Developmental Transformation of Ca2+ Channel-Vesicle Nanotopography at a Central GABAergic Synapse.” Neuron. Elsevier, n.d. https://doi.org/10.1016/j.neuron.2023.12.002.","ista":"Chen J, Kaufmann W, Chen C, Arai itaru, Kim O, Shigemoto R, Jonas PM. Developmental transformation of Ca2+ channel-vesicle nanotopography at a central GABAergic synapse. Neuron.","mla":"Chen, JingJing, et al. “Developmental Transformation of Ca2+ Channel-Vesicle Nanotopography at a Central GABAergic Synapse.” Neuron, Elsevier, doi:10.1016/j.neuron.2023.12.002.","ieee":"J. Chen et al., “Developmental transformation of Ca2+ channel-vesicle nanotopography at a central GABAergic synapse,” Neuron. Elsevier.","short":"J. Chen, W. Kaufmann, C. Chen, itaru Arai, O. Kim, R. Shigemoto, P.M. Jonas, Neuron (n.d.).","apa":"Chen, J., Kaufmann, W., Chen, C., Arai, itaru, Kim, O., Shigemoto, R., & Jonas, P. M. (n.d.). Developmental transformation of Ca2+ channel-vesicle nanotopography at a central GABAergic synapse. Neuron. Elsevier. https://doi.org/10.1016/j.neuron.2023.12.002","ama":"Chen J, Kaufmann W, Chen C, et al. Developmental transformation of Ca2+ channel-vesicle nanotopography at a central GABAergic synapse. Neuron. doi:10.1016/j.neuron.2023.12.002"},"user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87","scopus_import":"1","month":"01","acknowledged_ssus":[{"_id":"EM-Fac"},{"_id":"PreCl"},{"_id":"M-Shop"}],"abstract":[{"lang":"eng","text":"The coupling between Ca2+ channels and release sensors is a key factor defining the signaling properties of a synapse. However, the coupling nanotopography at many synapses remains unknown, and it is unclear how it changes during development. To address these questions, we examined coupling at the cerebellar inhibitory basket cell (BC)-Purkinje cell (PC) synapse. Biophysical analysis of transmission by paired recording and intracellular pipette perfusion revealed that the effects of exogenous Ca2+ chelators decreased during development, despite constant reliance of release on P/Q-type Ca2+ channels. Structural analysis by freeze-fracture replica labeling (FRL) and transmission electron microscopy (EM) indicated that presynaptic P/Q-type Ca2+ channels formed nanoclusters throughout development, whereas docked vesicles were only clustered at later developmental stages. Modeling suggested a developmental transformation from a more random to a more clustered coupling nanotopography. Thus, presynaptic signaling developmentally approaches a point-to-point configuration, optimizing speed, reliability, and energy efficiency of synaptic transmission."}],"oa_version":"None","pmid":1,"related_material":{"link":[{"description":"News on ISTA Website","url":"https://ista.ac.at/en/news/synapses-brought-to-the-point/","relation":"press_release"}],"record":[{"status":"public","id":"15101","relation":"dissertation_contains"}]},"ec_funded":1,"publication_identifier":{"issn":["0896-6273"],"eissn":["1097-4199"]},"publication_status":"inpress","language":[{"iso":"eng"}],"article_type":"original","type":"journal_article","status":"public","_id":"14843","department":[{"_id":"PeJo"},{"_id":"EM-Fac"},{"_id":"RySh"}],"date_updated":"2024-03-14T13:14:18Z"},{"project":[{"name":"Biophysics and circuit function of a giant cortical glumatergic synapse","grant_number":"692692","_id":"25B7EB9E-B435-11E9-9278-68D0E5697425","call_identifier":"H2020"},{"_id":"25C5A090-B435-11E9-9278-68D0E5697425","call_identifier":"FWF","grant_number":"Z00312","name":"The Wittgenstein Prize"},{"grant_number":"P36232","name":"Mechanisms of GABA release in hippocampal circuits","_id":"bd88be38-d553-11ed-ba76-81d5a70a6ef5"},{"_id":"26B66A3E-B435-11E9-9278-68D0E5697425","grant_number":"25383","name":"Development of nanodomain coupling between Ca2+ channels and release sensors at a central inhibitory synapse"}],"citation":{"short":"J. Chen, Developmental Transformation of Nanodomain Coupling between Ca2+ Channels and Release Sensors at a Central GABAergic Synapse, Institute of Science and Technology Austria, 2024.","ieee":"J. Chen, “Developmental transformation of nanodomain coupling between Ca2+ channels and release sensors at a central GABAergic synapse,” Institute of Science and Technology Austria, 2024.","ama":"Chen J. Developmental transformation of nanodomain coupling between Ca2+ channels and release sensors at a central GABAergic synapse. 2024. doi:10.15479/at:ista:15101","apa":"Chen, J. (2024). Developmental transformation of nanodomain coupling between Ca2+ channels and release sensors at a central GABAergic synapse. Institute of Science and Technology Austria. https://doi.org/10.15479/at:ista:15101","mla":"Chen, JingJing. Developmental Transformation of Nanodomain Coupling between Ca2+ Channels and Release Sensors at a Central GABAergic Synapse. Institute of Science and Technology Austria, 2024, doi:10.15479/at:ista:15101.","ista":"Chen J. 2024. Developmental transformation of nanodomain coupling between Ca2+ channels and release sensors at a central GABAergic synapse. Institute of Science and Technology Austria.","chicago":"Chen, JingJing. “Developmental Transformation of Nanodomain Coupling between Ca2+ Channels and Release Sensors at a Central GABAergic Synapse.” Institute of Science and Technology Austria, 2024. https://doi.org/10.15479/at:ista:15101."},"user_id":"8b945eb4-e2f2-11eb-945a-df72226e66a9","author":[{"last_name":"Chen","full_name":"Chen, JingJing","first_name":"JingJing","id":"2C4E65C8-F248-11E8-B48F-1D18A9856A87"}],"article_processing_charge":"No","title":"Developmental transformation of nanodomain coupling between Ca2+ channels and release sensors at a central GABAergic synapse","publisher":"Institute of Science and Technology Austria","has_accepted_license":"1","year":"2024","day":"11","page":"84","date_published":"2024-03-11T00:00:00Z","doi":"10.15479/at:ista:15101","date_created":"2024-03-11T10:09:54Z","_id":"15101","type":"dissertation","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","supervisor":[{"last_name":"Jonas","orcid":"0000-0001-5001-4804","full_name":"Jonas, Peter M","first_name":"Peter M","id":"353C1B58-F248-11E8-B48F-1D18A9856A87"}],"date_updated":"2024-03-14T13:14:19Z","ddc":["570"],"department":[{"_id":"GradSch"},{"_id":"PeJo"}],"file_date_updated":"2024-03-12T07:12:17Z","acknowledged_ssus":[{"_id":"EM-Fac"}],"oa_version":"Published Version","alternative_title":["ISTA Thesis"],"month":"03","publication_identifier":{"issn":["2663 - 337X"]},"publication_status":"published","degree_awarded":"PhD","file":[{"file_name":"Thesis_Jingjing CHEN.docx","date_created":"2024-03-11T14:10:58Z","file_size":11271363,"date_updated":"2024-03-12T07:12:17Z","creator":"jchen","file_id":"15104","checksum":"db4947474ffa271e66c254b6fe876a55","content_type":"application/vnd.openxmlformats-officedocument.wordprocessingml.document","relation":"source_file","access_level":"closed"},{"file_name":"Thesis_Jingjing CHEN_merged.pdf","date_created":"2024-03-11T14:11:06Z","creator":"jchen","file_size":16627311,"date_updated":"2024-03-11T14:11:06Z","embargo":"2024-04-01","file_id":"15105","checksum":"a5eeae8b5702cd540f5d03469bc33dde","relation":"main_file","access_level":"closed","embargo_to":"open_access","content_type":"application/pdf"}],"language":[{"iso":"eng"}],"related_material":{"record":[{"id":"14843","status":"public","relation":"part_of_dissertation"}]},"ec_funded":1},{"date_updated":"2024-03-20T07:42:52Z","department":[{"_id":"PeJo"}],"_id":"15117","article_type":"review","type":"journal_article","status":"public","publication_identifier":{"eissn":["1095-9203"]},"publication_status":"published","language":[{"iso":"eng"}],"issue":"6687","volume":383,"ec_funded":1,"abstract":[{"lang":"eng","text":"The hippocampal mossy fiber synapse, formed between axons of dentate gyrus granule cells and dendrites of CA3 pyramidal neurons, is a key synapse in the trisynaptic circuitry of the hippocampus. Because of its comparatively large size, this synapse is accessible to direct presynaptic recording, allowing a rigorous investigation of the biophysical mechanisms of synaptic transmission and plasticity. Furthermore, because of its placement in the very center of the hippocampal memory circuit, this synapse seems to be critically involved in several higher network functions, such as learning, memory, pattern separation, and pattern completion. Recent work based on new technologies in both nanoanatomy and nanophysiology, including presynaptic patch-clamp recording, paired recording, super-resolution light microscopy, and freeze-fracture and “flash-and-freeze” electron microscopy, has provided new insights into the structure, biophysics, and network function of this intriguing synapse. This brings us one step closer to answering a fundamental question in neuroscience: how basic synaptic properties shape higher network computations."}],"pmid":1,"oa_version":"None","scopus_import":"1","month":"03","intvolume":" 383","citation":{"ista":"Vandael DH, Jonas PM. 2024. Structure, biophysics, and circuit function of a ‘giant’ cortical presynaptic terminal. Science. 383(6687), eadg6757.","chicago":"Vandael, David H, and Peter M Jonas. “Structure, Biophysics, and Circuit Function of a ‘Giant’ Cortical Presynaptic Terminal.” Science. AAAS, 2024. https://doi.org/10.1126/science.adg6757.","apa":"Vandael, D. H., & Jonas, P. M. (2024). Structure, biophysics, and circuit function of a “giant” cortical presynaptic terminal. Science. AAAS. https://doi.org/10.1126/science.adg6757","ama":"Vandael DH, Jonas PM. Structure, biophysics, and circuit function of a “giant” cortical presynaptic terminal. Science. 2024;383(6687):eadg6757. doi:10.1126/science.adg6757","ieee":"D. H. Vandael and P. M. Jonas, “Structure, biophysics, and circuit function of a ‘giant’ cortical presynaptic terminal,” Science, vol. 383, no. 6687. AAAS, p. eadg6757, 2024.","short":"D.H. Vandael, P.M. Jonas, Science 383 (2024) eadg6757.","mla":"Vandael, David H., and Peter M. Jonas. “Structure, Biophysics, and Circuit Function of a ‘Giant’ Cortical Presynaptic Terminal.” Science, vol. 383, no. 6687, AAAS, 2024, p. eadg6757, doi:10.1126/science.adg6757."},"user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87","author":[{"full_name":"Vandael, David H","orcid":"0000-0001-7577-1676","last_name":"Vandael","first_name":"David H","id":"3AE48E0A-F248-11E8-B48F-1D18A9856A87"},{"id":"353C1B58-F248-11E8-B48F-1D18A9856A87","first_name":"Peter M","orcid":"0000-0001-5001-4804","full_name":"Jonas, Peter M","last_name":"Jonas"}],"article_processing_charge":"No","external_id":{"pmid":["38452088"]},"title":"Structure, biophysics, and circuit function of a \"giant\" cortical presynaptic terminal","project":[{"call_identifier":"H2020","_id":"25B7EB9E-B435-11E9-9278-68D0E5697425","grant_number":"692692","name":"Biophysics and circuit function of a giant cortical glumatergic synapse"},{"_id":"25C5A090-B435-11E9-9278-68D0E5697425","call_identifier":"FWF","name":"The Wittgenstein Prize","grant_number":"Z00312"},{"_id":"bd88be38-d553-11ed-ba76-81d5a70a6ef5","grant_number":"P36232","name":"Mechanisms of GABA release in hippocampal circuits"}],"year":"2024","day":"08","publication":"Science","page":"eadg6757","date_published":"2024-03-08T00:00:00Z","doi":"10.1126/science.adg6757","date_created":"2024-03-17T23:00:57Z","acknowledgement":"We thank previous students, postdocs, and collaborators, particularly J. Geiger, and (in alphabetical order) H. Alle, J. Bischofberger, C. Borges-Merjane, D. Engel, M. Frotscher, S. Hallermann, M. Heckmann, S. Jamrichova, O. Kim, L. Li, K. Lichter, P. Lin, J. Lübke, Y. Okamoto, C. Pawlu, C. Schmidt-Hieber, N. Spruston, and N. Vyleta for their outstanding experimental contributions. We also thank P. Castillo, J. Geiger, T. Sakaba, S. Siegert, T. Vogels, and J. Watson for critically reading the manuscript, E. Kralli-Beller for text editing, and J. Malikovic and L. Slomianka for useful discussions. We apologize that, due to space constraints, not all relevant papers could be cited.\r\nThis project was supported by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement 692692, AdG “GIANTSYN”) and the Fonds zur Förderung der Wissenschaftlichen Forschung (Z 312-B27, Wittgenstein Award; P 36232-B, stand-alone grant), both to P.J.","publisher":"AAAS","quality_controlled":"1"},{"department":[{"_id":"PeJo"}],"date_updated":"2023-03-16T08:34:24Z","type":"book_chapter","status":"public","series_title":"MIMB","_id":"12720","volume":2633,"publication_status":"published","publication_identifier":{"issn":["1064-3745"],"isbn":["978-1-0716-3003-7"],"eissn":["1940-6029"],"eisbn":["978-1-0716-3004-4"]},"language":[{"iso":"eng"}],"scopus_import":"1","alternative_title":["Methods in Molecular Biology"],"intvolume":" 2633","month":"03","place":"New York, NY, United States","abstract":[{"lang":"eng","text":"Here we describe the in vivo DNA assembly approach, where molecular cloning procedures are performed using an E. coli recA-independent recombination pathway, which assembles linear fragments of DNA with short homologous termini. This pathway is present in all standard laboratory E. coli strains and, by bypassing the need for in vitro DNA assembly, allows simplified molecular cloning to be performed without the plasmid instability issues associated with specialized recombination-cloning bacterial strains. The methodology requires specific primer design and can perform all standard plasmid modifications (insertions, deletions, mutagenesis, and sub-cloning) in a rapid, simple, and cost-efficient manner, as it does not require commercial kits or specialized bacterial strains. Additionally, this approach can be used to perform complex procedures such as multiple modifications to a plasmid, as up to 6 linear fragments can be assembled in vivo by this recombination pathway. Procedures generally require less than 3 h, involving PCR amplification, DpnI digestion of template DNA, and transformation, upon which circular plasmids are assembled. In this chapter we describe the requirements, procedure, and potential pitfalls when using this technique, as well as protocol variations to overcome the most common issues."}],"pmid":1,"oa_version":"None","article_processing_charge":"No","external_id":{"pmid":["36853454"]},"author":[{"first_name":"Sandra","last_name":"Arroyo-Urea","full_name":"Arroyo-Urea, Sandra"},{"last_name":"Watson","full_name":"Watson, Jake","orcid":"0000-0002-8698-3823","first_name":"Jake","id":"63836096-4690-11EA-BD4E-32803DDC885E"},{"last_name":"García-Nafría","full_name":"García-Nafría, Javier","first_name":"Javier"}],"title":"Molecular Cloning Using In Vivo DNA Assembly","editor":[{"first_name":"Garry","last_name":"Scarlett","full_name":"Scarlett, Garry"}],"citation":{"ieee":"S. Arroyo-Urea, J. Watson, and J. García-Nafría, “Molecular Cloning Using In Vivo DNA Assembly,” in DNA Manipulation and Analysis, vol. 2633, G. Scarlett, Ed. New York, NY, United States: Springer Nature, 2023, pp. 33–44.","short":"S. Arroyo-Urea, J. Watson, J. García-Nafría, in:, G. Scarlett (Ed.), DNA Manipulation and Analysis, Springer Nature, New York, NY, United States, 2023, pp. 33–44.","ama":"Arroyo-Urea S, Watson J, García-Nafría J. Molecular Cloning Using In Vivo DNA Assembly. In: Scarlett G, ed. DNA Manipulation and Analysis. Vol 2633. MIMB. New York, NY, United States: Springer Nature; 2023:33-44. doi:10.1007/978-1-0716-3004-4_3","apa":"Arroyo-Urea, S., Watson, J., & García-Nafría, J. (2023). Molecular Cloning Using In Vivo DNA Assembly. In G. Scarlett (Ed.), DNA Manipulation and Analysis (Vol. 2633, pp. 33–44). New York, NY, United States: Springer Nature. https://doi.org/10.1007/978-1-0716-3004-4_3","mla":"Arroyo-Urea, Sandra, et al. “Molecular Cloning Using In Vivo DNA Assembly.” DNA Manipulation and Analysis, edited by Garry Scarlett, vol. 2633, Springer Nature, 2023, pp. 33–44, doi:10.1007/978-1-0716-3004-4_3.","ista":"Arroyo-Urea S, Watson J, García-Nafría J. 2023.Molecular Cloning Using In Vivo DNA Assembly. In: DNA Manipulation and Analysis. Methods in Molecular Biology, vol. 2633, 33–44.","chicago":"Arroyo-Urea, Sandra, Jake Watson, and Javier García-Nafría. “Molecular Cloning Using In Vivo DNA Assembly.” In DNA Manipulation and Analysis, edited by Garry Scarlett, 2633:33–44. MIMB. New York, NY, United States: Springer Nature, 2023. https://doi.org/10.1007/978-1-0716-3004-4_3."},"user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87","page":"33-44","date_created":"2023-03-12T23:01:02Z","doi":"10.1007/978-1-0716-3004-4_3","date_published":"2023-03-01T00:00:00Z","year":"2023","publication":"DNA Manipulation and Analysis","day":"01","quality_controlled":"1","publisher":"Springer Nature"}]