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

Imagerie dynamique de la signalisation peptidergique VIP-CRF-PACAP dans le cortex cerébral

Le vasoactive intestinal peptide (VIP), le corticotropin releasing factor (CRF) et le pituitary adenylate cyclase-activating polypeptide (PACAP) sont des neuropeptides présents dans le cortex et impliqués dans de nombreux processus physiologiques. Bien qu ils activent la même voie AMPc/PKA, les réponses induites diffèrent. Mon travail de thèse apporte des éléments pour comprendre comment les cellules peuvent, via la modulation de la voie AMPc/PKA, répondre spécifiquement à divers signaux. L imagerie FRET de sondes fluorescentes codées génétiquement nous a permis d étudier la modulation de la voie AMPc/PKA en réponse au VIP, CRF et PACAP dans les cellules pyramidales du cortex de rat. Par single cell RT-PCR et par hybridation in situ, nous avons montré que ces cellules co-expriment les récepteurs VPAC1, CRF1 et PAC1 et que le PACAP-à l inverse de la plupart des peptides présents dans les interneurones-est majoritairement exprimé au niveau des cellules pyramidales. Par imagerie, nous avons montré que VIP, CRF et PACAP activent respectivement les récepteurs VPAC1, CRF1 et PAC1, induisant l activation de la voie AMPc/PKA avec des profils spécifiques à chaque peptide. Par une étude en patch-clamp, nous avons pu mettre en évidence l effet excitateur de ces peptides sur les cellules pyramidales, via l inhibition spécifique du courant IsAHP. Les résultats de ce travail montrent que les cellules pyramidales sont capables de discriminer différents signaux peptidergiques au travers de la modulation d une seule voie de signalisation et permettent de mieux comprendre comment la spécificité d action d un peptide puisse dépendre de la prise en compte de chacune des voies qu il active.PARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Noradrenalin and dopamine receptors both control cAMP-PKA signaling throughout the cerebral cortex

International audienceNoradrenergic fibers innervate the entire cerebral cortex, whereas the cortical distribution of dopaminergic fibers is more restricted. However, the relative functional impact of noradrenalin and dopamine receptors in various cortical regions is largely unknown. Using a specific genetic label, we first confirmed that noradrenergic fibers innervate the entire cortex whereas dopaminergic fibers were present in all layers of restricted medial and lateral areas but only in deep layers of other areas. Imaging of a genetically encoded sensor revealed that noradrenalin and dopamine widely activate PKA in cortical pyramidal neurons of frontal, parietal and occipital regions with scarce dopaminergic fibers. Responses to noradrenalin had higher amplitude, velocity and occurred at more than 10-fold lower dose than those elicited by dopamine, whose amplitude and velocity increased along the antero-posterior axis. The pharmacology of these responses was consistent with the involvement of Gs-coupled beta1 adrenergic and D1/D5 dopaminergic receptors, but the inhibition of both noradrenalin and dopamine responses by beta adrenergic antagonists was suggestive of the existence of beta1-D1/D5 heteromeric receptors. Responses also involved Gi-coupled alpha2 adrenergic and D2-like dopaminergic receptors that markedly reduced their amplitude and velocity and contributed to their cell-to-cell heterogeneity. Our results reveal that noradrenalin and dopamine receptors both control cAMP-PKA signaling throughout the cerebral cortex with moderate regional and laminar differences. These receptors can thus mediate widespread effects of both catecholamines, which are reportedly co-released by cortical noradrenergic fibers beyond the territory of dopaminergic fibers

Supragranular Pyramidal Cells Exhibit Early Metabolic Alterations in the 3xTg-AD Mouse Model of Alzheimer’s Disease

International audienceThe impairment of cerebral glucose utilization is an early and predictive biomarker of Alzheimer's disease (AD) that is likely to contribute to memory and cognition disorders during the progression of the pathology. Yet, the cellular and molecular mechanisms underlying these metabolic alterations remain poorly understood. Here we studied the glucose metabolism of supragranular pyramidal cells at an early presymptomatic developmental stage in non-transgenic (non-Tg) and 3xTg-AD mice, a mouse model of AD replicating numerous hallmarks of the disease. We performed both intracellular glucose imaging with a genetically encoded fluorescence resonance energy transfer (FRET)-based glucose biosensor and transcriptomic profiling of key molecular elements of glucose metabolism with single-cell multiplex RT-PCR (scRT-mPCR). We found that juvenile pyramidal cells exhibit active glycolysis and pentose phosphate pathway at rest that are respectively enhanced and impaired in 3xTg-AD mice without alteration of neuronal glucose uptake or transcriptional modification. Given the importance of glucose metabolism for neuronal survival, these early alterations could initiate or at least contribute to the later neuronal dysfunction of pyramidal cells in AD

VIP, CRF, and PACAP Act at Distinct Receptors to Elicit Different cAMP/PKA Dynamics in the Neocortex

The functional significance of diverse neuropeptide coexpression and convergence onto common second messenger pathways remains unclear. To address this question, we characterized responses to corticotropin-releasing factor (CRF), pituitary adenylate cyclase–activating peptide (PACAP), and vasoactive intestinal peptide (VIP) in rat neocortical slices using optical recordings of cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) sensors, patch-clamp, and single-cell reverse transcription–polymerase chain reaction. Responses of pyramidal neurons to the 3 neuropeptides markedly differed in time-course and amplitude. Effects of these neuropeptides on the PKA-sensitive slow afterhyperpolarization current were consistent with those observed with cAMP/PKA sensors. CRF-1 receptors, primarily expressed in pyramidal cells, reportedly mediate the neocortical effects of CRF. PACAP and VIP activated distinct PAC1 and VPAC1 receptors, respectively. Indeed, a selective VPAC1 antagonist prevented VIP responses but had a minor effect on PACAP responses, which were mimicked by a specific PAC1 agonist. While PAC1 and VPAC1 were coexpressed in pyramidal cells, PAC1 expression was also frequently detected in interneurons, suggesting that PACAP has widespread effects on the neuronal network. Our results suggest that VIP and CRF, originating from interneurons, and PACAP, expressed mainly by pyramidal cells, finely tune the excitability and gene expression in the neocortical network via distinct cAMP/PKA-mediated effects

Recovery from tachyphylaxis of TRPV1 coincides with recycling to the surface membrane

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