[{"acknowledged_ssus":[{"_id":"EM-Fac"}],"abstract":[{"lang":"eng","text":"AMPA receptors (AMPARs) mediate fast excitatory neurotransmission and their role is\r\nimplicated in complex processes such as learning and memory and various neurological\r\ndiseases. These receptors are composed of different subunits and the subunit composition can\r\naffect channel properties, receptor trafficking and interaction with other associated proteins.\r\nUsing the high sensitivity SDS-digested freeze-fracture replica labeling (SDS-FRL) for\r\nelectron microscopy I investigated the number, density, and localization of AMPAR subunits,\r\nGluA1, GluA2, GluA3, and GluA1-3 (panAMPA) in pyramidal cells in the CA1 area of mouse\r\nhippocampus. I have found that the immunogold labeling for all of these subunits in the\r\npostsynaptic sites was highest in stratum radiatum and lowest in stratum lacunosummoleculare. The labeling density for the all subunits in the extrasynaptic sites showed a gradual\r\nincrease from the pyramidal cell soma towards the distal part of stratum radiatum. The densities\r\nof extrasynaptic GluA1, GluA2 and panAMPA labeling reached 10-15% of synaptic densities,\r\nwhile the ratio of extrasynaptic labeling for GluA3 was significantly lower compared than those\r\nfor other subunits. The labeling patterns for GluA1, GluA2 and GluA1-3 are similar and their\r\ndensities were higher in the periphery than center of synapses. In contrast, the GluA3-\r\ncontaining receptors were more centrally localized compared to the GluA1- and GluA2-\r\ncontaining receptors.\r\nThe hippocampus plays a central role in learning and memory. Contextual learning has been\r\nshown to require the delivery of AMPA receptors to CA1 synapses in the dorsal hippocampus.\r\nHowever, proximodistal heterogeneity of this plasticity and particular contribution of different\r\nAMPA receptor subunits are not fully understood. By combining inhibitory avoidance task, a\r\nhippocampus-dependent contextual fear-learning paradigm, with SDS-FRL, I have revealed an\r\nincrease in synaptic density specific to GluA1-containing AMPA receptors in the CA1 area.\r\nThe intrasynaptic distribution of GluA1 also changed from the periphery to center-preferred\r\npattern. Furthermore, this synaptic plasticity was evident selectively in stratum radiatum but\r\nnot stratum oriens, and in the CA1 subregion proximal but not distal to CA2. These findings\r\nfurther contribute to our understanding of how specific hippocampal subregions and AMPA\r\nreceptor subunits are involved in physiological learning.\r\nAlthough the immunolabeling results above shed light on subunit-specific plasticity in\r\nAMPAR distribution, no tools to visualize and study the subunit composition at the single\r\nchannel level in situ have been available. Electron microscopy with conventional immunogold\r\nlabeling approaches has limitations in the single channel analysis because of the large size of\r\nantibodies and steric hindrance hampering multiple subunit labeling of single channels. I\r\nmanaged to develop a new chemical labeling system using a short peptide tag and small\r\nsynthetic probes, which form specific covalent bond with a cysteine residue in the tag fused to\r\nproteins of interest (reactive tag system). I additionally made substantial progress into adapting\r\nthis system for AMPA receptor subunits."}],"oa_version":"Published Version","alternative_title":["ISTA Thesis"],"month":"05","publication_identifier":{"issn":["2663-337X"]},"publication_status":"published","degree_awarded":"PhD","file":[{"creator":"cchlebak","file_size":56427603,"date_updated":"2023-05-17T22:30:03Z","file_name":"MJ thesis.docx","date_created":"2022-05-17T09:08:06Z","relation":"source_file","access_level":"closed","embargo_to":"open_access","content_type":"application/vnd.openxmlformats-officedocument.wordprocessingml.document","checksum":"8fc695d88020d70d231dad0e9f10b138","file_id":"11395"},{"content_type":"application/pdf","relation":"main_file","access_level":"open_access","embargo":"2023-05-16","file_id":"11397","checksum":"c1dd20a1aece521b3500607b00e463d6","file_size":4351981,"date_updated":"2023-05-17T22:30:03Z","creator":"cchlebak","file_name":"MJ_thesis_PDFA.pdf","date_created":"2022-05-17T12:09:25Z"}],"language":[{"iso":"eng"}],"related_material":{"record":[{"status":"public","id":"7391","relation":"part_of_dissertation"}]},"_id":"11393","type":"dissertation","status":"public","supervisor":[{"first_name":"Ryuichi","id":"499F3ABC-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0001-8761-9444","full_name":"Shigemoto, Ryuichi","last_name":"Shigemoto"}],"date_updated":"2023-09-07T14:53:44Z","ddc":["570"],"file_date_updated":"2023-05-17T22:30:03Z","department":[{"_id":"GradSch"},{"_id":"RySh"}],"publisher":"Institute of Science and Technology Austria","oa":1,"has_accepted_license":"1","year":"2022","day":"16","page":"108","date_published":"2022-05-16T00:00:00Z","doi":"10.15479/at:ista:11393","date_created":"2022-05-17T08:57:41Z","citation":{"mla":"Jevtic, Marijo. Contextual Fear Learning Induced Changes in AMPA Receptor Subtypes along the Proximodistal Axis in Dorsal Hippocampus. Institute of Science and Technology Austria, 2022, doi:10.15479/at:ista:11393.","apa":"Jevtic, M. (2022). Contextual fear learning induced changes in AMPA receptor subtypes along the proximodistal axis in dorsal hippocampus. Institute of Science and Technology Austria. https://doi.org/10.15479/at:ista:11393","ama":"Jevtic M. Contextual fear learning induced changes in AMPA receptor subtypes along the proximodistal axis in dorsal hippocampus. 2022. doi:10.15479/at:ista:11393","ieee":"M. Jevtic, “Contextual fear learning induced changes in AMPA receptor subtypes along the proximodistal axis in dorsal hippocampus,” Institute of Science and Technology Austria, 2022.","short":"M. Jevtic, Contextual Fear Learning Induced Changes in AMPA Receptor Subtypes along the Proximodistal Axis in Dorsal Hippocampus, Institute of Science and Technology Austria, 2022.","chicago":"Jevtic, Marijo. “Contextual Fear Learning Induced Changes in AMPA Receptor Subtypes along the Proximodistal Axis in Dorsal Hippocampus.” Institute of Science and Technology Austria, 2022. https://doi.org/10.15479/at:ista:11393.","ista":"Jevtic M. 2022. Contextual fear learning induced changes in AMPA receptor subtypes along the proximodistal axis in dorsal hippocampus. Institute of Science and Technology Austria."},"user_id":"8b945eb4-e2f2-11eb-945a-df72226e66a9","author":[{"first_name":"Marijo","id":"4BE3BC94-F248-11E8-B48F-1D18A9856A87","last_name":"Jevtic","full_name":"Jevtic, Marijo"}],"article_processing_charge":"No","title":"Contextual fear learning induced changes in AMPA receptor subtypes along the proximodistal axis in dorsal hippocampus"},{"scopus_import":"1","month":"12","intvolume":" 22","abstract":[{"text":"Electron microscopy (EM) is a technology that enables visualization of single proteins at a nanometer resolution. However, current protein analysis by EM mainly relies on immunolabeling with gold-particle-conjugated antibodies, which is compromised by large size of antibody, precluding precise detection of protein location in biological samples. Here, we develop a specific chemical labeling method for EM detection of proteins at single-molecular level. Rational design of α-helical peptide tag and probe structure provided a complementary reaction pair that enabled specific cysteine conjugation of the tag. The developed chemical labeling with gold-nanoparticle-conjugated probe showed significantly higher labeling efficiency and detectability of high-density clusters of tag-fused G protein-coupled receptors in freeze-fracture replicas compared with immunogold labeling. Furthermore, in ultrathin sections, the spatial resolution of the chemical labeling was significantly higher than that of antibody-mediated labeling. These results demonstrate substantial advantages of the chemical labeling approach for single protein visualization by EM.","lang":"eng"}],"oa_version":"Published Version","pmid":1,"volume":22,"issue":"12","related_material":{"record":[{"relation":"dissertation_contains","status":"public","id":"11393"}]},"ec_funded":1,"publication_identifier":{"issn":["2589-0042"]},"publication_status":"published","file":[{"date_updated":"2020-07-14T12:47:57Z","file_size":7197776,"creator":"dernst","date_created":"2020-02-04T10:48:36Z","file_name":"2019_iScience_Tabata.pdf","content_type":"application/pdf","access_level":"open_access","relation":"main_file","checksum":"f3e90056a49f09b205b1c4f8c739ffd1","file_id":"7448"}],"language":[{"iso":"eng"}],"type":"journal_article","article_type":"original","tmp":{"legal_code_url":"https://creativecommons.org/licenses/by/4.0/legalcode","image":"/images/cc_by.png","name":"Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)","short":"CC BY (4.0)"},"status":"public","_id":"7391","department":[{"_id":"RySh"}],"file_date_updated":"2020-07-14T12:47:57Z","date_updated":"2024-03-27T23:30:13Z","ddc":["570"],"publisher":"Elsevier","quality_controlled":"1","oa":1,"page":"256-268","doi":"10.1016/j.isci.2019.11.025","date_published":"2019-12-20T00:00:00Z","date_created":"2020-01-29T15:56:56Z","has_accepted_license":"1","year":"2019","day":"20","publication":"iScience","project":[{"call_identifier":"H2020","_id":"25CA28EA-B435-11E9-9278-68D0E5697425","name":"In situ analysis of single channel subunit composition in neurons: physiological implication in synaptic plasticity and behaviour","grant_number":"694539"},{"name":"Human Brain Project Specific Grant Agreement 1 (HBP SGA 1)","grant_number":"720270","_id":"25CBA828-B435-11E9-9278-68D0E5697425","call_identifier":"H2020"}],"author":[{"first_name":"Shigekazu","id":"4427179E-F248-11E8-B48F-1D18A9856A87","full_name":"Tabata, Shigekazu","last_name":"Tabata"},{"first_name":"Marijo","id":"4BE3BC94-F248-11E8-B48F-1D18A9856A87","last_name":"Jevtic","full_name":"Jevtic, Marijo"},{"first_name":"Nobutaka","last_name":"Kurashige","full_name":"Kurashige, Nobutaka"},{"last_name":"Fuchida","full_name":"Fuchida, Hirokazu","first_name":"Hirokazu"},{"first_name":"Munetsugu","full_name":"Kido, Munetsugu","last_name":"Kido"},{"first_name":"Kazushi","full_name":"Tani, Kazushi","last_name":"Tani"},{"last_name":"Zenmyo","full_name":"Zenmyo, Naoki","first_name":"Naoki"},{"first_name":"Shohei","full_name":"Uchinomiya, Shohei","last_name":"Uchinomiya"},{"first_name":"Harumi","id":"2E55CDF2-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0001-7429-7896","full_name":"Harada, Harumi","last_name":"Harada"},{"first_name":"Makoto","last_name":"Itakura","full_name":"Itakura, Makoto"},{"first_name":"Itaru","last_name":"Hamachi","full_name":"Hamachi, Itaru"},{"first_name":"Ryuichi","id":"499F3ABC-F248-11E8-B48F-1D18A9856A87","full_name":"Shigemoto, Ryuichi","orcid":"0000-0001-8761-9444","last_name":"Shigemoto"},{"first_name":"Akio","full_name":"Ojida, Akio","last_name":"Ojida"}],"external_id":{"pmid":["31786521"],"isi":[":000504652000020"]},"article_processing_charge":"No","title":"Electron microscopic detection of single membrane proteins by a specific chemical labeling","citation":{"mla":"Tabata, Shigekazu, et al. “Electron Microscopic Detection of Single Membrane Proteins by a Specific Chemical Labeling.” IScience, vol. 22, no. 12, Elsevier, 2019, pp. 256–68, doi:10.1016/j.isci.2019.11.025.","ieee":"S. Tabata et al., “Electron microscopic detection of single membrane proteins by a specific chemical labeling,” iScience, vol. 22, no. 12. Elsevier, pp. 256–268, 2019.","short":"S. Tabata, M. Jevtic, N. Kurashige, H. Fuchida, M. Kido, K. Tani, N. Zenmyo, S. Uchinomiya, H. Harada, M. Itakura, I. Hamachi, R. Shigemoto, A. Ojida, IScience 22 (2019) 256–268.","ama":"Tabata S, Jevtic M, Kurashige N, et al. Electron microscopic detection of single membrane proteins by a specific chemical labeling. iScience. 2019;22(12):256-268. doi:10.1016/j.isci.2019.11.025","apa":"Tabata, S., Jevtic, M., Kurashige, N., Fuchida, H., Kido, M., Tani, K., … Ojida, A. (2019). Electron microscopic detection of single membrane proteins by a specific chemical labeling. IScience. Elsevier. https://doi.org/10.1016/j.isci.2019.11.025","chicago":"Tabata, Shigekazu, Marijo Jevtic, Nobutaka Kurashige, Hirokazu Fuchida, Munetsugu Kido, Kazushi Tani, Naoki Zenmyo, et al. “Electron Microscopic Detection of Single Membrane Proteins by a Specific Chemical Labeling.” IScience. Elsevier, 2019. https://doi.org/10.1016/j.isci.2019.11.025.","ista":"Tabata S, Jevtic M, Kurashige N, Fuchida H, Kido M, Tani K, Zenmyo N, Uchinomiya S, Harada H, Itakura M, Hamachi I, Shigemoto R, Ojida A. 2019. Electron microscopic detection of single membrane proteins by a specific chemical labeling. iScience. 22(12), 256–268."},"user_id":"c635000d-4b10-11ee-a964-aac5a93f6ac1"}]