Most small unmyelinated neurons in adult rat dorsal root ganglia (DRG) express one or more of the coreceptors targeted by glial cell line-derived neurotrophic factor (GDNF), neurturin, and artemin (GFRalpha1, GFRalpha2, and GFRalpha3, respectively). The function of these GDNF family ligands (GFLs) is not fully elucidated but recent evidence suggests GFLs could function in sensory neuron regeneration after nerve injury and peripheral nociceptor sensitization. In this study we used immunohistochemistry to determine if the DRG neurons targeted by each GFL change after sciatic nerve injury. We compared complete sciatic nerve transection and the chronic constriction model and found that the pattern of changes incurred by each injury was broadly similar. In lumbar spinal cord there was a widespread increase in neuronal GFRalpha1 immunoreactivity (IR) in the L1-6 dorsal horn. GFRalpha3-IR also increased but in a more restricted area. In contrast, GFRalpha2-IR decreased in patches of superficial dorsal horn and this loss was more extensive after transection injury. No change in calcitonin gene-related peptide-IR was detected after either injury. Analysis of double-immunolabeled L5 DRG sections suggested the main effect of injury on GFRalpha1- and GFRalpha3-IR was to increase expression in both myelinated and unmyelinated neurons. In contrast, no change in basal expression of GFRalpha2-IR was detected in DRG by analysis of fluorescence intensity and there was a small but significant reduction in GFRalpha2-IR neurons. Our results suggest that the DRG neuronal populations targeted by GDNF, neurturin, or artemin and the effect of exogenous GFLs could change significantly after a peripheral nerve injury.

Bladder sensation is mediated by lumbosacral dorsal root ganglion neurons and is essential for normal voiding and nociception. Numerous electrophysiological, structural, and molecular changes occur in these neurons following inflammation. Defining which neurons undergo these changes is critical for understanding the mechanism underlying bladder pain and dysfunction. Our first aim was to define the chemical classes of bladder sensory neurons that express receptors for the endogenous modulators of nociceptor sensitivity, glial cell line-derived neurotrophic factor (GDNF), the related neurotrophic factor, artemin, and estrogens. Bladder sensory neurons of adult female Sprague-Dawley rats were identified with retrograde tracer. Diverse groups of neurons express these receptors, and some neurons express receptors for both neurotrophic factors and estrogens. Lumbar and sacral sensory neurons showed some distinct differences in their expression profile. We also distinguished the chemical profile of myelinated and unmyelinated bladder sensory neurons. Our second aim was to identify bladder sensory neurons likely to be undergoing structural remodeling during inflammation. Following systemic administration of cyclophosphamide (CYP), its renal metabolite acrolein causes transient urothelial loss, exposing local afferent terminals to a toxic environment. CYP induced expression of the injury-related immediate-early gene product, activating transcription factor-3 (ATF-3), in a small population of sacral nitrergic bladder sensory neurons. In conclusion, we have defined the bladder sensory neurons that express receptors for GDNF, artemin and estrogens. Our study has also identified a sub-population of sacral sensory neurons that are likely to be undergoing structural remodeling during acute inflammation of the bladder. Together these results contribute to increased understanding of the neurons that are known to be involved in pain modulation and hyperreflexia during inflammation.

Neuropathic pain caused by nerve damage is a common and severe class of chronic pain. Disease-modifying clinical therapies are needed as current treatments typically provide only symptomatic relief; show varying clinical efficacy; and most have significant adverse effects. One approach is targeting either neurotrophic factors or their receptors that normalize sensory neuron function and stimulate regeneration after nerve damage. Two candidate targets are glial cell line-derived neurotrophic factor (GDNF) and artemin (ARTN), as these GDNF family ligands (GFLs) show efficacy in animal models of neuropathic pain (Boucher et al., 2000; Gardell et al., 2003; Wang et al., 2008, 2014). As these protein ligands have poor drug-like properties and are expensive to produce for clinical use, we screened 18,400 drug-like compounds to develop small molecules that act similarly to GFLs (GDNF mimetics). This screening identified BT13 as a compound that selectively targeted GFL receptor RET to activate downstream signaling cascades. BT13 was similar to NGF and ARTN in selectively promoting neurite outgrowth from the peptidergic class of adult sensory neurons in culture, but was opposite to ARTN in causing neurite elongation without affecting initiation. When administered after spinal nerve ligation in a rat model of neuropathic pain, 20 and 25 mg/kg of BT13 decreased mechanical hypersensitivity and normalized expression of sensory neuron markers in dorsal root ganglia. In control rats, BT13 had no effect on baseline mechanical or thermal sensitivity, motor coordination, or weight gain. Thus, small molecule BT13 selectively activates RET and offers opportunities for developing novel disease-modifying medications to treat neuropathic pain.

GDNF (glial cell line-derived neurotrophic factor), neurturin and artemin use their co-receptors (GFRα1, GFRα2 and GFRα3, respectively) and the tyrosine kinase Ret for downstream signaling. In rodent dorsal root ganglia (DRG) most of the unmyelinated and some myelinated sensory afferents express at least one GFRα. The adult function of these receptors is not completely elucidated but their activity after peripheral nerve injury can facilitate peripheral and central axonal regeneration, recovery of sensation, and sensory hypersensitivity that contributes to pain. Our previous immunohistochemical studies of spinal cord and sciatic nerve injuries in adult rodents have identified characteristic changes in GFRα1, GFRα2 or GFRα3 in central spinal cord axons of sensory neurons located in DRG. Here we extend and contrast this analysis by studying injuries of the pelvic and hypogastric nerves that contain the majority of sensory axons projecting to the pelvic viscera (e.g., bladder and lower bowel). At 7 d, we detected some effects of pelvic but not hypogastric nerve transection on the ipsilateral spinal cord. In sacral (L6-S1) cord ipsilateral to nerve injury, GFRα1-immunoreactivity (IR) was increased in medial dorsal horn and CGRP-IR was decreased in lateral dorsal horn. Pelvic nerve injury also upregulated GFRα1- and GFRα3-IR terminals and GFRα1-IR neuronal cell bodies in the sacral parasympathetic nucleus that provides the spinal parasympathetic preganglionic output to the pelvic nerve. This evidence suggests peripheral axotomy has different effects on somatic and visceral sensory input to the spinal cord, and identifies sensory-autonomic interactions as a possible site of post-injury regulation.