Studying synaptic efficiency by post-hoc immunolabelling

Background In terms of vesicular recycling, synaptic efficiency is a key determinant of the fidelity of synaptic transmission. The ability of a presynaptic terminal to reuse its vesicular content is thought to be a signature of synaptic maturity and this process depends on the activity of several pr...

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Authors: Ramírez-Franco, Jorge, Alonso, Beatris, Bartolomé-Martín, David, Sánchez-Prieto Borja, José, Torres Molina, Magdalena Isabel
Format: article
Publication Date:2013
Country:España
Institution:Universidad Complutense de Madrid (UCM)
Repository:Docta Complutense
Language:English
OAI Identifier:oai:docta.ucm.es:20.500.14352/129690
Online Access:https://hdl.handle.net/20.500.14352/129690
Access Level:Open access
Keyword:612.8
Neurociencias (Biológicas)
2490.02 Neuroquímica
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spelling Studying synaptic efficiency by post-hoc immunolabellingRamírez-Franco, JorgeAlonso, BeatrisBartolomé-Martín, DavidSánchez-Prieto Borja, JoséTorres Molina, Magdalena Isabel612.8Neurociencias (Biológicas)2490.02 NeuroquímicaBackground In terms of vesicular recycling, synaptic efficiency is a key determinant of the fidelity of synaptic transmission. The ability of a presynaptic terminal to reuse its vesicular content is thought to be a signature of synaptic maturity and this process depends on the activity of several proteins that govern exo/endocytosis. Upon stimulation, individual terminals in networks of cultured cerebellar granule neurons exhibit heterogeneous exocytic responses, which reflect the distinct states of maturity and plasticity intrinsic to individual synaptic terminals. This dynamic scenario serves as the substrate for processes such as scaling, plasticity and synaptic weight redistribution. Presynaptic strength has been associated with the activity of several types of proteins, including the scaffolding proteins that form the active zone cytomatrix and the proteins involved in presynaptic exocytosis. Methods We have combined fluorescence imaging techniques using the styryl dye FM1-43 in primary cultures of cerebellar granule cells with subsequent post-hoc immunocytochemistry in order to study synaptic efficiency in terms of vesicular release. We describe a protocol to easily quantify these results with minimal user intervention. Results In this study we describe a technique that specifically correlates presynaptic activity with the levels of presynaptic markers. This method involves the use of the styryl dye FM1-43 to estimate the release capacity of a synaptic terminal, and the subsequent post-hoc immunolabelling of thousands of individual nerve terminals. We observed a strong correlation between the release capacity of the nerve terminal and the levels of the RIM1α but not the Munc13-1 protein in the active zone. Conclusions Our findings support those of previous studies and point out to RIM1α as a crucial factor in determining synaptic efficiency. These results also demonstrate that this technique is a useful tool to analyse the molecular differences underlying the heterogeneous responses exhibited by neuronal networks.Springer NatureUniversidad Complutense de Madrid20132013-01-0120132013-01-01journal articlehttp://purl.org/coar/resource_type/c_6501AMhttp://purl.org/coar/version/c_ab4af688f83e57aainfo:eu-repo/semantics/articleapplication/pdfhttps://hdl.handle.net/20.500.14352/129690reponame:Docta Complutenseinstname:Universidad Complutense de Madrid (UCM)Inglésengopen accesshttp://purl.org/coar/access_right/c_abf2Attribution 4.0 Internationalhttp://creativecommons.org/licenses/by/4.0/info:eu-repo/semantics/openAccessoai:docta.ucm.es:20.500.14352/1296902026-06-02T12:44:21Z
dc.title.none.fl_str_mv Studying synaptic efficiency by post-hoc immunolabelling
title Studying synaptic efficiency by post-hoc immunolabelling
spellingShingle Studying synaptic efficiency by post-hoc immunolabelling
Ramírez-Franco, Jorge
612.8
Neurociencias (Biológicas)
2490.02 Neuroquímica
title_short Studying synaptic efficiency by post-hoc immunolabelling
title_full Studying synaptic efficiency by post-hoc immunolabelling
title_fullStr Studying synaptic efficiency by post-hoc immunolabelling
title_full_unstemmed Studying synaptic efficiency by post-hoc immunolabelling
title_sort Studying synaptic efficiency by post-hoc immunolabelling
dc.creator.none.fl_str_mv Ramírez-Franco, Jorge
Alonso, Beatris
Bartolomé-Martín, David
Sánchez-Prieto Borja, José
Torres Molina, Magdalena Isabel
author Ramírez-Franco, Jorge
author_facet Ramírez-Franco, Jorge
Alonso, Beatris
Bartolomé-Martín, David
Sánchez-Prieto Borja, José
Torres Molina, Magdalena Isabel
author_role author
author2 Alonso, Beatris
Bartolomé-Martín, David
Sánchez-Prieto Borja, José
Torres Molina, Magdalena Isabel
author2_role author
author
author
author
dc.contributor.none.fl_str_mv Universidad Complutense de Madrid
dc.subject.none.fl_str_mv 612.8
Neurociencias (Biológicas)
2490.02 Neuroquímica
topic 612.8
Neurociencias (Biológicas)
2490.02 Neuroquímica
description Background In terms of vesicular recycling, synaptic efficiency is a key determinant of the fidelity of synaptic transmission. The ability of a presynaptic terminal to reuse its vesicular content is thought to be a signature of synaptic maturity and this process depends on the activity of several proteins that govern exo/endocytosis. Upon stimulation, individual terminals in networks of cultured cerebellar granule neurons exhibit heterogeneous exocytic responses, which reflect the distinct states of maturity and plasticity intrinsic to individual synaptic terminals. This dynamic scenario serves as the substrate for processes such as scaling, plasticity and synaptic weight redistribution. Presynaptic strength has been associated with the activity of several types of proteins, including the scaffolding proteins that form the active zone cytomatrix and the proteins involved in presynaptic exocytosis. Methods We have combined fluorescence imaging techniques using the styryl dye FM1-43 in primary cultures of cerebellar granule cells with subsequent post-hoc immunocytochemistry in order to study synaptic efficiency in terms of vesicular release. We describe a protocol to easily quantify these results with minimal user intervention. Results In this study we describe a technique that specifically correlates presynaptic activity with the levels of presynaptic markers. This method involves the use of the styryl dye FM1-43 to estimate the release capacity of a synaptic terminal, and the subsequent post-hoc immunolabelling of thousands of individual nerve terminals. We observed a strong correlation between the release capacity of the nerve terminal and the levels of the RIM1α but not the Munc13-1 protein in the active zone. Conclusions Our findings support those of previous studies and point out to RIM1α as a crucial factor in determining synaptic efficiency. These results also demonstrate that this technique is a useful tool to analyse the molecular differences underlying the heterogeneous responses exhibited by neuronal networks.
publishDate 2013
dc.date.none.fl_str_mv 2013
2013-01-01
2013
2013-01-01
dc.type.none.fl_str_mv journal article
http://purl.org/coar/resource_type/c_6501
AM
http://purl.org/coar/version/c_ab4af688f83e57aa
dc.type.openaire.fl_str_mv info:eu-repo/semantics/article
format article
dc.identifier.none.fl_str_mv https://hdl.handle.net/20.500.14352/129690
url https://hdl.handle.net/20.500.14352/129690
dc.language.none.fl_str_mv Inglés
eng
language_invalid_str_mv Inglés
language eng
dc.rights.none.fl_str_mv open access
http://purl.org/coar/access_right/c_abf2
Attribution 4.0 International
http://creativecommons.org/licenses/by/4.0/
dc.rights.openaire.fl_str_mv info:eu-repo/semantics/openAccess
rights_invalid_str_mv open access
http://purl.org/coar/access_right/c_abf2
Attribution 4.0 International
http://creativecommons.org/licenses/by/4.0/
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv Springer Nature
publisher.none.fl_str_mv Springer Nature
dc.source.none.fl_str_mv reponame:Docta Complutense
instname:Universidad Complutense de Madrid (UCM)
instname_str Universidad Complutense de Madrid (UCM)
reponame_str Docta Complutense
collection Docta Complutense
repository.name.fl_str_mv
repository.mail.fl_str_mv
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