Enhanced Artemin/GFRα3 Levels Regulate Mechanically Insensitive, Heat-Sensitive C-Fiber Recruitment after Axotomy and Regeneration

We have shown recently that following saphenous nerve transection and successful regeneration, cutaneous polymodal nociceptors (CPMs) lacking transient receptor potential vanilloid 1 (TRPV1) are sensitized to heat stimuli and that mechanically insensitive, heat-sensitive C-fibers (CHs) that contain TRPV1 increase in prevalence. Target-derived neurotrophic factor levels were also enhanced after axotomy and regeneration. In particular, the glial-cell line-derived neurotrophic factor (GDNF) family member artemin was found to be significantly enhanced in the hairy hindpaw skin and its receptor GDNF family receptor alpha 3 (GFR alpha 3) was increased in the L2/L3 dorsal root ganglia (DRGs) following nerve injury. In this study, we assessed the role of enhanced artemin/GFR alpha 3 levels on the changes in mouse cutaneous CH neurons following saphenous nerve regeneration. We used a newly developed siRNA-mediated in vivo knockdown strategy to specifically inhibit the injury-induced expression of GFR alpha 3 and coupled this with an ex vivo recording preparation to examine response characteristics and neurochemical phenotype of different types of functionally defined neurons after injury. We found that inhibition of GFR alpha 3 did not affect the axotomy-induced decrease in CPM threshold, but transiently prevented the recruitment of CH neurons. Western blot and real-time PCR analysis of hairy hindpaw skin and L2/L3 DRGs after saphenous nerve regeneration suggested that inhibition of the potential initial injury-induced increase in enhanced target-derived artemin signaling resulted in dynamic changes in TRPV1 expression after regeneration. These changes in TRPV1 expression may underlie the functional alterations observed in CH neurons after nerve regeneration.