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Chapitre D'ouvrage Année : 2014

Biosensor Imaging in Brain Slice Preparations

Marina Polito
  • Fonction : Auteur
Elvire Guiot
  • Fonction : Auteur

Résumé

Cyclic-AMP dependent protein kinase (PKA) is present in most branches of the animal kingdom, and is an example in the nervous system where a kinase effector integrates the cellular effects of various neuromodulators. The recent development of FRET-based biosensors, such as AKAR, now allows the direct measurement of PKA activation in living cells by simply measuring the ratio between the fluorescence emission at the CFP and YFP wavelengths upon CFP excitation. This novel approach provides data with a temporal resolution of a few seconds at the cellular and even subcellular level, opening a new avenue of understanding the integration processes in space and time. Our protocol has been optimized to study morphologically intact mature neurons and we describe how simple and cheap wide-field imaging, as well as more elaborate two-photon imaging, allows real-time monitoring of PKA activation in pyramidal cortical neurons in neonate rodent brain slices. In addition, many practical details presented here also pertain to image analysis in other cellular preparations, such as cultured cells. Finally, this protocol can also be applied to the various other CFP-YFP-based FRET biosensors that are available for other kinases or other intracellular signals. It is likely that this kind of approach will be generally applicable to a broad range of assays in the near future.
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Dates et versions

hal-01545401 , version 1 (22-06-2017)

Identifiants

Citer

Marina Polito, Pierre Vincent, Elvire Guiot. Biosensor Imaging in Brain Slice Preparations. Zhang, J and Ni, Q and Newman, RH. FLUORESCENT PROTEIN-BASED BIOSENSORS: METHODS AND PROTOCOLS, 1071, HUMANA PRESS INC, pp.175-194, 2014, Methods in Molecular Biology, 978-1-62703-621-4; 978-1-62703-622-1. ⟨10.1007/978-1-62703-622-1_14⟩. ⟨hal-01545401⟩
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