The mitogen-activated protein kinases (MAPKs) signal downstream from the small GTPases Rac/Rho/Cdc42 that are known to regulate axonal outgrowth from neurones. MAPKs may control sensory neurone axonal regeneration induced by nerve damage or axotomy via activation of transcription factors such as AP-1 and ATF2 and/or by direct interactions with cytoskeletal proteins. The aim of this study was to determine the role of MAPKs in sensory neurone regeneration using an in vitro system.

Levels of axonal regeneration in dissociated adult rat dorsal root ganglion (DRG) sensory neurone cultures were quantified by measuring total axon network and longest axon via immunocytochemistry and using the analysis program MetaMorph version 4.5. Data are expressed as means ± S.E.M. and statistical analysis was done using an ANOVA and a Tukey’s or Dunnett’s C post-hoc test.

Animals were killed according to current UK legislation. Dissociated DRG neurones were allowed to adhere and then treated with the ERK inhibitor U0126 (Promega) at a concentration of 10 mM, p38 inhibitor SB203580 (Calbiochem) at 2 mM or phosphinositide 3-kinase (PI 3-kinase) inhibitor LY294002 (Calbiochem) at 10 mM and nerve growth factor (NGF) at 10 ng ml^-1, then left overnight. The ERK inhibitor significantly reduced the level of the NGF-induced axonal network (2067 ± 1022 versus 8389 ± 2132 mm, P < 0.001, n = 3). The p38 and PI 3-kinase inhibitors did not significantly affect the level of NGF-induced axonal growth. The number of cells that responded to NGF was not affected by any inhibitor treatment.

Glial cell line-derived neurotrophic factor (GDNF) signals through a different subpopulation of neurones to NGF and can activate MAPKs. Dissociated DRG neurones were treated as above, using GDNF at a concentration of 10 ng ml^-1. The PI 3-kinase inhibitor significantly reduced the level of the GDNF-induced axonal network (2738.3 ± 500.5 versus 7941.7 ± 2169.1 mm, P < 0.05, n = 4). The p38 inhibitor did not significantly affect the level of the GDNF-induced axon network, nor did ERK inhibition. ERK inhibition did, however, cause a significant decrease in the GDNF-enhanced level of growth of the longest axon (515.6 ± 29.6 versus 646.1 ± 52.9 mm, P < 0.05, n = 12). Combined p38 and ERK inhibition significantly reduced the level of the GDNF-induced axon network (1955 ± 591.6 versus 6453.7 ± 2320.1 mm, P < 0.05, n = 5).

The results show that NGF signalling through ERK is required for maintenance of regenerative growth but not needed for initiation of axonal outgrowth. However, NGF signalling through p38 is not required for maintenance or initiation of axonal outgrowth. GDNF signalling through PI 3-kinase is required for maintenance of axon regeneration and GDNF activation of ERK enhances neurite elongation but does not affect total axon outgrowth. GDNF signalling through p38 and ERK in combination is required for maintenance and possibly initiation of regenerative axonal outgrowth.

This work was supported by BBSRC and Pfizer.

All procedures accord with current UK legislation.