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. 2000 Jan 1;20(1):427-37.
doi: 10.1523/JNEUROSCI.20-01-00427.2000.

The glial cell line-derived neurotrophic factor family receptor components are differentially regulated within sensory neurons after nerve injury

D L Bennett  1 , T J Boucher, M P Armanini, K T Poulsen, G J Michael, J V Priestley, H S Phillips, S B McMahon, D L Shelton
Affiliations

The glial cell line-derived neurotrophic factor family receptor components are differentially regulated within sensory neurons after nerve injury

D L Bennett et al. J Neurosci. .

Abstract

Glial cell line-derived neurotrophic factor (GDNF) has potent trophic effects on adult sensory neurons after nerve injury and is one of a family of proteins that includes neurturin, persephin, and artemin. Sensitivity to these factors is conferred by a receptor complex consisting of a ligand binding domain (GFRalpha1-GFRalpha4) and a signal transducing domain RET. We have investigated the normal expression of GDNF family receptor components within sensory neurons and the response to nerve injury. In normal rats, RET and GFRalpha1 were expressed in a subpopulation of both small- and large-diameter afferents projecting through the sciatic nerve [60 and 40% of FluoroGold (FG)-labeled cells, respectively]. GFRalpha2 and GFRalpha3 were both expressed principally within small-diameter DRG cells (30 and 40% of FG-labeled cells, respectively). Two weeks after sciatic axotomy, the expression of GFRalpha2 was markedly reduced (to 12% of sciatic afferents). In contrast, the proportion of sciatic afferents that expressed GFRalpha1 increased (to 66% of sciatic afferents) so that virtually all large-diameter afferents expressed this receptor component, and the expression of GFRalpha3 also increased (to 66% of sciatic afferents) so that almost all of the small-diameter afferents expressed this receptor component after axotomy. There was little change in RET expression. The changes in the proportions of DRG cells expressing different receptor components were mirrored by alterations in the total RNA levels within the DRG. The changes in GFRalpha1 and GFRalpha2 expression after axotomy could be largely reversed by treatment with GDNF.

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Figures

Fig. 1.
Fig. 1.
Dark-field photomicrographs of sections of L4–L5 DRG after hybridization with probes for RET (A), GFRα1 (B), GFRα2 (C), GFRα3 (D), or a sense strand control probe (E). Scale bar, 50 μm.
Fig. 2.
Fig. 2.
Determination of GDNF family receptor component expression in afferents projecting through the sciatic nerve.A, FluoroGold image of L5 after labeling of the sciatic nerve. B, The same section after hybridization with the probe to RET. Single arrows indicate cells that are labeled by both retrograde tracer and the in situhybridization. A double-headed arrow indicates a cell that is FG-labeled but that does not express RET. Anasterisk demonstrates a cell that is not FG-labeled but that does express RET. Scale bar, 50 μm.
Fig. 3.
Fig. 3.
Dual-color images showing FG labeling of DRG cells projecting through the sciatic nerve before hybridization (red) and silver grain deposition (green) after hybridization using probes for GFRα1 (A, C, E) or GFRα2 (B, D, F).Yellow indicates regions in which silver grains are deposited over FG-labeled cells. A subpopulation of sciatic afferents express GFRα1 (A) and GFRα2 (B) normally. After axotomy, the proportion of sciatic afferents that express GFRα1 increases (C), whereas the proportion that express GFRα2 declines (D). Administration of GDNF after axotomy can partially reverse the increased expression of GFRα1 (E) and can restore the expression of GFRα2 to normal (F). Scale bar, 50 μm.
Fig. 4.
Fig. 4.
Cell size distributions of all L4–L5 sciatic afferents and of those expressing GFRα1, GFRα2, GFRα3, or RET. Note that Total represents the combined cell size distribution of all the cells analyzed in each group. Distributions are shown for the normal animal (n = 5) and those that have undergone axotomy (n = 4) or axotomy in combination with an intrathecal infusion of GDNF at a dose of 12 μg/d (n = 4). Note that, in the normal L4–L5 DRG, GFRα1 and RET are expressed by neurons of both large and small cell diameter, whereas GFRα2 and GFRα3 are present principally within small-diameter cells. After axotomy, the expression of GFRα1 and RET is upregulated, and the majority of large-diameter cells express these receptor components; axotomy also induces an upregulation in GFRα3 expression, but this remains primarily confined to small-diameter cells. Provision of exogenous GDNF partially reverses the axotomy-induced changes in receptor distribution.
Fig. 5.
Fig. 5.
The percentage of FG-labeled cells in the L4–L5 DRG that also express mRNA for the GDNF receptor family components GFRα1, GFRα2, GFRα3, or RET. Values are shown (±SEM) for the normal animal (n = 5) and those that have undergone axotomy (n = 4) or axotomy in combination with an intrathecal infusion of GDNF at a dose of 12 μg/d (n = 4). Axotomy induced a significant increase in the proportion of cells expressing message for GFRα1, GFRα3, or RET, and a significant decrease in those expressing GFRα2 (*p < 0.05, significant difference from normal values; Tukey post hoc analysis; one-way ANOVA). The infusion of GDNF partially prevented these changes; the upregulation of GFRα1 and GFRα3 was partly reversed, whereas the level of GFRα2 expression returned to normal (#p < 0.05, significant difference between values in the axotomy and axotomy plus GDNF animals; Tukey post hoc analysis; one-way ANOVA). Intrathecal GDNF had no effect on the proportion of FG cells expressing RET after axotomy.
Fig. 6.
Fig. 6.
Double labeling for N52 (phosphorylated neurofilament heavy chain; A, C) and GFRα1 mRNA (B, D) in normal (A, B) and axotomized (C,D) animals. N52 is a marker for large-diameter DRG cells. Arrows denote cells that express both N52 and GFRα1, and asterisks denote cells that are N52-positive but that do not express GFRα1. After axotomy, the proportion of N52-positive cells that express GFRα1 increases. Scale bar, 50 μm.
Fig. 7.
Fig. 7.
RNA levels of RET, GFRα1–3, and GAPDH measured using the TaqMan technique in normal and axotomized L4–L5 DRGs. Axotomy resulted in a significant increase in GFRα1 and GFRα3 expression and a significant reduction in GFRα2 expression. There was also a small but significant reduction in RET RNA levels after axotomy. *p < 0.05, comparing normal with axotomized; Mann–Whitney rank sum test.
Fig. 8.
Fig. 8.
IB4 labeling within the dorsal horn of the spinal cord in a normal animal (A), an animal that has undergone sciatic axotomy (B), and an animal that has undergone sciatic axotomy combined with intrathecal treatment with GDNF (C; 12 μg/d). Axotomy results in a marked reduction in IB4 binding within the dorsal horn (B), which can be prevented by treatment with GDNF (C). Scale bar, 100 μm.
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