Axon Growth and Guidance

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Despite the tremendous effort put into developing 3D porous hydrogels as conduits for spinal cord repair, the effects of gel mechanical properties on axon growth remain largely unknown. This is due to the difficulty of isolating stiffness from other parameters, such as porosity and degradation rate Macaya and Spector, A promising new material is the self-assembling peptide nanofiber gels, which promote neural growth when embellished with laminin-like sequences Sur et al.

While stiffness has an immediate effect on the growth cone—substrate mechanical coupling, geometrically restraining focal adhesions offers alternative means of control.

Substrates decorated with patterns of adhesive regions Hardelauf et al. Patterns of adhesive regions can be created via microcontact printing, where molecules of interest are transferred to the substrate using an elastomeric stamp Vogt et al. Alternative approaches include reactive plasma etching, where the stamp is used to protect a pattern from being etched away due to oxygen plasma Kim et al.

The latter was used to generate intricate patterns of polylysine Figure 1A to the study cytoskeletal organization in axon collateral branching Withers et al. Multi-step patterning can be used to create complex patterns of multiple cell adhesion molecules CAMs , which selectively promote axonal or dendritic growth Shi et al.

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Submicron feature sizes can be achieved via photopatterning, crosslinking of functional groups on a non-adhesive monolayer using a focused femtosecond laser. Finally, micropatterning is not limited to adhesive molecules: axonal guidance cues von Philipsborn et al. Figure 1. Examples of novel growth cone microenvironments. Adapted with permission from Withers et al.

Adapted from Scott et al. Adapted with permission from Xie et al.

Optogenetic activation of axon guidance receptors controls direction of neurite outgrowth

Adapted with permission from Froeter et al. Copyright American Chemical Society. Anisotropic features, e. Forming contact with a structural handle is sufficient for an early stage neuron to polarize, as evidenced by N-cadherin clustering at the contact site, where an axon is subsequently initiated Micholt et al.

Contact-mediated signaling was also observed at isolated anchorages, e. Anava et al. Topography is suggested to modulate growth cone-substrate mechanical interactions at the molecular level Moore and Sheetz, Accordingly, interaction with nanoscale anchorages increased focal adhesion density thus traction force in growth cone filopodia, suggesting a curvature-sensing mechanism based on asymmetric torque generation across the growth cone Spedden et al. In 3D, nanotopography is readily introduced in electrospun nanofiber gels. Offering a variety of polymer formulations and biofunctionalization, these uniaxial scaffolds promote axon alignment and elongation Schnell et al.

Perpendicular ladder but not parallel rope growth was myosindependent, suggesting that nanoscale features are more than structural handles for growth cones. Microfluidic devices provide additional means of microenvironmental control, such as imposing chemical gradients or fluidically isolating subcellular structures. These are important aspects of axon guidance in vivo , where growth cones experience different chemical environments than their somata and are typically exposed to chemotactic gradients.

Gradients of substrate-bound cues can be imposed using flow-based devices Dertinger et al. The latter can be achieved by flanking a culture chamber with source and sink channels and connecting them with narrow microchannels. Importantly, if the microchannels are narrow enough to permit the crossing of axons but not somata, axons can be fluidically isolated Taylor et al.

Bicompartmental devices thus allow probing growth cone-substrate interactions independent of other factors Hur et al. Indeed, axon turning frequency decreases with increasing turning angles Francisco et al. Unlike passively modulating growth cone behavior through changing its microenvironment, external force application actively stimulates mechanosensitive pathways. Interestingly, over long timescales, central and peripheral neurons exhibited drastically different fluid-like vs. The last decade witnessed the development of microtechnologies for growth cone force application.

Force can be applied indirectly through simply counteracting the force exerted by the growth cone cytoskeleton. Here, the targeted CAM is coupled to the actin retrograde flow, the constant inward movement of F-actin as it polymerizes at the leading edge and depolymerizes at the inner regions of the growth cone. Restraining the bead interrupts the retrograde flow and diverts the growth cone through locally reorganizing its actin cytoskeleton, followed by the advance of microtubules towards the bead Schaefer et al.

Figure 2. Examples of growth cone force application and measurement. Note the cytoskeletal alignment and the actin-rich arc around the bead. Adapted with permission from Decourt et al. C Three-compartmental microfluidic device for isolating axons and exposing them to linear concentration gradients. Soma, somatic; Axon, axonal; Dist, distal. Growth cones of mouse cortical neurons targeted with neural cell adhesion molecule NCAM -functionalized 1.

Arrows indicate force direction. Overlay color indicates Semaphorin 3A gradient. Adapted from Kilinc et al.

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D Combination of adhesive patterning and force application. Broken line indicates magnet position. Arrow indicates force direction. Adapted with permission from Kunze et al. Classical MTW involves plasma membrane-bound targets.

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Recently, researchers started using internalized nanoparticles as MTW handles: upon exposure to magnetic field, nerve growth factor NGF -functionalized nanoparticles accumulated in the growing tips of PC12 neurites and affected their orientation Riggio et al. Nanoparticles have also been functionalized with agonists of signaling pathways to control axon growth. Localizing Tropomyosin receptor kinase B TrkB pathway agonists to growth cone periphery via MTW forces interfered with the local F-actin remodeling and affected growth cone advance Steketee et al.

Finally, axons with internalized nanoparticles can be stimulated in parallel, using micromagnet arrays embedded in the substrate Kunze et al. In summary, bead-based microtechnologies, MTW in particular, now offer a wide range of forces to be exerted on growth cones, externally or from within. MTW is becoming more and more accessible to neurobiologists, thanks to the recent progress in particle technology and the emergence of patterned micromagnets. A complete understanding of growth cone mechanics requires the characterization of mechanical properties of growth cones and the forces they generate.

Due to their smaller size, the visco elastic properties of mammalian growth cones cannot be characterized with this technique. Instead, local stresses can be estimated from the actin retrograde flow while the growth cone is being compressed Betz et al. Once the local stress distribution is known, the internal force field can be calculated. We mentioned that force can be exerted by restraining a microbead bound to a CAM that is coupled to the underlying cytoskeleton. If the bead is restrained using a calibrated optical trap, the exerted force can be determined Cojoc et al.

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Although this measurement can be made at multiple spots by employing optical holograms Mejean et al. It is, however, possible to map out traction forces, which the growth cones exert to the substrate via focal adhesions. Through time-lapse imaging of fluorescent markers embedded in a soft gel, the strain field thus the force field can be measured Koch et al. Alternatively, neurons can be cultured on micropillar arrays.

Force acting on each micropillar can be calculated from its bending, which is a function of its stiffness, height and diameter. Despite being available for over a decade Tan et al. As growth cones harness mechanical and biochemical stimuli alike, experimental models that separately control the two types of stimuli are sought-after. For example, growth behavior has been investigated in 2D by presenting neurons with competing topographical parallel grooves and chemotactic immobilized NGF cues, where the former dominated axon polarization Gomez et al.

Interestingly, topographical cues exhibited a strong effect on axon initiation, but a weaker effect on axon elongation after initiation Gomez et al.


As mentioned earlier, self-assembling peptide nanofiber gels now provide independent control on the matrix stiffness Sur et al. Effects of topography maybe also matrix stiffness and of haptotactic cues can also be compared in 3D by culturing cells on an electrospun nanofiber gel with a gradient of surface-bound laminin Zander and Beebe, When the gradient was parallel to the nanofibers, PC12 neurites grew preferentially up the laminin gradient; however, when the gradient was imposed in the perpendicular direction, they were unaffected.

These examples show that combinatorial approaches can be very effective in designing growth cone microenvironments. Direct force application has recently been combined with topographical, chemotactic, and adhesive cues to study neuron polarization and axon growth. Neural stem cells have been cultured on an elastic substrate with microgrooves to compare stretch-induced and microtopography-induced axon orientation and growth Chang et al.

Neurites stretched parallel to the microchannel geometry grew longer and displayed higher neuronal gene expression, compared to those stretched in the perpendicular direction. We have recently presented a microfluidic device that not only fluidically isolates axons from somata, but also exposes them to chemotactic gradients parallel to their elongation direction Kilinc et al. By subjecting axons and not somata to a panel of inhibitory molecules, we demonstrated that low pN forces can steer CNS axons towards potent axon repellents, when their molecular motors are locally inhibited.

A number of techniques for measuring and applying forces have emerged that can be implemented to study the mechanics of axon growth in the coming years. These sensors have already been incorporated into several focal adhesion proteins, including integrin Morimatsu et al.

In addition, nanoparticle tension probes, based on distance-dependent fluorescent quenching of substrate-bound gold nanoparticles, can report tension across individual integrins with submicron resolution Liu et al. An emerging technology for massively parallel force application is acoustic tweezers, the manipulation of microparticles using standing acoustic waves Ding et al.

Similarly, standing magnetic field waves can be generated using a micromagnet array to control the motion of individual magnetic beads in parallel Li et al. Our understanding of the mechanics of axon guidance is in its infancy compared to its biochemistry.

Recent and emerging microtechnologies now provide us with a range of tools that can be integrated into sophisticated experimental platforms for closing this gap. The ideal experimental platform will allow us to manipulate and precisely measure the mechanical interactions in and around a growth cone, in a tightly controlled microenvironment.

Bibliographic Information

Definition. Axon guidance is the process by which growing nerve fibers find their targets in the developing brain. Determining how these signaling pathways function to regulate axon growth and guidance is fundamentally important to understanding wiring specificity in the.

Such a high level of control will help us dissect the mechanotransduction mechanisms in growth cones downstream of other, e.