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. 2012;7(5):e36355.
doi: 10.1371/journal.pone.0036355. Epub 2012 May 1.

CGRPα-expressing sensory neurons respond to stimuli that evoke sensations of pain and itch

Eric S McCoy  1 , Bonnie Taylor-Blake, Mark J Zylka
Affiliations

CGRPα-expressing sensory neurons respond to stimuli that evoke sensations of pain and itch

Eric S McCoy et al. PLoS One. 2012.

Abstract

Calcitonin gene-related peptide (CGRPα, encoded by Calca) is a classic marker of nociceptive dorsal root ganglia (DRG) neurons. Despite years of research, it is unclear what stimuli these neurons detect in vitro or in vivo. To facilitate functional studies of these neurons, we genetically targeted an axonal tracer (farnesylated enhanced green fluorescent protein; GFP) and a LoxP-stopped cell ablation construct (human diphtheria toxin receptor; DTR) to the Calca locus. In culture, 10-50% (depending on ligand) of all CGRPα-GFP-positive (+) neurons responded to capsaicin, mustard oil, menthol, acidic pH, ATP, and pruritogens (histamine and chloroquine), suggesting a role for peptidergic neurons in detecting noxious stimuli and itch. In contrast, few (2.2±1.3%) CGRPα-GFP(+) neurons responded to the TRPM8-selective cooling agent icilin. In adult mice, CGRPα-GFP(+) cell bodies were located in the DRG, spinal cord (motor neurons and dorsal horn neurons), brain and thyroid-reproducibly marking all cell types known to express Calca. Half of all CGRPα-GFP(+) DRG neurons expressed TRPV1, ∼25% expressed neurofilament-200, <10% contained nonpeptidergic markers (IB4 and Prostatic acid phosphatase) and almost none (<1%) expressed TRPM8. CGRPα-GFP(+) neurons innervated the dorsal spinal cord and innervated cutaneous and visceral tissues. This included nerve endings in the epidermis and on guard hairs. Our study provides direct evidence that CGRPα(+) DRG neurons respond to agonists that evoke pain and itch and constitute a sensory circuit that is largely distinct from nonpeptidergic circuits and TRPM8(+)/cool temperature circuits. In future studies, it should be possible to conditionally ablate CGRPα-expressing neurons to evaluate sensory and non-sensory functions for these neurons.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. CGRPα-GFP+ DRG neurons respond to agonists that evoke pain and itch sensation.
(A) Farnesylated GFP-DTR knocked-in to the start codon of CGRPα. Expression of DTR is conditionally blocked by LoxP flanked GFP and three polyadenylation signals (not shown). LoxP sites are oriented so that the first ATG encountered is in GFP. (B) Representative images of cultured DRG neurons from a CGRPα-GFP+/− mouse after loading with Fura2-AM. Arrows point to CGRPα-GFP+ neurons. (C–J) Responses of CGRPα-GFP+ neurons from heterozygous mice to (C) capsaicin (1 µM), (D) mustard oil (100 µM), (E) menthol (200 µM), (F) icilin (4 µM), (G) histamine (100 µM), (H) chloroquine (1 mM), (I) ATP (100 µM) and (J) acidic pH, followed by stimulation with 100 mM KCl to identify healthy neurons. Scale bar in (B) right panel is 50 µm.
Figure 2
Figure 2. CGRPα-GFP colocalizes with peptidergic nociceptive neuron markers.
Sections of L4-L6 DRG from CGRPα-GFP+/− mice were stained with antibodies to GFP (A,D,G,J,M,P) and the indicated markers. Images were acquired by confocal microscopy. Scale bar in (R) is 50 µm.
Figure 3
Figure 3. CGRPα-GFP axons terminate in dorsal spinal cord.
Sections of lumbar spinal cord from CGRPα-GFP+/− mice were stained with antibodies to (A) GFP and (B) CGRP. (C) IB4-binding. (D) Merged image. Images were acquired by confocal microscopy and are representative of n = 3 mice. Scale bar in (D) is 100 µm.
Figure 4
Figure 4. CGRPα-GFP marks a small population of neurons in lamina II/III and motor neurons in spinal cord.
Sections of lumbar spinal cord from CGRPα-GFP+/− mice were stained with (A–F) antibodies to GFP (green) and PKCγ (red). IB4-binding (blue). Arrowheads point to GFP+ cells. (D–F) Single confocal scan image from box in (A) reveals a CGRPα-GFP+ neuron located in lamina II. (G–I) Motor neurons stained with antibodies to GFP (green) and CGRP (red). Images were acquired by confocal microscopy and are representative of n = 3 mice. Scale bar in (C and F) is 25 µm and in (I) is 100 µm.
Figure 5
Figure 5. Glabrous skin montage.
Sections from the glabrous skin of the hindpaw from CGRPα-GFP+/? mice were stained with antibodies to (A) GFP and (B) PGP9.5. (C) Merged images were stained with the nuclear marker DRAQ5. Images were acquired by confocal microscopy and are representative of n = 3 mice. Scale bar in (C) is 50 μm.
Figure 6
Figure 6. CGRP?-GFP+ axons innervate muscle and skin.
Sections of hindpaw from CGRPα-GFP+/? mice were stained with antibodies to (A,D,G,J) GFP and (B,E,H,K) the pan-nerve fiber marker PGP9.5. (C,F,I,L,G-inset) Merged images were stained with the nuclear marker DRAQ5 to visualize skin cells. (AC) Nerve bundle in the subdermis and motor end plates (ep). (DF) Epidermis (epi) and upper dermis (d) from glabrous skin, (GI) sweat gland (sg) in glabrous skin and (JL) guard hair follicle (hf) in hairy skin. Arrowheads point to putative transduction spheres. Images were acquired by confocal microscopy and are representative of n = 3 mice. Scale bar in (L) is 100 μm.
Figure 7
Figure 7. CGRPα-GFP axons and cells in visceral tissues.
Sections of (A–C) small intestine, (D–I), bladder and (J–L) thyroid from CGRPα-GFP+/− mice were stained with antibodies to GFP (A, D, G, J) and the indicated markers. Nuclei in (A–C) were labeled with DRAQ5. (C-inset) Section of wild-type mouse small intestine stained for GFP (green) and DRAQ5 (blue). Images were acquired by confocal microscopy and are representative of n = 3 mice. Scale bars in (C) and (C-inset) inset are 100 µm and apply to (A–C). Scale bar in (L) is 50 µm and applies to (D–L).
Figure 8
Figure 8. CGRPα-GFP labels neurons and axons in specific brain regions.
(A–F) Brain sections from CGRPα-GFP+/− mice were stained with antibodies to GFP. (A) Trigeminal spinal nucleus caudalis. Arrowheads point to labeled cells in lamina III, similar to those shown in Figure 4A–F. (B) Parabrachial nucleus. (C) Peripeduncular and posterior intralaminar thalamic nuclei. (D) Subparafascicular nucleus of the thalamus. (E) Nucleus accumbens. (F) Subiculum. Abbreviations: LPB = lateral parabrachial nucleus; MPB = medial parabrachial nucleus; scp = superior cerebral peduncle; MG = medial geniculate; SN = substantia nigra; fr = fasciculus retroflexus; mt = mammillothalamic tract; Sep = septum; aca = anterior commissure, anterior; S = subiculum; VC = visual cortex. Inset in each panel shows a lower magnification view. Scale bar in (A) inset is 1 mm and applies to all insets; (B) is 200 µm and applies to (B); (F) is 100 µm and applies to (A,C–F).
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