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. 2013 Jan 30;33(5):2060-70.
doi: 10.1523/JNEUROSCI.4012-12.2013.

Neurturin overexpression in skin enhances expression of TRPM8 in cutaneous sensory neurons and leads to behavioral sensitivity to cool and menthol

Ting Wang  1 , Xiaotang Jing, Jennifer J DeBerry, Erica S Schwartz, Derek C Molliver, Kathryn M Albers, Brian M Davis
Affiliations

Neurturin overexpression in skin enhances expression of TRPM8 in cutaneous sensory neurons and leads to behavioral sensitivity to cool and menthol

Ting Wang et al. J Neurosci. .

Abstract

Neurturin (NRTN) is a member of the glial cell line-derived neurotrophic factor family of ligands that exerts its actions via Ret tyrosine kinase and GFRα2. Expression of the Ret-GFRα2 coreceptor complex is primarily restricted to the peripheral nervous system and is selectively expressed by sensory neurons that bind the isolectin B(4) (IB(4)). To determine how target-derived NRTN affects sensory neuron properties, transgenic mice that overexpress NRTN in keratinocytes (NRTN-OE mice) were analyzed. Overexpression of NRTN increased the density of PGP9.5-positive, but not calcitonin gene-related peptide-positive, free nerve endings in footpad epidermis. GFRα2-immunopositive somata were hypertrophied in NRTN-OE mice. Electron microscopic analysis further revealed hypertrophy of unmyelinated sensory axons and a subset of myelinated axons. Overexpression of NRTN increased the relative level of mRNAs encoding GFRα2 and Ret, the ATP receptor P2X(3) (found in IB(4)-positive, GFRα2-expressing sensory neurons), the acid-sensing ion channel 2a, and transient receptor potential cation channel subfamily member M8 (TRPM8) in sensory ganglia. Behavioral testing of NRTN-OE mice revealed an increased sensitivity to mechanical stimuli in glabrous skin of the hindpaw. NRTN-OE mice also displayed increased behavioral sensitivity to cool temperature (17°C-20°C) and oral sensitivity to menthol. The increase in cool and menthol sensitivity correlated with a significant increase in TRPM8 expression and the percentage of menthol-responsive cutaneous sensory neurons. These data indicate that the expression level of NRTN in the skin modulates gene expression in cutaneous sensory afferents and behavioral sensitivity to thermal, chemical, and mechanical stimuli.

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Figures

Figure 1.
Figure 1.
Overexpression of NRTN in the skin is driven by the K14 keratin promoter. A, Diagram of the transgene construct used for isolation of NRTN-OE mice. The K14 promoter drives expression of the NRTN sequence (black boxes). The 3′ noncoding hGH sequence provides splice sites and a poly A (PA) addition signal. The arrow indicates transcription start site. B, RT-PCR analysis of RNA isolated from WT (n = 3) and NRTN-OE (n = 3) back skin showing relative level of NRTN mRNA in WT and NRTN-OE skin. Note the enhanced level of NRTN mRNA in NRTN-OE skin and the absence of transgene expression (hGH) in WT skin samples.
Figure 2.
Figure 2.
NRTN overexpression increases the density of PGP9.5-positive, but not CGRP-positive, nerve endings in the epidermis of footpad skin. Immunolabeling of glabrous skin from WT (n = 4; A, C) and NRTN-OE (n = 4; B, D) mice. The number and intensity of PGP9.5-positive fibers are increased in NRTN-OE (B) relative to WT (A) skin. No change was evident for CGRP-positive fibers between NRTN-OE (D) and WT (C). Scale bar, 25 μm.
Figure 3.
Figure 3.
NRTN overexpression increases the diameter of myelinated and unmyelinated axons in the saphenous nerve. Low-power magnification electron microscopic images of saphenous nerve cross-sections at mid-thigh level from a WT (A) and NRTN-OE (B) mouse. Although the total number of axons was unchanged (Table 2), nerves of NRTN-OE mice are larger than those from WT. Histograms show the distribution of axon diameters of unmyelinated (C) and myelinated (D) fibers in saphenous nerves from WT (n = 4) and NRTN-OE (n = 4) mice. The average diameter of myelinated and unmyelinated axons is significantly increased in NRTN-OE mice (Table 2). For unmyelinated axons, a significant rightward shift of the entire population occurs (two-way ANOVA, p < 0.05). For myelinated axons, an enlargement of the largest-diameter axons (>4.5 μm) occurs. Scale bar, 20 μm.
Figure 4.
Figure 4.
Somas of NRTN-responsive neurons are hypertrophied. GFRα2-positive/IB4-binding neurons (arrows) from NRTN-OE mice (n = 4) appeared larger relative to WT neurons (n = 4) in the TG (compare A–C with D–F) and DRG (compare G–I with J–L). In WT DRG, GFRα2-positive/IB4-negative neurons were rare, whereas in the NRTN-OE mice, numerous GFRα2-positive/IB4-negative neurons could be found (J–L; arrowheads). The size distribution of somal areas of GFRα2-positive neurons for TG and DRG is shown in M and N, respectively. A significant rightward shift in TG and DRG populations in NRTN-OE ganglia occurs, indicating hypertrophy of GFRα2-positive/IB4-binding neurons. *NRTN-OE > WT (χ2 test, p < 0.05).
Figure 5.
Figure 5.
NRTN overexpression increases the number and size of TRPM8-positive/IB4-binding neurons. In WT DRG (A–C), TRPM8 staining was not detectable in IB4-binding neurons, whereas in NRTN-OE DRG (D–F), most IB4-binding neurons express TRPM8 (arrows). Immunoblot analysis of pooled L2–L4 DRG from WT (n = 3) and NRTN-OE (n = 3) mice (G). TRPM8 protein level was greater in DRG from NRTN-OE mice. TRPM8 protein was undetectable in WT ganglia under the conditions used. Scale bar, 20 μm.
Figure 6.
Figure 6.
NRTN-OE mice have increased sensitivity to innocuous cold temperature and oral menthol. A, A two-temperature choice test was performed in which mice explored two adjacent temperature-controlled keys for 5 min. The control key was held at a constant 32°C, whereas the test key ranged from 4°C to 32°C. Data are plotted as percentage of time spent on the test key (y-axis) as a function of test temperature (x-axis). No preference was displayed by NRTN-OE (n = 20) or WT (n = 20) mice at test temperatures of 29°C and 32°C (i.e., they spent ∼50% of the time on both keys). When the temperature of the test key was <29°C, both NRTN-OE and WT mice spent less time on the test key relative to the control key, indicating a preference for 32°C. NRTN-OE mice spent significantly less time than WT mice at the 17°C and 20°C test temperatures. *NRTN-OE < WT (t tests; at each temperature). B, NRTN-OE mice have decreased sensitivity to noxious cold on wet ice block. Plot shows that NRTN-OE mice placed on a −20°C surface have a longer latency to their first nocifensive response than WT mice (p < 0.05; t test). C, NRTN-OE mice also had fewer responses during the ice block trial period (p < 0.05; t test). D, WT (n = 6) and NRTN-OE (n = 6) mice were tested for oral sensitivity to menthol using a two-bottle drinking aversion assay. One bottle contained water with vehicle (0.07% ethanol), and the other bottle contained water with menthol (0.1, 1, and 5 mm). At the lowest concentration (0.1 mm), both WT and NRTN-OE mice drank equal amounts of vehicle and menthol solutions, exhibiting no preference. At menthol concentrations of 1 and 5 mm, both WT and NRTN-OE mice drank less menthol solution compared with baseline, and NRTN-OE mice drank significantly less menthol-water than WT mice. *NRTN-OE < WT (p < 0.05; two-way ANOVA and Bonferroni post hoc test).
Figure 7.
Figure 7.
Examples of calcium imaging traces of IB4–488 back-labeled neurons in response to (A) menthol, (B) capsaicin, and (C) mustard oil. There was no difference between WT and NRTN-OE neurons in the response to any of these agonists.
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