NanoPaint: a tool for rapid and dynamic imaging of membrane structural plasticity at the nanoscale

Single-particle tracking with quantum dots (QDs) constitutes a powerful tool to track the nanoscopic dynamics of individual cell membrane components unveiling their membrane diffusion characteristics. Here, the nano-resolved population dynamics of QDs is exploited to reconstruct the topography and structural changes of the cell membrane surface with high temporal and spatial resolution. For this proof-of-concept study, bright, small, and stable biofunctional QD nanoconstructs are utilized recognizing the endogenous neuronal cannabinoid receptor 1, a highly expressed and fast-diffusing membrane protein, together with a commercial point-localization microscope. Rapid QD diffusion on the axonal plasma membrane of cultured hippocampal neurons allows precise reconstruction of the membrane surface in less than 1 min with a spatial resolution of tens of nanometers. Access of the QD nanoconstructs to the synaptic cleft enables rapid 3D topological reconstruction of the entire presynaptic component. Successful reconstruction of membrane nano-topology and deformation at the second time-scale is also demonstrated for HEK293 cell filopodia and axons. Named "nanoPaint," this super-resolution imaging technique amenable to any endogenous transmembrane target represents a versatile platform to rapidly and accurately reconstruct the cell membrane nano-topography, thereby enabling the study of the rapid dynamic phenomena involved in neuronal membrane plasticity.Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicada

A highly soluble, non-phototoxic, non-fluorescent blebbistatin derivative

Blebbistatin is a commonly used molecular tool for the specific inhibition of various myosin II isoforms both in vitro and in vivo. Despite its popularity, the use of blebbistatin is hindered by its poor water-solubility (below 10 micromolar in aqueous buffer) and blue-light sensitivity, resulting in the photoconversion of the molecule, causing severe cellular phototoxicity in addition to its cytotoxicity. Furthermore, blebbistatin forms insoluble aggregates in water-based media above 10 micromolar with extremely high fluorescence and also high adherence to different types of surfaces, which biases its experimental usage. Here, we report a highly soluble (440 micromolar in aqueous buffer), non-fluorescent and photostable C15 amino-substituted derivative of blebbistatin, called para-aminoblebbistatin. Importantly, it is neither photo- nor cytotoxic, as demonstrated on HeLa cells and zebrafish embryos. Additionally, para-aminoblebbistatin bears similar myosin II inhibitory properties to blebbistatin or para-nitroblebbistatin (not to be confused with the C7 substituted nitroblebbistatin), tested on rabbit skeletal muscle myosin S1 and on M2 and HeLa cells. Due to its drastically improved solubility and photochemical feature, as well as lack of photo- or cytotoxicity, para-aminoblebbistatin may become a feasible replacement for blebbistatin, especially at applications when high concentrations of the inhibitor or blue light irradiation is required

Microglia modulate blood flow, neurovascular coupling, and hypoperfusion via purinergic actions

Microglia, the main immunocompetent cells of the brain, regulate neuronal function, but their contribution to cerebral blood flow (CBF) regulation has remained elusive. Here, we identify microglia as important modulators of CBF both under physiological conditions and during hypoperfusion. Microglia establish direct, dynamic purinergic contacts with cells in the neurovascular unit that shape CBF in both mice and humans. Surprisingly, the absence of microglia or blockade of microglial P2Y12 receptor (P2Y12R) substantially impairs neurovascular coupling in mice, which is reiterated by chemogenetically induced microglial dysfunction associated with impaired ATP sensitivity. Hypercapnia induces rapid microglial calcium changes, P2Y12R-mediated formation of perivascular phylopodia, and microglial adenosine production, while depletion of microglia reduces brain pH and impairs hypercapnia-induced vasodilation. Microglial actions modulate vascular cyclic GMP levels but are partially independent of nitric oxide. Finally, microglial dysfunction markedly impairs P2Y12R-mediated cerebrovascular adaptation to common carotid artery occlusion resulting in hypoperfusion. Thus, our data reveal a previously unrecognized role for microglia in CBF regulation, with broad implications for common neurological diseases

Crowdsourced traffic information in traffic management : Evaluation of traffic information from Waze

The early observation and elimination of non-recurring incidents is a crucial task in trafficmanagement. The performance of the conventional incident detection methods (trafficcameras and other sensory technologies) is limited and there are still challenges inobtaining an accurate picture of the traffic conditions in real time. During the last decade,the technical development of mobile platforms and the growing online connectivity made itpossible to obtain traffic information from social media and applications based on spatialcrowdsourcing. Utilizing the benefits of crowdsourcing, traffic authorities can receiveinformation about a more comprehensive number of incidents and can monitor areaswhich are not covered by the conventional incident detection systems. The crowdsourcedtraffic data can provide supplementary information for incidents already reported throughother sources and it can contribute to earlier detection of incidents, which can lead tofaster response and clearance time. Furthermore, spatial crowdsourcing can help to detectincident types, which are not collected systematically yet (e.g. potholes, traffic light faults,missing road signs). However, before exploiting crowdsourced traffic data in trafficmanagement, numerous challenges need to be resolved, such as verification of the incidentreports, predicting the severity of the crowdsourced incidents and integration with trafficdata obtained from other sources.During this thesis, the possibilities and challenges of utilizing spatial crowdsourcingtechnologies to detect non-recurring incidents were examined in form of a case study.Traffic incident alerts obtained from Waze, a navigation application using the concept ofcrowdsourcing, were analyzed and compared with officially verified incident reports inStockholm. The thesis provides insight into the spatial and temporal characteristics of theWaze data. Moreover, a method to identify related Waze alerts and to determine matchingincident reports from different sources is presented. The results showed that the number ofreported incidents in Waze is 4,5 times higher than the number of registered incidents bythe Swedish authorities. Furthermore, 27,5 % of the incidents could have been detectedfaster by using the traffic alerts from Waze. In addition, the severity of Waze alerts isexamined depending on the attributes of the alerts

Crowdsourced traffic information in traffic management : Evaluation of traffic information from Waze

The early observation and elimination of non-recurring incidents is a crucial task in trafficmanagement. The performance of the conventional incident detection methods (trafficcameras and other sensory technologies) is limited and there are still challenges inobtaining an accurate picture of the traffic conditions in real time. During the last decade,the technical development of mobile platforms and the growing online connectivity made itpossible to obtain traffic information from social media and applications based on spatialcrowdsourcing. Utilizing the benefits of crowdsourcing, traffic authorities can receiveinformation about a more comprehensive number of incidents and can monitor areaswhich are not covered by the conventional incident detection systems. The crowdsourcedtraffic data can provide supplementary information for incidents already reported throughother sources and it can contribute to earlier detection of incidents, which can lead tofaster response and clearance time. Furthermore, spatial crowdsourcing can help to detectincident types, which are not collected systematically yet (e.g. potholes, traffic light faults,missing road signs). However, before exploiting crowdsourced traffic data in trafficmanagement, numerous challenges need to be resolved, such as verification of the incidentreports, predicting the severity of the crowdsourced incidents and integration with trafficdata obtained from other sources.During this thesis, the possibilities and challenges of utilizing spatial crowdsourcingtechnologies to detect non-recurring incidents were examined in form of a case study.Traffic incident alerts obtained from Waze, a navigation application using the concept ofcrowdsourcing, were analyzed and compared with officially verified incident reports inStockholm. The thesis provides insight into the spatial and temporal characteristics of theWaze data. Moreover, a method to identify related Waze alerts and to determine matchingincident reports from different sources is presented. The results showed that the number ofreported incidents in Waze is 4,5 times higher than the number of registered incidents bythe Swedish authorities. Furthermore, 27,5 % of the incidents could have been detectedfaster by using the traffic alerts from Waze. In addition, the severity of Waze alerts isexamined depending on the attributes of the alerts

Drosophila melanogaster, un organisme modèle pour l'étude de la biologie cellulaire des RCPGs neuronaux

Le récepteur cannabinoique de type 1 (CB1R) est l un des récepteurs couplés aux protéines G les plus abondants du cerveau mammifère. CB1R a longtemps été décrit comme un récepteur présynaptique régulant de manière rétrograde la transmission synaptique. Cependant, depuis les vingt dernières années, de nouveaux rôles ont été découverts et il est maintenant clairement admis que l action des endocannabinoides (eCBs) ne se limite pas à la régulationde la neurotransmission au niveau de synapses adultes déjà établies. En effet, les eCBs et le CB1R sont des acteurs majeurs de l ensemble des phases du développement cérébral. Cependant, les mécanismes moléculaires impliqués n ont toujours pas été identifiés. Les mécanismes cellulaires auxquels nous nous intéressons ne dépendant pas de l environnement cellulaire, nous proposons donc de combiner la puissance génétique du modèle drosophile à l accessibilité et la haute résolution offerte par la culture primaire de neurones. De plus, le récepteur CB1 ne possédant pas d orthologue parmi les invertébrés, ce système offre la possibilité d étudier la biologie du récepteur en s affranchissant de la machinerie endocannabinoide. Cependant, actuellement, aucun protocole de culture primaire de neurones de drosophile ne permet d obtenir des cellules hautement différenciées et polarisées à basse densité. Ainsi, nous avons tout d abord développé, optimisé et validé un nouveau protocole permettant de d obtenir des neurones fonctionnels, hautement différenciés et polarisés en culture de basse densité. Dans un second temps, nous avons démontré que l activation durécepteur CB1, exprimé ectopiquement dans les neurones de drosophile, entrainait son internalisation, de manière identique à ce qui avait déjà été observé chez les mammifères. Puis, nous avons étudié l effet de l expression et de l activation ectopique de CB1R sur le développement neuronal chez la drosophile. Ainsi, nous avons démontré que l activation du récepteur module directement la dendritogénèse. Afin de compléter la caractérisation de notremodèle, nous avons démontré que l activation transitoire du récepteur dans les corps pédonculés (le centre de la mémoire olfactive chez la drosophile) altérait spécifiquement la formation d une forme consolidée de mémoire après un conditionnement aversif. En conclusion, la validation du modèle drosophile dans l étude de la biologie cellulaire durécepteur CB1 ouvre de nouvelles perspectives quant à la détermination des mécanismes moléculaires régissant l action du récepteur sur le fonctionnement neuronal.The type-1 cannabinoid receptor (CB1R), the neuronal receptor for the major psychoactive substance of marijuana, is one, of the most abundant G-protein coupled receptors in the mammalian central nervous system. CB1R is traditionally described as a presynaptic receptor that retrogradely regulates synaptic transmission. In addition to this now relatively wellcharacterized function, in the last two decades it has become widely recognized that endocannabinoid (eCB) actions in the brain are not limited to the regulation of neurotransmission at established adult synapses. Indeed, eCB and CB1R are now recognized to be involved in brain development at the synaptic, neuronal and network levels. However, precise mechanisms underlying these processes remain poorly described. Since cellular mechanisms that mediate CB1R-activition dependent neuronal remodeling and subneuronal targeting have been demonstrated to be cell-autonomous, we aimed to combine the power of Drosophila genetics with the experimental accessibility and single-cell resolution of lowdensity primary neuronal cultures, a tool currently lacking in Drosophila. Moreover, becauseDrosophila does not have a CB1R ortholog, CB1R cell biology may be observed independently from eCB machinery. Thus, we first developed and validated an in vitro culture protocol that yields mature and fully differentiated Drosophila neurons. Secondly, we showed that activation-dependent endocytosis of ectopically expressed CB1R is conserved in Drosophila neurons. Next, we investigated whether ectopic expression and activation of CB1R in Drosophila modulate neuronal development. As observed in mammals, we observed that activation of CB1R impairs dendritogenesis in a cell-autonomous manner. For further characterization of our model, we showed that, as with mammals, transient ectopic CB1R expression and activation in mushroom body neurons (the center of olfactory memory in Drosophila) modulate the formation of a consolidated form of aversive memory. In conclusion, the validation of this new animal model opens new perspectives to better characterize mechanisms underlying modulation of neuronal functions induced by CB1RactivityPARIS5-Bibliotheque electronique (751069902) / SudocSudocFranceF

Functional imaging evidence for task-induced deactivation and disconnection of a major default mode network hub in the mouse brain

International audienceThe default mode network (DMN) has been defined in functional brain imaging studies as a set of highly connected brain areas, which are active during wakeful rest and inactivated during task-based stimulation. DMN function is characteristically impaired in major neuropsychiatric diseases, emphasizing its interest for translational research. However, in the mouse, a major preclinical rodent model, there is still no functional imaging evidence supporting DMN deactivation and deconnection during high-demanding cognitive/sensory tasks. Here we have developed functional ultrasound (fUS) imaging to properly visualize both activation levels and functional connectivity patterns, in head-restrained awake and behaving mice, and investigated their modulation during a sensory-task, whisker stimulation. We identified reproducible and highly symmetric resting-state networks, with overall connectivity strength directly proportional to the wakefulness level of the animal. We show that unilateral whisker stimulation leads to the expected activation of the contralateral barrel cortex in lightly sedated mice, while interhemispheric inhibition reduces activity in the ipsilateral barrel cortex. Whisker stimulation also leads to elevated bilateral connectivity in the hippocampus. Importantly, in addition to functional changes in these major hubs of tactile information processing, whisker stimulation during genuine awake resting-state periods leads to highly specific reductions both in activation and interhemispheric correlation within the restrosplenial cortex, a major hub of the DMN. These results validate an imaging technique for the study of activation and connectivity in the lightly sedated awake mouse brain and provide evidence supporting an evolutionary preserved function of the DMN, putatively improving translational relevance of preclinical models of neuropsychiatric diseases

Orofacial Neuropathic Pain Leads to a Hyporesponsive Barrel Cortex with Enhanced Structural Synaptic Plasticity

International audienceChronic pain is a long-lasting debilitating condition that is particularly difficult to treat due to the lack of identified underlying mechanisms. Although several key contributing processes have been described at the level of the spinal cord, very few studies have investigated the supraspinal mechanisms underlying chronic pain. Using a combination of approaches (cortical intrinsic imaging, immunohistochemical and behavioural analysis), our study aimed to decipher the nature of functional and structural changes in a mouse model of orofacial neuropathic pain, focusing on cortical areas involved in various pain components. Our results show that chronic neuropathic orofacial pain is associated with decreased haemodynamic responsiveness to whisker stimulation in the barrel field cortex. This reduced functional activation is likely due to the increased basal neuronal activity (measured indirectly using cFos and phospho-ERK immunoreactivity) observed in several cortical areas, including the contralateral barrel field, motor and cingulate cortices. In the same animals, immunohistochemical analysis of markers for active pre-or postsynaptic elements (Piccolo and phosphoCofilin, respectively) revealed an increased immunofluorescence in deep cortical layers of the contralateral barrel field, motor and cingulate cortices. These results suggest that longlasting orofacial neuropathic pain is associated with exacerbated neuronal activity and synaptic plasticity at the cortical level

Functional ultrasound imaging of intrinsic connectivity in the living rat brain with high spatiotemporal resolution

International audienceLong-range coherences in spontaneous brain activity reflect functional connectivity. Here we propose a novel, highly resolved connectivity mapping approach, using ultrafast functional ultrasound (fUS), which enables imaging of cerebral microvascular haemodynamics deep in the anaesthetized rodent brain, through a large thinned-skull cranial window, with pixel dimensions of 100 μm × 100 μm in-plane. The millisecond-range temporal resolution allows unambiguous cancellation of low-frequency cardio-respiratory noise. Both seed-based and singular value decomposition analysis of spatial coherences in the low-frequency (<0.1 Hz) spontaneous fUS signal fluctuations reproducibly report, at different coronal planes, overlapping high-contrast, intrinsic functional connectivity patterns. These patterns are similar to major functional networks described in humans by resting-state fMRI, such as the lateral task-dependent network putatively anticorrelated with the midline default-mode network. These results introduce fUS as a powerful novel neuroimaging method, which could be extended to portable systems for three-dimensional functional connectivity imaging in awake and freely moving rodents

The type 1 cannabinoid receptor is highly expressed in embryonic cortical projection neurons and negatively regulates neurite growth in vitro

International audienceIn the rodent and human embryonic brains, the cerebral cortex and hippocampus transiently express high levels of type 1 cannabinoid receptors (CB 1 Rs), at a developmental stage when these areas are composed mainly of glutamatergic neurons. However, the precise cellular and subcellular localization of CB 1 R expression as well as effects of CB 1 R modulation in this cell population remain largely unknown. We report that, starting from embryonic day 12.5, CB 1 Rs are strongly expressed in both reelin-expressing Cajal-Retzius cells and newly differentiated postmitotic glutamatergic neurons of the mouse telencephalon. CB 1 R protein is localized first to somato-dendritic endosomes and at later developmental stages it localizes mostly to developing axons. In young axons, CB 1 Rs are localized both to the axolemma and to large, often multivesicular endosomes. Acute maternal injection of agonist CP-55940 results in the relocation of receptors from axons to somato-dendritic endosomes, indicating the functional competence of embryonic CB 1 Rs. The adult phenotype of CB 1 R expression is established around postnatal day 5. By using pharmacological and mutational modulation of CB 1 R activity in isolated cultured rat hippocampal neurons, we also show that basal activation of CB 1 R acts as a negative regulatory signal for dendritogenesis, dendritic and axonal outgrowth, and branching. Together, the overall negative regulatory role in neurite development suggests that embryonic CB 1 R signaling may participate in the correct establishment of neuronal connectivity and suggests a possible mechanism for the development of reported glutamatergic dysfunction in the offspring following maternal cannabis consumption