Polarization of hippocampal neurons with competitive surface stimuli: contact guidance cues are preferred over chemical ligands

Abstract

Neuronal behaviour is profoundly influenced by extracellular stimuli in many developmental and regeneration processes. Understanding neuron responses and integration of environmental signals could impact the design of successful therapies for neurodegenerative diseases and nerve injuries. Here, we have investigated the influence of localized extracellular cues on polarization (i.e. axon formation) of hippocampal neurons. Electron-beam lithography, microfabrication techniques and protein immobilization were used to create a unique system that provided simultaneous and independent chemical and physical cues to individual neurons. In particular, we analysed competitive responses between simultaneous stimulation with chemical ligands, including immobilized nerve growth factor and laminin, and contact guidance cues mediated by surface topography (i.e. microchannels). Contact guidance cues were preferred 70% of the time over chemical ligands by neurons extending axons, which suggests a stronger stimulation mechanism triggered by topography. This investigation contributes to the understanding of neuronal behaviour on artificial substrates, which is applicable to the creation of artificial environments for neural engineering applications.

Selective axonal growth of embryonic hippocampal neurons according to topographic features of various sizes and shapes

“…Due to a relatively small sample size in the comparison of the holes, power analysis revealed that Type II error could have precluded us from obtaining a significant result. The data for the lines is consistent with results published by Gomez et al 13 who found that axons preferred smaller lines of 1 µm width over larger lines of 2 µm width.…”

“…Topographies consisted of single structures arrayed in either one (lines) or two dimensions (holes) and were strategically chosen based on results obtained by Gomez et al 13 and on additional design rules. Gomez et al showed that 2 µm lines were more effective at stimulating axon polarization in hippocampal neurons than chemical ligands; they also showed that 1 µm lines were more stimulative than 2 µm lines.…”

“…They showed that topography and its dimensions heavily affect axonal alignment. Gomez et al 13 performed novel competition axon guidance assays by culturing individual hippocampal neurons between two micropatterned polydimethylsiloxane surfaces, one containing microscale lines and the other various neuroactive biomolecules, such as nerve growth factor and laminin. They found that embryonic hippocampal neurons extended their axons preferentially toward the 2 µm line topographies relative to smooth polydimethylsiloxane surfaces or polydimethylsiloxane patterned with nerve growth factor and laminin, emphasizing the relative ability of topography to stimulate axonal growth.…”

Interaction of leech neurons with topographical gratings: comparison with rodent and human neuronal lines and primary cells

“…Extracellular nano/microtopography signals are locally retrieved through a complex phenomenon called contact guidance and can drive many neuronal activities, such as differentiation, polarization, neurite pathfinding, nucleokinesis and the final CNS wiring [10][11][12][13][14][15][16]. These processes are tightly regulated and involve coordinated interactions between microtubules and the actin cytoskeleton [16,17] as well as the establishment and maturation of focal adhesions (FAs) [18].…”

Laser-based three-dimensional multiscale micropatterning of biocompatible hydrogels for customized tissue engineering scaffolds

“…3 A-D). This contact guidance phenomenon is well-known and has previously been used to induce alignment of various cell types (25,26). Because features can be machined onto the gel through a sealed dish, we hypothesize that this could be a convenient method to reorient or disrupt already established cell cultures.…”