Surface Tension and Neuronal Sorting in Magnetically Engineered Brain-Like Tissue.

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.

Parallelized Manipulation of Adherent Living Cells by Magnetic Nanoparticles-Mediated Forces.

The remote actuation of cellular processes such as migration or neuronal outgrowth is a challenge for future therapeutic applications in regenerative medicine. Among the different methods that have been proposed, the use of magnetic nanoparticles appears to be promising, since magnetic fields can act at a distance without interactions with the surrounding biological system. To control biological processes at a subcellular spatial resolution, magnetic nanoparticles can be used either to induce biochemical reactions locally or to apply forces on different elements of the cell. Here, we show that cell migration and neurite outgrowth can be directed by the forces produced by a switchable parallelized array of micro-magnetic pillars, following the passive uptake of nanoparticles. Using live cell imaging, we first demonstrate that adherent cell migration can be biased toward magnetic pillars and that cells can be reversibly trapped onto these pillars. Second, using differentiated neuronal cells we were able to induce events of neurite outgrowth in the direction of the pillars without impending cell viability. Our results show that the range of forces applied needs to be adapted precisely to the cellular process under consideration. We propose that cellular actuation is the result of the force on the plasma membrane caused by magnetically filled endo-compartments, which exert a pulling force on the cell periphery.

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

Microfluidic 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%.

Sequences Flanking the Gephyrin-Binding Site of GlyRß 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 ß-subunit (ß-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 ß-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 ß-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.