By quantitatively comparing a variety of macromolecular surface coating agents, we discovered that surface coating strongly modulates the adhesion and morphogenesis of primary hippocampal neurons and serves as a switch of somata clustering and neurite fasciculation in vitro. The kinetics of neuronal adhesion on poly-lysine-coated surfaces is much faster than that on laminin and Matrigel-coated surfaces, and the distribution of adhesion is more homogenous on poly-lysine. Matrigel and laminin, on the other hand, facilitate neuritogenesis more than poly-lysine does. Eventually, on Matrigel-coated surfaces of self-assembled monolayers, neurons tend to undergo somata clustering and neurite fasciculation. By replacing coating proteins with cerebral astrocytes, and patterning neurons on astrocytes through self-assembled monolayers, microfluidics and micro-contact printing, we found that astrocyte promotes soma adhesion and astrocyte processes guide neurites. There, astrocytes could be a versatile substrate in engineering neuronal networks in vitro. Besides, quantitative measurements of cellular responses on various coatings would be valuable information for the neurobiology community in the choice of the most appropriate coating strategy.

Axon specification during neuronal polarization is closely associated with increased microtubule stabilization in one of the neurites of unpolarized neuron, but how this increased microtubule stability is achieved is unclear. Here, we show that extracellular matrix (ECM) component laminin promotes neuronal polarization via regulating directional microtubule assembly through β1 integrin (Itgb1). Contact with laminin coated on culture substrate or polystyrene beads was sufficient for axon specification of undifferentiated neurites in cultured hippocampal neurons and cortical slices. Active Itgb1 was found to be concentrated in laminin-contacting neurites. Axon formation was promoted and abolished by enhancing and attenuating Itgb1 signaling, respectively. Interestingly, laminin contact promoted plus-end microtubule assembly in a manner that required Itgb1. Moreover, stabilizing microtubules partially prevented polarization defects caused by Itgb1 downregulation. Finally, genetic ablation of Itgb1 in dorsal telencephalic progenitors caused deficits in axon development of cortical pyramidal neurons. Thus, laminin/Itgb1 signaling plays an instructive role in axon initiation and growth, both in vitro and in vivo, through the regulation of microtubule assembly. This study has established a linkage between an extrinsic factor and intrinsic cytoskeletal dynamics during neuronal polarization.

In this report we fabricated laminin (LN) stripes on a background of poly-L-lysine as substrates for the growth of rat hippocampal neurons, and found that a sharp change of the concentration of LN guides the growth of neurites by leading the growth cones in a time- and space-dependent manner. The percentage of neurites that grow along the edge of LN stripes (where there is a sharp change of concentration) decreases as a function of the concentration of LN under a threshold value. The actin cytoskeleton plays an important role in the process of growth cone's response to the sharp change of concentration of LN on micropatterns. We believe that the findings here are useful for not only fundamental studies in neuroscience, but also helpful for the design of devices or chips for nervous prosthesis.

Axon branching enables neurons to contact with multiple targets and respond to their microenvironment. Owing to its importance in neuronal network formation, axon branching has been studied extensively during the past decades. The chemical properties of extracellular matrices have been proposed to regulate axonal development, but the effects of their density changes on axon branching are not well understood. Here, we demonstrate that both the sharp broadening of substrate geometry and the sharp change of laminin density stimulate axon branching by using microcontact printing (μCP) and microfluidic printing (μFP) techniques. We also found that the change of axon branching stimulated by laminin density depends on myosin II activity. The change of laminin density induces asymmetric extensions of filopodia on the growth cone, which is the precondition for axon branching. These previously unknown mechanisms of change of laminin density-stimulated axon branching may explain how the extracellular matrices regulate axon branching in vivo and facilitate the establishment of neuronal networks in vitro.