a b s t r a c tThe ability to construct easily in vitro networks of primary neurons organized with imposed topologies is required for neural tissue engineering as well as for the development of neuronal interfaces with desirable characteristics. However, accumulating evidence suggests that the mechanical properties of the culture matrix can modulate important neuronal functions such as growth, extension, branching and activity. Here we designed robust and reproducible laminin-polylysine grid micropatterns on cell culture substrates that have similar biochemical properties but a 100-fold difference in Young's modulus to investigate the role of the matrix rigidity on the formation and activity of cortical neuronal networks. We found that cell bodies of primary cortical neurons gradually accumulate in circular islands, whereas axonal extensions spread on linear tracks to connect circular islands. Our findings indicate that migration of cortical neurons is enhanced on soft substrates, leading to a faster formation of neuronal networks. Furthermore, the pre-synaptic density was two times higher on stiff substrates and consistently the number of action potentials and miniature synaptic currents was enhanced on stiff substrates. Taken together, our results provide compelling evidence to indicate that matrix stiffness is a key parameter to modulate the growth dynamics, synaptic density and electrophysiological activity of cortical neuronal networks, thus providing useful information on scaffold design for neural tissue engineering.

Collective cell migration is fundamental throughout development, wound healing and in many diseases. Although much effort has focused on cell-cell junctions, a role for physical confinement in collective cell migration remains unclear. Here we used adhesive microstripes of varying widths to mimic the spatial confinement experienced by follower cells within epithelial tissues. Our results reveal that the substrate area confinement is sufficient to modulate the three-dimensional (3D) cellular morphology without the need for intercellular adhesive cues. Our findings show a direct correlation between the migration velocity of confined cells and their cell-substrate adhesive area. Closer examination revealed that adhesive area confinement reduces lamellipodial protrusive forces, decreases the number of focal complexes at the leading edge and prevents the maturation of focal adhesions at the trailing edge, leading together to less effective

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Alterations of the axonal morphology are key signatures of traumatic brain injury (TBI). Although the pathobiology of axonal injury has been extensively investigated, the vulnerability of the axonal microcompartment over the soma was still misunderstood. We hypothesized that the soma and the axon of neurons display opposite mechanical behaviors, rendering the axon more sensitive to a mechanical stress. To test this hypothesis, we used a microcontact printing method to control the growth of cortical neuron in a bipolar morphology and the viscoelastic properties of soma and axon microcompartments were measured with magnetic tweezers. Creep experiments showed that neuronal microcompartments exhibit distinct mechanical behaviors: the soma is softer and characterized by an elastic-like behavior, while the neurite is stiffer and viscous-like. By altering cytoskeletal filaments with pharmacological agents, we determined the origin of the compartmentalization of mechanical behaviors within cortical neurons. The nucleus determines the elastic and stress stiffening behavior of the soma, while the sliding of neurofilaments determines the viscous-like state of the neurite. In addition, our results revealed that at the contrary of the soma, the neurite can sense its mechanical environment and becomes softer and more viscous on soft surfaces, showing that, as for the mechanical behavior, the mechanosensitivity is localized to the neuronal microcompartments. Our findings shed light on the importance of the regionalization of neuronal properties to their microcompartments in response to a mechanical insult. Future works will need to investigate the relationship between the mechanical differences of neuronal microcompartments and their functions. In this context, we suggest to consider microprinted neuronal networks as an efficient tool for investigating the effect of the propagation of injury forces on the behavior of neuronal circuits.Keywords: Neuron; microcompartment; rheology; diffuse axonal injury; mechanosensitivity To cite this article: Thomas Grevesse, et al. Dissecting the mechanical behavior of neuronal microcompartments by combining magnetic tweezers and protein micropatterns. Neurosci Commun 2015; 1: e1003.