Low Piconewton Towing of CNS Axons against Diffusing and Surface-Bound Repellents Requires the Inhibition of Motor Protein-Associated Pathways

Abstract

Growth cones, dynamic structures at axon tips, integrate chemical and physical stimuli and translate them into coordinated axon behaviour, e.g., elongation or turning. External force application to growth cones directs and enhances axon elongation in vitro; however, direct mechanical stimulation is rarely combined with chemotactic stimulation. We describe a microfluidic device that exposes isolated cortical axons to gradients of diffusing and substrate-bound molecules, and permits the simultaneous application of piconewton (pN) forces to multiple individual growth cones via magnetic tweezers. Axons treated with Y-27632, a RhoA kinase inhibitor, were successfully towed against Semaphorin 3A gradients, which repel untreated axons, with less than 12 pN acting on a small number of neural cell adhesion molecules. Treatment with Y-27632 or monastrol, a kinesin-5 inhibitor, promoted axon towing on substrates coated with chondroitin sulfate proteoglycans, potent axon repellents. Thus, modulating key molecular pathways that regulate contractile stress generation in axons counteracts the effects of repellent molecules and promotes tension-induced growth. The demonstration of parallel towing of axons towards inhibitory environments with minute forces suggests that mechanochemical stimulation may be a promising therapeutic approach for the repair of the damaged central nervous system, where regenerating axons face repellent factors over-expressed in the glial scar.

Neuron Subpopulations with Different Elongation Rates and DCC Dynamics Exhibit Distinct Responses to Isolated Netrin-1 Treatment

“…(ii) Does soma-axon communication coordinate axonal Netrin-1 response? The former question can be addressed with different microfluidic designs involving concentration gradients that are parallel 56 or orthogonal 33 to the direction of initial axonal growth. The latter question can be addressed by using the circuit design presented in this study by treating fluidically-isolated somata and subsequently observing axon behavior.…”

“…Mouse cortical neurons were obtained from embryonic day 14 CD-1 outbred mouse and dissociated according to established protocols. 56 Neurons were seeded in the somatic chamber at a density of approximately 8×10 5 cells/cm 2 and maintained in a tissue culture incubator (5% CO 2 , 37°C) in DMEM-Glutamax medium containing 10% fetal bovine serum, Pen/Strep, and B 27 neuron supplement (all from Gibco, Dun Laoghaire, Ireland). At 3 DIV, the medium was replaced with Neurobasal medium (Gibco) containing Pen/Strep and B 27 .…”

Charge and topography patterned lithium niobate provides physical cues to fluidically isolated cortical axons

“…Immunocytochemistry in these devices followed the same protocol with minor adaptations as described previously. 30 Substrates that were bonded to microfluidic chips were also recovered and reused. Substrates were first sonicated in detergent solution for 30 min, and the chips were carefully removed by pushing from edges.…”

“…Finally, to test microfluidic integration with microtopography, we bonded a bicompartmental neuron culture device described. 30 The devices consist of two compartmentssomatic and axonal-interconnected by parallel microchannels that permit axonal growth. The "hexagonal pits" pattern was manually aligned with the axonal compartment during bonding such that axons emanating from the microchannels encountered an array of pits.…”

Acute and Chronic Neural Stimulation via Mechano-Sensitive Ion Channels

“…4C). 114 The inhibition of kinesin-5 motor promoted axonal towing on surfaces coated with axon repellents, suggesting the possibility of using mechano-chemical stimulation for nerve repair such as in a glia scar environment that overexpresses axonal repellents. 115 Gu and colleagues integrated nano-pumps into microfluidic culture to create cross-flows and found that the direction of fluid flow could influence axonal migration and cause axonal turning with an angle more than 90 o (Fig.…”