Adaptive Movement Compensation for In Vivo Imaging of Fast Cellular Dynamics within a Moving Tissue

In vivo non-linear optical microscopy has been essential to advance our knowledge of how intact biological systems work. It has been particularly enabling to decipher fast spatiotemporal cellular dynamics in neural networks. The power of the technique stems from its optical sectioning capability that in turn also limits its application to essentially immobile tissue. Only tissue not affected by movement or in which movement can be physically constrained can be imaged fast enough to conduct functional studies at high temporal resolution. Here, we show dynamic two-photon Ca2+ imaging in the spinal cord of a living rat at millisecond time scale, free of motion artifacts using an optical stabilization system. We describe a fast, non-contact adaptive movement compensation approach, applicable to rough and weakly reflective surfaces, allowing real-time functional imaging from intrinsically moving tissue in live animals. The strategy involves enslaving the position of the microscope objective to that of the tissue surface in real-time through optical monitoring and a closed feedback loop. The performance of the system allows for efficient image locking even in conditions of random or irregular movements

Mécanismes spinaux des douleurs neuropathiques (déréglement du contole inhibiteur GABAB)

Les récepteurs couplés aux protéines G (RCPG) existent généralement sous forme d'oligomères. Dans le système nerveux central, le récepteur inhibiteur GABAB, formé des deux sous-unités GABAB1 et GABAB2, est un archétype de RCPG hétérodimérique. Cependant, les mécanismes qui régulent sa dimérisation et leurs conséquences fonctionnelles sont encore méconnus. Nous avons montré, qu'en condition de douleur chronique, la surexpression de la protéine cytosolique 14-3-3, induisait la dissociation de l'hétérodimère et sa perte de fonctionnalité en se liant à la sous-unité GABAB1. Des siRNA anti-14-3-3, ou des peptides de compétition sélectifs de l'interaction 14-3-3/GABAB1, permettent de potentialiser les effets antinociceptifs d'un agoniste du récepteur sur un modèle animal de neuropathie. L'ensemble de ces résultats suggère que l'état d'oligomérisation des RCPGs peut être modulé par des mécanismes endogènes impliqués dans le développement ou le maintien d'états pathologiques chroniques.G-protein-coupled receptors (GPCRs) generally exist as oligomers. In the central nervous system, the inhibitory GABAB receptor is an obligate heterodimeric GPCR that requires the association between GABAB1 and GABAB2 subunits. However, the mechanisms that regulate GABAB receptor oligomerization in physiological conditions, and their functional consequences remain largely unknown. Our results demonstrate that in chronic pain state, the overexpression of the cytosolic 14-3-3 protein induced the dissociation of the GABAB heterodimer, and a loss of GABAB signalling by trapping the GABAB1 subunit. Moreover, anti 14-3-3 siRNA, or competing peptides, specific of 14-3-3/GABAB1 interaction, potentiate the antinociceptive effects of GABAB agonist in an animal model of neuropathic pain. Taken together, our data suggest that GCPR oligomeric state can be modulated in vivo by endogenous mechanisms involved in the development and the maintenance of pain sensitization.BORDEAUX2-BU Santé (330632101) / SudocSudocFranceF

T-type calcium channels in neuropathic pain

Review articles from the 5th International Meeting of the IASP Special Interest Group on Neuropathic Pain (NeuPSIG)International audiencePain is a quite frequent complaint accompanying numerous pathologies. Among these pathological cases, numerous neuropathies are retrieved with identified etiologies (chemotherapies, diabetes, surgeries…) and also more diffuse syndromes such as fibromyalgia. More broadly, pain is one of the first consequences of most inherited diseases. Despite its importance for the quality of life, current pain management is limited to drugs that are either old or with a limited efficacy or that possess a bad risk benefit ratio. As no new pharmacological concept has led to new analgesics in the last decades, the discovery of new medications is needed, and to this aim, the identification of new druggable targets in pain transmission is a first step. Therefore, studies of ion channels in pain pathways are extremely active. This is particularly true with ion channels in peripheral sensory neurons in dorsal root ganglia known how to express unique sets of these channels. Moreover, both spinal and supraspinal levels are clearly important in pain modulation. Among these ion channels, we and others revealed the important role of low voltage-gated calcium channels in cellular excitability in different steps of the pain pathways. These channels, by being activated nearby resting membrane potential, have biophysical characteristics suited to facilitate action potential generation and rhythmicity. In this review, we will present the current knowledge on the role of these channels in the perception and modulation of pain

T-type calcium channels in chronic pain: mouse models and specific blockers

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Dissociation and trafficking of rat GABAB receptor heterodimer upon chronic capsaicin stimulation

Gamma-aminobutyric acid type B receptors (GABAB) are G-protein-coupled receptors that mediate GABAergic inhibition in the brain. Their functional expression is dependent upon the formation of heterodimers between GABAB1 and GABAB2 subunits, a process that occurs within the endoplasmic reticulum. However, the mechanisms that regulate GABAB receptor oligomerization at the plasma membrane remain largely unknown. We first characterized the functional cytoarchitecture of an organotypic co-culture model of rat dorsal root ganglia and spinal cord. Subsequently, we studied the interactions between GABAB subunits after chronic stimulation of sensory fibres with capsaicin. Surface labelling of recombinant proteins showed a decrease in subunit co-localization and GABAB2 labelling, after capsaicin treatment. In these conditions, fluorescence lifetime imaging measurements further demonstrated a loss of interactions between green fluorescent protein-GABAB1b and t-dimer discosoma sp red fluorescent protein-GABAB2 subunits. Finally, we established that the GABAB receptor undergoes clathrin-dependent internalization and rapid recycling to the plasma membrane following activation with baclofen, a GABAB agonist. However, in cultures chronically stimulated with capsaicin, the agonist-induced endocytosis was decreased, reflecting changes in the dimeric state of the receptor. Taken together, our results indicate that the chronic stimulation of sensory fibres can dissociate the GABAB heterodimer and alters its responsiveness to the endogenous ligand. Chronic stimulation thus modulates receptor oligomerization, providing additional levels of control of signalling

Transcriptional Profiling of Cutaneous MRGPRD Free Nerve Endings and C-LTMRs

Cutaneous C-unmyelinated MRGPRD+ free nerve endings and C-LTMRs innervating hair follicles convey two opposite aspects of touch sensation: a sensation of pain and a sensation of pleasant touch. The molecular mechanisms underlying these diametrically opposite functions are unknown. Here, we used a mouse model that genetically marks C-LTMRs and MRGPRD+ neurons in combination with fluorescent cell surface labeling, flow cytometry, and RNA deep-sequencing technology (RNA-seq). Cluster analysis of RNA-seq profiles of the purified neuronal subsets revealed 486 and 549 genes differentially expressed in MRGPRD-expressing neurons and C-LTMRs, respectively. We validated 48 MRGPD- and 68 C-LTMRs-enriched genes using a triple-staining approach, and the Cav3.3 channel, found to be exclusively expressed in C-LTMRs, was validated using electrophysiology. Our study greatly expands the molecular characterization of C-LTMRs and suggests that this particular population of neurons shares some molecular features with Aβ and Aδ low-threshold mechanoreceptors

INMCancer : Base de données ouverte des publications d’études interventionnelles en prévention et soin des cancers sur les interventions non médicamenteuses (INM): Revues avant publié une étude interventionnelle sur une INM Publications

Connaître les publications d’études comparatives sur les INM en prévention et soin des cancersInternational audienceLes professionnels de la lutte contre le cancer recourent de plus en plus aux interventions non médicamenteuses (INM). Elles se distinguent des médecines alternatives, des pratiques socioculturelles et des campagnes de santé publique (Ninot, 2019). Ces méthodes ciblées et personnalisées s'intègrent dans le parcours de soin oncologique. Elles sont fondées sur la science, et en particulier sur des études interventionnelles (Ninot, 2020). Elles visent à (1) atténuer les symptômes du cancer et de ses traitements (fatigue, anxiété, douleur…), (2) prévenir la survenue ou la récidive d'un cancer, (3) prévenir des comorbidités, (4) améliorer l'état général de santé et la qualité de vie, (5) et à augmenter la durée de vie sans perte de qualité de vie. De nombreux chercheurs constatent des difficultés à identifier les publications d'études interventionnelles sur les INM dans la littérature (HAS, 2011). Les principales bases de données mondiales sont spécialisées dans une approche disciplinaire (biomédicale pour Pubmed, psychologique pour PsyInfo…), alors que par essence les INM font appel à des mécanismes biopsychosociaux et des contenus associant plusieurs composants (techniques, ingrédients…). Par ailleurs, la transformation numérique de l'édition scientifique ces 10 dernières années a diversifié l'offre de publication d'articles (Open Journals, Archive Ouverte…) de telle manière que de nombreuses études et publications échappent aux bases de données classiques. Ce système inédit, dédié à la prévention des cancers et aux soins de support en oncologie, et spécifique aux INM, se nomme INMCancer

INMCancer : Base de données ouverte des publications d’études interventionnelles en prévention et soin des cancers sur les interventions non médicamenteuses (INM): Revues avant publié une étude interventionnelle sur une INM Publications

Connaître les publications d’études comparatives sur les INM en prévention et soin des cancersInternational audienceLes professionnels de la lutte contre le cancer recourent de plus en plus aux interventions non médicamenteuses (INM). Elles se distinguent des médecines alternatives, des pratiques socioculturelles et des campagnes de santé publique (Ninot, 2019). Ces méthodes ciblées et personnalisées s'intègrent dans le parcours de soin oncologique. Elles sont fondées sur la science, et en particulier sur des études interventionnelles (Ninot, 2020). Elles visent à (1) atténuer les symptômes du cancer et de ses traitements (fatigue, anxiété, douleur…), (2) prévenir la survenue ou la récidive d'un cancer, (3) prévenir des comorbidités, (4) améliorer l'état général de santé et la qualité de vie, (5) et à augmenter la durée de vie sans perte de qualité de vie. De nombreux chercheurs constatent des difficultés à identifier les publications d'études interventionnelles sur les INM dans la littérature (HAS, 2011). Les principales bases de données mondiales sont spécialisées dans une approche disciplinaire (biomédicale pour Pubmed, psychologique pour PsyInfo…), alors que par essence les INM font appel à des mécanismes biopsychosociaux et des contenus associant plusieurs composants (techniques, ingrédients…). Par ailleurs, la transformation numérique de l'édition scientifique ces 10 dernières années a diversifié l'offre de publication d'articles (Open Journals, Archive Ouverte…) de telle manière que de nombreuses études et publications échappent aux bases de données classiques. Ce système inédit, dédié à la prévention des cancers et aux soins de support en oncologie, et spécifique aux INM, se nomme INMCancer

Centrally expressed Cav3.2 T-type calcium channel is critical for the initiation and maintenance of neuropathic pain

Cav3.2 T-type calcium channel is a major molecular actor of neuropathic pain in peripheral sensory neurons, but its involvement at the supra-spinal level is almost unknown. In the Anterior Pretectum (APT), a hub of connectivity of the somatosensory system involved in pain perception, we show that Cav3.2 channels are expressed in a sub-population of GABAergic neurons co-expressing parvalbumin (PV). In these PV-expressing neurons, Cav3.2 channels contribute to a high frequency bursting activity, which is increased in the spared nerve injury model of neuropathy. Specific deletion of Cav3.2 channels in APT neurons reduced both the initiation and maintenance of mechanical and cold allodynia. These data are a direct demonstration that centrally expressed Cav3.2 channels also play a fundamental role in pain pathophysiology

Matrix therapy combined with mesenchymal stem cells-based approach in the context of brain ischemia

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