Deficient nonpeptidergic epidermis innervation and reduced inflammatory pain in glial cell line-derived neurotrophic factor family receptor alpha2 knock-out mice

Abstract

Most unmyelinated nociceptive neurons that mediate pain and temperature sensation from the skin bind isolectin B4 (IB4)-lectin and express Ret, the common signaling component of glial cell line-derived neurotrophic factor (GDNF) family. One of these factors, neurturin, is expressed in the epidermis, whereas its GDNF family receptor alpha2 (GFRalpha2) is expressed in the majority of unmyelinated Ret-positive sensory neurons. However, the physiological roles of endogenous neurturin signaling in primary sensory neurons are poorly understood. Here, we show that the vast majority (approximately 85%) of IB4 binding and P2X3 purinoreceptor-positive neurons, but virtually none of the calcitonin gene-related peptide (CGRP) or vanilloid receptor transient receptor potential vanilloid 1-positive neurons in mouse dorsal root ganglion (DRG) express GFRalpha2. In GFRalpha2 knock-out (KO) mice, the IB4-binding and P2X3-positive DRG neurons were present but reduced in size, consistent with normal number but reduced caliber of unmyelinated axons in a cutaneous nerve. Strikingly, nonpeptidergic (CGRP-negative) free nerve endings in footpad epidermis were >70% fewer in GFRalpha2-KO mice than in their wild-type littermates. In contrast, the density of CGRP-positive epidermal innervation remained unaffected. In the formalin test, the KO mice showed a normal acute response but a markedly attenuated persistent phase, indicating a deficit in inflammatory pain response. Behavioral responses of GFRalpha2-KO mice to innocuous warm and noxious heat were not blunted; the mice were actually markedly hypersensitive to noxious cold in tail immersion test. Overall, our results indicate a critical role for endogenous GFRalpha2 signaling in maintaining the size and terminal innervation of the nonpeptidergic class of cutaneous nociceptors in vivo.

Figure 1.

Colocalization of GFRα2 protein with markers of unmyelinated neurons in mouse DRG. Sections of wild-type (A–C, G–R) and GFRα2-KO (D–F) mouse lumbar L4 DRG are stained for IB4-binding (B, E), peripherin (H), P2X₃ (K), CGRP (N), and TRPV1 (Q) to estimate colocalization with GFRα2 protein (left; green). The yellow color in the merged images (right) indicates colocalization. No specific GFRα2 staining is seen in the DRG section from the GFRα2-KO mouse (D). Note that GFRα2-expressing neurons that are peripherin negative (arrowheads) appear to be similarly sized to neurons that are peripherin positive (arrows). Scale bars, 50 μm.

Figure 2.

IB4-lectin binding and P2X₃-positive neurons are smaller in GFRα2-KO mice. Representative images of IB4-binding (A, B), P2X₃-positive (D, E), and CGRP-positive (G, H) neurons in adult lumbar DRG from wild-type (A, D, G) and GFRα2-KO (B, E, H) mice, which were used to count the neurons and to generate the size distribution histograms (C, F, I), are shown. Note the atrophy of IB4-binding and P2X₃-positive but not CGRP-positive neurons in GFRα2-KO mice. Scale bars, 50 μm.

Figure 3.

Loss of PGP9.5-positive but not CGRP-positive free nerve endings in the footpad epidermis in GFRα2-KO mice. A, B, Conventional microscopic images of sections through the forepaw footpad skin from wild-type (A) and GFRα2-KO (B) mice stained for pan-neuronal marker PGP9.5. C, Quantification of PGP9.5- and CGRP-positive nerve fibers per unit length of epidermis indicates profound loss of PGP9.5 fibers (***p < 0.001; n = 8 mice in both genotypes), whereas the density of CGRP fibers is unchanged (n = 4 mice in both genotypes). Error bars represent SEM. D, E, Confocal microscopic images from sections through the footpad skin from wild-type (D) and GFRα2-KO (E) mice double stained for pan-neuronal marker PGP9.5 (green) and CGRP (red). Nuclei are counterstained with DAPI (blue). Arrowheads indicate PGP9.5 and CGRP double-positive fibers. Scale bars, 20 μm.

Figure 4.

IB4 binding is unaltered in the GFRα2-KO spinal cord. Cryosections of wild-type (A) and GFRα2-KO (B) mouse spinal cords were labeled with IB4 (green) and CGRP (red). Scale bar, 100 μm.

Figure 5.

Behavioral analysis of cutaneous sensory responses in GFRα2-KO mice. Results using mice in 129B6 hybrid and in congenic B6 or 129 backgrounds are shown. The number of mice in each test is shown in parentheses. A, B, GFRα2-KO mice do not differ from their wild-type littermates in the 52°C hot plate (A; 129B6, F_(1,70) = 1.26, p = 0.3; B6, F_(1,20) = 0.21, p = 0.7) or tail-withdrawal tests (B; 129B6, F_(1,23) = 0.17, p = 0.7; B6, F_(1,20) = 0.73, p = 0.4). C, D, Response latencies in GFRα2-KO mice are shorter than in their wild-type littermates at 49°C (C; 129B6, F_(1,48) = 5.15, *p = 0.03; 129, F_(1,12) = 5.49, *p = 0.04) and 4°C (D; 129B6, F_(1,23) = 42.58, ***p < 0.001; B6, F_(1,20) = 7.95, **p = 0.01). E, GFRα2-KO mice do not differ from their wild-type littermates in responses to the warm two-temperature choice test (36 vs 34°C, F_(1,14) = 0.05, p = 0.8; 35 vs 22°C, F_(1,14) = 0.24, p = 0.6). F, The second but not the first phase of formalin-induced pain behavior is impaired in GFRα2-KO mice. Hindpaw licking and shaking responses in wild-type (n = 10) and GFRα2-KO mice (n = 9) in a B6 background after intraplantar injection of 5% formalin are shown. ***p = 0.0001. Error bars represent SEM.