Regeneration of embryonic and adult dorsal root ganglion (DRG) sensory axons is highly impeded when they encounter neuronal growth cone-collapsing factor semaphorin3A (Sema3A). On the other hand, increasing evidence shows that DRG axon's regeneration can be stimulated by nerve growth factor (NGF). In this study, we aimed to evaluate whether increased NGF concentrations can counterweight Sema3A-induced inhibitory responses in 15-day-old mouse embryo (E15) DRG axons. The DRG explants were grown in Neurobasal-based medium with different NGF concentrations ranging from 0 to 100 ng/mL and then treated with Sema3A at constant 10 ng/mL concentration. To evaluate interplay between NGF and Sema3A number of DRG axons, axon outgrowth distance and collapse rate were measured. We found that the increased NGF concentrations abolish Sema3A-induced inhibitory effect on axon outgrowth, while they have no effect on Sema3A-induced collapse rate.

Semaphorin 3A (Sema3A) is a secreted protein involved in axon path-finding during nervous system development. Calcium signaling plays an important role during axonal growth in response to different guidance cues; however it remains unclear whether this is also the case for Sema3A. In this study we used intracellular calcium imaging to figure out whether Sema3A-induced growth cone collapse is a Ca2+ dependent process. Intracellular Ca2+ imaging results using Fura-2 AM showed Ca2+ increase in E15 mice dorsal root ganglia neurons upon Sema3A treatment. Consequently we analyzed Sema3A effect on growth cones after blocking or modifying intracellular and extracellular Ca2+ channels that are expressed in E15 mouse embryos. Our results demonstrate that Sema3A increased growth cone collapse rate is blocked by the non-selective R- and T- type Ca2+ channel blocker NiCl2 and by the selective R-type Ca2+ channel blocker SNX482. These Ca2+ channel blockers consistently decreased the Sema3A-induced intracellular Ca2+ concentration elevation. Overall, our results demonstrate that Sema3A-induced growth cone collapses are intimately related with increase in intracellular calcium concentration mediated by R-type calcium channels.

Development of adult mammal central nervous system (CNS) is closely related to loosing the ability spontaneously regenerate after injuries. On the other hand, peripheral nervous system (PNS) maintains its capability to regenerate after injuries entire lifespan. Ability to regenerate successfully is mainly determined by the balance of growth promoting and growth inhibiting factors, expressed by both neuronal and non-neuronal cells found in the injury site. Some of signaling cues involved in regeneration are expressed in adult CNS constantly, although expression of other factors occurs only in the injury site of adult mammal. Ephrins, Semaphorins, Slits and Netrins are among most important molecules involved in lack of success in regeneration of CNS. PNS neurons initiate reparation mechanisms right after development of injury, and are capable to recover functional activity even if an area of injury is more than several centimeters wide. Understanding of differences between CNS and PNS regeneration and factors involved in functional nervous system recovery are crucial for both in depth analysis of plasticity of adult mammal neural system, and for developing new treatment strategies.

The axonal growth cone is a specialized structure enabling axon extension and proper guidance to its target by sensing the extracellular environment. A growth cone collapse assay is a popular approach designed to characterize the inhibitory effect of secreted guidance cues in vitro. However, the actin cytoskeleton of the growth cone is very sensitive to various factors like physical impact, temperature, and acidity of environment that may also induce responses resembling those of guidance signals. Herein, we provide an easy and reproducible method to analyze growth cone sensitivity to the prototypic guidance molecule family class 3 semaphorin. This protocol is intended to present a systematic approach that is easy to apply to any soluble factors with a potential to impact axon elongation.

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