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. 2006 Aug 16;26(33):8588-99.
doi: 10.1523/JNEUROSCI.1726-06.2006.

Glial cell line-derived neurotrophic factor family members sensitize nociceptors in vitro and produce thermal hyperalgesia in vivo

Sacha A Malin  1 , Derek C Molliver, H Richard Koerber, Pamela Cornuet, Rebecca Frye, Kathryn M Albers, Brian M Davis
Affiliations

Glial cell line-derived neurotrophic factor family members sensitize nociceptors in vitro and produce thermal hyperalgesia in vivo

Sacha A Malin et al. J Neurosci. .

Abstract

Nerve growth factor (NGF) has been implicated as an effector of inflammatory pain because it sensitizes primary afferents to noxious thermal, mechanical, and chemical [e.g., capsaicin, a transient receptor potential vanilloid receptor 1 (TRPV1) agonist] stimuli and because NGF levels increase during inflammation. Here, we report the ability of glial cell line-derived neurotrophic factor (GDNF) family members artemin, neurturin and GDNF to potentiate TRPV1 signaling and to induce behavioral hyperalgesia. Analysis of capsaicin-evoked Ca2+ transients in dissociated mouse dorsal root ganglion (DRG) neurons revealed that a 7 min exposure to GDNF, neurturin, or artemin potentiated TRPV1 function at doses 10-100 times lower than NGF. Moreover, GDNF family members induced capsaicin responses in a subset of neurons that were previously insensitive to capsaicin. Using reverse transcriptase-PCR, we found that artemin mRNA was profoundly upregulated in response to inflammation induced by hindpaw injection of complete Freund's adjuvant (CFA): artemin expression increased 10-fold 1 d after CFA injection, whereas NGF expression doubled by day 7. No increase was seen in neurturin or GDNF. A corresponding increase in mRNA for the artemin coreceptor GFRalpha3 (for GDNF family receptor alpha) was seen in DRG, and GFRalpha3 immunoreactivity was widely colocalized with TRPV1 in epidermal afferents. Finally, hindpaw injection of artemin, neurturin, GDNF, or NGF produced acute thermal hyperalgesia that lasted up to 4 h; combined injection of artemin and NGF produced hyperalgesia that lasted for 6 d. These results indicate that GDNF family members regulate the sensitivity of thermal nociceptors and implicate artemin in particular as an important effector in inflammatory hyperalgesia.

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Figures

Figure 1.
Figure 1.
GFRα2 and GFRα3 colocalize with TRPV1. Images show immunohistochemical staining for GFRα2 or GFRα3 (red) with TRPV1 (green) in sections of mouse L4 DRG. A, TRPV1 labeled small- and medium-sized neurons with a range of staining intensity, as widely described in the literature. Staining for GFRα2 was intense in axons and neuronal cell bodies of small and medium size. Only a minority of TRPV1-positive neurons was also GFRα2 positive. Arrows in the merged panel indicate double-labeled neurons. B, GFRα3 stained cell bodies and axons of a subset of small neurons. In contrast to GFRα2 staining, most GFRα3-positive neurons also expressed TRPV1 (see arrows in merged panels). Scale bar, 50 μm. Quantification of staining is provided in Table 1.
Figure 2.
Figure 2.
Acute potentiation of TRPV1 by growth factors. A, Ca2+ influx after 1 μm capsaicin (Cap) treatment before and after 7 min perfusion of NGF, GDNF, artemin (ART), or neurturin (NTN) (all at 100 ng/ml) was examined in isolated DRG sensory neurons. Each coverslip received a single growth factor perfusion. Growth factor treatment increased capsaicin responses (both peak and area measurements) in many cells (see Table 1); percentage of capsaicin responders potentiated is given above each record. The red bar indicates capsaicin application. B, Fold change in capsaicin response area (ΔFarea) measurements for each cell after treatment with NGF, artemin, neurturin, GDNF, or buffer (Untreated).
Figure 3.
Figure 3.
Time course of TRPV1 potentiation varies with growth factor. Capsaicin-induced Ca2+ influx was recorded before and 10, 20, and 30 min after 7 min perfusion of growth factors (GF) on isolated DRG neurons. Potentiation of capsaicin-evoked Ca2+ transients by growth factors are presented as the percentage of cells at each time point at peak potentiation (A) and the percentage magnitude of potentiation at each time point (B). The greatest effects of NGF, artemin (ART), and GDNF on TRPV1 occur at 10 min, whereas the action of neurturin (NTN) on TRPV1 is more delayed. Interestingly, although the magnitude of the potentiation by NGF (in potentiated cells) remains relatively constant over 20 min, the percentage of neurons affected decreases substantially over this time period.
Figure 4.
Figure 4.
GDNF family members are more potent than NGF in TRPV1 potentiation. Capsaicin-evoked Ca2+ influx was recorded before and 10 min after perfusion of growth factors on isolated DRG neurons. Each growth factor concentration (0.1, 1, 10, and 100 ng/ml) was tested on individual coverslips, cells were allowed to recover for 30 min, and maximal doses of each growth factor were given to identify all potential responders in the field and to determine maximal potentiation magnitude for each responsive cell. Representative traces are shown in A. Note that the magnitude of potentiation increases with increasing growth factor concentration (A, C), as does the number of cells potentiated (B). Although all four factors potentiated capsaicin-evoked responses, all GDNF family members were substantially more potent than NGF. ART, Artemin; NTN, neurturin.
Figure 5.
Figure 5.
Growth factors affect overlapping subsets of capsaicin-responsive neurons. Growth factors were given serially to investigate the action(s) of multiple growth factors in a single cell. Capsaicin-induced Ca2+ influx was recorded before and 10 min after perfusion of artemin (ART), neurturin (NTN), or GDNF (each at 100 ng/ml) on isolated DRG neurons. Cells were allowed to recover for 30 min and tested for NGF-induced potentiation of capsaicin responses. Capsaicin responses obtained from a dually potentiated cell are shown in A. Percentage of overlap in NGF/artemin, NGF/GDNF, and NGF/neurturin responses are shown in B. There is substantial overlap in the actions of NGF and artemin, as predicted by the overlap in their receptor expression. However, the overlap between NGF and neurturin or GDNF is low, suggesting that these growth factors act on distinct subsets of neurons (B). These data are graphed as a Venn diagram in C. Neurturin and GDNF populations are graphed together because they showed similar overlap with NGF. D, ΔFarea of capsaicin responses after NGF application is graphed versus ΔFarea after artemin, neurturin, or GDNF application in the same cell. Potentiation magnitudes were not correlated for these growth factor pairs.
Figure 6.
Figure 6.
GDNF family members potentiate in the presence of maximal NGF. Potentiation of capsaicin responses by NGF alone and by combined NGF, artemin, neurturin, and GDNF (each at 100 ng/ml; Mix) were examined in single cells to determine whether GDNF family members provide additional sensitization of TRPV1 in the presence of maximal doses of NGF. NGF was given for 7 min, capsaicin responses were examined for 30 min (at 10 min intervals), the growth factor mix was given for 7 min, and capsaicin responses were examined for another 30 min. Representative traces are shown in A and B. A, Most capsaicin responses (74%) are potentiated by both NGF and GDNF family members. B, Some cells (24%) were not responsive to NGF but were potentiated by GDNF family members. C, ΔFarea values for all capsaicin responses in this experiment after initial tachyphylaxis (Untreated), NGF treatment, and treatment with all four growth factors (Mix). In all cells examined, combined NGF and GDNF family members resulted in greater potentiation of TRPV1 than was achieved with NGF alone. Notably, the effect of 400 ng/ml NGF was not greater than that of 100 ng/ml NGF (data not shown).
Figure 7.
Figure 7.
TRPV1 potentiation occurs in cutaneous afferents. A, Immunohistochemical staining for GFRα3 and TRPV1 in hindpaw skin. GFRα3 staining identified numerous fibers entering the epidermis and was extensively colocalized with staining for TRPV1. Scale bar, 50 μm. B, Cutaneous afferents were identified in vitro by injecting the retrograde tracer WGA 488 into the saphenous nerve before dissociation of sensory neurons. The top panel is a phase-contrast image of dissociated sensory neurons; the bottom panel is an epifluorescence image of the same field. Arrows indicate the WGA 488-labeled cutaneous afferent. C, Capsaicin responses recorded from the retrogradely labeled cutaneous afferent in B; this response is representative of cutaneous afferents examined in this study. Fold potentiation of the capsaicin response by artemin (100 ng/ml) is given above the trace. Application of artemin substantially increased the magnitude of the capsaicin response in most capsaicin-responsive cutaneous afferents (see Results).
Figure 8.
Figure 8.
Growth factors are upregulated in the skin during inflammation. Time course and magnitude of changes in growth factor mRNA levels in hindpaw skin during inflammation evoked by injection of CFA and measured by 32P-labeled RT-PCR (A). The shaded box indicates the period of thermal hyperalgesia recorded in a previous experiment (Zwick et al., 2003). Artemin is most dramatically upregulated (10-fold) at day 1 after CFA injection; this immediately precedes the time period of greatest thermal hyperalgesia in CFA-induced inflammation (1–4 d). NGF was also upregulated, but at later time points (4, 7, and 15 d) and to a lesser degree (2-fold) than artemin. Neurturin mRNA also doubled by day 7 after showing an initial decrease at day 1. B, Expression levels of growth factor receptors and TRPV1 were measured using real-time PCR in L3–L5 DRG during inflammation evoked by CFA injection. GFRα2, the neurturin receptor, is upregulated during the first week of inflammation; GFRα3, the artemin receptor, is upregulated at day 7, during the period of intense thermal hyperalgesia. Expression of TrkA, the NGF receptor, and GFRα1 are not changed until day 14. *p < 0.05 versus naive.
Figure 9.
Figure 9.
Growth factors acutely potentiate heat responses in vivo. Latency to thermal nociceptive response was measured in mice using the Hargreaves apparatus before and after injection of 0.2 μg/20 μl growth factor (or saline control) into the glabrous skin of the hindpaw. All growth factors tested [NGF, artemin (ART), neurturin (NTN), and GDNF] caused significant thermal hyperalgesia during the first 4 h; these effects of single growth factor injection resolved by 24 h after injection. Injection of NGF plus artemin (0.2 μg of NGF/0.2 μg of artemin/20 μl of PBS) resulted in persistent thermal hyperalgesia lasting 6 d (last time point not shown). These data suggest that GDNF family members, as well as NGF, directly potentiate nociceptive thermal signaling in vivo. Additionally, the effects of growth factors on thermal signaling are additive in vivo. *p < 0.05 versus saline control injection.
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