---
_id: '11393'
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."
acknowledged_ssus:
- _id: EM-Fac
alternative_title:
- ISTA Thesis
article_processing_charge: No
author:
- first_name: Marijo
  full_name: Jevtic, Marijo
  id: 4BE3BC94-F248-11E8-B48F-1D18A9856A87
  last_name: Jevtic
citation:
  ama: Jevtic M. Contextual fear learning induced changes in AMPA receptor subtypes
    along the proximodistal axis in dorsal hippocampus. 2022. doi:<a href="https://doi.org/10.15479/at:ista:11393">10.15479/at:ista:11393</a>
  apa: Jevtic, M. (2022). <i>Contextual fear learning induced changes in AMPA receptor
    subtypes along the proximodistal axis in dorsal hippocampus</i>. Institute of
    Science and Technology Austria. <a href="https://doi.org/10.15479/at:ista:11393">https://doi.org/10.15479/at:ista:11393</a>
  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. <a href="https://doi.org/10.15479/at:ista:11393">https://doi.org/10.15479/at:ista:11393</a>.
  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.
  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.
  mla: Jevtic, Marijo. <i>Contextual Fear Learning Induced Changes in AMPA Receptor
    Subtypes along the Proximodistal Axis in Dorsal Hippocampus</i>. Institute of
    Science and Technology Austria, 2022, doi:<a href="https://doi.org/10.15479/at:ista:11393">10.15479/at:ista:11393</a>.
  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.
date_created: 2022-05-17T08:57:41Z
date_published: 2022-05-16T00:00:00Z
date_updated: 2023-09-07T14:53:44Z
day: '16'
ddc:
- '570'
degree_awarded: PhD
department:
- _id: GradSch
- _id: RySh
doi: 10.15479/at:ista:11393
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  date_created: 2022-05-17T09:08:06Z
  date_updated: 2023-05-17T22:30:03Z
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  file_name: MJ thesis.docx
  file_size: 56427603
  relation: source_file
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  date_created: 2022-05-17T12:09:25Z
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  embargo: 2023-05-16
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file_date_updated: 2023-05-17T22:30:03Z
has_accepted_license: '1'
language:
- iso: eng
month: '05'
oa: 1
oa_version: Published Version
page: '108'
publication_identifier:
  issn:
  - 2663-337X
publication_status: published
publisher: Institute of Science and Technology Austria
related_material:
  record:
  - id: '7391'
    relation: part_of_dissertation
    status: public
status: public
supervisor:
- first_name: Ryuichi
  full_name: Shigemoto, Ryuichi
  id: 499F3ABC-F248-11E8-B48F-1D18A9856A87
  last_name: Shigemoto
  orcid: 0000-0001-8761-9444
title: Contextual fear learning induced changes in AMPA receptor subtypes along the
  proximodistal axis in dorsal hippocampus
type: dissertation
user_id: 8b945eb4-e2f2-11eb-945a-df72226e66a9
year: '2022'
...
---
_id: '7391'
abstract:
- lang: eng
  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.
article_processing_charge: No
article_type: original
author:
- first_name: Shigekazu
  full_name: Tabata, Shigekazu
  id: 4427179E-F248-11E8-B48F-1D18A9856A87
  last_name: Tabata
- first_name: Marijo
  full_name: Jevtic, Marijo
  id: 4BE3BC94-F248-11E8-B48F-1D18A9856A87
  last_name: Jevtic
- first_name: Nobutaka
  full_name: Kurashige, Nobutaka
  last_name: Kurashige
- first_name: Hirokazu
  full_name: Fuchida, Hirokazu
  last_name: Fuchida
- first_name: Munetsugu
  full_name: Kido, Munetsugu
  last_name: Kido
- first_name: Kazushi
  full_name: Tani, Kazushi
  last_name: Tani
- first_name: Naoki
  full_name: Zenmyo, Naoki
  last_name: Zenmyo
- first_name: Shohei
  full_name: Uchinomiya, Shohei
  last_name: Uchinomiya
- first_name: Harumi
  full_name: Harada, Harumi
  id: 2E55CDF2-F248-11E8-B48F-1D18A9856A87
  last_name: Harada
  orcid: 0000-0001-7429-7896
- first_name: Makoto
  full_name: Itakura, Makoto
  last_name: Itakura
- first_name: Itaru
  full_name: Hamachi, Itaru
  last_name: Hamachi
- first_name: Ryuichi
  full_name: Shigemoto, Ryuichi
  id: 499F3ABC-F248-11E8-B48F-1D18A9856A87
  last_name: Shigemoto
  orcid: 0000-0001-8761-9444
- first_name: Akio
  full_name: Ojida, Akio
  last_name: Ojida
citation:
  ama: Tabata S, Jevtic M, Kurashige N, et al. Electron microscopic detection of single
    membrane proteins by a specific chemical labeling. <i>iScience</i>. 2019;22(12):256-268.
    doi:<a href="https://doi.org/10.1016/j.isci.2019.11.025">10.1016/j.isci.2019.11.025</a>
  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. <i>IScience</i>. Elsevier. <a href="https://doi.org/10.1016/j.isci.2019.11.025">https://doi.org/10.1016/j.isci.2019.11.025</a>
  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.” <i>IScience</i>.
    Elsevier, 2019. <a href="https://doi.org/10.1016/j.isci.2019.11.025">https://doi.org/10.1016/j.isci.2019.11.025</a>.
  ieee: S. Tabata <i>et al.</i>, “Electron microscopic detection of single membrane
    proteins by a specific chemical labeling,” <i>iScience</i>, vol. 22, no. 12. Elsevier,
    pp. 256–268, 2019.
  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.
  mla: Tabata, Shigekazu, et al. “Electron Microscopic Detection of Single Membrane
    Proteins by a Specific Chemical Labeling.” <i>IScience</i>, vol. 22, no. 12, Elsevier,
    2019, pp. 256–68, doi:<a href="https://doi.org/10.1016/j.isci.2019.11.025">10.1016/j.isci.2019.11.025</a>.
  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.
date_created: 2020-01-29T15:56:56Z
date_published: 2019-12-20T00:00:00Z
date_updated: 2024-03-25T23:30:07Z
day: '20'
ddc:
- '570'
department:
- _id: RySh
doi: 10.1016/j.isci.2019.11.025
ec_funded: 1
external_id:
  isi:
  - :000504652000020
  pmid:
  - '31786521'
file:
- access_level: open_access
  checksum: f3e90056a49f09b205b1c4f8c739ffd1
  content_type: application/pdf
  creator: dernst
  date_created: 2020-02-04T10:48:36Z
  date_updated: 2020-07-14T12:47:57Z
  file_id: '7448'
  file_name: 2019_iScience_Tabata.pdf
  file_size: 7197776
  relation: main_file
file_date_updated: 2020-07-14T12:47:57Z
has_accepted_license: '1'
intvolume: '        22'
issue: '12'
language:
- iso: eng
month: '12'
oa: 1
oa_version: Published Version
page: 256-268
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: 25CBA828-B435-11E9-9278-68D0E5697425
  call_identifier: H2020
  grant_number: '720270'
  name: Human Brain Project Specific Grant Agreement 1 (HBP SGA 1)
publication: iScience
publication_identifier:
  issn:
  - 2589-0042
publication_status: published
publisher: Elsevier
quality_controlled: '1'
related_material:
  record:
  - id: '11393'
    relation: dissertation_contains
    status: public
scopus_import: '1'
status: public
title: Electron microscopic detection of single membrane proteins by a specific chemical
  labeling
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: c635000d-4b10-11ee-a964-aac5a93f6ac1
volume: 22
year: '2019'
...