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%

Fasciculation axonale et dynamique du cône de croissance en environnement confiné

Precise navigation of axons is crucial to establish in vivo neuronal networks. It relies on proper guidance of the growth cone (GC), an actin-rich dynamic structure located at the axonal tip. In vivo, axons ofter progress toward their target by forming bundles. In this study, we use a microfluidic chip composed of axonal microchannels of various widths to induce and study the phenomenon of fasciculation. We show that bundles are built from two populations of axons, i.e. pioneers and followers, characterized by distinct behaviors and GC morphologies. Pioneer axons display an explorative behavior, characterized by a discontinuous growth due to alternance of advancing and pausing periods, while follower axons exhibit a higher growth rate with less frequent pauses. Besides, morphological analysis of GCs reveals that these structures are wider in pioneer axons, while GCs of followers display more elongated shapes. Curiously, confined pioneer GCs into 2 μm wide channels adopt both follower morphologies and dynamics. These results prompting us to wonder about the nature of the link between GC shape and dynamics, we adress this question by investigating the organization and dynamics of the actin filaments (AF) using Spt-PALM. Preliminary results suggest that filament orientation defines a narrower angle in followers and highly confined pioneers than in unconfined pioneers. In addition, measurements of the actin retrograde flow rate (ARF) in GCs reveal a faster flow for unconfined pioneers. The speed of the ARF being inversely proportional to the cell migration speed, these results would explain the respective behaviors of pioneers and followers. Further investigations into quantifying the quantity or density of AF, as well as their coupling to cell adhesion molecules, will allow us to improve our understanding of the molecular mechanisms involved in these two axon populations.La navigation des axones est une étape cruciale du développement des réseaux neuronaux. Elle s’opère via divers mécanismes de guidage du cône de croissance (GC), une structure motile et riche en actine localisée à l’extrémité des neurites. Dans de nombreux cas, les axones progressent vers leur destination en formant des faisceaux. Dans cette étude, nous utilisons des puces microfluidiques composées de micro-canaux axonaux de différentes largeurs pour induire et étudier la fasciculation. Nous montrons que les faisceaux sont constitués de deux populations d’axones, les pionniers et les suiveurs, qui se distinguent par leur comportement et la morphologie du GC. Les pionniers adoptent un comportement exploratoire caractérisé par une alternance d’avancées et de pauses alors que les suiveurs ont une croissance plus rapide et des pauses moins fréquentes. L’analyse morphologique des GC révèle que ces structures sont plus larges chez les pionniers alors que ceux des suiveurs ont une forme plus allongée. Curieusement, lorsque les GC des pionniers sont fortement confinés et ainsi contraints d’adopter la morphologie des suiveurs, ils en acquièrent également le comportement et la dynamique. Ces résultats nous ont amené à nous interroger sur la nature du lien existant entre la forme et la dynamique du GC. Nous avons entrepris d’investiguer cette question par une étude de l’organisation et de la dynamique du cytosquelette d’actine en microscopie super-résolutive. Les résultats obtenus semblent indiquer que l’orientation des filaments définit un angle plus étroit chez les suiveurs et les pionniers très confinés que chez les pionniers non confinés. De plus, les mesures de vitesse du flux rétrograde d’actine (ARF) dans les GC révèlent un flux plus rapide pour les pionniers non confinés. La vitesse de l’ARF étant inversement proportionnelle à la vitesse de migration des cellules, ces résultats expliqueraient les différentes dynamiques de croissance des pionniers et des suiveurs. De prochaines investigations visant à quantifier la quantité ou la densité de filaments d’actine, ainsi que leur couplage à des molécules d’adhésion cellulaire, nous permettrons d’améliorer notre compréhension des mécanismes moléculaires mis en oeuvre dans ces deux populations d’axones

Axonal fasciculation and growth cone dynamics in confined micro-environments

La navigation des axones est une étape cruciale du développement des réseaux neuronaux. Elle s’opère via divers mécanismes de guidage du cône de croissance (GC), une structure motile et riche en actine localisée à l’extrémité des neurites. Dans de nombreux cas, les axones progressent vers leur destination en formant des faisceaux. Dans cette étude, nous utilisons des puces microfluidiques composées de micro-canaux axonaux de différentes largeurs pour induire et étudier la fasciculation. Nous montrons que les faisceaux sont constitués de deux populations d’axones, les pionniers et les suiveurs, qui se distinguent par leur comportement et la morphologie du GC. Les pionniers adoptent un comportement exploratoire caractérisé par une alternance d’avancées et de pauses alors que les suiveurs ont une croissance plus rapide et des pauses moins fréquentes. L’analyse morphologique des GC révèle que ces structures sont plus larges chez les pionniers alors que ceux des suiveurs ont une forme plus allongée. Curieusement, lorsque les GC des pionniers sont fortement confinés et ainsi contraints d’adopter la morphologie des suiveurs, ils en acquièrent également le comportement et la dynamique. Ces résultats nous ont amené à nous interroger sur la nature du lien existant entre la forme et la dynamique du GC. Nous avons entrepris d’investiguer cette question par une étude de l’organisation et de la dynamique du cytosquelette d’actine en microscopie super-résolutive. Les résultats obtenus semblent indiquer que l’orientation des filaments définit un angle plus étroit chez les suiveurs et les pionniers très confinés que chez les pionniers non confinés. De plus, les mesures de vitesse du flux rétrograde d’actine (ARF) dans les GC révèlent un flux plus rapide pour les pionniers non confinés. La vitesse de l’ARF étant inversement proportionnelle à la vitesse de migration des cellules, ces résultats expliqueraient les différentes dynamiques de croissance des pionniers et des suiveurs. De prochaines investigations visant à quantifier la quantité ou la densité de filaments d’actine, ainsi que leur couplage à des molécules d’adhésion cellulaire, nous permettrons d’améliorer notre compréhension des mécanismes moléculaires mis en oeuvre dans ces deux populations d’axones.Precise navigation of axons is crucial to establish in vivo neuronal networks. It relies on proper guidance of the growth cone (GC), an actin-rich dynamic structure located at the axonal tip. In vivo, axons ofter progress toward their target by forming bundles. In this study, we use a microfluidic chip composed of axonal microchannels of various widths to induce and study the phenomenon of fasciculation. We show that bundles are built from two populations of axons, i.e. pioneers and followers, characterized by distinct behaviors and GC morphologies. Pioneer axons display an explorative behavior, characterized by a discontinuous growth due to alternance of advancing and pausing periods, while follower axons exhibit a higher growth rate with less frequent pauses. Besides, morphological analysis of GCs reveals that these structures are wider in pioneer axons, while GCs of followers display more elongated shapes. Curiously, confined pioneer GCs into 2 μm wide channels adopt both follower morphologies and dynamics. These results prompting us to wonder about the nature of the link between GC shape and dynamics, we adress this question by investigating the organization and dynamics of the actin filaments (AF) using Spt-PALM. Preliminary results suggest that filament orientation defines a narrower angle in followers and highly confined pioneers than in unconfined pioneers. In addition, measurements of the actin retrograde flow rate (ARF) in GCs reveal a faster flow for unconfined pioneers. The speed of the ARF being inversely proportional to the cell migration speed, these results would explain the respective behaviors of pioneers and followers. Further investigations into quantifying the quantity or density of AF, as well as their coupling to cell adhesion molecules, will allow us to improve our understanding of the molecular mechanisms involved in these two axon populations

Surface Tension and Neuronal Sorting in Magnetically Engineered Brain‐Like Tissue

Abstract Engineered 3D brain‐like models have advanced the understanding of neurological mechanisms and disease, yet their mechanical signature, while fundamental for brain function, remains understudied. The surface tension for instance controls brain development and is a marker of cell‐cell interactions. Here, 3D magnetic brain‐like tissue spheroids composed of intermixed primary glial and neuronal cells at different ratios are engineered. Remarkably, the two cell types self‐assemble into a functional tissue, with the sorting of the neuronal cells toward the periphery of the spheroids, whereas the glial cells constitute the core. The magnetic fingerprint of the spheroids then allows their deformation when placed under a magnetic field gradient, at a force equivalent to a 70 g increased gravity at the spheroid level. The tissue surface tension and elasticity can be directly inferred from the resulting deformation, revealing a transitional dependence on the glia/neuron ratio, with the surface tension of neuronal tissue being much lower. The results suggest an underlying mechanical contribution to the exclusion of the neurons toward the outer spheroid region, and depict the glia/neuron organization as a sophisticated mechanism that should in turn influence tissue development and homeostasis relevant in the neuroengineering field

Sequences Flanking the Gephyrin-Binding Site of GlyR beta Tune Receptor Stabilization at Synapses

The efficacy of synaptic transmission is determined by the number of neurotransmitter receptors at synapses. Their recruitment depends upon the availability of postsynaptic scaffolding molecules that interact with specific binding sequences of the receptor. At inhibitory synapses, gephyrin is the major scaffold protein that mediates the accumulation of heteromeric glycine receptors (GlyRs) via the cytoplasmic loop in the beta-subunit (beta-loop). This binding involves high- and low-affinity interactions, but the molecular mechanism of this bimodal binding and its implication in GlyR stabilization at synapses remain unknown. We have approached this question using a combination of quantitative biochemical tools and high-density single molecule tracking in cultured rat spinal cord neurons. The high-affinity binding site could be identified and was shown to rely on the formation of a 310-helix C-terminal to the beta-loop core gephyrin-binding motif. This site plays a structural role in shaping the core motif and represents the major contributor to the synaptic confinement of GlyRs by gephyrin. The N-terminal flanking sequence promotes lower affinity interactions by occupying newly identified binding sites on gephyrin. Despite its low affinity, this binding site plays a modulatory role in tuning the mobility of the receptor. Together, the GlyR beta-loop sequences flanking the core-binding site differentially regulate the affinity of the receptor for gephyrin and its trapping at synapses. Our experimental approach thus bridges the gap between thermodynamic aspects of receptor-scaffold interactions and functional receptor stabilization at synapses in living cells