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. 2002 Oct 18;545(Pt 1):43–50. doi: 10.1113/jphysiol.2002.027656

GFR α2/neurturin signalling regulates noxious heat transduction in isolectin B4-binding mouse sensory neurons

Cheryl L Stucky *,†, Jari Rossi ‡, Matti S Airaksinen ‡, Gary R Lewin *
PMCID: PMC2290664  PMID: 12433948

Abstract

The GFR α2 receptor is the cognate co-receptor for the neurotrophic factor neurturin and GFR α2 is selectively expressed by isolectin B4 (IB4)-binding nociceptive sensory neurons. Here, we used two physiological approaches in combination with mice that have a targeted deletion of the GFR α2 gene (GFR α2 −/− mice) in order to determine whether GFR α2/neurturin signalling regulates the functional properties or the survival of IB4-binding nociceptors. Because 50 % of IB4-binding neurons respond to noxious heat and because patch clamp recordings of isolated dorsal root ganglion sensory neurons allow one to neurochemically identify subpopulations of neurons, we analysed the noxious heat responsiveness of IB4-positive and -negative small-diameter neurons isolated from adult GFR α2 −/− and littermate control mice. The percentage of IB4-positive neurons that had large (> 100 pA) heat-evoked inward currents was severely reduced in GFR α2 −/− mice (12 %) compared to wild-type littermates (47 %), and this loss in large-magnitude heat currents was accounted for by an increase in neurons with very small (< 100 pA) heat-evoked currents as well as an increase in neurons with no detectable heat current. Counts of IB4-positive and -negative neurons, as well as counts of unmyelinated axons in the saphenous nerve, confirmed that the loss in neurons with large-amplitude heat currents was due to a deficit in heat transduction and not a decrease in cell survival. The effect was modality specific for heat because mechanical transduction of all fibre types, including IB4-positive C fibres, was normal. Our data are the first to indicate a transduction-function role for GFR α2/neurturin signalling in a specific class of sensory neurons.

Nociceptive sensory neurons with unmyelinated axons (C fibres) have been divided into two distinct classes on the basis of their neurochemical, anatomical and functional properties (Snider & McMahon, 1998; Stucky & Lewin, 1999). One group expresses a surface carbohydrate group that binds the plant lectin isolectin B4 (IB4) but expresses relatively few neuropeptides. The second group does not bind IB4 but is rich in expression of neuropeptides such as calcitonin gene-related peptide (CGRP) and substance P (Snider & McMahon, 1998). Sensory neurons that bind IB4 acquire a dependence on glial cell line-derived neurotrophic factor (GDNF) for survival in the immediate postnatal period and lose their receptors for the more classical neurotrophin nerve growth factor (NGF) (Molliver et al. 1997). GDNF and the related molecule neurturin (NRTN) exert their actions by binding to the glycosyl phosphatidyl inositol (GPI)-anchored receptors GFR α1 and GFR α2, respectively, and these receptors together with their respective ligands then form signalling receptor complexes with the common transmembrane re-arranged in transformation (RET) tyrosine kinase (Baloh et al. 2000). GFR α1, GFR α2 and RET receptors are expressed extensively by the IB4-binding GDNF-dependent population but are virtually absent in the trkA-expressing NGF-dependent nociceptors. The GFR α2 receptor in particular is tightly co-localized with the IB4-positive class of nociceptors as nearly 80 % of GFRα2-expressing neurons bind IB4 (Bennett et al. 1998).

The fact that GFR α2 expression overlaps extensively with the IB4-positive population of nociceptors suggested to us that GFR α2 in combination with RET may regulate functional properties of these nociceptors. Approximately one-half of IB4-positive nociceptors are normally sensitive to noxious heat (Stucky & Lewin, 1999) and many are sensitive to noxious mechanical stimuli (Gerke & Plenderleith, 2001; Vulchanova et al. 2001). Therefore, we studied the thermal and mechanosensitive functional properties of small-diameter sensory neurons in mice with a targeted disruption of GFR α2 (Rossi et al. 1999). We found that endogenous signalling via the GFR α2 receptor is specifically required to sustain noxious heat sensitivity in IB4-positive nociceptors but is not required for the survival of these neurons or any other class of cutaneous sensory neurons. Furthermore, our results indicate that GFR α2 receptor/NTRN signalling plays a classical trophic role in the maintenance of axon diameter.

Methods

Neuronal cultures

Adult mice heterozygous for deletion of the GFR α2 receptor were bred and genotyped as previously described (Rossi et al. 1999), and adult GFR α2 −/− and GFR α2 +/+ offspring were used for this study. Animal housing and killing, and all experiments were carried out according to the German national guidelines. To analyse noxious heat currents in sensory neurons, GFR α2 +/+ (n = 5) and GFR α2 −/− (n = 6) mice were rapidly killed by inhalation of a rising concentration of CO2. Dorsal root ganglia (DRG) from all spinal levels were removed and incubated with 1 mg ml−1 collagenase IV (Sigma) for 30 min at 37 °C followed by 0.05 % trypsin (Sigma) for 30 min at 37 °C. DRGs were resuspended in DMEM/Ham's F12 media supplemented with 10 % heat-inactivated horse serum (Biochrom, UK), 20 mm glutamine, 0.8 % glucose, 100 units penicillin and 100 mg ml−1 streptomycin (Gibco). DRGs were dissociated into single cells by passing them through fire-polished Pasteur pipettes of decreasing diameter, spot-plated on poly-l-lysine (200 mg ml−1)-coated coverslips at a density of 1000–2000 cells per coverslip and maintained at 37 °C in 5 % CO2. The median time in culture was 24 h (range 4–48 h). An approximately equal number of IB4-positive and -negative neurons were recorded on each experimental day.

Electrophysiology

Whole cell electrophysiological recordings were made using fire-polished, glass electrodes of 3–5 MΩ resistance pulled from borosilicate glass (Hilgenberg, Malsfeld, Germany) on a laser micropipette puller (P-2000, Sutter Instrument Co., Novato, CA, USA). The recording chamber (volume 500 μl) containing a coverglass with adherent neurons was continuously superfused (2-3 ml min−1) with extracellular solution containing (mm): NaCl, 154; KCl, 5.6; CaCl2, 2; MgCl2, 1; Hepes, 10; glucose, 8. pH was adjusted to 7.4 with NaOH and osmolarity was adjusted to 325 mosmol l−1 with sucrose. The electrodes were filled with solution containing (mm): KCl, 122; Na+, 10; MgCl2, 1; EGTA, 1; Hepes, 10. pH was adjusted to 7.3 with KOH and osmolarity was adjusted to 290 mosmol l−1 with sucrose. Neurons were visualized using phase contrast illumination at × 63 magnification on a Leica DMIRB inverted microscope. The diameter of each neuron was calculated from the mean of the longest and shortest diameters measured with a calibrated reticule. Only small-diameter neurons (< 26 μm), which are predominantly unmyelinated nociceptors, were used (Stucky & Lewin, 1999). For heat tests, temperature in the recording bath was monitored using a miniature thermocouple (response time constant of 5 ms; Physitemp, Clifton, NJ, USA) placed 1 mm from the recorded neuron. Heat ramp stimuli (from 24 to 49 °C in 10 ± 1 s) were applied by heating the extracellular solution immediately before it entered the bath. Bath temperature was maintained at room temperature (22-24 °C) except during heat tests. One neuron per coverglass was characterized and after each recording, neurons on the coverglass were incubated with 10 μg ml−1 IB4 conjugated directly to fluorescein isothiocyanate (IB4-FITC) for 10 min and then rinsed for 5 min in normal extracellular solution.

Data recording and analysis

Membrane voltage or current was clamped using an EPC-9 amplifier run by Tida 4.1 software for Windows 95 (HEKA Electronic, Lambrecht, Germany). Data were filtered using a 4-pole Bessel filter (5.0 kHz) and sampled at 20 kHz. Whole cell configuration was maintained at a holding potential of −60 mV. Seals ranged from 1.5 to 6.0 GΩ. Neurons were discarded if they did not exhibit an action potential overshoot, had resting potentials more positive than −45 mV in current clamp mode, or exhibited a reduction in magnitude of whole cell inward currents greater than 20 % after a heat test. For generating a family of whole cell voltage currents, neurons were pre-pulsed to −120 mV for 150 ms and then depolarized from −50 to +50 mV in increments of 5 mV (40 ms for each depolarizing test pulse). Voltage errors were minimized by using 70 % series resistance compensation, and pipette and cell capacitance artifacts were cancelled by using the computer-controlled circuitry of the patch clamp amplifier. For generation of action potentials (APs), currents from 0.02 to 1.2 nA were injected for 40 ms at each step. AP threshold was taken as the lowest current injected that evoked an AP with an overshoot. The duration of the AP was taken between 50 % and 75 % of the peak amplitude from resting potential because the 50 % amplitude was close to the base of the AP whereas 75 % was near the inflection on the falling phase of the AP.

IB4 staining of fixed cultures

Cultures of neurons from adult wild-type (n = 3) and GFR α2 (n = 3) mutant mice were prepared and fixed 24 h after isolation with 4 % paraformaldehyde for 20 min. Neurons were incubated with 10 mg ml−1 FITC-labelled IB4 in 0.1 m PBS containing 0.1 mm CaCl2, 0.1 mm MgCl2 and 0.1 mm MnCl2 for 1 h and then rinsed and inverted on a slide over a drop of Mowiol. Openlab software (Improvision, UK) was used for analysis. Images of fields of cells (6 fields per culture) were randomly collected at a magnification of × 20. A total of 645 neurons were analysed for mean brightness intensity and mean soma diameter. To determine non-specific staining per culture, brightness intensities were sampled and averaged from six large-diameter neurons that were clearly negative for IB4. Our previous results show that the largest IB4-positive neurons from adult mouse are 26 μm in diameter (Stucky & Lewin, 1999). Neurons with staining intensities 40 % or more above this value were considered IB4 positive.

Skin-nerve preparation

Mice were rapidly killed by inhalation of a rising concentration of CO2 and the hindlimb was shaved. The skin from the territory innervated by the saphenous nerve, a purely cutaneous nerve, was removed with the nerve intact. The preparation was placed in an organ bath and superfused with an oxygen-saturated modified interstitial fluid solution containing (mm): NaCl, 123; KCl, 3.5; MgSO4, 0.7; NaH2PO4, 1.7; CaCl2, 2.0; sodium gluconate, 9.5; glucose, 5.5; sucrose, 7.5; Hepes, 10 (pH 7.4 + 0.05); temperature, 32 ± 0.5 °C. Recordings were made from functionally single myelinated fibres in teased filaments of the nerve and characterized according to previously described criteria (Koltzenburg et al. 1997).

Nerve histology

The saphenous nerve from wild-type (n = 4) and GFR α2 mutant (n = 4) mice was exposed at mid-thigh level, fixed with 2.5 % glutaraldehyde in 0.15 m PB for 10 min and a 3 mm segment was removed and post-fixed in 2.5 % glutaraldehyde. Nerve segments were then fixed in 1 % OsO4 in 0.1 m cacodylate for 1 h at room temperature, washed in cacodylate buffer, dehydrated in graded alchohols, infiltrated with propylene oxide and embedded in epoxy resin and polymerized at 60 °C for 24 h. Ultrathin sections (0.7-0.8 nm) were cut on an ultramicrotome, stained with lead citrate and uranyl acetate and photographed on an electron microscope. Myelinated and unmyelinated axons were counted using Metamorph image software. For myelinated axons, the entire nerve cross-section was counted; for unmyelinated axons, a sample of the nerve was analysed and extrapolated to determine the total number of axons in each nerve.

For statistical measures in all experiments, groups were compared using a two-tailed, unpaired t test or Fisher's exact test. All error bars shown represent the standard error of the mean (s.e.m.).

Results

The responsiveness of isolated small-diameter (< 26 μm) sensory neurons to noxious heat was measured using whole cell patch clamp methods. Immediately after recordings, neurons were identified as IB4-positive or -negative by using a fluorescent-tagged isolectin B4. Noxious heat-induced inward current was measured in 110 isolated small-diameter (< 26 μm) dorsal root ganglion neurons from GFR α2 −/− mice and GFR α2 +/+ controls. Figure 1A shows noxious heat-evoked inward current in an IB4-positive and an IB4-negative small-diameter neuron from a wild-type mouse. The percentage of IB4-negative neurons that responded to noxious heat with a large inward current > 100 pA was normal in GFR α2 −/− mice (47 % 16/34) compared to GFR α2 +/+ littermates (38 % 9/24) (Fig. 1B). In contrast, only 12 % (4/33) of IB4-positive neurons from GFR α2 −/− mice exhibited a heat-evoked inward current larger than 100 pA compared to 47 % (9/19) from GFR α2 +/+ mice (P = 0.008; Fisher's exact test). Figure 1C shows that the decrease in the number of IB4-positive neurons with heat currents larger than 100 pA was due to a shift in more neurons with smaller heat currents (< 100 pA) as well as to an increase in neurons with no detectable heat current (0 pA). Other properties of IB4-positive and -negative neurons from GFR α2 −/− mice including heat threshold, cell diameter, capacitance, input resistance and amplitude, duration and threshold of the somal action potential were not different from wild-type controls (data not shown).

Figure 1. IB4-positive neurons with large heat currents are markedly reduced in GFRα2 −/− mice.

Figure 1

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A, examples of noxious heat-induced inward currents in an IB4-positive and an IB4-negative neuron from a wild-type mouse. B, the percentage of IB4-positive neurons with heat-evoked currents > 100 pA was substantially reduced in GFR α2 −/− mice compared to wild-type mice (** P = 0.008, Fisher's exact test) whereas the percentage of heat-sensitive IB4-negative neurons was unaltered. C, the loss in IB4-positive neurons with large-magnitude (> 100 pA) heat currents was due to a shift toward neurons with small heat currents (< 100 pA) and to more neurons with no detectable heat current (0 pA).

To determine whether the loss of IB4-positive neurons with large magnitude heat-evoked currents was due to selective death of these neurons in GFR α2 −/− mice, we counted the number of unmyelinated fibres in the saphenous nerve as previously described (Stucky et al. 1999). There was no loss in the number of unmyelinated axons in GFR α2 mutant mice (2899 ± 428; n =4) compared to wild-type littermates (2868 ± 246; n =4), indicating that the axons of the unmyelinated IB4-positive neurons were present in normal numbers. There was also no change in the number of myelinated axons in the mutant mice (531 ± 32; n = 4) compared to controls (542 ± 15; n =4).

Interestingly, however, the diameter of the entire saphenous nerve was smaller in GFR α2 −/− mice (Fig. 2A and B), and this was due to the finding that the individual axon diameters of both unmyelinated and myelinated axons were significantly smaller in GFR α2 −/− mice (Fig. 2C and D). The mean diameter of unmyelinated axons in GFR α2 −/− mice was only 0.45 ± 0.00 μm compared to 0.70 ± 0.00 μm in wild-type littermates (P < 0.001; t test), and the diameter of myelinated axons in GFR α2 −/− mice was 3.10 ± 0.03 μm compared to 3.76 ± 0.03 μm in wild-type controls (P < 0.001; t test). These findings indicate that GFR α2/neurturin signalling plays a classic neurotrophic factor role in regulating axon diameter, particularly for the unmyelinated axons from GFR α2 −/− mice which showed a substantial shift to the left in Fig. 2C.

Figure 2. GFR α2 −/− mice have smaller-diameter unmyelinated and myelinated axons in cutaneous nerves.

Figure 2

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A and B, cross-sections of the saphenous nerve at mid-thigh level from a wild-type (A) and a GFR α2 −/− (B) mouse. Calibration bar, 50 μm. Note that although no axons were lost (see Results), the nerves from GFR α2 −/− mice were considerably smaller than those from wild-type littermates. C-D, histograms show the distribution of axon diameters of unmyelinated (C) and myelinated (D) axons in the saphenous nerves from GFR α2 −/− (n = 4) and wild-type (n = 4) mice. Note the leftward shift in both classes of axons from GFR α2 −/− mutants.

Furthermore, if the reduction in the number of IB4-positive neurons with large heat currents was due to selective cell death, one would expect that a significant proportion of IB4-positive neurons would be missing. Therefore, we quantified the proportion of small-diameter neurons that were positive for IB4 among the total neurons isolated from the ganglia. We found no indication that there was a selective loss of IB4-positive neurons, as the percentage of IB4-positive neurons present in short term cultures of isolated dorsal root ganglion neurons from GFR α2 −/− mice (48.8 %; n =3 animals) was identical to that in wild-type mice (48.8 %; n = 3 animals) (Fig. 3A and B). Unlike the decreased axon diameter of all axons in GFR α2 −/− mice, the average soma diameter of DRG neurons from these mice was not different from controls (IB4 positive: 20.0 ± 0.4 μm in GFR α2 −/− (n =125) vs. 20.3 ± 0.4 μm in GFR α2 +/+ (n =190); IB4 negative: 24.3 ± 0.8 μm in GFR α2 −/− (n =131) vs. 22.8 ± 0.6 μm in GFR α2 +/+ (n =199)). Together, our results indicate that the GFR α2 receptor is required for setting the heat sensitivity of IB4-positive neurons but the receptor is not required at any time in development for their survival.

Figure 3. GFR α2 −/− mice have normal numbers of IB4-positive DRG neurons.

Figure 3

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A-B, histograms illustrate the distribution of cell bodies of IB4-positive and -negative neurons in DRG cultures taken from GFR α2 +/+ (n = 3) and wild-type (n = 3) mice. There was no difference in the percentage of neurons that were IB4 positive in the GFR α2 −/− mutants (48.8 %; 125/256) compared to the wild-type controls (48.8 %; 190/389).

Because heat sensitivity depends on the presence of GFRα2, we then determined whether the GFR α2 receptor is also required for other functional modalities besides heat. Since over 80 % of cutaneous sensory neurons, including IB4-positive nociceptors, are responsive to mechanical stimuli (Kress et al. 1992; Gerke & Plenderleith, 2001; Vulchanova et al. 2001), we determined whether the absence of the GFR α2 receptor affects mechanotransduction. The in vitro skin nerve preparation is particularly well-suited for testing the mechanical responsiveness of the terminals of cutaneous afferents in situ (Koltzenburg et al. 1997). Thus, we used this approach and recorded from 178 cutaneous sensory neurons in GFR α2 −/− and GFR α2 +/+ mice and found that all subtypes of cutaneous neurons with myelinated axons were present in normal numbers and had normal mechanical response properties (Table 1). Large-diameter, low-threshold mechanoreceptors (Aβ fibres), which are classified as either slowly adapting (SA) or rapidly adapting (RA) were encountered with a normal frequency and their mechanosensitivity in GFR α2 −/− mice was indistinguishable from SA and RA fibres recorded in GFR α2 +/+ wild-type littermates (Table 1). Similarly, small-diameter, thinly myelinated mechanoreceptors (Aδ fibres), which are classified as either A-fibre myelinated nociceptors (AM) or down-hair follicle receptors (D-hairs), were unaffected in GFR α2 −/− mice. The finding that all four classes of myelinated fibres were functionally normal in GFR α2 mutants is consistent with evidence that myelinated sensory neurons largely do not express mRNA for the GFR α2 receptor (Bennett et al. 1998).

Table 1.

Mechanical response properties of subclasses of cutaneous neurones in situ

Von Frey threshold Conduction velocity (m s−1)
Class GFRα2 +/+ GFRα2 −/− GFRα2 +/+ GFRα2 −/−
SA % Response of Aβs 42% 33% — —
VFT (mN) 1.4 2.0 — —
Interquartile range 1.0 1.65 16.6 ± 1.4 15.6 ± 0.9
n 10 24 — —
RA % Response Aβs 58% 67% — —
VFT (mN) 1.7 1.4 — —
Interquartile range 2.3 1.0 19.5 ± 0.8 17.3 ± 0.5
n 14 49 — —
AM % Response of Aδs 64% 64% — —
VFT (mN) 7.0 6.3 — —
Interquartile range 6.7 3.0 5.8 ± 0.7 5.7 ± 0.7
n 16 23 — —
D-hair % Response of Aδs 36% 36% — —
VFT (mN) 0.8 0.8 — —
Interquartile range 0.0 0.4 4.8 ± 0.6 6.7 ± 0.5*
n 9 13 — —
C-fibre VFT(mN) 6.3 6.3 0.88 ± 0.09 0.82 ± 0.05
Interquartile range 8.0 3.0 — —
n 11 9 — —
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None of the von Frey thresholds were different as tested by a Mann—Whitney U test; P > 0.25 for all comparisons.

*

Significantly different from wild-type controls by a two-tailed t test; P < 0.05.

Since approximately one-half of the unmyelinated C fibres (the IB4-binding population) do express GFR α2, we examined the mechanical responsiveness of C fibres and found that the mechanical von Frey thresholds and the maximum action potential discharge (not shown) were unaltered in the GFR α2 mutants (Table 1). Therefore, the absence of GFR α2 has a modality-specific effect on heat transduction in unmyelinated nociceptors. Surprisingly, in spite of the smaller diameter of both unmyelinated and myelinated axons from GFR α2 −/− mice, there was no significant slowing in the conduction velocity of any subclass of these fibres in GFR α2 −/− mice (Table 1).

Discussion

The major findings in our study are: (1) the GFR α2 co-receptor which preferentially binds neurturin is required for the proper function of a heat transducer that is specifically localized to IB4-positive nociceptors; (2) whereas the GFR α2 receptor is necessary for heat transduction, it is not required for the survival of these IB4-positive neurons, nor is it essential for the survival of any other functionally or neurochemically defined class of cutaneous sensory neurons; (3) the GFR α2-mediated regulation of heat transduction is modality specific because the mechanical response properties of unmyelinated C fibres, including those responsive to GDNF family ligands, were unaffected in the absence of GFR α2. Furthermore, the mechanical function of all other subtypes of cutaneous sensory neurons was completely normal in the absence of GFR α2.

GFR α2 receptor signalling regulates heat transduction in IB4-positive nociceptors

Our results clearly show that GFR α2 signalling is necessary for the large-magnitude heat responses in IB4-positive unmyelinated nociceptors. The finding that the effect on heat transduction is localized to IB4-positive nociceptors is consistent with evidence that GFR α2 and RET receptors are expressed specifically by one-half of the small-diameter neurons that bind IB4 (Molliver et al. 1997; Bennett et al. 1998) and by our previous studies showing that 50 % of IB4-positive neurons respond to noxious heat (Stucky & Lewin, 1999; Pesquero et al. 2000).

The ligand that binds to the GFR α2 co-receptor and, together with RET, regulates the heat responsiveness of IB4-positive nociceptors is most likely to be neurturin (NRTN). Among the GDNF family ligands, NRTN has the highest selectivity for GFR α2, although GDNF at high concentrations has also been shown to activate GFR α2 (Heuckeroth et al. 1999; Rossi et al. 1999; Baloh et al. 2000). In addition, we previously showed that exogenous GDNF has no effect on the heat responsiveness of IB4-positive nociceptors (Stucky & Lewin, 1999), a result that is consistent with the hypothesis that NTRN but not GDNF is probably the ligand that mediates the effect of GFR α2 on heat transduction.

The heat receptor that mediates the large heat-evoked inward currents in IB4-positive neurons and that requires GFR α2 signalling may be the vanilloid receptor-1 (VR1). VR1 is abundantly expressed in nociceptors and confers heat responsiveness to isolated cells (Caterina et al. 1997, 2000). Furthermore, evidence shows that neurotrophic factors can regulate VR1 because NGF rapidly sensitizes DRG neurons to capsaicin (Shu & Mendell, 1999, 2001), possibly by NGF/trkA releasing VR1 from phosphotidylinositol-mediated inhibition (Chuang et al. 2001). However, other lines of evidence suggest that non-VR1 heat receptors may be involved. First, only 2–3 % of IB4-positive neurons in mouse appear to stain with VR1 antibodies (Zwick et al. 2002), even though 45–50 % of IB4-positive neurons respond to noxious heat (Stucky & Lewin, 1999). Second, animals lacking VR1 surprisingly retain much of their responsiveness to acute noxious heat, suggesting that heat receptors other than VR1 exist (Caterina et al. 2000; Davis et al. 2000). Indeed, other heat-sensitive ion channel receptors have recently been identified that include TRPV3 and TRPV4 (Güler et al. 2002; Peier et al. 2002; Smith et al. 2002) and TRPV3 has been found in small-diameter sensory neurons (Smith et al. 2002). Our finding that many small-magnitude heat-evoked currents (< 100 pA) remain in IB4-positive neurons from GFR α2 −/− mice suggests that the GFR α2 receptor regulates some but not all of the noxious heat receptors in IB4-positive nociceptors.

GFR α2 receptor signalling is not required for the survival of any subclass of cutaneous sensory neuron

Together, our quantification of the numbers of myelinated and unmyelinated cutaneous axons, IB4-positive and -negative DRG neurons and proportions of functionally classified cutaneous fibres demonstrates that no class of cutaneous sensory neurons depends on GFR α2 signalling for survival during embryonic or postnatal development. This finding is consistent with an earlier report that there is no significant loss (≤ 10 %) in cell soma number in the trigeminal ganglia from GFR α2 −/− mice (Rossi et al. 1999). In addition, a recent study showed that motor neurons depend upon the presence of the GFR α1 receptor but not GFR α2 for survival during development (Garcés et al. 2000). Thus, the survival of both cutaneous and motor neurons during development is independent of GFR α2 signalling.

Although no axons were lost in GFR α2 mutants, the size of the saphenous nerve was notably smaller and the diameters of individual myelinated and unmyelinated axons were significantly smaller. These data indicate that, similar to the actions of other neurotrophins such as NGF and brain-derived neurotrophic factor, GFR α2 signalling plays a classic role in developing or maintaining the axon diameter of cutaneous sensory neurons (Cellerino et al. 1997; Stucky et al. 1999).

Our data identify a novel and specific role for GFR α2 receptor signalling in regulating heat transduction. Since NTRN is the preferred ligand for the GFR α2 receptor, the available data suggest that the target-derived GDNF family ligands play quite different roles in regulating IB4-positive nociceptors. GDNF signalling regulates the postnatal survival of IB4-positive neurons (Molliver et al. 1997; Bennett et al. 1998) whereas NTRN signalling regulates the noxious heat response function of these neurons. In summary, along with pharmaceutical therapies that inhibit NGF/TrkA-mediated sensitization of IB4 negative, peptidergic nociceptors (Shu & Mendell, 1999, 2001; Heppenstall & Lewin, 2000), it may be appropriate to consider the neurturin-GFR α2-RET signalling pathway as a potential target for analgesic therapy.

Acknowledgments

This work was supported by a DFG grant SP 1026 to G.R.L., an Academy of Finland grant to M.S.A. and a NIH grant NS40538 to C.L.S. The technical assistance of Emily McGinley and Anke Kanehl is gratefully acknowledged.

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