Gradients of substrate-bound laminin orient axonal specification of neurons

Abstract

Little is known about the influence of substrate-bound gradients on neuronal development, since it has been difficult to fabricate gradients over the distances typically required for biological studies (a few hundred micrometers). This article demonstrates a generally applicable technique for the fabrication of substratebound gradients of proteins with complex shapes, using laminar flows in microchannels. Gradients that range from pure laminin to pure BSA were formed in solution by using a network of microchannels, and these proteins were allowed to adsorb onto a homogeneous layer of poly-L-lysine. Rat hippocampal neurons were cultivated on these substrate-bound gradients. Analysis of optical images of these neurons showed that axon specification is oriented in the direction of increasing surface density of laminin. Linear gradients in laminin adsorbed from a gradient in solution having a slope of ١[laminin] > about 0.06 g (ml⅐m) ؊1 (defined by dividing the change of concentration of laminin in solution over the distance of the gradient) orient axon specification, whereas those with ١[laminin] < about 0.06 g (ml⅐m) ؊1 have no effect. microfluidics ͉ neuronal polarity ͉ hippocampal G radients of chemoattractant and chemorepellent substances play central roles in controlling the development of the brain (1). Although the influence of gradients of soluble substances on neuronal behavior has been studied extensively and has been used to unravel the molecular and cellular mechanisms of axon guidance (2), much less is known about gradients of substrate-bound substances (3). Generating consistent substrate-bound gradients over the distances required for biological studies (a few hundred micrometers) has been difficult, and this difficulty hindered the investigation of the role of immobilized gradients in neuronal development (e.g., establishment of cellular polarity and axon guidance) and cell migration. This article demonstrates a generally applicable technique for the fabrication of substrate-bound gradients that uses laminar flow of fluids in microchannels; we have described the microfluidic systems used to form these gradients (4). By generating gradients in the concentration of proteins in solution and allowing them to adsorb on a surface, we fabricated substratebound gradients that range from pure laminin to pure BSA immobilized on a uniform layer of poly-L-lysine (PLL) over distances of a few hundred micrometers. We demonstrate that axon specification of rat hippocampal neurons cultivated on these gradients is oriented in the direction of increasing concentration of laminin. We demonstrate that gradients of extracellular molecules adsorbed on the surface are capable of orienting the polarity of cultured hippocampal neurons.Hippocampal neurons in culture develop their characteristic structural and functional polarity in a stereotyped sequence of developmental events (5). Cells first attach to the substrate and then develop processes. When new processes start to form, they cannot be distinguished as either axons o...

Biology on a Chip: Microfabrication for Studying the Behavior of Cultured Cells

“…Linear gradients in laminin adsorbed from a gradient in solution having a slope of laminin concentration larger than ∼0.06 μg/(mL × μm) (defined by dividing the change of laminin concentration in solution over the distance of the gradient) oriented axon specification, whereas those with laminin concentration smaller than ∼0.06 μg/(mL × μm) had no effect. 68 …”

“…Recently Dertinger et al 68 studied the influence of laminin gradients on neuronal guidance. Complicated gradient shapes could be created on polyL-lysine-coated PDMS substrates using microfluidic patterning with a soluble-gradient-generating device.…”

Surface Coating as a Key Parameter in Engineering Neuronal Network Structures In Vitro

“…Development in micro-nano-technology has brought in several new tools for engineering the patterning of cell adhesion and morphogenesis [9][10][11][12][13]. Many cell types have been successfully patterned with microfluidics [14][15][16], lCP [17], inkjet printing [18,19], plasma treatment [20], self-assembled monolayers [21][22][23][24], self-assembled constructs [25], laser scanning lithography [26], atomic force microscope lithography, dip-pen nanolithography [27], topography [28,29], carbon nano-tubes [30], or their combinations [31,32].…”

Laminin/β1 integrin signal triggers axon formation by promoting microtubule assembly and stabilization

“…Cultured hippocampal neurons tend to form axons preferentially on the substrates coated with extracellular matrix (ECM) component laminin or neuron-glia cell adhesion molecule (NgCAM/L1) than on poly-l-lysine [17,18], suggesting that ECM or cell surface components may serve as extrinsic cues for neuronal polarization. A recent report shows that laminin contact correlates with the emergence of oriented axon of retinal ganglion cells in the zebrafish larvae [16].…”