Reconstruction of directed neuronal networks in a microfluidic device with asymmetric microchannels

International audienceMicrofluidic devices for controlling neuronal connectivity in vitro are extremely useful tools for deciphering pathological and physiological processes occurring in neuronal networks. These devices allow the connection between different neuronal populations located into separate culture chambers through axon-selective microchannels. In order to implement specific features of brain connectivity such as directionality, it is necessary to control axonal growth orientation in these devices. Among the various strategies proposed to achieve this goal, one of the most promising and easily reproducible is the use of asymmetric microchannels. We present here a general protocol and several guidelines for the design, production and testing of a new paradigm of asymmetric microchannels geometries based on a “return to sender” strategy. In this method, axons are either allowed to travel between the emitting and receiving chambers within straight microchannels (forward direction), or are rerouted toward their initial location through curved microchannels (reverse direction). We introduce variations of these “arches” microchannels and evaluate their respective axonal filtering capacities. Importantly, one of these variants presents an almost complete filtration of axonal growth in the non-permissive direction while allowing robust axonal invasion in the other one, with a selectivity ratio as high as 99.7%

Retinotopic and lateralized processing of spatial frequencies in human visual cortex during scene categorization.

International audienceUsing large natural scenes filtered in spatial frequencies, we aimed to demonstrate that spatial frequency processing could not only be retinotopically mapped but could also be lateralized in both hemispheres. For this purpose, participants performed a categorization task using large black and white photographs of natural scenes (indoors vs. outdoors, with a visual angle of 24° × 18°) filtered in low spatial frequencies (LSF), high spatial frequencies (HSF), and nonfiltered scenes, in block-designed fMRI recording sessions. At the group level, the comparison between the spatial frequency content of scenes revealed first that, compared with HSF, LSF scene categorization elicited activation in the anterior half of the calcarine fissures linked to the peripheral visual field, whereas, compared with LSF, HSF scene categorization elicited activation in the posterior part of the occipital lobes, which are linked to the fovea, according to the retinotopic property of visual areas. At the individual level, functional activations projected on retinotopic maps revealed that LSF processing was mapped in the anterior part of V1, whereas HSF processing was mapped in the posterior and ventral part of V2, V3, and V4. Moreover, at the group level, direct interhemispheric comparisons performed on the same fMRI data highlighted a right-sided occipito-temporal predominance for LSF processing and a left-sided temporal cortex predominance for HSF processing, in accordance with hemispheric specialization theories. By using suitable method of analysis on the same data, our results enabled us to demonstrate for the first time that spatial frequencies processing is mapped retinotopically and lateralized in human occipital cortex

Combining Microfluidics, Optogenetics and Calcium Imaging to Study Neuronal Communication In Vitro

International audienceIn this paper we report the combination of microfluidics, optogenetics and calcium imaging as a cheap and convenient platform to study synaptic communication between neuronal populations in vitro. We first show that Calcium Orange indicator is compatible in vitro with a commonly used Channelrhodopsine-2 (ChR2) variant, as standard calcium imaging conditions did not alter significantly the activity of transduced cultures of rodent primary neurons. A fast, robust and scalable process for micro-chip fabrication was developed in parallel to build micro-compartmented cultures. Coupling optical fibers to each micro-compartment allowed for the independent control of ChR2 activation in the different populations without crosstalk. By analyzing the post-stimuli activity across the different populations, we finally show how this platform can be used to evaluate quantitatively the effective connectivity between connected neuronal populations

Synapto-protective drugs evaluation in reconstructed neuronal network

Chronic neurodegenerative syndromes such as Alzheimer’s and Parkinson’s diseases, or acute syndromes such as ischemic stroke or traumatic brain injuries are characterized by early synaptic collapse which precedes axonal and neuronal cell body degeneration and promotes early cognitive impairment in patients. Until now, neuroprotective strategies have failed to impede the progression of neurodegenerative syndromes. Drugs preventing the loss of cell body do not prevent the cognitive decline, probably because they lack synapto-protective effects. The absence of physiologically realistic neuronal network models which can be easily handled has hindered the development of synapto-protective drugs suitable for therapies. Here we describe a new microfluidic platform which makes it possible to study the consequences of axonal trauma of reconstructed oriented mouse neuronal networks. Each neuronal population and sub-compartment can be chemically addressed individually. The somatic, mid axon, presynaptic and postsynaptic effects of local pathological stresses or putative protective molecules can thus be evaluated with the help of this versatile “brain on chip” platform. We show that presynaptic loss is the earliest event observed following axotomy of cortical fibers, before any sign of axonal fragmentation or post-synaptic spine alteration. This platform can be used to screen and evaluate the synapto-protective potential of several drugs. For instance, NAD+ and the Rho-kinase inhibitor Y27632 can efficiently prevent synaptic disconnection, whereas the broad-spectrum caspase inhibitor zVAD-fmk and the stilbenoid resveratrol do not prevent presynaptic degeneration. Hence, this platform is a promising tool for fundamental research in the field of developmental and neurodegenerative neurosciences, and also offers the opportunity to set up pharmacological screening of axon-protective and synapto-protective drugs

Wallerian-Like Degeneration of Central Neurons After Synchronized and Geometrically Registered Mass Axotomy in a Three-Compartmental Microfluidic Chip

Degeneration of central axons may occur following injury or due to various diseases and it involves complex molecular mechanisms that need to be elucidated. Existing in vitro axotomy models are difficult to perform, and they provide limited information on the localization of events along the axon. We present here a novel experimental model system, based on microfluidic isolation, which consists of three distinct compartments, interconnected by parallel microchannels allowing axon outgrowth. Neurons cultured in one compartment successfully elongated their axons to cross a short central compartment and invade the outermost compartment. This design provides an interesting model system for studying axonal degeneration and death mechanisms, with a previously impossible spatial and temporal control on specific molecular pathways. We provide a proof-of-concept of the system by reporting its application to a well-characterized experimental paradigm, axotomy-induced Wallerian degeneration in primary central neurons. Using this model, we applied localized central axotomy by a brief, isolated flux of detergent. We report that mouse embryonic cortical neurons exhibit rapid Wallerian-like distal degeneration but no somatic death following central axotomy. Distal axons show progressive degeneration leading to axonal beading and cytoskeletal fragmentation within a few hours after axotomy. Degeneration is asynchronous, reminiscent of in vivo Wallerian degeneration. Axonal cytoskeletal fragmentation is significantly delayed with nicotinamide adenine dinucleotide pretreatment, but it does not change when distal calpain or caspase activity is inhibited. These findings, consistent with previous experiments in vivo, confirm the power and biological relevance of this microfluidic architecture

Les effets des indices non-verbaux sur les activités de communication à distance (deux études expérimentales sur le dialogue tutoriel)

Notre travail s'insère dans le domaine de la communication audio-vidéo médiatisée. Les recherches de ce domaine ont l'objectif de comprendre l'impact des technologies de la communication sur les différentes activités de communication et de réaliser des dispositifs adéquats pour ces activités. Quelle que soit la situation de communication, l'interface n'est plus un simple objet d'adressage et de traduction pratique des commandes de l'opérateur, comme dans la vision classique de l'interaction homme-machine, mais devient plutôt un espace de communication interpersonnelle L'action de communiquer avec autrui ne se limite ni à une simple adéquation de codes verbaux et non-verbaux, ni à un processus de production d'inférences et d'interprétation d'indices contextuels. Les interlocuteurs agissent selon une logique coopérative (Clark, 1996) : cela signifie que les interlocuteurs doivent réciproquement accepter chacune de leurs contributions pendant un échange, afin de pouvoir partager des connaissances ou des faits relatifs à leur activité. Une communication réussie demande que les interlocuteurs co-construisent mutuellement leurs référentiels, s'appuyant sur les indices verbaux et non-verbaux. Parmi ces derniers, on se doit de distinguer les indices non-verbaux kinésiques (e.g., les expressions faciales, les regards, les gestes du corps, etc.) et les indices non-verbaux ostensifs-inférentiels (e.g. les gestes déictiques, les actions physiques). Nous avançons deux hypothèses de travail principales : A) les indices non-verbaux kinésiques facilitent surtout le processus de communication et favorisent un sentiment de proximité sociale entre les partenaires distants ; B) les indices non-verbaux ostensif-inférentiels facilitent surtout l'intercompréhension et la réalisation de la tâche commune. Pour vérifier ces hypothèses, nous avons construit une tâche expérimentale dans laquelle un tuteur se doit d'assister deux apprenants distants impliqués dans la réalisation d'un travail pratique (la réalisation d'une page en notation html) et qui ne peuvent pas communiquer entre eux. Dans la première expérimentation (n tutor=12, N étudiants = 48), nous étudions les effets des indices kinésiques, véhiculés par l'image-vidéo montrant les partenaires distants. Les résultats indiquent que les indices kinésiques facilitent le processus de compréhension mutuelle et l'activité de suivi du tuteur, sans pour autant améliorer la qualité du travail des apprenants. Dans la deuxième expérimentation (n tutors = 12, étudiants = 72), nous nous intéressons aux indices ostensifs-inférentiels, véhiculés par l'image-vidéo relative aux actions effectuées par les partenaires distants sur les objets appartenant à leur espace de travail. Les indices ostensifs-inférentiels favorisent la construction des référentiels communs, facilitent le suivi du tuteur et induisent une amélioration du travail des apprenants. Nous discutons les résultats de deux expérimentations et nous avançons des propositions pour la conception des dispositifs consacrés à la communication tutorielle.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Pour quelques approches de plus...

Sommaire du numéro :http://archive-edutice.ccsd.cnrs.fr/edutice-00000840En revenant de son cours, un professeur de programmation est perdu dans ses pensées... Des difficultés ont surgi ce matin-là. Il a essayé plusieurs approches, mais aucune ne convenait à tous ; il a consommé beaucoup de craie, les étudiants étaient passifs et ne savaient pas comment poser des questions. Il aurait aimé disposer d'autres moyens, rendre les étudiants plus actifs, leur permettre un travail personnel et des expérimentations de l'objet enseigné. Et il aurait été plus disponible pour les problèmes propres de chacun. La didactique se préoccupe à la fois de la théorie et de la méthode de l'enseignement. Notre travail nous a permis de tester cette complémentarité, mais nous y ajoutons l'idée que l'ordre « a priori » de présentation d'un concept ou d'une méthode n'est pas foncièrement déterminant, les étudiants (et l'enseignant) ayant leurs propres schémas mentaux de compréhension. L'ordinateur peut offrir cette part de liberté, au même titre que les livres, les films, les rencontres, les expériences de vie, etc. qui appartiennent à chacun. Nous souhaitons que l'étudiant puisse effectuer les activités de programmation en manipulant, en observant, en écoutant, en jouant, et qu'il structure ainsi de façon personnelle ses acquisitions

Prion Strain- and Species-Dependent Effects of Antiprion Molecules in Primary Neuronal Cultures▿

Transmissible spongiform encephalopathies (TSE) arise as a consequence of infection of the central nervous system by prions and are incurable. To date, most antiprion compounds identified by in vitro screening failed to exhibit therapeutic activity in animals, thus calling for new assays that could more accurately predict their in vivo potency. Primary nerve cell cultures are routinely used to assess neurotoxicity of chemical compounds. Here, we report that prion strains from different species can propagate in primary neuronal cultures derived from transgenic mouse lines overexpressing ovine, murine, hamster, or human prion protein. Using this newly developed cell system, the activity of three generic compounds known to cure prion-infected cell lines was evaluated. We show that the antiprion activity observed in neuronal cultures is species or strain dependent and recapitulates to some extent the activity reported in vivo in rodent models. Therefore, infected primary neuronal cultures may be a relevant system in which to investigate the efficacy and mode of action of antiprion drugs, including toward human transmissible spongiform encephalopathy agents