[{"year":"2023","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).","_id":"12759","abstract":[{"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.","lang":"eng"}],"date_updated":"2023-08-01T13:46:39Z","month":"03","oa_version":"Published Version","type":"journal_article","file_date_updated":"2023-03-27T06:51:09Z","date_created":"2023-03-26T22:01:07Z","volume":18,"external_id":{"isi":["001024737400001"]},"status":"public","citation":{"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.” <i>PLoS ONE</i>. Public Library of Science, 2023. <a href=\"https://doi.org/10.1371/journal.pone.0277148\">https://doi.org/10.1371/journal.pone.0277148</a>.","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,” <i>PLoS ONE</i>, vol. 18, no. 3 March. Public Library of Science, 2023.","short":"J.S. Rothman, C. Borges Merjane, N. Holderith, P.M. Jonas, R. Angus Silver, PLoS ONE 18 (2023).","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. <i>PLoS ONE</i>. 2023;18(3 March). doi:<a href=\"https://doi.org/10.1371/journal.pone.0277148\">10.1371/journal.pone.0277148</a>","apa":"Rothman, J. S., Borges Merjane, C., Holderith, N., Jonas, P. M., &#38; Angus Silver, R. (2023). Validation of a stereological method for estimating particle size and density from 2D projections with high accuracy. <i>PLoS ONE</i>. Public Library of Science. <a href=\"https://doi.org/10.1371/journal.pone.0277148\">https://doi.org/10.1371/journal.pone.0277148</a>","mla":"Rothman, Jason Seth, et al. “Validation of a Stereological Method for Estimating Particle Size and Density from 2D Projections with High Accuracy.” <i>PLoS ONE</i>, vol. 18, no. 3 March, e0277148, Public Library of Science, 2023, doi:<a href=\"https://doi.org/10.1371/journal.pone.0277148\">10.1371/journal.pone.0277148</a>.","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."},"intvolume":"        18","has_accepted_license":"1","publication_status":"published","oa":1,"acknowledged_ssus":[{"_id":"EM-Fac"}],"ddc":["570"],"date_published":"2023-03-17T00:00:00Z","department":[{"_id":"PeJo"}],"user_id":"4359f0d1-fa6c-11eb-b949-802e58b17ae8","publisher":"Public Library of Science","scopus_import":"1","article_processing_charge":"No","ec_funded":1,"article_type":"original","tmp":{"legal_code_url":"https://creativecommons.org/licenses/by/4.0/legalcode","image":"/images/cc_by.png","name":"Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)","short":"CC BY (4.0)"},"publication":"PLoS ONE","file":[{"file_id":"12770","date_updated":"2023-03-27T06:51:09Z","checksum":"2380331ec27cc87808826fc64419ac1c","date_created":"2023-03-27T06:51:09Z","access_level":"open_access","file_name":"2023_PLoSOne_Rothman.pdf","success":1,"file_size":7290413,"relation":"main_file","content_type":"application/pdf","creator":"dernst"}],"day":"17","author":[{"last_name":"Rothman","first_name":"Jason Seth","full_name":"Rothman, Jason Seth"},{"first_name":"Carolina","last_name":"Borges Merjane","full_name":"Borges Merjane, Carolina","orcid":"0000-0003-0005-401X","id":"4305C450-F248-11E8-B48F-1D18A9856A87"},{"full_name":"Holderith, Noemi","last_name":"Holderith","first_name":"Noemi"},{"orcid":"0000-0001-5001-4804","id":"353C1B58-F248-11E8-B48F-1D18A9856A87","full_name":"Jonas, Peter M","first_name":"Peter M","last_name":"Jonas"},{"last_name":"Angus Silver","first_name":"R.","full_name":"Angus Silver, R."}],"title":"Validation of a stereological method for estimating particle size and density from 2D projections with high accuracy","article_number":"e0277148","project":[{"call_identifier":"H2020","_id":"25B7EB9E-B435-11E9-9278-68D0E5697425","name":"Biophysics and circuit function of a giant cortical glumatergic synapse","grant_number":"692692"},{"name":"The Wittgenstein Prize","call_identifier":"FWF","_id":"25C5A090-B435-11E9-9278-68D0E5697425","grant_number":"Z00312"},{"grant_number":"V00739","call_identifier":"FWF","_id":"2696E7FE-B435-11E9-9278-68D0E5697425","name":"Structural plasticity at mossy fiber-CA3 synapses"}],"language":[{"iso":"eng"}],"issue":"3 March","isi":1,"publication_identifier":{"eissn":["1932-6203"]},"quality_controlled":"1","doi":"10.1371/journal.pone.0277148"},{"doi":"10.1038/s41596-021-00526-0","quality_controlled":"1","publication_identifier":{"issn":["17542189"],"eissn":["17502799"]},"isi":1,"language":[{"iso":"eng"}],"issue":"6","project":[{"grant_number":"692692","call_identifier":"H2020","_id":"25B7EB9E-B435-11E9-9278-68D0E5697425","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"},{"grant_number":"V00739","name":"Structural plasticity at mossy fiber-CA3 synapses","_id":"2696E7FE-B435-11E9-9278-68D0E5697425","call_identifier":"FWF"}],"title":"Subcellular patch-clamp techniques for single-bouton stimulation and simultaneous pre- and postsynaptic recording at cortical synapses","author":[{"last_name":"Vandael","first_name":"David H","orcid":"0000-0001-7577-1676","id":"3AE48E0A-F248-11E8-B48F-1D18A9856A87","full_name":"Vandael, David H"},{"first_name":"Yuji","last_name":"Okamoto","id":"3337E116-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0003-0408-6094","full_name":"Okamoto, Yuji"},{"full_name":"Borges Merjane, Carolina","id":"4305C450-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0003-0005-401X","last_name":"Borges Merjane","first_name":"Carolina"},{"first_name":"Victor M","last_name":"Vargas Barroso","id":"2F55A9DE-F248-11E8-B48F-1D18A9856A87","full_name":"Vargas Barroso, Victor M"},{"id":"4952F31E-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0002-9885-6936","full_name":"Suter, Benjamin","first_name":"Benjamin","last_name":"Suter"},{"full_name":"Jonas, Peter M","orcid":"0000-0001-5001-4804","id":"353C1B58-F248-11E8-B48F-1D18A9856A87","last_name":"Jonas","first_name":"Peter M"}],"day":"01","file":[{"file_name":"VandaeletalAuthorVersion2021.pdf","creator":"cziletti","embargo":"2021-12-01","content_type":"application/pdf","relation":"main_file","file_size":38574802,"checksum":"7eb580abd8893cdb0b410cf41bc8c263","date_updated":"2021-12-02T23:30:05Z","file_id":"9639","access_level":"open_access","date_created":"2021-07-08T12:27:55Z"}],"publication":"Nature Protocols","scopus_import":"1","ec_funded":1,"article_processing_charge":"No","article_type":"original","user_id":"4359f0d1-fa6c-11eb-b949-802e58b17ae8","publisher":"Springer Nature","pmid":1,"department":[{"_id":"PeJo"}],"ddc":["570"],"date_published":"2021-06-01T00:00:00Z","acknowledged_ssus":[{"_id":"M-Shop"}],"oa":1,"publication_status":"published","has_accepted_license":"1","intvolume":"        16","citation":{"apa":"Vandael, D. H., Okamoto, Y., Borges Merjane, C., Vargas Barroso, V. M., Suter, B., &#38; Jonas, P. M. (2021). Subcellular patch-clamp techniques for single-bouton stimulation and simultaneous pre- and postsynaptic recording at cortical synapses. <i>Nature Protocols</i>. Springer Nature. <a href=\"https://doi.org/10.1038/s41596-021-00526-0\">https://doi.org/10.1038/s41596-021-00526-0</a>","mla":"Vandael, David H., et al. “Subcellular Patch-Clamp Techniques for Single-Bouton Stimulation and Simultaneous Pre- and Postsynaptic Recording at Cortical Synapses.” <i>Nature Protocols</i>, vol. 16, no. 6, Springer Nature, 2021, pp. 2947–2967, doi:<a href=\"https://doi.org/10.1038/s41596-021-00526-0\">10.1038/s41596-021-00526-0</a>.","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.","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. <i>Nature Protocols</i>. 2021;16(6):2947–2967. doi:<a href=\"https://doi.org/10.1038/s41596-021-00526-0\">10.1038/s41596-021-00526-0</a>","short":"D.H. Vandael, Y. Okamoto, C. Borges Merjane, V.M. Vargas Barroso, B. Suter, P.M. Jonas, Nature Protocols 16 (2021) 2947–2967.","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.” <i>Nature Protocols</i>. Springer Nature, 2021. <a href=\"https://doi.org/10.1038/s41596-021-00526-0\">https://doi.org/10.1038/s41596-021-00526-0</a>.","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,” <i>Nature Protocols</i>, vol. 16, no. 6. Springer Nature, pp. 2947–2967, 2021."},"external_id":{"pmid":["33990799"],"isi":["000650528700003"]},"status":"public","volume":16,"file_date_updated":"2021-12-02T23:30:05Z","date_created":"2021-05-30T22:01:24Z","page":"2947–2967","date_updated":"2023-08-10T22:30:51Z","abstract":[{"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.","lang":"eng"}],"month":"06","type":"journal_article","oa_version":"Submitted Version","_id":"9438","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.","year":"2021"},{"publication_identifier":{"eissn":["10974199"],"issn":["0896-6273"]},"quality_controlled":"1","doi":"10.1016/j.neuron.2020.05.013","project":[{"grant_number":"692692","_id":"25B7EB9E-B435-11E9-9278-68D0E5697425","call_identifier":"H2020","name":"Biophysics and circuit function of a giant cortical glumatergic synapse"},{"name":"The Wittgenstein Prize","call_identifier":"FWF","_id":"25C5A090-B435-11E9-9278-68D0E5697425","grant_number":"Z00312"},{"name":"Structural plasticity at mossy fiber-CA3 synapses","_id":"2696E7FE-B435-11E9-9278-68D0E5697425","call_identifier":"FWF","grant_number":"V00739"}],"issue":"3","language":[{"iso":"eng"}],"isi":1,"day":"05","file":[{"success":1,"file_name":"2020_Neuron_Vandael.pdf","creator":"dernst","content_type":"application/pdf","relation":"main_file","file_size":4390833,"checksum":"4030b2be0c9625d54694a1e9fb00305e","date_updated":"2020-11-25T11:23:02Z","file_id":"8811","access_level":"open_access","date_created":"2020-11-25T11:23:02Z"}],"author":[{"last_name":"Vandael","first_name":"David H","id":"3AE48E0A-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0001-7577-1676","full_name":"Vandael, David H"},{"first_name":"Carolina","last_name":"Borges Merjane","id":"4305C450-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0003-0005-401X","full_name":"Borges Merjane, Carolina"},{"full_name":"Zhang, Xiaomin","id":"423EC9C2-F248-11E8-B48F-1D18A9856A87","first_name":"Xiaomin","last_name":"Zhang"},{"first_name":"Peter M","last_name":"Jonas","id":"353C1B58-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0001-5001-4804","full_name":"Jonas, Peter M"}],"title":"Short-term plasticity at hippocampal mossy fiber synapses is induced by natural activity patterns and associated with vesicle pool engram formation","department":[{"_id":"PeJo"}],"pmid":1,"publisher":"Elsevier","user_id":"4359f0d1-fa6c-11eb-b949-802e58b17ae8","article_type":"original","tmp":{"name":"Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0)","short":"CC BY-NC-ND (4.0)","image":"/images/cc_by_nc_nd.png","legal_code_url":"https://creativecommons.org/licenses/by-nc-nd/4.0/legalcode"},"ec_funded":1,"scopus_import":"1","article_processing_charge":"No","publication":"Neuron","has_accepted_license":"1","oa":1,"publication_status":"published","acknowledged_ssus":[{"_id":"SSU"}],"ddc":["570"],"date_published":"2020-08-05T00:00:00Z","external_id":{"pmid":["32492366"],"isi":["000556135600004"]},"status":"public","citation":{"ista":"Vandael DH, Borges Merjane C, Zhang X, Jonas PM. 2020. Short-term plasticity at hippocampal mossy fiber synapses is induced by natural activity patterns and associated with vesicle pool engram formation. Neuron. 107(3), 509–521.","mla":"Vandael, David H., et al. “Short-Term Plasticity at Hippocampal Mossy Fiber Synapses Is Induced by Natural Activity Patterns and Associated with Vesicle Pool Engram Formation.” <i>Neuron</i>, vol. 107, no. 3, Elsevier, 2020, pp. 509–21, doi:<a href=\"https://doi.org/10.1016/j.neuron.2020.05.013\">10.1016/j.neuron.2020.05.013</a>.","apa":"Vandael, D. H., Borges Merjane, C., Zhang, X., &#38; Jonas, P. M. (2020). Short-term plasticity at hippocampal mossy fiber synapses is induced by natural activity patterns and associated with vesicle pool engram formation. <i>Neuron</i>. Elsevier. <a href=\"https://doi.org/10.1016/j.neuron.2020.05.013\">https://doi.org/10.1016/j.neuron.2020.05.013</a>","ama":"Vandael DH, Borges Merjane C, Zhang X, Jonas PM. Short-term plasticity at hippocampal mossy fiber synapses is induced by natural activity patterns and associated with vesicle pool engram formation. <i>Neuron</i>. 2020;107(3):509-521. doi:<a href=\"https://doi.org/10.1016/j.neuron.2020.05.013\">10.1016/j.neuron.2020.05.013</a>","short":"D.H. Vandael, C. Borges Merjane, X. Zhang, P.M. Jonas, Neuron 107 (2020) 509–521.","ieee":"D. H. Vandael, C. Borges Merjane, X. Zhang, and P. M. Jonas, “Short-term plasticity at hippocampal mossy fiber synapses is induced by natural activity patterns and associated with vesicle pool engram formation,” <i>Neuron</i>, vol. 107, no. 3. Elsevier, pp. 509–521, 2020.","chicago":"Vandael, David H, Carolina Borges Merjane, Xiaomin Zhang, and Peter M Jonas. “Short-Term Plasticity at Hippocampal Mossy Fiber Synapses Is Induced by Natural Activity Patterns and Associated with Vesicle Pool Engram Formation.” <i>Neuron</i>. Elsevier, 2020. <a href=\"https://doi.org/10.1016/j.neuron.2020.05.013\">https://doi.org/10.1016/j.neuron.2020.05.013</a>."},"related_material":{"link":[{"relation":"press_release","url":"https://ist.ac.at/en/news/possible-physical-trace-of-short-term-memory-found/","description":"News on IST Homepage"}]},"intvolume":"       107","type":"journal_article","oa_version":"Published Version","month":"08","abstract":[{"lang":"eng","text":"Post-tetanic potentiation (PTP) is an attractive candidate mechanism for hippocampus-dependent short-term memory. Although PTP has a uniquely large magnitude at hippocampal mossy fiber-CA3 pyramidal neuron synapses, it is unclear whether it can be induced by natural activity and whether its lifetime is sufficient to support short-term memory. We combined in vivo recordings from granule cells (GCs), in vitro paired recordings from mossy fiber terminals and postsynaptic CA3 neurons, and “flash and freeze” electron microscopy. PTP was induced at single synapses and showed a low induction threshold adapted to sparse GC activity in vivo. PTP was mainly generated by enlargement of the readily releasable pool of synaptic vesicles, allowing multiplicative interaction with other plasticity forms. PTP was associated with an increase in the docked vesicle pool, suggesting formation of structural “pool engrams.” Absence of presynaptic activity extended the lifetime of the potentiation, enabling prolonged information storage in the hippocampal network."}],"date_updated":"2023-08-22T07:45:25Z","page":"509-521","file_date_updated":"2020-11-25T11:23:02Z","date_created":"2020-06-22T13:29:05Z","volume":107,"year":"2020","acknowledgement":"This project received funding from the European Research Council (ERC) under the European Union Horizon 2020 Research and Innovation Program (grant agreement 692692 to P.J.) and the Fond zur Förderung der Wissenschaftlichen Forschung ( Z 312-B27 , Wittgenstein award to P.J. and V 739-B27 to C.B.-M.). We thank Drs. Jozsef Csicsvari, Jose Guzman, Erwin Neher, and Ryuichi Shigemoto for commenting on earlier versions of the manuscript. We are grateful to Walter Kaufmann, Daniel Gütl, and Vanessa Zheden for EM training; Alois Schlögl for programming; Florian Marr for excellent technical assistance and cell reconstruction; Christina Altmutter for technical help; Eleftheria Kralli-Beller for manuscript editing; Taija Makinen for providing the Prox1-CreERT2 mouse line; and the Scientific Service Units of IST Austria for support.","_id":"8001"}]