---
_id: '9437'
abstract:
- lang: eng
text: The synaptic connection from medial habenula (MHb) to interpeduncular nucleus
(IPN) is critical for emotion-related behaviors and uniquely expresses R-type
Ca2+ channels (Cav2.3) and auxiliary GABAB receptor (GBR) subunits, the K+-channel
tetramerization domain-containing proteins (KCTDs). Activation of GBRs facilitates
or inhibits transmitter release from MHb terminals depending on the IPN subnucleus,
but the role of KCTDs is unknown. We therefore examined the localization and function
of Cav2.3, GBRs, and KCTDs in this pathway in mice. We show in heterologous cells
that KCTD8 and KCTD12b directly bind to Cav2.3 and that KCTD8 potentiates Cav2.3
currents in the absence of GBRs. In the rostral IPN, KCTD8, KCTD12b, and Cav2.3
co-localize at the presynaptic active zone. Genetic deletion indicated a bidirectional
modulation of Cav2.3-mediated release by these KCTDs with a compensatory increase
of KCTD8 in the active zone in KCTD12b-deficient mice. The interaction of Cav2.3
with KCTDs therefore scales synaptic strength independent of GBR activation.
acknowledgement: We are grateful to Akari Hagiwara and Toshihisa Ohtsuka for CAST
antibody, and Masahiko Watanabe for neurexin antibody. We thank David Adams for
kindly providing the stable Cav2.3 cell line. Cav2.3 KO mice were kindly provided
by Tsutomu Tanabe. This project has received funding from the European Research
Council (ERC) and European Commission (EC), under the European Union’s Horizon 2020
research and innovation programme (ERC grant agreement no. 694539 to Ryuichi Shigemoto,
no. 692692 to Peter Jonas, and the Marie Skłodowska-Curie grant agreement no. 665385
to Cihan Önal), the Swiss National Science Foundation Grant 31003A-172881 to Bernhard
Bettler and Deutsche Forschungsgemeinschaft (For 2143) and BIOSS-2 to Akos Kulik.
article_number: e68274
article_processing_charge: No
article_type: original
author:
- first_name: Pradeep
full_name: Bhandari, Pradeep
id: 45EDD1BC-F248-11E8-B48F-1D18A9856A87
last_name: Bhandari
orcid: 0000-0003-0863-4481
- 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: Diego
full_name: Fernández-Fernández, Diego
last_name: Fernández-Fernández
- first_name: Thorsten
full_name: Fritzius, Thorsten
last_name: Fritzius
- first_name: David
full_name: Kleindienst, David
id: 42E121A4-F248-11E8-B48F-1D18A9856A87
last_name: Kleindienst
- 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: Jacqueline-Claire
full_name: Montanaro-Punzengruber, Jacqueline-Claire
id: 3786AB44-F248-11E8-B48F-1D18A9856A87
last_name: Montanaro-Punzengruber
- first_name: Martin
full_name: Gassmann, Martin
last_name: Gassmann
- 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: Akos
full_name: Kulik, Akos
last_name: Kulik
- first_name: Bernhard
full_name: Bettler, Bernhard
last_name: Bettler
- first_name: Ryuichi
full_name: Shigemoto, Ryuichi
id: 499F3ABC-F248-11E8-B48F-1D18A9856A87
last_name: Shigemoto
orcid: 0000-0001-8761-9444
- first_name: Peter
full_name: Koppensteiner, Peter
id: 3B8B25A8-F248-11E8-B48F-1D18A9856A87
last_name: Koppensteiner
orcid: 0000-0002-3509-1948
citation:
ama: Bhandari P, Vandael DH, Fernández-Fernández D, et al. GABAB receptor auxiliary
subunits modulate Cav2.3-mediated release from medial habenula terminals. eLife.
2021;10. doi:10.7554/ELIFE.68274
apa: Bhandari, P., Vandael, D. H., Fernández-Fernández, D., Fritzius, T., Kleindienst,
D., Önal, H. C., … Koppensteiner, P. (2021). GABAB receptor auxiliary subunits
modulate Cav2.3-mediated release from medial habenula terminals. ELife.
eLife Sciences Publications. https://doi.org/10.7554/ELIFE.68274
chicago: Bhandari, Pradeep, David H Vandael, Diego Fernández-Fernández, Thorsten
Fritzius, David Kleindienst, Hüseyin C Önal, Jacqueline-Claire Montanaro-Punzengruber,
et al. “GABAB Receptor Auxiliary Subunits Modulate Cav2.3-Mediated Release from
Medial Habenula Terminals.” ELife. eLife Sciences Publications, 2021. https://doi.org/10.7554/ELIFE.68274.
ieee: P. Bhandari et al., “GABAB receptor auxiliary subunits modulate Cav2.3-mediated
release from medial habenula terminals,” eLife, vol. 10. eLife Sciences
Publications, 2021.
ista: Bhandari P, Vandael DH, Fernández-Fernández D, Fritzius T, Kleindienst D,
Önal HC, Montanaro-Punzengruber J-C, Gassmann M, Jonas PM, Kulik A, Bettler B,
Shigemoto R, Koppensteiner P. 2021. GABAB receptor auxiliary subunits modulate
Cav2.3-mediated release from medial habenula terminals. eLife. 10, e68274.
mla: Bhandari, Pradeep, et al. “GABAB Receptor Auxiliary Subunits Modulate Cav2.3-Mediated
Release from Medial Habenula Terminals.” ELife, vol. 10, e68274, eLife
Sciences Publications, 2021, doi:10.7554/ELIFE.68274.
short: P. Bhandari, D.H. Vandael, D. Fernández-Fernández, T. Fritzius, D. Kleindienst,
H.C. Önal, J.-C. Montanaro-Punzengruber, M. Gassmann, P.M. Jonas, A. Kulik, B.
Bettler, R. Shigemoto, P. Koppensteiner, ELife 10 (2021).
date_created: 2021-05-30T22:01:23Z
date_published: 2021-04-29T00:00:00Z
date_updated: 2024-03-25T23:30:16Z
day: '29'
ddc:
- '570'
department:
- _id: RySh
- _id: PeJo
doi: 10.7554/ELIFE.68274
ec_funded: 1
external_id:
isi:
- '000651761700001'
file:
- access_level: open_access
checksum: 6ebcb79999f889766f7cd79ee134ad28
content_type: application/pdf
creator: cziletti
date_created: 2021-05-31T09:43:09Z
date_updated: 2021-05-31T09:43:09Z
file_id: '9440'
file_name: 2021_eLife_Bhandari.pdf
file_size: 8174719
relation: main_file
success: 1
file_date_updated: 2021-05-31T09:43:09Z
has_accepted_license: '1'
intvolume: ' 10'
isi: 1
language:
- iso: eng
month: '04'
oa: 1
oa_version: Published Version
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: 25B7EB9E-B435-11E9-9278-68D0E5697425
call_identifier: H2020
grant_number: '692692'
name: Biophysics and circuit function of a giant cortical glumatergic synapse
- _id: 2564DBCA-B435-11E9-9278-68D0E5697425
call_identifier: H2020
grant_number: '665385'
name: International IST Doctoral Program
publication: eLife
publication_identifier:
eissn:
- 2050-084X
publication_status: published
publisher: eLife Sciences Publications
quality_controlled: '1'
related_material:
link:
- relation: earlier_version
url: https://doi.org/10.1101/2020.04.16.045112
record:
- id: '9562'
relation: dissertation_contains
status: public
scopus_import: '1'
status: public
title: GABAB receptor auxiliary subunits modulate Cav2.3-mediated release from medial
habenula terminals
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: 10
year: '2021'
...
---
_id: '9562'
abstract:
- lang: eng
text: Left-right asymmetries can be considered a fundamental organizational principle
of the vertebrate central nervous system. The hippocampal CA3-CA1 pyramidal cell
synaptic connection shows an input-side dependent asymmetry where the hemispheric
location of the presynaptic CA3 neuron determines the synaptic properties. Left-input
synapses terminating on apical dendrites in stratum radiatum have a higher density
of NMDA receptor subunit GluN2B, a lower density of AMPA receptor subunit GluA1
and smaller areas with less often perforated PSDs. On the other hand, left-input
synapses terminating on basal dendrites in stratum oriens have lower GluN2B densities
than right-input ones. Apical and basal synapses further employ different signaling
pathways involved in LTP. SDS-digested freeze-fracture replica labeling can visualize
synaptic membrane proteins with high sensitivity and resolution, and has been
used to reveal the asymmetry at the electron microscopic level. However, it requires
time-consuming manual demarcation of the synaptic surface for quantitative measurements.
To facilitate the analysis of replica labeling, I first developed a software named
Darea, which utilizes deep-learning to automatize this demarcation. With Darea
I characterized the synaptic distribution of NMDA and AMPA receptors as well as
the voltage-gated Ca2+ channels in CA1 stratum radiatum and oriens. Second, I
explored the role of GluN2B and its carboxy-terminus in the establishment of input-side
dependent hippocampal asymmetry. In conditional knock-out mice lacking GluN2B
expression in CA1 and GluN2B-2A swap mice, where GluN2B carboxy-terminus was exchanged
to that of GluN2A, no significant asymmetries of GluN2B, GluA1 and PSD area were
detected. We further discovered a previously unknown functional asymmetry of GluN2A,
which was also lost in the swap mouse. These results demonstrate that GluN2B carboxy-terminus
plays a critical role in normal formation of input-side dependent asymmetry.
acknowledged_ssus:
- _id: EM-Fac
alternative_title:
- ISTA Thesis
article_processing_charge: No
author:
- first_name: David
full_name: Kleindienst, David
id: 42E121A4-F248-11E8-B48F-1D18A9856A87
last_name: Kleindienst
citation:
ama: 'Kleindienst D. 2B or not 2B: Hippocampal asymmetries mediated by NMDA receptor
subunit GluN2B C-terminus and high-throughput image analysis by Deep-Learning.
2021. doi:10.15479/at:ista:9562'
apa: 'Kleindienst, D. (2021). 2B or not 2B: Hippocampal asymmetries mediated
by NMDA receptor subunit GluN2B C-terminus and high-throughput image analysis
by Deep-Learning. Institute of Science and Technology Austria. https://doi.org/10.15479/at:ista:9562'
chicago: 'Kleindienst, David. “2B or Not 2B: Hippocampal Asymmetries Mediated by
NMDA Receptor Subunit GluN2B C-Terminus and High-Throughput Image Analysis by
Deep-Learning.” Institute of Science and Technology Austria, 2021. https://doi.org/10.15479/at:ista:9562.'
ieee: 'D. Kleindienst, “2B or not 2B: Hippocampal asymmetries mediated by NMDA receptor
subunit GluN2B C-terminus and high-throughput image analysis by Deep-Learning,”
Institute of Science and Technology Austria, 2021.'
ista: 'Kleindienst D. 2021. 2B or not 2B: Hippocampal asymmetries mediated by NMDA
receptor subunit GluN2B C-terminus and high-throughput image analysis by Deep-Learning.
Institute of Science and Technology Austria.'
mla: 'Kleindienst, David. 2B or Not 2B: Hippocampal Asymmetries Mediated by NMDA
Receptor Subunit GluN2B C-Terminus and High-Throughput Image Analysis by Deep-Learning.
Institute of Science and Technology Austria, 2021, doi:10.15479/at:ista:9562.'
short: 'D. Kleindienst, 2B or Not 2B: Hippocampal Asymmetries Mediated by NMDA Receptor
Subunit GluN2B C-Terminus and High-Throughput Image Analysis by Deep-Learning,
Institute of Science and Technology Austria, 2021.'
date_created: 2021-06-17T14:10:47Z
date_published: 2021-06-01T00:00:00Z
date_updated: 2023-09-11T12:55:53Z
day: '01'
ddc:
- '570'
degree_awarded: PhD
department:
- _id: GradSch
- _id: RySh
doi: 10.15479/at:ista:9562
file:
- access_level: open_access
checksum: 659df5518db495f679cb1df9e9bd1d94
content_type: application/pdf
creator: dkleindienst
date_created: 2021-06-17T14:03:14Z
date_updated: 2022-07-02T22:30:04Z
embargo: 2022-07-01
file_id: '9563'
file_name: Thesis.pdf
file_size: 77299142
relation: main_file
- access_level: closed
checksum: 3bcf63a2b19e5b6663be051bea332748
content_type: application/zip
creator: dkleindienst
date_created: 2021-06-17T14:04:30Z
date_updated: 2022-07-02T22:30:04Z
embargo_to: open_access
file_id: '9564'
file_name: Thesis_source.zip
file_size: 369804895
relation: source_file
file_date_updated: 2022-07-02T22:30:04Z
has_accepted_license: '1'
language:
- iso: eng
month: '06'
oa: 1
oa_version: Published Version
page: '124'
publication_identifier:
issn:
- 2663-337X
publication_status: published
publisher: Institute of Science and Technology Austria
related_material:
record:
- id: '9756'
relation: part_of_dissertation
status: public
- id: '9437'
relation: part_of_dissertation
status: public
- id: '8532'
relation: part_of_dissertation
status: public
- id: '612'
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: '2B or not 2B: Hippocampal asymmetries mediated by NMDA receptor subunit GluN2B
C-terminus and high-throughput image analysis by Deep-Learning'
type: dissertation
user_id: c635000d-4b10-11ee-a964-aac5a93f6ac1
year: '2021'
...
---
_id: '10051'
abstract:
- lang: eng
text: 'Rab-interacting molecule (RIM)-binding protein 2 (BP2) is a multidomain protein
of the presynaptic active zone (AZ). By binding to RIM, bassoon (Bsn), and voltage-gated
Ca2+ channels (CaV), it is considered to be a central organizer of the topography
of CaV and release sites of synaptic vesicles (SVs) at the AZ. Here, we used RIM-BP2
knock-out (KO) mice and their wild-type (WT) littermates of either sex to investigate
the role of RIM-BP2 at the endbulb of Held synapse of auditory nerve fibers (ANFs)
with bushy cells (BCs) of the cochlear nucleus, a fast relay of the auditory pathway
with high release probability. Disruption of RIM-BP2 lowered release probability
altering short-term plasticity and reduced evoked EPSCs. Analysis of SV pool dynamics
during high-frequency train stimulation indicated a reduction of SVs with high
release probability but an overall normal size of the readily releasable SV pool
(RRP). The Ca2+-dependent fast component of SV replenishment after RRP depletion
was slowed. Ultrastructural analysis by superresolution light and electron microscopy
revealed an impaired topography of presynaptic CaV and a reduction of docked and
membrane-proximal SVs at the AZ. We conclude that RIM-BP2 organizes the topography
of CaV, and promotes SV tethering and docking. This way RIM-BP2 is critical for
establishing a high initial release probability as required to reliably signal
sound onset information that we found to be degraded in BCs of RIM-BP2-deficient
mice in vivo. SIGNIFICANCE STATEMENT: Rab-interacting molecule (RIM)-binding proteins
(BPs) are key organizers of the active zone (AZ). Using a multidisciplinary approach
to the calyceal endbulb of Held synapse that transmits auditory information at
rates of up to hundreds of Hertz with submillisecond precision we demonstrate
a requirement for RIM-BP2 for normal auditory signaling. Endbulb synapses lacking
RIM-BP2 show a reduced release probability despite normal whole-terminal Ca2+
influx and abundance of the key priming protein Munc13-1, a reduced rate of SV
replenishment, as well as an altered topography of voltage-gated (CaV)2.1 Ca2+
channels, and fewer docked and membrane proximal synaptic vesicles (SVs). This
hampers transmission of sound onset information likely affecting downstream neural
computations such as of sound localization.'
acknowledgement: This work was supported by the Deutsche Forschungsgemeinschaft (DFG,
German Research Foundation) through the Collaborative Sensory Research Center 1286
[to C.W. (A4) and T.M. (B5)] and under Germany’s Excellence Strategy Grant EXC 2067/1-390729940.
We thank S. Gerke, A.J. Goldak, and C. Senger-Freitag for expert technical assistance;
G. Hoch for developing image analysis routines; and S. Chepurwar and N. Strenzke
for technical support and discussion regarding in vivo experiments. We also thank
Dr. Christian Rosenmund, Dr. Katharina Grauel, and Dr. Stephan Sigrist for providing
RIM-BP2 KO mice and Dr. Masahiko Watanabe for providing the anti-neurexin-antibody,
and Dr. Toshihisa Ohtsuka for the anti-ELKS-antibody. J. Neef for help with the
STED imaging and image analysis; E. Neher and S. Rizzoli for discussion and comments
on the manuscript; K. Eguchi for help with the statistical analysis; and C. H. Huang
and J. Neef for constant support and scientific discussion.
article_processing_charge: No
article_type: original
author:
- first_name: Tanvi
full_name: Butola, Tanvi
last_name: Butola
- first_name: Theocharis
full_name: Alvanos, Theocharis
last_name: Alvanos
- first_name: Anika
full_name: Hintze, Anika
last_name: Hintze
- first_name: Peter
full_name: Koppensteiner, Peter
id: 3B8B25A8-F248-11E8-B48F-1D18A9856A87
last_name: Koppensteiner
orcid: 0000-0002-3509-1948
- first_name: David
full_name: Kleindienst, David
id: 42E121A4-F248-11E8-B48F-1D18A9856A87
last_name: Kleindienst
- first_name: Ryuichi
full_name: Shigemoto, Ryuichi
id: 499F3ABC-F248-11E8-B48F-1D18A9856A87
last_name: Shigemoto
orcid: 0000-0001-8761-9444
- first_name: Carolin
full_name: Wichmann, Carolin
last_name: Wichmann
- first_name: Tobias
full_name: Moser, Tobias
last_name: Moser
citation:
ama: Butola T, Alvanos T, Hintze A, et al. RIM-binding protein 2 organizes Ca21
channel topography and regulates release probability and vesicle replenishment
at a fast central synapse. Journal of Neuroscience. 2021;41(37):7742-7767.
doi:10.1523/JNEUROSCI.0586-21.2021
apa: Butola, T., Alvanos, T., Hintze, A., Koppensteiner, P., Kleindienst, D., Shigemoto,
R., … Moser, T. (2021). RIM-binding protein 2 organizes Ca21 channel
topography and regulates release probability and vesicle replenishment at a fast
central synapse. Journal of Neuroscience. Society for Neuroscience. https://doi.org/10.1523/JNEUROSCI.0586-21.2021
chicago: Butola, Tanvi, Theocharis Alvanos, Anika Hintze, Peter Koppensteiner, David
Kleindienst, Ryuichi Shigemoto, Carolin Wichmann, and Tobias Moser. “RIM-Binding
Protein 2 Organizes Ca21 Channel Topography and Regulates Release Probability
and Vesicle Replenishment at a Fast Central Synapse.” Journal of Neuroscience.
Society for Neuroscience, 2021. https://doi.org/10.1523/JNEUROSCI.0586-21.2021.
ieee: T. Butola et al., “RIM-binding protein 2 organizes Ca21
channel topography and regulates release probability and vesicle replenishment
at a fast central synapse,” Journal of Neuroscience, vol. 41, no. 37. Society
for Neuroscience, pp. 7742–7767, 2021.
ista: Butola T, Alvanos T, Hintze A, Koppensteiner P, Kleindienst D, Shigemoto R,
Wichmann C, Moser T. 2021. RIM-binding protein 2 organizes Ca21 channel
topography and regulates release probability and vesicle replenishment at a fast
central synapse. Journal of Neuroscience. 41(37), 7742–7767.
mla: Butola, Tanvi, et al. “RIM-Binding Protein 2 Organizes Ca21 Channel
Topography and Regulates Release Probability and Vesicle Replenishment at a Fast
Central Synapse.” Journal of Neuroscience, vol. 41, no. 37, Society for
Neuroscience, 2021, pp. 7742–67, doi:10.1523/JNEUROSCI.0586-21.2021.
short: T. Butola, T. Alvanos, A. Hintze, P. Koppensteiner, D. Kleindienst, R. Shigemoto,
C. Wichmann, T. Moser, Journal of Neuroscience 41 (2021) 7742–7767.
date_created: 2021-09-27T14:33:13Z
date_published: 2021-09-15T00:00:00Z
date_updated: 2023-08-14T06:56:30Z
day: '15'
ddc:
- '570'
department:
- _id: RySh
doi: 10.1523/JNEUROSCI.0586-21.2021
external_id:
isi:
- '000752287700005'
pmid:
- '34353898'
file:
- access_level: open_access
checksum: 769ab627c7355a50ccfd445e43a5f351
content_type: application/pdf
creator: dernst
date_created: 2022-05-31T09:10:15Z
date_updated: 2022-05-31T09:10:15Z
file_id: '11423'
file_name: 2021_JourNeuroscience_Butola.pdf
file_size: 11571961
relation: main_file
success: 1
file_date_updated: 2022-05-31T09:10:15Z
has_accepted_license: '1'
intvolume: ' 41'
isi: 1
issue: '37'
language:
- iso: eng
month: '09'
oa: 1
oa_version: Published Version
page: 7742-7767
pmid: 1
publication: Journal of Neuroscience
publication_identifier:
eissn:
- 1529-2401
issn:
- 0270-6474
publication_status: published
publisher: Society for Neuroscience
quality_controlled: '1'
scopus_import: '1'
status: public
title: RIM-binding protein 2 organizes Ca21 channel topography and regulates
release probability and vesicle replenishment at a fast central 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: journal_article
user_id: 4359f0d1-fa6c-11eb-b949-802e58b17ae8
volume: 41
year: '2021'
...
---
_id: '9756'
abstract:
- lang: eng
text: High-resolution visualization and quantification of membrane proteins contribute
to the understanding of their functions and the roles they play in physiological
and pathological conditions. Sodium dodecyl sulfate-digested freeze-fracture replica
labeling (SDS-FRL) is a powerful electron microscopy method to study quantitatively
the two-dimensional distribution of transmembrane proteins and their tightly associated
proteins. During treatment with SDS, intracellular organelles and proteins not
anchored to the replica are dissolved, whereas integral membrane proteins captured
and stabilized by carbon/platinum deposition remain on the replica. Their intra-
and extracellular domains become exposed on the surface of the replica, facilitating
the accessibility of antibodies and, therefore, providing higher labeling efficiency
than those obtained with other immunoelectron microscopy techniques. In this chapter,
we describe the protocols of SDS-FRL adapted for mammalian brain samples, and
optimization of the SDS treatment to increase the labeling efficiency for quantification
of Cav2.1, the alpha subunit of P/Q-type voltage-dependent calcium channels utilizing
deep learning algorithms.
acknowledgement: This work was supported by the European Union (European Research
Council Advanced grant no. 694539 and Human Brain Project Ref. 720270 to R. S.)
and the Austrian Academy of Sciences (DOC fellowship to D.K.).
alternative_title:
- Neuromethods
article_processing_charge: No
author:
- first_name: Walter
full_name: Kaufmann, Walter
id: 3F99E422-F248-11E8-B48F-1D18A9856A87
last_name: Kaufmann
orcid: 0000-0001-9735-5315
- first_name: David
full_name: Kleindienst, David
id: 42E121A4-F248-11E8-B48F-1D18A9856A87
last_name: Kleindienst
- first_name: Harumi
full_name: Harada, Harumi
id: 2E55CDF2-F248-11E8-B48F-1D18A9856A87
last_name: Harada
orcid: 0000-0001-7429-7896
- first_name: Ryuichi
full_name: Shigemoto, Ryuichi
id: 499F3ABC-F248-11E8-B48F-1D18A9856A87
last_name: Shigemoto
orcid: 0000-0001-8761-9444
citation:
ama: 'Kaufmann W, Kleindienst D, Harada H, Shigemoto R. High-Resolution localization
and quantitation of membrane proteins by SDS-digested freeze-fracture replica
labeling (SDS-FRL). In: Receptor and Ion Channel Detection in the Brain.
Vol 169. Neuromethods. New York: Humana; 2021:267-283. doi:10.1007/978-1-0716-1522-5_19'
apa: 'Kaufmann, W., Kleindienst, D., Harada, H., & Shigemoto, R. (2021). High-Resolution
localization and quantitation of membrane proteins by SDS-digested freeze-fracture
replica labeling (SDS-FRL). In Receptor and Ion Channel Detection in the Brain
(Vol. 169, pp. 267–283). New York: Humana. https://doi.org/10.1007/978-1-0716-1522-5_19'
chicago: 'Kaufmann, Walter, David Kleindienst, Harumi Harada, and Ryuichi Shigemoto.
“High-Resolution Localization and Quantitation of Membrane Proteins by SDS-Digested
Freeze-Fracture Replica Labeling (SDS-FRL).” In Receptor and Ion Channel Detection
in the Brain, 169:267–83. Neuromethods. New York: Humana, 2021. https://doi.org/10.1007/978-1-0716-1522-5_19.'
ieee: 'W. Kaufmann, D. Kleindienst, H. Harada, and R. Shigemoto, “High-Resolution
localization and quantitation of membrane proteins by SDS-digested freeze-fracture
replica labeling (SDS-FRL),” in Receptor and Ion Channel Detection in the
Brain, vol. 169, New York: Humana, 2021, pp. 267–283.'
ista: 'Kaufmann W, Kleindienst D, Harada H, Shigemoto R. 2021.High-Resolution localization
and quantitation of membrane proteins by SDS-digested freeze-fracture replica
labeling (SDS-FRL). In: Receptor and Ion Channel Detection in the Brain. Neuromethods,
vol. 169, 267–283.'
mla: Kaufmann, Walter, et al. “High-Resolution Localization and Quantitation of
Membrane Proteins by SDS-Digested Freeze-Fracture Replica Labeling (SDS-FRL).”
Receptor and Ion Channel Detection in the Brain, vol. 169, Humana, 2021,
pp. 267–83, doi:10.1007/978-1-0716-1522-5_19.
short: W. Kaufmann, D. Kleindienst, H. Harada, R. Shigemoto, in:, Receptor and
Ion Channel Detection in the Brain, Humana, New York, 2021, pp. 267–283.
date_created: 2021-07-30T09:34:56Z
date_published: 2021-07-27T00:00:00Z
date_updated: 2024-03-25T23:30:16Z
day: '27'
ddc:
- '573'
department:
- _id: RySh
- _id: EM-Fac
doi: 10.1007/978-1-0716-1522-5_19
ec_funded: 1
has_accepted_license: '1'
intvolume: ' 169'
keyword:
- 'Freeze-fracture replica: Deep learning'
- Immunogold labeling
- Integral membrane protein
- Electron microscopy
language:
- iso: eng
month: '07'
oa_version: None
page: 267-283
place: New York
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: ' Receptor and Ion Channel Detection in the Brain'
publication_identifier:
eisbn:
- '9781071615225'
isbn:
- '9781071615218'
publication_status: published
publisher: Humana
quality_controlled: '1'
related_material:
record:
- id: '9562'
relation: dissertation_contains
status: public
series_title: Neuromethods
status: public
title: High-Resolution localization and quantitation of membrane proteins by SDS-digested
freeze-fracture replica labeling (SDS-FRL)
type: book_chapter
user_id: D865714E-FA4E-11E9-B85B-F5C5E5697425
volume: 169
year: '2021'
...
---
_id: '8532'
abstract:
- lang: eng
text: The molecular anatomy of synapses defines their characteristics in transmission
and plasticity. Precise measurements of the number and distribution of synaptic
proteins are important for our understanding of synapse heterogeneity within and
between brain regions. Freeze–fracture replica immunogold electron microscopy
enables us to analyze them quantitatively on a two-dimensional membrane surface.
Here, we introduce Darea software, which utilizes deep learning for analysis of
replica images and demonstrate its usefulness for quick measurements of the pre-
and postsynaptic areas, density and distribution of gold particles at synapses
in a reproducible manner. We used Darea for comparing glutamate receptor and calcium
channel distributions between hippocampal CA3-CA1 spine synapses on apical and
basal dendrites, which differ in signaling pathways involved in synaptic plasticity.
We found that apical synapses express a higher density of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic
acid (AMPA) receptors and a stronger increase of AMPA receptors with synaptic
size, while basal synapses show a larger increase in N-methyl-D-aspartate (NMDA)
receptors with size. Interestingly, AMPA and NMDA receptors are segregated within
postsynaptic sites and negatively correlated in density among both apical and
basal synapses. In the presynaptic sites, Cav2.1 voltage-gated calcium channels
show similar densities in apical and basal synapses with distributions consistent
with an exclusion zone model of calcium channel-release site topography.
acknowledgement: "This research was funded by Austrian Academy of Sciences, DOC fellowship
to D.K., European Research\r\nCouncil Advanced Grant 694539 and European Union Human
Brain Project (HBP) SGA2 785907 to R.S.\r\nWe acknowledge Elena Hollergschwandtner
for technical support."
article_number: '6737'
article_processing_charge: No
article_type: original
author:
- first_name: David
full_name: Kleindienst, David
id: 42E121A4-F248-11E8-B48F-1D18A9856A87
last_name: Kleindienst
- first_name: Jacqueline-Claire
full_name: Montanaro-Punzengruber, Jacqueline-Claire
id: 3786AB44-F248-11E8-B48F-1D18A9856A87
last_name: Montanaro-Punzengruber
- first_name: Pradeep
full_name: Bhandari, Pradeep
id: 45EDD1BC-F248-11E8-B48F-1D18A9856A87
last_name: Bhandari
orcid: 0000-0003-0863-4481
- first_name: Matthew J
full_name: Case, Matthew J
id: 44B7CA5A-F248-11E8-B48F-1D18A9856A87
last_name: Case
- first_name: Yugo
full_name: Fukazawa, Yugo
last_name: Fukazawa
- first_name: Ryuichi
full_name: Shigemoto, Ryuichi
id: 499F3ABC-F248-11E8-B48F-1D18A9856A87
last_name: Shigemoto
orcid: 0000-0001-8761-9444
citation:
ama: Kleindienst D, Montanaro-Punzengruber J-C, Bhandari P, Case MJ, Fukazawa Y,
Shigemoto R. Deep learning-assisted high-throughput analysis of freeze-fracture
replica images applied to glutamate receptors and calcium channels at hippocampal
synapses. International Journal of Molecular Sciences. 2020;21(18). doi:10.3390/ijms21186737
apa: Kleindienst, D., Montanaro-Punzengruber, J.-C., Bhandari, P., Case, M. J.,
Fukazawa, Y., & Shigemoto, R. (2020). Deep learning-assisted high-throughput
analysis of freeze-fracture replica images applied to glutamate receptors and
calcium channels at hippocampal synapses. International Journal of Molecular
Sciences. MDPI. https://doi.org/10.3390/ijms21186737
chicago: Kleindienst, David, Jacqueline-Claire Montanaro-Punzengruber, Pradeep Bhandari,
Matthew J Case, Yugo Fukazawa, and Ryuichi Shigemoto. “Deep Learning-Assisted
High-Throughput Analysis of Freeze-Fracture Replica Images Applied to Glutamate
Receptors and Calcium Channels at Hippocampal Synapses.” International Journal
of Molecular Sciences. MDPI, 2020. https://doi.org/10.3390/ijms21186737.
ieee: D. Kleindienst, J.-C. Montanaro-Punzengruber, P. Bhandari, M. J. Case, Y.
Fukazawa, and R. Shigemoto, “Deep learning-assisted high-throughput analysis of
freeze-fracture replica images applied to glutamate receptors and calcium channels
at hippocampal synapses,” International Journal of Molecular Sciences,
vol. 21, no. 18. MDPI, 2020.
ista: Kleindienst D, Montanaro-Punzengruber J-C, Bhandari P, Case MJ, Fukazawa Y,
Shigemoto R. 2020. Deep learning-assisted high-throughput analysis of freeze-fracture
replica images applied to glutamate receptors and calcium channels at hippocampal
synapses. International Journal of Molecular Sciences. 21(18), 6737.
mla: Kleindienst, David, et al. “Deep Learning-Assisted High-Throughput Analysis
of Freeze-Fracture Replica Images Applied to Glutamate Receptors and Calcium Channels
at Hippocampal Synapses.” International Journal of Molecular Sciences,
vol. 21, no. 18, 6737, MDPI, 2020, doi:10.3390/ijms21186737.
short: D. Kleindienst, J.-C. Montanaro-Punzengruber, P. Bhandari, M.J. Case, Y.
Fukazawa, R. Shigemoto, International Journal of Molecular Sciences 21 (2020).
date_created: 2020-09-20T22:01:35Z
date_published: 2020-09-14T00:00:00Z
date_updated: 2024-03-25T23:30:16Z
day: '14'
ddc:
- '570'
department:
- _id: RySh
doi: 10.3390/ijms21186737
ec_funded: 1
external_id:
isi:
- '000579945300001'
file:
- access_level: open_access
checksum: 2e4f62f3cfe945b7391fc3070e5a289f
content_type: application/pdf
creator: dernst
date_created: 2020-09-21T14:08:58Z
date_updated: 2020-09-21T14:08:58Z
file_id: '8551'
file_name: 2020_JournMolecSciences_Kleindienst.pdf
file_size: 5748456
relation: main_file
success: 1
file_date_updated: 2020-09-21T14:08:58Z
has_accepted_license: '1'
intvolume: ' 21'
isi: 1
issue: '18'
language:
- iso: eng
month: '09'
oa: 1
oa_version: Published Version
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: 25D32BC0-B435-11E9-9278-68D0E5697425
name: Mechanism of formation and maintenance of input side-dependent asymmetry in
the hippocampus
- _id: 26436750-B435-11E9-9278-68D0E5697425
call_identifier: H2020
grant_number: '785907'
name: Human Brain Project Specific Grant Agreement 2 (HBP SGA 2)
publication: International Journal of Molecular Sciences
publication_identifier:
eissn:
- '14220067'
issn:
- '16616596'
publication_status: published
publisher: MDPI
quality_controlled: '1'
related_material:
record:
- id: '9562'
relation: dissertation_contains
status: public
scopus_import: '1'
status: public
title: Deep learning-assisted high-throughput analysis of freeze-fracture replica
images applied to glutamate receptors and calcium channels at hippocampal synapses
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: 21
year: '2020'
...
---
_id: '612'
abstract:
- lang: eng
text: Metabotropic GABAB receptors mediate slow inhibitory effects presynaptically
and postsynaptically through the modulation of different effector signalling pathways.
Here, we analysed the distribution of GABAB receptors using highly sensitive SDS-digested
freeze-fracture replica labelling in mouse cerebellar Purkinje cells. Immunoreactivity
for GABAB1 was observed on presynaptic and, more abundantly, on postsynaptic compartments,
showing both scattered and clustered distribution patterns. Quantitative analysis
of immunoparticles revealed a somato-dendritic gradient, with the density of immunoparticles
increasing 26-fold from somata to dendritic spines. To understand the spatial
relationship of GABAB receptors with two key effector ion channels, the G protein-gated
inwardly rectifying K+ (GIRK/Kir3) channel and the voltage-dependent Ca2+ channel,
biochemical and immunohistochemical approaches were performed. Co-immunoprecipitation
analysis demonstrated that GABAB receptors co-assembled with GIRK and CaV2.1 channels
in the cerebellum. Using double-labelling immunoelectron microscopic techniques,
co-clustering between GABAB1 and GIRK2 was detected in dendritic spines, whereas
they were mainly segregated in the dendritic shafts. In contrast, co-clustering
of GABAB1 and CaV2.1 was detected in dendritic shafts but not spines. Presynaptically,
although no significant co-clustering of GABAB1 and GIRK2 or CaV2.1 channels was
detected, inter-cluster distance for GABAB1 and GIRK2 was significantly smaller
in the active zone than in the dendritic shafts, and that for GABAB1 and CaV2.1
was significantly smaller in the active zone than in the dendritic shafts and
spines. Thus, GABAB receptors are associated with GIRK and CaV2.1 channels in
different subcellular compartments. These data provide a better framework for
understanding the different roles played by GABAB receptors and their effector
ion channels in the cerebellar network.
article_processing_charge: No
article_type: original
author:
- first_name: Rafael
full_name: Luján, Rafael
last_name: Luján
- first_name: Carolina
full_name: Aguado, Carolina
last_name: Aguado
- first_name: Francisco
full_name: Ciruela, Francisco
last_name: Ciruela
- first_name: Javier
full_name: Cózar, Javier
last_name: Cózar
- first_name: David
full_name: Kleindienst, David
id: 42E121A4-F248-11E8-B48F-1D18A9856A87
last_name: Kleindienst
- first_name: Luis
full_name: De La Ossa, Luis
last_name: De La Ossa
- first_name: Bernhard
full_name: Bettler, Bernhard
last_name: Bettler
- first_name: Kevin
full_name: Wickman, Kevin
last_name: Wickman
- first_name: Masahiko
full_name: Watanabe, Masahiko
last_name: Watanabe
- first_name: Ryuichi
full_name: Shigemoto, Ryuichi
id: 499F3ABC-F248-11E8-B48F-1D18A9856A87
last_name: Shigemoto
orcid: 0000-0001-8761-9444
- first_name: Yugo
full_name: Fukazawa, Yugo
last_name: Fukazawa
citation:
ama: Luján R, Aguado C, Ciruela F, et al. Differential association of GABAB receptors
with their effector ion channels in Purkinje cells. Brain Structure and Function.
2018;223(3):1565-1587. doi:10.1007/s00429-017-1568-y
apa: Luján, R., Aguado, C., Ciruela, F., Cózar, J., Kleindienst, D., De La Ossa,
L., … Fukazawa, Y. (2018). Differential association of GABAB receptors with their
effector ion channels in Purkinje cells. Brain Structure and Function.
Springer. https://doi.org/10.1007/s00429-017-1568-y
chicago: Luján, Rafael, Carolina Aguado, Francisco Ciruela, Javier Cózar, David
Kleindienst, Luis De La Ossa, Bernhard Bettler, et al. “Differential Association
of GABAB Receptors with Their Effector Ion Channels in Purkinje Cells.” Brain
Structure and Function. Springer, 2018. https://doi.org/10.1007/s00429-017-1568-y.
ieee: R. Luján et al., “Differential association of GABAB receptors with
their effector ion channels in Purkinje cells,” Brain Structure and Function,
vol. 223, no. 3. Springer, pp. 1565–1587, 2018.
ista: Luján R, Aguado C, Ciruela F, Cózar J, Kleindienst D, De La Ossa L, Bettler
B, Wickman K, Watanabe M, Shigemoto R, Fukazawa Y. 2018. Differential association
of GABAB receptors with their effector ion channels in Purkinje cells. Brain Structure
and Function. 223(3), 1565–1587.
mla: Luján, Rafael, et al. “Differential Association of GABAB Receptors with Their
Effector Ion Channels in Purkinje Cells.” Brain Structure and Function,
vol. 223, no. 3, Springer, 2018, pp. 1565–87, doi:10.1007/s00429-017-1568-y.
short: R. Luján, C. Aguado, F. Ciruela, J. Cózar, D. Kleindienst, L. De La Ossa,
B. Bettler, K. Wickman, M. Watanabe, R. Shigemoto, Y. Fukazawa, Brain Structure
and Function 223 (2018) 1565–1587.
date_created: 2018-12-11T11:47:29Z
date_published: 2018-04-01T00:00:00Z
date_updated: 2024-03-25T23:30:16Z
day: '01'
ddc:
- '571'
department:
- _id: RySh
doi: 10.1007/s00429-017-1568-y
ec_funded: 1
external_id:
isi:
- '000428419500030'
file:
- access_level: open_access
checksum: a55b3103476ecb5f4f983d8801807e8b
content_type: application/pdf
creator: system
date_created: 2018-12-12T10:15:36Z
date_updated: 2020-07-14T12:47:20Z
file_id: '5157'
file_name: IST-2018-1013-v1+1_2018_Kleindienst_Differential.pdf
file_size: 5542926
relation: main_file
file_date_updated: 2020-07-14T12:47:20Z
has_accepted_license: '1'
intvolume: ' 223'
isi: 1
issue: '3'
language:
- iso: eng
month: '04'
oa: 1
oa_version: Published Version
page: 1565 - 1587
project:
- _id: 25CBA828-B435-11E9-9278-68D0E5697425
call_identifier: H2020
grant_number: '720270'
name: Human Brain Project Specific Grant Agreement 1 (HBP SGA 1)
- _id: 25681D80-B435-11E9-9278-68D0E5697425
call_identifier: FP7
grant_number: '291734'
name: International IST Postdoc Fellowship Programme
publication: Brain Structure and Function
publication_status: published
publisher: Springer
publist_id: '7192'
pubrep_id: '1013'
quality_controlled: '1'
related_material:
record:
- id: '9562'
relation: dissertation_contains
status: public
scopus_import: '1'
status: public
title: Differential association of GABAB receptors with their effector ion channels
in Purkinje cells
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: 223
year: '2018'
...