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.

Figure 1

Fabrication and characterization of competitive scheme between physical and chemical cues for neuron polarization. (a) Microchannels are fabricated in PDMS using microlithographic techniques. After this, a PDMS well is aligned along the edge of microchannels under the microscope (1). NGF immobilization using aryl-azido photolinkers (2A) or laminin coating (2B) is performed inside the PDMS well. After extensive washing, the PDMS well is removed (3), which finally renders an area modified with chemical ligands that is parallel to microchannels. Hippocampal neurons are subsequently cultured on the modified PDMS substrates for competition studies (4). Random cell culture is used to test the effect of an individual stimulus on polarization (dotted ovals show cells analysed). Micropositioning is used when testing competition between two cues. Note: parallel and perpendicular are defined based on the position of the cell body relative to the long axis of microchannels as illustrated in the schematic; (b) and (c) phase-contrast and fluorescence images, respectively, of interface between microchannels and immobilized NGF–FITC; (d) higher magnification image (overlay of phase-contrast and fluorescence images) of the interface between microchannels and immobilized NGF (enclosed area in b and c). The two stimuli were designed to be simultaneous but spatially independent for studying competition in neuronal polarization. Scale bars, (b,c) 100 μm, (d) 25 μm.

Figure 2

Effect of individual stimuli on neuron polarization. (a–c) Hippocampal neurons that were adjacent to the interface of microchannels were analysed for direction of axon formation. (a,b) Phase-contrast and fluorescence (tau-1-labelled) images, respectively, of a neuron next to 2 μm microchannels. Axon is established on the surface with topographical features. Scale bar, 25 μm; (c) quantitative analysis of polarization of neurons next to the microchannels. The x-axis represents the dimensions and the orientation of the microchannels (perpendicular and parallel definitions are illustrated in figure 1a). The y-axis represents the percentage of cells that establish axons on either the microchannels (physical) or smooth areas (no stimulus). The majority of neurons polarized towards the micropatterned area (p=0.003, 0.02, 0.003 and 0.008 for each dimension from left to right in the x-axis, respectively). (d–f) Hippocampal neurons that were adjacent to the interface of immobilized NGF or laminin were analysed for direction of axon formation. (d) Phase-contrast image; (e) overlay of tau-1 and NGF labelling images of a neuron next to immobilized NGF. Axon is established on the surface with immobilized NGF. Scale bar, 25 μm; (f) quantitative analysis of polarization of neurons next to chemical stimuli. The x-axis represents the type of ligand at the interface. The y-axis represents the percentage of cells that establish axons on either the chemical ligand or unmodified areas. The majority of neurons polarized towards the chemical stimulus (p<0.001 for immobilized NGF; p=0.01 for laminin).

Figure 3

Competition between chemical and physical stimuli on neuron polarization. Hippocampal neurons were micropositioned between immobilized NGF or laminin and microchannels, allowed to extend multiple premature neurites in all directions and then analysed for the orientation and formation of a single axon (tau-1 positive labelling) after 44 h in culture. (a) Overlay of phase-contrast and fluorescence (green labelling for tau-1, red labelling for laminin) images of neuron between 2 μm microchannels and laminin. Axon was established on the surface with microchannels. Scale bar, 25 μm; (b) SEM image of neuron in (a) (arrowhead; pseudo-coloured for visualization). Scale bar, 12 μm; (c) quantitative analysis of polarization of neurons between chemical and physical stimuli. The x-axis represents the specific competition pair. The y-axis represents the percentage of cells that establish axons on either the chemical or physical stimuli. The majority of neurons polarized towards the physical stimulus (p=0.2, 0.02 and 0.03 for each combination from left to right in the x-axis, respectively).

Figure 4

Competition between combined microchannels and immobilized NGF and microchannels alone. Hippocampal neurons were micropositioned between microchannels with immobilized NGF and microchannels only, and analysed for the direction of axon formation. (a) Overlay of phase-contrast and fluorescence (green labelling for tau-1; red labelling for NGF) images of neuron between 2 μm microchannels and combined 2 μm microchannels with immobilized NGF. Axon was established on the surface with the combined cues. Scale bar, 25 μm; (b) SEM image of neuron in (a) (arrowhead; pseudo-coloured for visualization). Scale bar, 12 μm; (c) quantitative analysis of polarization of neurons between combined stimuli and microchannels only. The x-axis represents the specific competition pair. The y-axis represents the percentage of cells that established axons on either the combination or microchannels only (p=0.35 and 1 for combination and microchannels only, respectively).