Caractérisation fonctionnelle de senseurs calciques bioluminescents et utilisation pour l imagerie des activités neuronales dans le cortex cérébral

Mes travaux de doctorat ont porté sur la caractérisation fonctionnelle de senseurs calciques bioluminescents et leur utilisation pour l imagerie des activités neuronales dans le cortex cérébral de rat, in vitro. J ai étudié les cinétiques d émission de bioluminescence de trois senseurs qui sont des protéines de fusion unissant la Green Fluorescent Protein (GFP) à la photoprotéine aequorine, obeline ou un mutant d aequorine à haute affinité calcique. J ai montré que les propriétés cinétiques intrinsèques de ces trois senseurs diffèrent largement. Puis, en utilisant une approche couplant l imagerie de bioluminescence et l enregistrement électrophysiologique en patch-clamp dans les neurones pyramidaux de la couche V du néocortex exprimant ces senseurs par transfert viral, j ai caractérisé la sensibilité de ces senseurs aux transitoires calciques associés à l activité électrique neuronale. J ai montré que ces chimères GFP-photoprotéine sont des indicateurs supralinéaires de l activité électrique neuronale et que leurs propriétés biophysiques intrinsèques ont peu d influence sur leur capacité à détecter des transitoires calciques neuronaux.Parallèlement au travail de caractérisation fonctionnelle, nous avons utilisé la protéine GFP-aequorine pour étudier la modulation des activités du réseau néocortical par l acétylcholine. Par imagerie de bioluminescence, nous avons ainsi pu montrer que l acétylcholine, via les récepteurs muscariniques, modifie l organisation spatio-temporelle d activités évoquées électriquement au sein du réseau néocortical en induisant une réponse prolongée de plusieurs dizaines de secondes dans les neurones de la couche V.PARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Calcium imaging in single neurons from brain slices using bioluminescent reporters.

International audienceResponses 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

Bioluminescence calcium imaging of network dynamics and their cholinergic modulation in slices of cerebral cortex from male rats

International audienceThe activity of neuronal ensembles was monitored in neocortical slices from male rats using wide‐field bioluminescence imaging of a calcium sensor formed with the fusion of green fluorescent protein and aequorin (GA) and expressed through viral transfer. GA expression was restricted to pyramidal neurons and did not conspicuously alter neuronal morphology or neocortical cytoarchitecture. Removal of extracellular magnesium or addition of GABA receptor antagonists triggered epileptiform flashes of variable amplitude and spatial extent, indicating that the excitatory and inhibitory networks were functionally preserved in GA‐expressing slices. We found that agonists of muscarinic acetylcholine receptors largely increased the peak bioluminescence response to local electrical stimulation in layer I or white matter, and gave rise to a slowly decaying response persisting for tens of seconds. The peak increase involved layers II/III and V and did not result in marked alteration of response spatial properties. The persistent response involved essentially layer V and followed the time course of the muscarinic afterdischarge depolarizing plateau in layer V pyramidal cells. This plateau potential triggered spike firing in layer V, but not layer II/III pyramidal cells, and was accompanied by recurrent synaptic excitation in layer V. Our results indicate that wide‐field imaging of GA bioluminescence is well suited to monitor local and global network activity patterns, involving different mechanisms of intracellular calcium increase, and occurring on various timescales

Early Functional Impairment of Sensory-Motor Connectivity in a Mouse Model of Spinal Muscular Atrophy

To define alterations of neuronal connectivity that occur during motor neuron degeneration, we characterized the function and structure of spinal circuitry in spinal muscular atrophy (SMA) model mice. SMA motor neurons show reduced proprioceptive reflexes that correlate with decreased number and function of synapses on motor neuron somata and proximal dendrites. These abnormalities occur at an early stage of disease in motor neurons innervating proximal hindlimb muscles and medial motor neurons innervating axial muscles, but only at end-stage disease in motor neurons innervating distal hindlimb muscles. Motor neuron loss follows afferent synapse loss with the same temporal and topographical pattern. Trichostatin A, which improves motor behavior and survival of SMA mice, partially restores spinal reflexes illustrating the reversibility of these synaptic defects. Deafferentation of motor neurons is an early event in SMA and may be a primary cause of motor dysfunction that is amenable to therapeutic intervention

Early Functional Impairment of Sensory-Motor Connectivity in a Mouse Model of Spinal Muscular Atrophy

To define alterations of neuronal connectivity that occur during motor neuron degeneration, we characterized the function and structure of spinal circuitry in spinal muscular atrophy (SMA) model mice. SMA motor neurons show reduced proprioceptive reflexes that correlate with decreased number and function of synapses on motor neuron somata and proximal dendrites. These abnormalities occur at an early stage of disease in motor neurons innervating proximal hindlimb muscles and medial motor neurons innervating axial muscles, but only at end-stage disease in motor neurons innervating distal hindlimb muscles. Motor neuron loss follows afferent synapse loss with the same temporal and topographical pattern. Trichostatin A, which improves motor behavior and survival of SMA mice, partially restores spinal reflexes illustrating the reversibility of these synaptic defects. Deafferentation of motor neurons is an early event in SMA and may be a primary cause of motor dysfunction that is amenable to therapeutic intervention