Combined Optogenetic Approaches Reveal Quantitative Dynamics of Endogenous Noradrenergic Transmission in the Brain

Little is known about the real-time cellular dynamics triggered by endogenous catecholamine release despite their importance in brain functions. To address this issue, we expressed channelrhodopsin in locus coeruleus neurons and protein kinase-A activity biosensors in cortical pyramidal neurons and combined two-photon imaging of biosensors with photostimulation of locus coeruleus cortical axons, in acute slices and in vivo. Burst photostimulation of axons for 5–10 s elicited robust, minutes-lasting kinase-A activation in individual neurons, indicating that a single burst firing episode of synchronized locus coeruleus neurons has rapid and lasting effects on cortical network. Responses were mediated by β1 adrenoceptors, dampened by co-activation of α2 adrenoceptors, and dramatically increased upon inhibition of noradrenaline reuptake transporter. Dopamine receptors were not involved, showing that kinase-A activation was due to noradrenaline release. Our study shows that noradrenergic transmission can be characterized with high spatiotemporal resolution in brain slices and in vivo using optogenetic tools

GluD1, linked to schizophrenia, controls the burst firing of dopamine neurons

Human mutations of the GRID1 gene encoding the orphan delta1 glutamate receptor-channel (GluD1) are associated with schizophrenia but the explicit role of GluD1 in brain circuits is unknown. Based on the known function of its paralog GluD2 in cerebellum, we searched for a role of GluD1 in slow glutamatergic transmission mediated by metabotropic receptor mGlu1 in midbrain dopamine neurons, whose dysfunction is a hallmark of schizophrenia. We found that an mGlu1 agonist elicits a slow depolarizing current in HEK cells co-expressing mGlu1 and GluD1, but not in cells expressing mGlu1 or GluD1 alone. This current is abolished by additional co-expression of a dominant-negative GluD1 dead pore mutant. We then characterized mGlu1-dependent currents in dopamine neurons from midbrain slices. Both the agonist-evoked and the slow postsynaptic currents are abolished by expression of the dominant-negative GluD1 mutant, pointing to the involvement of native GluD1 channels in these currents. Likewise, both mGlu1-dependent currents are suppressed in GRID1 knockout mice, which reportedly display endophenotypes relevant for schizophrenia. It is known that mGlu1 activation triggers the transition from tonic to burst firing of dopamine neurons, which signals salient stimuli and encodes reward prediction. In vivo recordings of dopamine neurons showed that their spontaneous burst firing is abolished in GRID1 knockout mice or upon targeted expression of the dominant-negative GluD1 mutant in wild-type mice. Our results de-orphanize GluD1, unravel its key role in slow glutamatergic transmission and provide insights into how GRID1 gene alterations can lead to dopaminergic dysfunctions in schizophrenia

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

La bioluminescence de l'aequorine en réponse au calcium in vitro et dans le cortex cérébral

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Neuronal Network Imaging in Acute Slices Using Ca2+ Sensitive Bioluminescent Reporter

International audienceGenetically encoded indicators are valuable tools to study intracellular signaling cascades in real time using fluorescent or bioluminescent imaging techniques. Imaging of Ca2+ indicators is widely used to record transient intracellular Ca2+ increases associated with bioelectrical activity. The natural bioluminescent Ca2+ sensor aequorin has been historically the first Ca2+ indicator used to address biological questions. Aequorin imaging offers several advantages over fluorescent reporters: it is virtually devoid of background signal; it does not require light excitation and interferes little with intracellular processes. Genetically encoded sensors such as aequorin are commonly used in dissociated cultured cells; however it becomes more challenging to express them in differentiated intact specimen such as brain tissue. Here we describe a method to express a GFP-aequorin (GA) fusion protein in pyramidal cells of neocortical acute slices using recombinant Sindbis virus. This technique allows expressing GA in several hundreds of neurons on the same slice and to perform the bioluminescence recording of Ca2+ transients in single neurons or multiple neurons simultaneously

Bioluminescence Imaging of Neuronal Network Dynamics Using Aequorin-Based Calcium Sensors

International audienceOptogenetic calcium sensors enable the imaging in real-time of the activities of single or multiple neurons in brain slices and in vivo. Bioluminescent probes engineered from the natural calcium sensor aequorin do not require illumination, are virtually devoid of background signal, and exhibit large dynamic range and low cytotoxicity. These probes are thus well-suited for long-duration, whole-field recordings of multiple neurons simultaneously. Here we describe a protocol for monitoring and analyzing the dynamics of neuronal ensembles using whole-field bioluminescence imaging of an aequorin-based sensor in brain slice

Etude des propriétés fonctionnelles d'une population d'interneurones gabaergiques inhibiteurs du Néocortex

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Nicotinic Transmission onto Layer 6 Cortical Neurons Relies on Synaptic Activation of Non-alpha 7 Receptors

International audienceNicotinic excitation in neocortex is mediated by low-affinity alpha 7 receptors and by high-affinity alpha 4 beta 2 receptors. There is evidence that alpha 7 receptors are synaptic, but it is unclear whether high-affinity receptors are activated by volume transmission or synaptic transmission. To address this issue, we characterized responses of excitatory layer 6 (L6) neurons to optogenetic release of acetylcholine (ACh) in cortical slices. L6 responses consisted in a slowly decaying alpha 4 beta 2 current and were devoid of alpha 7 component. Evidence that these responses were mediated by synapses was 4-fold. 1) Channelrhodopsin-positive cholinergic varicosities made close appositions onto responsive neurons. 2) Inhibition of ACh degradation failed to alter onset kinetics and amplitude of currents. 3) Quasi-saturation of alpha 4 beta 2 receptors occurred upon ACh release. 4) Response kinetics were unchanged in low release probability conditions. Train stimulations increased amplitude and decay time of responses and these effects appeared to involve recruitment of extrasynaptic receptors. Finally, we found that the alpha 5 subunit, known to be associated with alpha 4 beta 2 in L6, regulates short-term plasticity at L6 synapses. Our results are consistent with previous anatomical observations of widespread cholinergic synapses and suggest that a significant proportion of these small synapses operate via high-affinity nicotinic receptors