--- _id: '15084' 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." acknowledged_ssus: - _id: M-Shop - _id: PreCl - _id: EM-Fac 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. article_number: e2301449121 article_processing_charge: Yes (in subscription journal) article_type: original author: - first_name: Peter full_name: Koppensteiner, Peter id: 3B8B25A8-F248-11E8-B48F-1D18A9856A87 last_name: Koppensteiner orcid: 0000-0002-3509-1948 - first_name: Pradeep full_name: Bhandari, Pradeep id: 45EDD1BC-F248-11E8-B48F-1D18A9856A87 last_name: Bhandari orcid: 0000-0003-0863-4481 - first_name: Hüseyin C full_name: Önal, Hüseyin C id: 4659D740-F248-11E8-B48F-1D18A9856A87 last_name: Önal orcid: 0000-0002-2771-2011 - first_name: Carolina full_name: Borges Merjane, Carolina id: 4305C450-F248-11E8-B48F-1D18A9856A87 last_name: Borges Merjane orcid: 0000-0003-0005-401X - first_name: Elodie full_name: Le Monnier, Elodie id: 3B59276A-F248-11E8-B48F-1D18A9856A87 last_name: Le Monnier - first_name: Utsa full_name: Roy, Utsa id: 4d26cf11-5355-11ee-ae5a-eb05e255b9b2 last_name: Roy - first_name: Yukihiro full_name: Nakamura, Yukihiro last_name: Nakamura - first_name: Tetsushi full_name: Sadakata, Tetsushi last_name: Sadakata - first_name: Makoto full_name: Sanbo, Makoto last_name: Sanbo - first_name: Masumi full_name: Hirabayashi, Masumi last_name: Hirabayashi - first_name: JeongSeop full_name: Rhee, JeongSeop last_name: Rhee - first_name: Nils full_name: Brose, Nils last_name: Brose - first_name: Peter M full_name: Jonas, Peter M id: 353C1B58-F248-11E8-B48F-1D18A9856A87 last_name: Jonas orcid: 0000-0001-5001-4804 - first_name: Ryuichi full_name: Shigemoto, Ryuichi id: 499F3ABC-F248-11E8-B48F-1D18A9856A87 last_name: Shigemoto orcid: 0000-0001-8761-9444 citation: 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 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 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. 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. 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. 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. 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). date_created: 2024-03-05T09:23:55Z date_published: 2024-02-20T00:00:00Z date_updated: 2024-03-12T13:44:18Z day: '20' ddc: - '570' department: - _id: RySh - _id: PeJo doi: 10.1073/pnas.2301449121 ec_funded: 1 external_id: pmid: - '38346189' file: - access_level: open_access checksum: b25b2a057c266ff317a48b0d54d6fc8a content_type: application/pdf creator: dernst date_created: 2024-03-12T13:42:42Z date_updated: 2024-03-12T13:42:42Z file_id: '15110' file_name: 2024_PNAS_Koppensteiner.pdf file_size: 13648221 relation: main_file success: 1 file_date_updated: 2024-03-12T13:42:42Z has_accepted_license: '1' intvolume: ' 121' issue: '8' language: - iso: eng month: '02' oa: 1 oa_version: Published Version 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: 2564DBCA-B435-11E9-9278-68D0E5697425 call_identifier: H2020 grant_number: '665385' name: International IST Doctoral Program publication: Proceedings of the National Academy of Sciences publication_identifier: eissn: - 1091-6490 issn: - 0027-8424 publication_status: published publisher: Proceedings of the National Academy of Sciences quality_controlled: '1' 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' relation: research_data status: public status: public title: GABAB receptors induce phasic release from medial habenula terminals through activity-dependent recruitment of release-ready vesicles tmp: image: /images/cc_by_nc_nd.png legal_code_url: https://creativecommons.org/licenses/by-nc-nd/4.0/legalcode name: Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) short: CC BY-NC-ND (4.0) type: journal_article user_id: 2DF688A6-F248-11E8-B48F-1D18A9856A87 volume: 121 year: '2024' ... --- _id: '14843' 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. acknowledged_ssus: - _id: EM-Fac - _id: PreCl - _id: M-Shop 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. article_processing_charge: No article_type: original author: - first_name: JingJing full_name: Chen, JingJing id: 2C4E65C8-F248-11E8-B48F-1D18A9856A87 last_name: Chen - first_name: Walter full_name: Kaufmann, Walter id: 3F99E422-F248-11E8-B48F-1D18A9856A87 last_name: Kaufmann orcid: 0000-0001-9735-5315 - first_name: Chong full_name: Chen, Chong id: 3DFD581A-F248-11E8-B48F-1D18A9856A87 last_name: Chen - first_name: Itaru full_name: Arai, Itaru id: 32A73F6C-F248-11E8-B48F-1D18A9856A87 last_name: Arai - first_name: Olena full_name: Kim, Olena id: 3F8ABDDA-F248-11E8-B48F-1D18A9856A87 last_name: Kim - first_name: Ryuichi full_name: Shigemoto, Ryuichi id: 499F3ABC-F248-11E8-B48F-1D18A9856A87 last_name: Shigemoto orcid: 0000-0001-8761-9444 - first_name: Peter M full_name: Jonas, Peter M id: 353C1B58-F248-11E8-B48F-1D18A9856A87 last_name: Jonas orcid: 0000-0001-5001-4804 citation: 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 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 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. ieee: J. Chen et al., “Developmental transformation of Ca2+ channel-vesicle nanotopography at a central GABAergic synapse,” Neuron. Elsevier. 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. short: J. Chen, W. Kaufmann, C. Chen, itaru Arai, O. Kim, R. Shigemoto, P.M. Jonas, Neuron (n.d.). date_created: 2024-01-21T23:00:56Z date_published: 2024-01-11T00:00:00Z date_updated: 2024-03-14T13:14:18Z day: '11' department: - _id: PeJo - _id: EM-Fac - _id: RySh doi: 10.1016/j.neuron.2023.12.002 ec_funded: 1 external_id: pmid: - '38215739' language: - iso: eng month: '01' oa_version: None pmid: 1 project: - _id: 25B7EB9E-B435-11E9-9278-68D0E5697425 call_identifier: H2020 grant_number: '692692' name: Biophysics and circuit function of a giant cortical glumatergic synapse - _id: 25C5A090-B435-11E9-9278-68D0E5697425 call_identifier: FWF grant_number: Z00312 name: The Wittgenstein Prize - _id: bd88be38-d553-11ed-ba76-81d5a70a6ef5 grant_number: P36232 name: Mechanisms of GABA release in hippocampal circuits - _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 publication: Neuron publication_identifier: eissn: - 1097-4199 issn: - 0896-6273 publication_status: inpress publisher: Elsevier quality_controlled: '1' related_material: link: - description: News on ISTA Website relation: press_release url: https://ista.ac.at/en/news/synapses-brought-to-the-point/ record: - id: '15101' relation: dissertation_contains status: public scopus_import: '1' status: public title: Developmental transformation of Ca2+ channel-vesicle nanotopography at a central GABAergic synapse type: journal_article user_id: 2DF688A6-F248-11E8-B48F-1D18A9856A87 year: '2024' ... --- _id: '15101' acknowledged_ssus: - _id: EM-Fac alternative_title: - ISTA Thesis article_processing_charge: No author: - first_name: JingJing full_name: Chen, JingJing id: 2C4E65C8-F248-11E8-B48F-1D18A9856A87 last_name: Chen citation: 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 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. 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. 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. 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. 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. date_created: 2024-03-11T10:09:54Z date_published: 2024-03-11T00:00:00Z date_updated: 2024-03-14T13:14:19Z day: '11' ddc: - '570' degree_awarded: PhD department: - _id: GradSch - _id: PeJo doi: 10.15479/at:ista:15101 ec_funded: 1 file: - access_level: closed checksum: db4947474ffa271e66c254b6fe876a55 content_type: application/vnd.openxmlformats-officedocument.wordprocessingml.document creator: jchen date_created: 2024-03-11T14:10:58Z date_updated: 2024-03-12T07:12:17Z file_id: '15104' file_name: Thesis_Jingjing CHEN.docx file_size: 11271363 relation: source_file - access_level: closed checksum: a5eeae8b5702cd540f5d03469bc33dde content_type: application/pdf creator: jchen date_created: 2024-03-11T14:11:06Z date_updated: 2024-03-11T14:11:06Z embargo: 2024-04-01 embargo_to: open_access file_id: '15105' file_name: Thesis_Jingjing CHEN_merged.pdf file_size: 16627311 relation: main_file file_date_updated: 2024-03-12T07:12:17Z has_accepted_license: '1' language: - iso: eng month: '03' oa_version: Published Version page: '84' project: - _id: 25B7EB9E-B435-11E9-9278-68D0E5697425 call_identifier: H2020 grant_number: '692692' name: Biophysics and circuit function of a giant cortical glumatergic synapse - _id: 25C5A090-B435-11E9-9278-68D0E5697425 call_identifier: FWF grant_number: Z00312 name: The Wittgenstein Prize - _id: bd88be38-d553-11ed-ba76-81d5a70a6ef5 grant_number: P36232 name: Mechanisms of GABA release in hippocampal circuits - _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 publication_identifier: issn: - 2663 - 337X publication_status: published publisher: Institute of Science and Technology Austria related_material: record: - id: '14843' relation: part_of_dissertation status: public status: public supervisor: - first_name: Peter M full_name: Jonas, Peter M id: 353C1B58-F248-11E8-B48F-1D18A9856A87 last_name: Jonas orcid: 0000-0001-5001-4804 title: Developmental transformation of nanodomain coupling between Ca2+ channels and release sensors at a central GABAergic synapse 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: dissertation user_id: 8b945eb4-e2f2-11eb-945a-df72226e66a9 year: '2024' ... --- _id: '15117' 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.' 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." article_processing_charge: No article_type: review author: - first_name: David H full_name: Vandael, David H id: 3AE48E0A-F248-11E8-B48F-1D18A9856A87 last_name: Vandael orcid: 0000-0001-7577-1676 - first_name: Peter M full_name: Jonas, Peter M id: 353C1B58-F248-11E8-B48F-1D18A9856A87 last_name: Jonas orcid: 0000-0001-5001-4804 citation: 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 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 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. 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. ista: Vandael DH, Jonas PM. 2024. Structure, biophysics, and circuit function of a ‘giant’ cortical presynaptic terminal. Science. 383(6687), 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. short: D.H. Vandael, P.M. Jonas, Science 383 (2024) eadg6757. date_created: 2024-03-17T23:00:57Z date_published: 2024-03-08T00:00:00Z date_updated: 2024-03-20T07:42:52Z day: '08' department: - _id: PeJo doi: 10.1126/science.adg6757 ec_funded: 1 external_id: pmid: - '38452088' intvolume: ' 383' issue: '6687' language: - iso: eng month: '03' oa_version: None page: eadg6757 pmid: 1 project: - _id: 25B7EB9E-B435-11E9-9278-68D0E5697425 call_identifier: H2020 grant_number: '692692' name: Biophysics and circuit function of a giant cortical glumatergic synapse - _id: 25C5A090-B435-11E9-9278-68D0E5697425 call_identifier: FWF grant_number: Z00312 name: The Wittgenstein Prize - _id: bd88be38-d553-11ed-ba76-81d5a70a6ef5 grant_number: P36232 name: Mechanisms of GABA release in hippocampal circuits publication: Science publication_identifier: eissn: - 1095-9203 publication_status: published publisher: AAAS quality_controlled: '1' scopus_import: '1' status: public title: Structure, biophysics, and circuit function of a "giant" cortical presynaptic terminal type: journal_article user_id: 2DF688A6-F248-11E8-B48F-1D18A9856A87 volume: 383 year: '2024' ... --- _id: '12720' 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. alternative_title: - Methods in Molecular Biology article_processing_charge: No author: - first_name: Sandra full_name: Arroyo-Urea, Sandra last_name: Arroyo-Urea - first_name: Jake full_name: Watson, Jake id: 63836096-4690-11EA-BD4E-32803DDC885E last_name: Watson orcid: 0000-0002-8698-3823 - first_name: Javier full_name: García-Nafría, Javier last_name: García-Nafría citation: 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' 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.' 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.' 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.' 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. 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. date_created: 2023-03-12T23:01:02Z date_published: 2023-03-01T00:00:00Z date_updated: 2023-03-16T08:34:24Z day: '01' department: - _id: PeJo doi: 10.1007/978-1-0716-3004-4_3 editor: - first_name: Garry full_name: Scarlett, Garry last_name: Scarlett external_id: pmid: - '36853454' intvolume: ' 2633' language: - iso: eng month: '03' oa_version: None page: 33-44 place: New York, NY, United States pmid: 1 publication: DNA Manipulation and Analysis publication_identifier: eisbn: - 978-1-0716-3004-4 eissn: - 1940-6029 isbn: - 978-1-0716-3003-7 issn: - 1064-3745 publication_status: published publisher: Springer Nature quality_controlled: '1' scopus_import: '1' series_title: MIMB status: public title: Molecular Cloning Using In Vivo DNA Assembly type: book_chapter user_id: 2DF688A6-F248-11E8-B48F-1D18A9856A87 volume: 2633 year: '2023' ... --- _id: '12567' abstract: - lang: eng text: Single-molecule localization microscopy (SMLM) greatly advances structural studies of diverse biological tissues. For example, presynaptic active zone (AZ) nanotopology is resolved in increasing detail. Immunofluorescence imaging of AZ proteins usually relies on epitope preservation using aldehyde-based immunocompetent fixation. Cryofixation techniques, such as high-pressure freezing (HPF) and freeze substitution (FS), are widely used for ultrastructural studies of presynaptic architecture in electron microscopy (EM). HPF/FS demonstrated nearer-to-native preservation of AZ ultrastructure, e.g., by facilitating single filamentous structures. Here, we present a protocol combining the advantages of HPF/FS and direct stochastic optical reconstruction microscopy (dSTORM) to quantify nanotopology of the AZ scaffold protein Bruchpilot (Brp) at neuromuscular junctions (NMJs) of Drosophila melanogaster. Using this standardized model, we tested for preservation of Brp clusters in different FS protocols compared to classical aldehyde fixation. In HPF/FS samples, presynaptic boutons were structurally well preserved with ~22% smaller Brp clusters that allowed quantification of subcluster topology. In summary, we established a standardized near-to-native preparation and immunohistochemistry protocol for SMLM analyses of AZ protein clusters in a defined model synapse. Our protocol could be adapted to study protein arrangements at single-molecule resolution in other intact tissue preparations. acknowledgement: This work has been supported by funding of the German Research Foundation (Deutsche Forschungsgemeinschaft [DFG], CRC 166, Project B06 to M.H. and A.-L.S., FOR 3004 SYNABS P1 to M.H.) and by the Interdisciplinary Clinical Research Center (IZKF) Würzburg (Z-3/69 to M.M.P., N-229 to M.H. and A.-L.S.). A.M. is funded by the University of Leipzig Clinician Scientist Program. article_number: '2128' article_processing_charge: No article_type: original author: - first_name: Achmed full_name: Mrestani, Achmed last_name: Mrestani - first_name: Katharina full_name: Lichter, Katharina id: 39302e62-fcfc-11ec-8196-8b01447dbd3d last_name: Lichter - first_name: Anna Leena full_name: Sirén, Anna Leena last_name: Sirén - first_name: Manfred full_name: Heckmann, Manfred last_name: Heckmann - first_name: Mila M. full_name: Paul, Mila M. last_name: Paul - first_name: Martin full_name: Pauli, Martin last_name: Pauli citation: ama: Mrestani A, Lichter K, Sirén AL, Heckmann M, Paul MM, Pauli M. Single-molecule localization microscopy of presynaptic active zones in Drosophila melanogaster after rapid cryofixation. International Journal of Molecular Sciences. 2023;24(3). doi:10.3390/ijms24032128 apa: Mrestani, A., Lichter, K., Sirén, A. L., Heckmann, M., Paul, M. M., & Pauli, M. (2023). Single-molecule localization microscopy of presynaptic active zones in Drosophila melanogaster after rapid cryofixation. International Journal of Molecular Sciences. MDPI. https://doi.org/10.3390/ijms24032128 chicago: Mrestani, Achmed, Katharina Lichter, Anna Leena Sirén, Manfred Heckmann, Mila M. Paul, and Martin Pauli. “Single-Molecule Localization Microscopy of Presynaptic Active Zones in Drosophila Melanogaster after Rapid Cryofixation.” International Journal of Molecular Sciences. MDPI, 2023. https://doi.org/10.3390/ijms24032128. ieee: A. Mrestani, K. Lichter, A. L. Sirén, M. Heckmann, M. M. Paul, and M. Pauli, “Single-molecule localization microscopy of presynaptic active zones in Drosophila melanogaster after rapid cryofixation,” International Journal of Molecular Sciences, vol. 24, no. 3. MDPI, 2023. ista: Mrestani A, Lichter K, Sirén AL, Heckmann M, Paul MM, Pauli M. 2023. Single-molecule localization microscopy of presynaptic active zones in Drosophila melanogaster after rapid cryofixation. International Journal of Molecular Sciences. 24(3), 2128. mla: Mrestani, Achmed, et al. “Single-Molecule Localization Microscopy of Presynaptic Active Zones in Drosophila Melanogaster after Rapid Cryofixation.” International Journal of Molecular Sciences, vol. 24, no. 3, 2128, MDPI, 2023, doi:10.3390/ijms24032128. short: A. Mrestani, K. Lichter, A.L. Sirén, M. Heckmann, M.M. Paul, M. Pauli, International Journal of Molecular Sciences 24 (2023). date_created: 2023-02-19T23:00:56Z date_published: 2023-01-21T00:00:00Z date_updated: 2023-08-01T13:16:36Z day: '21' ddc: - '570' department: - _id: PeJo doi: 10.3390/ijms24032128 external_id: isi: - '000930324700001' file: - access_level: open_access checksum: 69a35dcd3e0249f902ab881b06ee2e58 content_type: application/pdf creator: dernst date_created: 2023-02-20T07:09:27Z date_updated: 2023-02-20T07:09:27Z file_id: '12569' file_name: 2023_IJMS_Mrestani.pdf file_size: 2823025 relation: main_file success: 1 file_date_updated: 2023-02-20T07:09:27Z has_accepted_license: '1' intvolume: ' 24' isi: 1 issue: '3' language: - iso: eng month: '01' oa: 1 oa_version: Published Version publication: International Journal of Molecular Sciences publication_identifier: eissn: - 1422-0067 publication_status: published publisher: MDPI quality_controlled: '1' scopus_import: '1' status: public title: Single-molecule localization microscopy of presynaptic active zones in Drosophila melanogaster after rapid cryofixation 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: 4359f0d1-fa6c-11eb-b949-802e58b17ae8 volume: 24 year: '2023' ... --- _id: '12759' abstract: - lang: eng text: Stereological methods for estimating the 3D particle size and density from 2D projections are essential to many research fields. These methods are, however, prone to errors arising from undetected particle profiles due to sectioning and limited resolution, known as ‘lost caps’. A potential solution developed by Keiding, Jensen, and Ranek in 1972, which we refer to as the Keiding model, accounts for lost caps by quantifying the smallest detectable profile in terms of its limiting ‘cap angle’ (ϕ), a size-independent measure of a particle’s distance from the section surface. However, this simple solution has not been widely adopted nor tested. Rather, model-independent design-based stereological methods, which do not explicitly account for lost caps, have come to the fore. Here, we provide the first experimental validation of the Keiding model by comparing the size and density of particles estimated from 2D projections with direct measurement from 3D EM reconstructions of the same tissue. We applied the Keiding model to estimate the size and density of somata, nuclei and vesicles in the cerebellum of mice and rats, where high packing density can be problematic for design-based methods. Our analysis reveals a Gaussian distribution for ϕ rather than a single value. Nevertheless, curve fits of the Keiding model to the 2D diameter distribution accurately estimate the mean ϕ and 3D diameter distribution. While systematic testing using simulations revealed an upper limit to determining ϕ, our analysis shows that estimated ϕ can be used to determine the 3D particle density from the 2D density under a wide range of conditions, and this method is potentially more accurate than minimum-size-based lost-cap corrections and disector methods. Our results show the Keiding model provides an efficient means of accurately estimating the size and density of particles from 2D projections even under conditions of a high density. acknowledged_ssus: - _id: EM-Fac acknowledgement: "We thank the IST Austria Electron Microscopy Facility for technical support, and Diccon Coyle, Andrea Lőrincz and Zoltan Nusser for their helpful comments and discussions.\r\nFunding for JSR and RAS was from the Wellcome Trust (203048; 224499; https://\r\nwellcome.org/). RAS is in receipt of a Wellcome Trust Principal Research Fellowship (224499).\r\nFunding for CBM and PJ was from Fond zur Förderung der Wissenschaftlichen Forschung (V\r\n739-B27 Elise-Richter Programme to CBM, Z 312-B27 Wittgenstein Award to PJ; \r\nhttps://www.fwf.ac.at). PJ received funding from the European Research Council (ERC; https://erc.europa.eu) under the European Union’s Horizon 2020 research and innovation programme (grant agreement no. 692692). NH was supported by a European\r\nResearch Council Advanced Grant (ERC-AG787157)." article_number: e0277148 article_processing_charge: No article_type: original author: - first_name: Jason Seth full_name: Rothman, Jason Seth last_name: Rothman - first_name: Carolina full_name: Borges Merjane, Carolina id: 4305C450-F248-11E8-B48F-1D18A9856A87 last_name: Borges Merjane orcid: 0000-0003-0005-401X - first_name: Noemi full_name: Holderith, Noemi last_name: Holderith - first_name: Peter M full_name: Jonas, Peter M id: 353C1B58-F248-11E8-B48F-1D18A9856A87 last_name: Jonas orcid: 0000-0001-5001-4804 - first_name: R. full_name: Angus Silver, R. last_name: Angus Silver citation: ama: Rothman JS, Borges Merjane C, Holderith N, Jonas PM, Angus Silver R. Validation of a stereological method for estimating particle size and density from 2D projections with high accuracy. PLoS ONE. 2023;18(3 March). doi:10.1371/journal.pone.0277148 apa: Rothman, J. S., Borges Merjane, C., Holderith, N., Jonas, P. M., & Angus Silver, R. (2023). Validation of a stereological method for estimating particle size and density from 2D projections with high accuracy. PLoS ONE. Public Library of Science. https://doi.org/10.1371/journal.pone.0277148 chicago: Rothman, Jason Seth, Carolina Borges Merjane, Noemi Holderith, Peter M Jonas, and R. Angus Silver. “Validation of a Stereological Method for Estimating Particle Size and Density from 2D Projections with High Accuracy.” PLoS ONE. Public Library of Science, 2023. https://doi.org/10.1371/journal.pone.0277148. ieee: J. S. Rothman, C. Borges Merjane, N. Holderith, P. M. Jonas, and R. Angus Silver, “Validation of a stereological method for estimating particle size and density from 2D projections with high accuracy,” PLoS ONE, vol. 18, no. 3 March. Public Library of Science, 2023. ista: Rothman JS, Borges Merjane C, Holderith N, Jonas PM, Angus Silver R. 2023. Validation of a stereological method for estimating particle size and density from 2D projections with high accuracy. PLoS ONE. 18(3 March), e0277148. mla: Rothman, Jason Seth, et al. “Validation of a Stereological Method for Estimating Particle Size and Density from 2D Projections with High Accuracy.” PLoS ONE, vol. 18, no. 3 March, e0277148, Public Library of Science, 2023, doi:10.1371/journal.pone.0277148. short: J.S. Rothman, C. Borges Merjane, N. Holderith, P.M. Jonas, R. Angus Silver, PLoS ONE 18 (2023). date_created: 2023-03-26T22:01:07Z date_published: 2023-03-17T00:00:00Z date_updated: 2023-08-01T13:46:39Z day: '17' ddc: - '570' department: - _id: PeJo doi: 10.1371/journal.pone.0277148 ec_funded: 1 external_id: isi: - '001024737400001' file: - access_level: open_access checksum: 2380331ec27cc87808826fc64419ac1c content_type: application/pdf creator: dernst date_created: 2023-03-27T06:51:09Z date_updated: 2023-03-27T06:51:09Z file_id: '12770' file_name: 2023_PLoSOne_Rothman.pdf file_size: 7290413 relation: main_file success: 1 file_date_updated: 2023-03-27T06:51:09Z has_accepted_license: '1' intvolume: ' 18' isi: 1 issue: 3 March language: - iso: eng month: '03' oa: 1 oa_version: Published Version project: - _id: 25B7EB9E-B435-11E9-9278-68D0E5697425 call_identifier: H2020 grant_number: '692692' name: Biophysics and circuit function of a giant cortical glumatergic synapse - _id: 25C5A090-B435-11E9-9278-68D0E5697425 call_identifier: FWF grant_number: Z00312 name: The Wittgenstein Prize - _id: 2696E7FE-B435-11E9-9278-68D0E5697425 call_identifier: FWF grant_number: V00739 name: Structural plasticity at mossy fiber-CA3 synapses publication: PLoS ONE publication_identifier: eissn: - 1932-6203 publication_status: published publisher: Public Library of Science quality_controlled: '1' scopus_import: '1' status: public title: Validation of a stereological method for estimating particle size and density from 2D projections with high accuracy 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: 4359f0d1-fa6c-11eb-b949-802e58b17ae8 volume: 18 year: '2023' ... --- _id: '12515' abstract: - lang: eng text: "Introduction: The olfactory system in most mammals is divided into several subsystems based on the anatomical locations of the neuroreceptor cells involved and the receptor families that are expressed. In addition to the main olfactory system and the vomeronasal system, a range of olfactory subsystems converge onto the transition zone located between the main olfactory bulb (MOB) and the accessory olfactory bulb (AOB), which has been termed the olfactory limbus (OL). The OL contains specialized glomeruli that receive noncanonical sensory afferences and which interact with the MOB and AOB. Little is known regarding the olfactory subsystems of mammals other than laboratory rodents.\r\nMethods: We have focused on characterizing the OL in the red fox by performing general and specific histological stainings on serial sections, using both single and double immunohistochemical and lectin-histochemical labeling techniques.\r\nResults: As a result, we have been able to determine that the OL of the red fox (Vulpes vulpes) displays an uncommonly high degree of development and complexity.\r\nDiscussion: This makes this species a novel mammalian model, the study of which could improve our understanding of the noncanonical pathways involved in the processing of chemosensory cues." acknowledgement: This work was partially supported by a grant from “Consello Social Universidade de Santiago de Compostela” 2022-PU004.We would like to show special gratitude to Prof. Ludwig Wagner (Medical University, Vienna) for kindly providing us with the secretagogin antibody. We thank the Wildlife Recovery Centres of Galicia, Dirección Xeral de Patrimonio Natural (Xunta de Galicia, Spain), and Federación Galega de Caza for providing the red foxes used in this study. article_number: '1097467' article_processing_charge: No article_type: original author: - first_name: Irene full_name: Ortiz-Leal, Irene last_name: Ortiz-Leal - first_name: Mateo V. full_name: Torres, Mateo V. last_name: Torres - first_name: Victor M full_name: Vargas Barroso, Victor M id: 2F55A9DE-F248-11E8-B48F-1D18A9856A87 last_name: Vargas Barroso - first_name: Luis Eusebio full_name: Fidalgo, Luis Eusebio last_name: Fidalgo - first_name: Ana María full_name: López-Beceiro, Ana María last_name: López-Beceiro - first_name: Jorge A. full_name: Larriva-Sahd, Jorge A. last_name: Larriva-Sahd - first_name: Pablo full_name: Sánchez-Quinteiro, Pablo last_name: Sánchez-Quinteiro citation: ama: Ortiz-Leal I, Torres MV, Vargas Barroso VM, et al. The olfactory limbus of the red fox (Vulpes vulpes). New insights regarding a noncanonical olfactory bulb pathway. Frontiers in Neuroanatomy. 2023;16. doi:10.3389/fnana.2022.1097467 apa: Ortiz-Leal, I., Torres, M. V., Vargas Barroso, V. M., Fidalgo, L. E., López-Beceiro, A. M., Larriva-Sahd, J. A., & Sánchez-Quinteiro, P. (2023). The olfactory limbus of the red fox (Vulpes vulpes). New insights regarding a noncanonical olfactory bulb pathway. Frontiers in Neuroanatomy. Frontiers. https://doi.org/10.3389/fnana.2022.1097467 chicago: Ortiz-Leal, Irene, Mateo V. Torres, Victor M Vargas Barroso, Luis Eusebio Fidalgo, Ana María López-Beceiro, Jorge A. Larriva-Sahd, and Pablo Sánchez-Quinteiro. “The Olfactory Limbus of the Red Fox (Vulpes Vulpes). New Insights Regarding a Noncanonical Olfactory Bulb Pathway.” Frontiers in Neuroanatomy. Frontiers, 2023. https://doi.org/10.3389/fnana.2022.1097467. ieee: I. Ortiz-Leal et al., “The olfactory limbus of the red fox (Vulpes vulpes). New insights regarding a noncanonical olfactory bulb pathway,” Frontiers in Neuroanatomy, vol. 16. Frontiers, 2023. ista: Ortiz-Leal I, Torres MV, Vargas Barroso VM, Fidalgo LE, López-Beceiro AM, Larriva-Sahd JA, Sánchez-Quinteiro P. 2023. The olfactory limbus of the red fox (Vulpes vulpes). New insights regarding a noncanonical olfactory bulb pathway. Frontiers in Neuroanatomy. 16, 1097467. mla: Ortiz-Leal, Irene, et al. “The Olfactory Limbus of the Red Fox (Vulpes Vulpes). New Insights Regarding a Noncanonical Olfactory Bulb Pathway.” Frontiers in Neuroanatomy, vol. 16, 1097467, Frontiers, 2023, doi:10.3389/fnana.2022.1097467. short: I. Ortiz-Leal, M.V. Torres, V.M. Vargas Barroso, L.E. Fidalgo, A.M. López-Beceiro, J.A. Larriva-Sahd, P. Sánchez-Quinteiro, Frontiers in Neuroanatomy 16 (2023). date_created: 2023-02-05T23:01:00Z date_published: 2023-01-10T00:00:00Z date_updated: 2023-08-16T11:37:52Z day: '10' ddc: - '570' department: - _id: PeJo doi: 10.3389/fnana.2022.1097467 external_id: isi: - '000919786900001' pmid: - '36704406' file: - access_level: open_access checksum: 49cd40f3bda6f267079427042e7d15e3 content_type: application/pdf creator: dernst date_created: 2023-02-06T07:56:14Z date_updated: 2023-02-06T07:56:14Z file_id: '12518' file_name: 2022_FrontiersNeuroanatomy_OrtizLeal.pdf file_size: 21943473 relation: main_file success: 1 file_date_updated: 2023-02-06T07:56:14Z has_accepted_license: '1' intvolume: ' 16' isi: 1 language: - iso: eng month: '01' oa: 1 oa_version: Published Version pmid: 1 publication: Frontiers in Neuroanatomy publication_identifier: eissn: - 1662-5129 publication_status: published publisher: Frontiers quality_controlled: '1' scopus_import: '1' status: public title: The olfactory limbus of the red fox (Vulpes vulpes). New insights regarding a noncanonical olfactory bulb pathway 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: 16 year: '2023' ... --- _id: '12786' abstract: - lang: eng text: AMPA glutamate receptors (AMPARs) mediate excitatory neurotransmission throughout the brain. Their signalling is uniquely diversified by brain region-specific auxiliary subunits, providing an opportunity for the development of selective therapeutics. AMPARs associated with TARP γ8 are enriched in the hippocampus, and are targets of emerging anti-epileptic drugs. To understand their therapeutic activity, we determined cryo-EM structures of the GluA1/2-γ8 receptor associated with three potent, chemically diverse ligands. We find that despite sharing a lipid-exposed and water-accessible binding pocket, drug action is differentially affected by binding-site mutants. Together with patch-clamp recordings and MD simulations we also demonstrate that ligand-triggered reorganisation of the AMPAR-TARP interface contributes to modulation. Unexpectedly, one ligand (JNJ-61432059) acts bifunctionally, negatively affecting GluA1 but exerting positive modulatory action on GluA2-containing AMPARs, in a TARP stoichiometry-dependent manner. These results further illuminate the action of TARPs, demonstrate the sensitive balance between positive and negative modulatory action, and provide a mechanistic platform for development of both positive and negative selective AMPAR modulators. acknowledgement: We thank James Krieger for generating the ‘proDy’ interaction maps in Fig. 5B and S7C, and Jan-Niklas Dohrke for critically reading the manuscript. We thank members of the Greger lab for insightful comments during this study. We acknowledge Trevor Rutherford for confirming ligand integrity by NMR. We are also grateful to LMB scientific computing and the EM facility for their support. This research was funded in part by the Wellcome Trust (223194/Z/21/Z) to I.H.G. For the purpose of Open Access, the MRC Laboratory of Molecular Biology has applied a CC BY public copyright licence to any Author Accepted Manuscript (AAM) version arising from this submission. Further funding came from the Medical Research Council (MRU105174197) to I.H.G, and NIH grant (R56/R01MH123474) to T.N. article_number: '1659' article_processing_charge: No article_type: original author: - first_name: Danyang full_name: Zhang, Danyang last_name: Zhang - first_name: Remigijus full_name: Lape, Remigijus last_name: Lape - first_name: Saher A. full_name: Shaikh, Saher A. last_name: Shaikh - first_name: Bianka K. full_name: Kohegyi, Bianka K. last_name: Kohegyi - first_name: Jake full_name: Watson, Jake id: 63836096-4690-11EA-BD4E-32803DDC885E last_name: Watson orcid: 0000-0002-8698-3823 - first_name: Ondrej full_name: Cais, Ondrej last_name: Cais - first_name: Terunaga full_name: Nakagawa, Terunaga last_name: Nakagawa - first_name: Ingo H. full_name: Greger, Ingo H. last_name: Greger citation: ama: Zhang D, Lape R, Shaikh SA, et al. Modulatory mechanisms of TARP γ8-selective AMPA receptor therapeutics. Nature Communications. 2023;14. doi:10.1038/s41467-023-37259-5 apa: Zhang, D., Lape, R., Shaikh, S. A., Kohegyi, B. K., Watson, J., Cais, O., … Greger, I. H. (2023). Modulatory mechanisms of TARP γ8-selective AMPA receptor therapeutics. Nature Communications. Springer Nature. https://doi.org/10.1038/s41467-023-37259-5 chicago: Zhang, Danyang, Remigijus Lape, Saher A. Shaikh, Bianka K. Kohegyi, Jake Watson, Ondrej Cais, Terunaga Nakagawa, and Ingo H. Greger. “Modulatory Mechanisms of TARP Γ8-Selective AMPA Receptor Therapeutics.” Nature Communications. Springer Nature, 2023. https://doi.org/10.1038/s41467-023-37259-5. ieee: D. Zhang et al., “Modulatory mechanisms of TARP γ8-selective AMPA receptor therapeutics,” Nature Communications, vol. 14. Springer Nature, 2023. ista: Zhang D, Lape R, Shaikh SA, Kohegyi BK, Watson J, Cais O, Nakagawa T, Greger IH. 2023. Modulatory mechanisms of TARP γ8-selective AMPA receptor therapeutics. Nature Communications. 14, 1659. mla: Zhang, Danyang, et al. “Modulatory Mechanisms of TARP Γ8-Selective AMPA Receptor Therapeutics.” Nature Communications, vol. 14, 1659, Springer Nature, 2023, doi:10.1038/s41467-023-37259-5. short: D. Zhang, R. Lape, S.A. Shaikh, B.K. Kohegyi, J. Watson, O. Cais, T. Nakagawa, I.H. Greger, Nature Communications 14 (2023). date_created: 2023-04-02T22:01:09Z date_published: 2023-03-25T00:00:00Z date_updated: 2023-12-13T11:15:58Z day: '25' ddc: - '570' department: - _id: PeJo doi: 10.1038/s41467-023-37259-5 external_id: isi: - '001066658700003' file: - access_level: open_access checksum: 0a97b31191432dae5853bbb5ccb7698d content_type: application/pdf creator: dernst date_created: 2023-04-03T06:38:56Z date_updated: 2023-04-03T06:38:56Z file_id: '12797' file_name: 2023_NatureComm_Zhang.pdf file_size: 2613996 relation: main_file success: 1 file_date_updated: 2023-04-03T06:38:56Z has_accepted_license: '1' intvolume: ' 14' isi: 1 language: - iso: eng month: '03' oa: 1 oa_version: Published Version publication: Nature Communications publication_identifier: eissn: - 2041-1723 publication_status: published publisher: Springer Nature quality_controlled: '1' scopus_import: '1' status: public title: Modulatory mechanisms of TARP γ8-selective AMPA receptor therapeutics 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: 14 year: '2023' ... --- _id: '13267' abstract: - lang: eng text: Three-dimensional (3D) reconstruction of living brain tissue down to an individual synapse level would create opportunities for decoding the dynamics and structure–function relationships of the brain’s complex and dense information processing network; however, this has been hindered by insufficient 3D resolution, inadequate signal-to-noise ratio and prohibitive light burden in optical imaging, whereas electron microscopy is inherently static. Here we solved these challenges by developing an integrated optical/machine-learning technology, LIONESS (live information-optimized nanoscopy enabling saturated segmentation). This leverages optical modifications to stimulated emission depletion microscopy in comprehensively, extracellularly labeled tissue and previous information on sample structure via machine learning to simultaneously achieve isotropic super-resolution, high signal-to-noise ratio and compatibility with living tissue. This allows dense deep-learning-based instance segmentation and 3D reconstruction at a synapse level, incorporating molecular, activity and morphodynamic information. LIONESS opens up avenues for studying the dynamic functional (nano-)architecture of living brain tissue. acknowledged_ssus: - _id: ScienComp - _id: Bio - _id: PreCl - _id: E-Lib - _id: LifeSc - _id: M-Shop acknowledgement: "We thank J. Vorlaufer, N. Agudelo and A. Wartak for microscope maintenance and troubleshooting, C. Kreuzinger and A. Freeman for technical assistance, M. Šuplata for hardware control support and M. Cunha dos Santos for initial exploration of software. We\r\nthank P. Henderson for advice on deep-learning training and M. Sixt, S. Boyd and T. Weiss for discussions and critical reading of the manuscript. L. Lavis (Janelia Research Campus) generously provided the JF585-HaloTag ligand. We acknowledge expert support by IST\r\nAustria’s scientific computing, imaging and optics, preclinical, library and laboratory support facilities and by the Miba machine shop. We gratefully acknowledge funding by the following sources: Austrian Science Fund (F.W.F.) grant no. I3600-B27 (J.G.D.), grant no. DK W1232\r\n(J.G.D. and J.M.M.) and grant no. Z 312-B27, Wittgenstein award (P.J.); the Gesellschaft für Forschungsförderung NÖ grant no. LSC18-022 (J.G.D.); an ISTA Interdisciplinary project grant (J.G.D. and B.B.); the European Union’s Horizon 2020 research and innovation programme,\r\nMarie-Skłodowska Curie grant 665385 (J.M.M. and J.L.); the European Union’s Horizon 2020 research and innovation programme, European Research Council grant no. 715767, MATERIALIZABLE (B.B.); grant no. 715508, REVERSEAUTISM (G.N.); grant no. 695568, SYNNOVATE (S.G.N.G.); and grant no. 692692, GIANTSYN (P.J.); the Simons\r\nFoundation Autism Research Initiative grant no. 529085 (S.G.N.G.); the Wellcome Trust Technology Development grant no. 202932 (S.G.N.G.); the Marie Skłodowska-Curie Actions Individual Fellowship no. 101026635 under the EU Horizon 2020 program (J.F.W.);\r\nthe Human Frontier Science Program postdoctoral fellowship LT000557/2018 (W.J.); and the National Science Foundation grant no. IIS-1835231 (H.P.) and NCS-FO-2124179 (H.P.)." article_processing_charge: Yes article_type: original author: - first_name: Philipp full_name: Velicky, Philipp id: 39BDC62C-F248-11E8-B48F-1D18A9856A87 last_name: Velicky orcid: 0000-0002-2340-7431 - first_name: Eder full_name: Miguel Villalba, Eder id: 3FB91342-F248-11E8-B48F-1D18A9856A87 last_name: Miguel Villalba orcid: 0000-0001-5665-0430 - first_name: Julia M full_name: Michalska, Julia M id: 443DB6DE-F248-11E8-B48F-1D18A9856A87 last_name: Michalska orcid: 0000-0003-3862-1235 - first_name: Julia full_name: Lyudchik, Julia id: 46E28B80-F248-11E8-B48F-1D18A9856A87 last_name: Lyudchik - first_name: Donglai full_name: Wei, Donglai last_name: Wei - first_name: Zudi full_name: Lin, Zudi last_name: Lin - first_name: Jake full_name: Watson, Jake id: 63836096-4690-11EA-BD4E-32803DDC885E last_name: Watson orcid: 0000-0002-8698-3823 - first_name: Jakob full_name: Troidl, Jakob last_name: Troidl - first_name: Johanna full_name: Beyer, Johanna last_name: Beyer - first_name: Yoav full_name: Ben Simon, Yoav id: 43DF3136-F248-11E8-B48F-1D18A9856A87 last_name: Ben Simon - first_name: Christoph M full_name: Sommer, Christoph M id: 4DF26D8C-F248-11E8-B48F-1D18A9856A87 last_name: Sommer orcid: 0000-0003-1216-9105 - first_name: Wiebke full_name: Jahr, Wiebke id: 425C1CE8-F248-11E8-B48F-1D18A9856A87 last_name: Jahr - first_name: Alban full_name: Cenameri, Alban id: 9ac8f577-2357-11eb-997a-e566c5550886 last_name: Cenameri - first_name: Johannes full_name: Broichhagen, Johannes last_name: Broichhagen - first_name: Seth G.N. full_name: Grant, Seth G.N. last_name: Grant - first_name: Peter M full_name: Jonas, Peter M id: 353C1B58-F248-11E8-B48F-1D18A9856A87 last_name: Jonas orcid: 0000-0001-5001-4804 - first_name: Gaia full_name: Novarino, Gaia id: 3E57A680-F248-11E8-B48F-1D18A9856A87 last_name: Novarino orcid: 0000-0002-7673-7178 - first_name: Hanspeter full_name: Pfister, Hanspeter last_name: Pfister - first_name: Bernd full_name: Bickel, Bernd id: 49876194-F248-11E8-B48F-1D18A9856A87 last_name: Bickel orcid: 0000-0001-6511-9385 - first_name: Johann G full_name: Danzl, Johann G id: 42EFD3B6-F248-11E8-B48F-1D18A9856A87 last_name: Danzl orcid: 0000-0001-8559-3973 citation: ama: Velicky P, Miguel Villalba E, Michalska JM, et al. Dense 4D nanoscale reconstruction of living brain tissue. Nature Methods. 2023;20:1256-1265. doi:10.1038/s41592-023-01936-6 apa: Velicky, P., Miguel Villalba, E., Michalska, J. M., Lyudchik, J., Wei, D., Lin, Z., … Danzl, J. G. (2023). Dense 4D nanoscale reconstruction of living brain tissue. Nature Methods. Springer Nature. https://doi.org/10.1038/s41592-023-01936-6 chicago: Velicky, Philipp, Eder Miguel Villalba, Julia M Michalska, Julia Lyudchik, Donglai Wei, Zudi Lin, Jake Watson, et al. “Dense 4D Nanoscale Reconstruction of Living Brain Tissue.” Nature Methods. Springer Nature, 2023. https://doi.org/10.1038/s41592-023-01936-6. ieee: P. Velicky et al., “Dense 4D nanoscale reconstruction of living brain tissue,” Nature Methods, vol. 20. Springer Nature, pp. 1256–1265, 2023. ista: Velicky P, Miguel Villalba E, Michalska JM, Lyudchik J, Wei D, Lin Z, Watson J, Troidl J, Beyer J, Ben Simon Y, Sommer CM, Jahr W, Cenameri A, Broichhagen J, Grant SGN, Jonas PM, Novarino G, Pfister H, Bickel B, Danzl JG. 2023. Dense 4D nanoscale reconstruction of living brain tissue. Nature Methods. 20, 1256–1265. mla: Velicky, Philipp, et al. “Dense 4D Nanoscale Reconstruction of Living Brain Tissue.” Nature Methods, vol. 20, Springer Nature, 2023, pp. 1256–65, doi:10.1038/s41592-023-01936-6. short: P. Velicky, E. Miguel Villalba, J.M. Michalska, J. Lyudchik, D. Wei, Z. Lin, J. Watson, J. Troidl, J. Beyer, Y. Ben Simon, C.M. Sommer, W. Jahr, A. Cenameri, J. Broichhagen, S.G.N. Grant, P.M. Jonas, G. Novarino, H. Pfister, B. Bickel, J.G. Danzl, Nature Methods 20 (2023) 1256–1265. date_created: 2023-07-23T22:01:13Z date_published: 2023-08-01T00:00:00Z date_updated: 2024-01-10T08:37:48Z day: '01' department: - _id: PeJo - _id: GaNo - _id: BeBi - _id: JoDa - _id: Bio doi: 10.1038/s41592-023-01936-6 ec_funded: 1 external_id: isi: - '001025621500001' pmid: - '37429995' intvolume: ' 20' isi: 1 language: - iso: eng main_file_link: - open_access: '1' url: https://doi.org/10.1038/s41592-023-01936-6 month: '08' oa: 1 oa_version: Published Version page: 1256-1265 pmid: 1 project: - _id: 265CB4D0-B435-11E9-9278-68D0E5697425 call_identifier: FWF grant_number: I03600 name: Optical control of synaptic function via adhesion molecules - _id: 2548AE96-B435-11E9-9278-68D0E5697425 call_identifier: FWF grant_number: W1232-B24 name: Molecular Drug Targets - _id: 25C5A090-B435-11E9-9278-68D0E5697425 call_identifier: FWF grant_number: Z00312 name: The Wittgenstein Prize - _id: 23889792-32DE-11EA-91FC-C7463DDC885E name: High content imaging to decode human immune cell interactions in health and allergic disease - _id: 2564DBCA-B435-11E9-9278-68D0E5697425 call_identifier: H2020 grant_number: '665385' name: International IST Doctoral Program - _id: 24F9549A-B435-11E9-9278-68D0E5697425 call_identifier: H2020 grant_number: '715767' name: 'MATERIALIZABLE: Intelligent fabrication-oriented Computational Design and Modeling' - _id: 25444568-B435-11E9-9278-68D0E5697425 call_identifier: H2020 grant_number: '715508' name: Probing the Reversibility of Autism Spectrum Disorders by Employing in vivo and in vitro Models - _id: 25B7EB9E-B435-11E9-9278-68D0E5697425 call_identifier: H2020 grant_number: '692692' name: Biophysics and circuit function of a giant cortical glumatergic synapse - _id: fc2be41b-9c52-11eb-aca3-faa90aa144e9 call_identifier: H2020 grant_number: '101026635' name: Synaptic computations of the hippocampal CA3 circuitry - _id: 2668BFA0-B435-11E9-9278-68D0E5697425 grant_number: LT00057 name: High-speed 3D-nanoscopy to study the role of adhesion during 3D cell migration publication: Nature Methods publication_identifier: eissn: - 1548-7105 issn: - 1548-7091 publication_status: published publisher: Springer Nature quality_controlled: '1' related_material: link: - relation: software url: https://github.com/danzllab/LIONESS record: - id: '12817' relation: research_data status: public - id: '14770' relation: shorter_version status: public scopus_import: '1' status: public title: Dense 4D nanoscale reconstruction of living brain tissue type: journal_article user_id: 2DF688A6-F248-11E8-B48F-1D18A9856A87 volume: 20 year: '2023' ...