--- _id: '9438' abstract: - lang: eng text: Rigorous investigation of synaptic transmission requires analysis of unitary synaptic events by simultaneous recording from presynaptic terminals and postsynaptic target neurons. However, this has been achieved at only a limited number of model synapses, including the squid giant synapse and the mammalian calyx of Held. Cortical presynaptic terminals have been largely inaccessible to direct presynaptic recording, due to their small size. Here, we describe a protocol for improved subcellular patch-clamp recording in rat and mouse brain slices, with the synapse in a largely intact environment. Slice preparation takes ~2 h, recording ~3 h and post hoc morphological analysis 2 d. Single presynaptic hippocampal mossy fiber terminals are stimulated minimally invasively in the bouton-attached configuration, in which the cytoplasmic content remains unperturbed, or in the whole-bouton configuration, in which the cytoplasmic composition can be precisely controlled. Paired pre–postsynaptic recordings can be integrated with biocytin labeling and morphological analysis, allowing correlative investigation of synapse structure and function. Paired recordings can be obtained from mossy fiber terminals in slices from both rats and mice, implying applicability to genetically modified synapses. Paired recordings can also be performed together with axon tract stimulation or optogenetic activation, allowing comparison of unitary and compound synaptic events in the same target cell. Finally, paired recordings can be combined with spontaneous event analysis, permitting collection of miniature events generated at a single identified synapse. In conclusion, the subcellular patch-clamp techniques detailed here should facilitate analysis of biophysics, plasticity and circuit function of cortical synapses in the mammalian central nervous system. acknowledged_ssus: - _id: M-Shop acknowledgement: This project received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement no. 692692 to P.J.) and the Fond zur Förderung der Wissenschaftlichen Forschung (Z 312-B27, Wittgenstein award to P.J., V 739-B27 to C.B.M.). We are grateful to F. Marr and C. Altmutter for excellent technical assistance and cell reconstruction, E. Kralli-Beller for manuscript editing, and the Scientific Service Units of IST Austria, especially T. Asenov and Miba machine shop, for maximally efficient support. article_processing_charge: No article_type: original 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: Yuji full_name: Okamoto, Yuji id: 3337E116-F248-11E8-B48F-1D18A9856A87 last_name: Okamoto orcid: 0000-0003-0408-6094 - 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: Victor M full_name: Vargas Barroso, Victor M id: 2F55A9DE-F248-11E8-B48F-1D18A9856A87 last_name: Vargas Barroso - first_name: Benjamin full_name: Suter, Benjamin id: 4952F31E-F248-11E8-B48F-1D18A9856A87 last_name: Suter orcid: 0000-0002-9885-6936 - 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, Okamoto Y, Borges Merjane C, Vargas Barroso VM, Suter B, Jonas PM. Subcellular patch-clamp techniques for single-bouton stimulation and simultaneous pre- and postsynaptic recording at cortical synapses. Nature Protocols. 2021;16(6):2947–2967. doi:10.1038/s41596-021-00526-0 apa: Vandael, D. H., Okamoto, Y., Borges Merjane, C., Vargas Barroso, V. M., Suter, B., & Jonas, P. M. (2021). Subcellular patch-clamp techniques for single-bouton stimulation and simultaneous pre- and postsynaptic recording at cortical synapses. Nature Protocols. Springer Nature. https://doi.org/10.1038/s41596-021-00526-0 chicago: Vandael, David H, Yuji Okamoto, Carolina Borges Merjane, Victor M Vargas Barroso, Benjamin Suter, and Peter M Jonas. “Subcellular Patch-Clamp Techniques for Single-Bouton Stimulation and Simultaneous Pre- and Postsynaptic Recording at Cortical Synapses.” Nature Protocols. Springer Nature, 2021. https://doi.org/10.1038/s41596-021-00526-0. ieee: D. H. Vandael, Y. Okamoto, C. Borges Merjane, V. M. Vargas Barroso, B. Suter, and P. M. Jonas, “Subcellular patch-clamp techniques for single-bouton stimulation and simultaneous pre- and postsynaptic recording at cortical synapses,” Nature Protocols, vol. 16, no. 6. Springer Nature, pp. 2947–2967, 2021. ista: Vandael DH, Okamoto Y, Borges Merjane C, Vargas Barroso VM, Suter B, Jonas PM. 2021. Subcellular patch-clamp techniques for single-bouton stimulation and simultaneous pre- and postsynaptic recording at cortical synapses. Nature Protocols. 16(6), 2947–2967. mla: Vandael, David H., et al. “Subcellular Patch-Clamp Techniques for Single-Bouton Stimulation and Simultaneous Pre- and Postsynaptic Recording at Cortical Synapses.” Nature Protocols, vol. 16, no. 6, Springer Nature, 2021, pp. 2947–2967, doi:10.1038/s41596-021-00526-0. short: D.H. Vandael, Y. Okamoto, C. Borges Merjane, V.M. Vargas Barroso, B. Suter, P.M. Jonas, Nature Protocols 16 (2021) 2947–2967. date_created: 2021-05-30T22:01:24Z date_published: 2021-06-01T00:00:00Z date_updated: 2023-08-10T22:30:51Z day: '01' ddc: - '570' department: - _id: PeJo doi: 10.1038/s41596-021-00526-0 ec_funded: 1 external_id: isi: - '000650528700003' pmid: - '33990799' file: - access_level: open_access checksum: 7eb580abd8893cdb0b410cf41bc8c263 content_type: application/pdf creator: cziletti date_created: 2021-07-08T12:27:55Z date_updated: 2021-12-02T23:30:05Z embargo: 2021-12-01 file_id: '9639' file_name: VandaeletalAuthorVersion2021.pdf file_size: 38574802 relation: main_file file_date_updated: 2021-12-02T23:30:05Z has_accepted_license: '1' intvolume: ' 16' isi: 1 issue: '6' language: - iso: eng month: '06' oa: 1 oa_version: Submitted Version page: 2947–2967 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: 2696E7FE-B435-11E9-9278-68D0E5697425 call_identifier: FWF grant_number: V00739 name: Structural plasticity at mossy fiber-CA3 synapses publication: Nature Protocols publication_identifier: eissn: - '17502799' issn: - '17542189' publication_status: published publisher: Springer Nature quality_controlled: '1' scopus_import: '1' status: public title: Subcellular patch-clamp techniques for single-bouton stimulation and simultaneous pre- and postsynaptic recording at cortical synapses type: journal_article user_id: 4359f0d1-fa6c-11eb-b949-802e58b17ae8 volume: 16 year: '2021' ...