RET signaling is required for survival and normal function of nonpeptidergic nociceptors

Abstract

Small unmyelinated sensory neurons classified as nociceptors are divided into two subpopulations based on phenotypic differences, including expression of neurotrophic factor receptors. Approximately half of unmyelinated nociceptors express the NGF receptor TrkA, and half express the GDNF family ligand (GFL) receptor Ret. The function of NGF/TrkA signaling in the TrkA population of nociceptors has been extensively studied, and NGF/TrkA signaling is a well established mediator of pain. The GFLs are analgesic in models of neuropathic pain emphasizing the importance of understanding the physiological function of GFL/Ret signaling in nociceptors. However, perinatal lethality of Ret-null mice has precluded the study of the physiological role of GFL/Ret signaling in the survival, maintenance, and function of nociceptors in viable mice. We deleted Ret exclusively in nociceptors by crossing nociceptor-specific Na(v)1.8 Cre and Ret conditional mice to produce Ret-Na(v)1.8 conditional knock-out (CKO) mice. Loss of Ret exclusively in nociceptors results in a reduction in nociceptor number and size, indicating that Ret signaling is important for the survival and trophic support of these cells. Ret-Na(v)1.8 CKO mice exhibit reduced epidermal innervation but normal central projections. In addition, Ret-Na(v)1.8 CKO mice have increased sensitivity to cold and increased formalin-induced pain, demonstrating that Ret signaling modulates the function of nociceptors in vivo. Enhanced inflammation-induced pain may be mediated by decreased prostatic acid phosphatase (PAP), as PAP levels are markedly reduced in Ret-Na(v)1.8 CKO mice. The results of this study identify the physiological role of endogenous Ret signaling in the survival and function of nociceptors.

Figure 1.

The conditional Ret allele is deleted with high efficiency and specificity in nonpeptidergic nociceptors by Cre expressed from the Na_v1.8 locus. A, C, D, Transverse sections of spinal cord and DRG from PND 0 mice showing EGFP expression restricted to the DRG (A) and to afferents in the superficial laminae of the dorsal horn (C) and no EGFP in motor neurons (D) in the Ret-Na_v1.8 Het mouse. B, E, F, EGFP expression in DRG neurons (B), motor neurons in the ventral horn (F), and cell bodies in the dorsal horn (E) of the Ret ^EGFP/+ reporter mouse. G–I, PND 1 DRG from a Ret-Na_v1.8 Het mouse showing virtually complete overlap of EGFP expression (G) with IB4 label (H). Images in G–I are representative of 3 mice. Scale bars: A, B, 100 μm; G–I, 30 μm.

Figure 2.

Ret expression is not detected in small-diameter DRG neurons in Ret-Na_v1.8 CKO mice. A, B, DRG from a PND 60 WT mouse (A) and a Ret-Na_v1.8 CKO mouse (B) labeled with an anti-Ret antibody showing loss of Ret exclusively in small-diameter DRG neurons in the Ret-Na_v1.8 CKO mouse (B). Photographs are representative of the staining pattern observed in three mice of each genotype. C–E, DRG from a PND 10 Ret-Na_v1.8 CKO mouse showing no overlap of EGFP (C) with anti-Ret antibody (D). Merged image of C and D is shown in E. There are two cells in E in which EGFP and Ret appear to overlap. The cell on the right is a large-diameter Ret-positive neuron with an EGFP-positive neuron on top of it. It can be seen that EGFP is not present in the same cell, since the EGFP label fills a smaller area than the Ret label. In addition, the cells are not in the same plane of focus. The cell on the left is not EGFP positive, but exhibits autofluorescence, which is clearly different from the very bright EGFP labeling seen in the cell immediately to the left. Scale bars: (in B) A, B, 20 μm; (in E) C–E, 30 μm.

Figure 3.

The number and size of nonpeptidergic nociceptors is decreased in Ret-Na_v1.8 CKO mice. A–C, The total number of neurons is decreased in the L4 DRG of adult Ret-Na_v1.8 CKO mice (B, C; n = 4) compared to control mice (A, C; n = 8) (*p < 0.05). D–F, The number of EGFP-positive DRG neurons is decreased in L4 DRG of Ret-Na_v1.8 CKO mice (E, F) compared to Ret-Na_v1.8 Het control mice (D, F) (F, n = 3 of each; *p = 0.01). D, E, G, H, The soma area of EGFP-positive DRG neurons is decreased in Ret-Na_v1.8 CKO mice (E) compared to Ret-Na_v1.8 Het control mice (D) (G, n = 3 of each; *p < 0.05). H, Size frequency analysis of EGFP-positive neurons. I–K, EGFP (Ret) and GFRα3 do not overlap extensively in lumbar DRG of Ret-Na_v1.8 Het control mice (I). Overlap of EGFP and GFRα3 is also not extensive in Ret-Na_v1.8 CKO mice (J) but is significantly increased compared to Ret-Na_v1.8 Het control mice (K, n = 4 Ret-Na_v1.8 CKO mice, n = 5 control mice, **p < 0.01). In C, F, G, and K, data are represented as mean ± SEM. Scale bars: (in E) D, E, 30 μm; (in J) I, J, 50 μm.

Figure 4.

The density of nonpeptidergic fibers is decreased in the epidermis of Ret-Na_v1.8 CKO mice. The density of EGFP-positive fibers (A, D, G) in the epidermis is significantly decreased in Ret-Na_v1.8 CKO mice (D) compared to Ret-Na_v1.8 Het control mice (A) (n = 4 for each genotype, **p < 0.005). Total epidermal innervation, as measured by the density of βIII tubulin-positive fibers (B, E, G), is not different in Ret-Na_v1.8 CKO mice (E) from that in Ret-Na_v1.8 Het controls (B) (n = 8 for each genotype). The density of CGRP-positive fibers (C, F, G) is also not different in Ret-Na_v1.8 CKO mice (F) from that in Ret-Na_v1.8 Het control mice (C) (n = 7 for each genotype). In G, data are represented as mean ± SEM. Scale bar: (in F) A–F, 50 μm.

Figure 5.

The density and topography of the central projections of nonpeptidergic nociceptors appear normal in Ret-Na_v1.8 CKO mice. The density and topography of the central projections from nonpeptidergic DRG neurons in the superficial laminae of the lumbar dorsal horn of Ret-Na_v1.8 CKO mice (A, C, E) are not changed compared with Ret-Na_v1.8 Het control mice (B, D, F). Photos are representative of four mice of each genotype. Scale bar: (in F) A–F, 50 μm.

Figure 6.

Ret-Na_v1.8 CKO mice have normal responses to mechanical and noxious heat stimuli but are hypersensitive to a cold stimulus. A, Mechanical sensitivity is not changed in Ret-Na_v1.8 CKO mice compared to control mice. Left, von Frey, Ret-Na_v1.8 CKO mice, n = 10; control mice n = 17. Right, Randall–Selitto, n = 14 of each genotype. B, Noxious heat sensitivity is unchanged in female and male Ret-Na_v1.8 CKO mice compared with female and male control mice (females: Ret-Na_v1.8 CKO mice, n = 11; control, n = 38; males: Ret-Na_v1.8 CKO mice, n = 8; control, n = 17). Female Ret-Na_v1.8 CKO and control mice have a significantly shorter withdrawal latency than male Ret-Na_v1.8 CKO and control mice (female vs male Ret-Na_v1.8 CKO mice, p < 0.05; female vs male control, p < 0.000001). C, D, Female Ret-Na_v1.8 CKO mice are hyper-responsive to acetone compared to female control mice. C, Female Ret-Na_v1.8 CKO mice (n = 11) respond to a significantly greater percentage of acetone applications than female control mice (n = 24) (**p < 0.001). Male Ret-Na_v1.8 CKO mice (n = 10) are not different from male controls (n = 18) in percentage of responses to acetone applications. Female control mice (n = 24) respond to a significantly lower percentage of acetone applications than male control mice (n = 18) (*p < 0.05). D, Female Ret-Na_v1.8 CKO mice (n = 7) spend significantly more time engaged in pain behavior than female control mice (n = 11) following acetone application (*p = 0.05). Male Ret-Na_v1.8 CKO mice (n = 4) are not different from male control mice (n = 9) in the amount of time spent in pain behavior. Female control mice (n = 11) are not different from male control mice (n = 9) in the amount of time spent in pain behavior. Data are represented as mean ± SEM.

Figure 7.

Ret-Na_v1.8 CKO mice exhibit increased formalin-induced pain behavior. Ret-Na_v1.8 CKO (n = 9) mice show increased sensitivity to formalin compared to control mice (n = 11). A, Time course of spontaneous pain behavior following intraplantar injection of formalin. B, Total time spent in spontaneous pain behavior in phase 1 (5–10 min) and phase 2 (10–60 min) of the formalin test. Ret-Na_v1.8 CKO mice are different from control mice in both phases (phase 1, **p < 0.01; phase 2, *p < 0.05). Data are represented as mean ± SEM.

Figure 8.

The number of TrkA-positive neurons is unchanged in lumbar DRG from Ret-Na_v1.8 CKO and TMP (PAP) expression is decreased in the dorsal horn of the spinal cord of Ret-Na_v1.8 CKO mice. The percentage of EGFP-positive neurons that coexpress TrkA is increased in Ret-Na_v1.8 CKO mice (B, C, left) compared with Ret-Na_v1.8 Het control mice (A, C, left) (*p < 0.05, n = 4 of each genotype). Arrows indicate EGFP/TrkA double-labeled neurons. C, Right, The total number of TrkA-positive neurons is not different in Ret-Na_v1.8 CKO mice from that in Ret-Na_v1.8 Het control mice (n = 4 of each genotype). D, E, TMP (PAP) is decreased in the superficial laminae of the dorsal horn of the spinal cord in Ret-Na_v1.8 CKO mice (E) compared with Ret-Na_v1.8 Het control mice (D). IB4 labeling is the same in Ret-Na_v1.8 Het control (F) and Ret-Na_v1.8 CKO mice (G). Images shown in D and E are representative of eight mice of each genotype, F and G are representative of 6 mice of each genotype. In C, data are represented as mean ± SEM. Scale bars: B (for A, B), G (for D–G), 50 μm.