Biolistic transfection and expression analysis of acute cortical slices.
Acute slices
Calcium imaging
Gene-gun transfection
Genetically encoded calcium indicators
Neocortex
Journal
Journal of neuroscience methods
ISSN: 1872-678X
Titre abrégé: J Neurosci Methods
Pays: Netherlands
ID NLM: 7905558
Informations de publication
Date de publication:
01 05 2020
01 05 2020
Historique:
received:
14
09
2019
revised:
21
02
2020
accepted:
27
02
2020
pubmed:
3
3
2020
medline:
22
6
2021
entrez:
3
3
2020
Statut:
ppublish
Résumé
Biolistic gene gun transfection has been used to transfect organotypic cultures (OTCs) or dissociated cultures in vitro. Here, we modified this technique to allow successful transfection of acute brain slices, followed by measurement of neuronal activity within a few hours. We established biolistic transfection of murine acute cortical slices to measure calcium signals. Acute slices are mounted on plasma/thrombin coagulate and transfected with a calcium sensor. Imaging can be performed within 4 h post transfection without affecting cell viability. Four hours after GCaMP6s transfection, acute slices display remarkable fluorescent protein expression level allowing to study spontaneous activity and receptor pharmacology. While optimal gas pressure (150 psi) and gold particle size used (1 μm) confirm previously published protocols, the amount of 5 μg DNA was found to be optimal for particle coating. The major advantage of this technique is the rapid disposition of acute slices for calcium imaging. No transgenic GECI expressing animals or OTC for long periods are required. In acute slices, network interaction and connectivity are preserved. The method allows to obtain physiological readouts within 4 h, before functional tissue modifications might come into effect. Limitations of this technique are random transfection, low expression efficiency when using specific promotors, and preclusion or genetic manipulations that require a prolonged time before physiological changes become measurable, such as expression of recombinant proteins that require transport to distant subcellular localizations. The method is optimal for short-time investigation of calcium signals in acute slices.
Sections du résumé
BACKGROUND
Biolistic gene gun transfection has been used to transfect organotypic cultures (OTCs) or dissociated cultures in vitro. Here, we modified this technique to allow successful transfection of acute brain slices, followed by measurement of neuronal activity within a few hours.
NEW METHOD
We established biolistic transfection of murine acute cortical slices to measure calcium signals. Acute slices are mounted on plasma/thrombin coagulate and transfected with a calcium sensor. Imaging can be performed within 4 h post transfection without affecting cell viability.
RESULTS
Four hours after GCaMP6s transfection, acute slices display remarkable fluorescent protein expression level allowing to study spontaneous activity and receptor pharmacology. While optimal gas pressure (150 psi) and gold particle size used (1 μm) confirm previously published protocols, the amount of 5 μg DNA was found to be optimal for particle coating.
COMPARISON WITH EXISTING METHODS
The major advantage of this technique is the rapid disposition of acute slices for calcium imaging. No transgenic GECI expressing animals or OTC for long periods are required. In acute slices, network interaction and connectivity are preserved. The method allows to obtain physiological readouts within 4 h, before functional tissue modifications might come into effect. Limitations of this technique are random transfection, low expression efficiency when using specific promotors, and preclusion or genetic manipulations that require a prolonged time before physiological changes become measurable, such as expression of recombinant proteins that require transport to distant subcellular localizations.
CONCLUSION
The method is optimal for short-time investigation of calcium signals in acute slices.
Identifiants
pubmed: 32119875
pii: S0165-0270(20)30088-1
doi: 10.1016/j.jneumeth.2020.108666
pmc: PMC7125026
mid: NIHMS1572047
pii:
doi:
Substances chimiques
DNA
9007-49-2
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
108666Subventions
Organisme : NINDS NIH HHS
ID : R01 NS093382
Pays : United States
Organisme : NINDS NIH HHS
ID : R01 NS108115
Pays : United States
Organisme : NHLBI NIH HHS
ID : R37 HL063762
Pays : United States
Organisme : NIA NIH HHS
ID : RF1 AG053391
Pays : United States
Informations de copyright
Copyright © 2020 Elsevier B.V. All rights reserved.
Déclaration de conflit d'intérêts
Declaration of Competing Interest None.
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