Calcium imaging in single neurons from brain slices using bioluminescent reporters

Autor: Elvire Guiot, Ludovic Tricoire, Alain-François Chaffotte, Bertrand Lambolez, Estelle Drobac
Přispěvatelé: Neurobiologie des processus adaptatifs (NPA), Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)
Jazyk: angličtina
Rok vydání: 2009
Předmět:
Zdroj: Journal of Neuroscience Research
Journal of Neuroscience Research, Wiley, 2009, 88 (4), pp.695-711 ⟨10.1002/jnr.22249⟩
Journal of Neuroscience Research, 2009, 88 (4), pp.695-711 ⟨10.1002/jnr.22249⟩
ISSN: 0360-4012
1097-4547
Popis: International audience; Responses of three bioluminescent Ca(2+) sensors were studied in vitro and in neurons from brain slices. These sensors consisted of tandem fusions of green fluorescent protein (GFP) with the photoproteins aequorin, obelin, or a mutant aequorin with high Ca(2+) sensitivity. Kinetics of GFP-obelin responses to a saturating Ca(2+) concentration were faster than those of GFP-aequorin at all Mg(2+) concentrations tested, whereas GFP-mutant aequorin responses were the slowest. GFP-photoproteins were efficiently expressed in pyramidal neurons following overnight incubation of acute neocortical slices with recombinant Sindbis viruses. Expression of GFP-photoproteins did not result in conspicuous modification of morphological or electrophysiological properties of layer V pyramidal cells. The three sensors allowed the detection of Ca(2+) transients associated with action potential discharge in single layer V pyramidal neurons. In these neurons, depolarizing steps of increasing amplitude elicited action potential discharge of increasing frequency. Bioluminescent responses of the three sensors were similar in several respects: detection thresholds, an exponential increase with stimulus intensity, photoprotein consumptions, and kinetic properties. These responses, which were markedly slower than kinetics measured in vitro, increased linearly during the action potential discharge and decayed exponentially at the end of the discharge. Onset slopes increased with stimulus intensity, whereas decay kinetics remained constant. Dendritic light emission contributed to whole-field responses, but the spatial resolution of bioluminescence imaging was limited to the soma and proximal apical dendrite. Nonetheless, the high signal-to-background ratio of GFP-photoproteins allowed the detection of Ca(2+) transients associated with 5 action potentials in single neurons upon whole-field bioluminescence recordings. (c) 2009 Wiley-Liss, Inc.
Databáze: OpenAIRE
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