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Correction for Cavanaugh et al., Distinct subsets of unmyelinated primary sensory fibers mediate behavioral responses to noxious thermal and mechanical stimuli

2009; National Academy of Sciences; Volume: 106; Issue: 27 Linguagem: Inglês

10.1073/pnas.0906213106

1091-6490

Pain Mechanisms and Treatments

Distinct subsets of unmyelinated primary sensory fibers mediate behavioral responses to noxious thermal and mechanical stimuli Daniel J. Cavanaugh a,1 , Hyosang Lee b,c,1 , Liching Lo b,c,1 , Shannon D. Shields a,1 , Mark J. Zylka d , Allan I. Basbaum a,2 , and David J. Anderson b,c,2 a Department of Anatomy, University of California, San Francisco CA 94158; b Division of Biology 216-76 and c Howard Hughes Medical Institute, California Institute of Technology, Pasadena, CA 91125; and d Department of Cell and Molecular Physiology, University of North Carolina, Chapel Hill, NC 27599 Contributed by David J. Anderson, February 12, 2009 (sent for review December 22, 2008) Mrgprd 兩 nociception 兩 TRPV1 N ociceptors are heterogeneous by a variety of molecular criteria (1–4), but the functional significance of this heterogeneity is unclear. Electrophysiological recordings of unmyelinated primary afferent (C) fibers from dorsal root ganglia (DRG) show that most (⬎70%) nociceptors are polymodal: they can be activated by multiple types of painful stimuli, such as mechanical or thermal (5–7). This has led to a prevailing view that the brain’s ability to discriminate different noxious stimulus modalities is unlikely at- tributable to modality-specific primary nociceptor subsets. Rather, it is believed that modality discrimination occurs by the decoding of nociceptor inputs in higher order spinal cord or brain areas (8, 9). A prediction of this hypothesis is that targeted ablation of any single specific nociceptor subpopulation should cause deficits in behav- ioral responses to noxious stimuli of multiple modalities. Indeed, some previous studies using genetic- or immunotoxin-based meth- ods support this prediction (10–12). A critical issue in such experiments, however, regards the cellular specificity and timing of administration of the markers/reagents used for ablation. For example, if ablation of a population of DRG neurons using a given marker caused deficits in behavioral sensi- tivity to multiple pain modalities (10–12), it could reflect the expression of this marker either in a homogeneous population of polymodal nociceptors or in multiple modality-specific subpopula- tions. Modality specificity of nociceptor subpopulations could also be difficult to detect if ablation is performed constitutively using markers that, although specific in adulthood, are transiently ex- pressed more broadly during development (13). We have examined the behavioral consequences of selectively eliminating 2 nonoverlapping subsets of nociceptors, based on their expression of specific receptors, in the adult. Genetic ablation of neurons that express the sensory neuron-specific G www.pnas.org兾cgi兾doi兾10.1073兾pnas.0901507106 protein-coupled receptor Mrgprd (2, 14) caused specific deficits in the behavioral response to noxious mechanical stimuli but not to noxious heat or cold stimuli. Conversely, pharmacological ablation of the central projections of neurons that express the heat-sensitive channel TRPV1 (15) caused a complete loss of heat pain sensitivity, without affecting responses to noxious mechanical or cold stimuli. Combined elimination of both populations yielded an additive phenotype with no further behavioral deficits. These data reveal the existence of distinct subsets of primary sensory neurons that selectively mediate behavioral responses to different noxious stimulus modalities. Results Conditional Ablation of Mrgprd ⴙ Nociceptors. Mrgprd ⫹ afferents exclusively innervate the epidermis and constitute ⬎90% of all nonpeptidergic cutaneous C-fibers (2, 14, 16). These neurons bind isolectin IB4 and terminate in inner lamina II of the spinal cord dorsal horn (4). In vitro, Mrgprd ⫹ neurons exhibit elec- trophysiological properties characteristic of nociceptors (17) and behave as C-polymodal units in ex vivo recordings (K. K. Rau, S. L. McIlwrath, H. Wang, J. J. Lawson, M. P. Jankowski, M.J.Z., D.J.A., H. R. Koerber, unpublished data). To determine the behavioral consequences of ablating Mrg- prd ⫹ neurons, we used a conditional strategy (18, 19), in which the human diphtheria toxin receptor (DTR) was inserted in the Mrgprd locus (Fig. 1A) by homologous recombination in murine embryonic stem cells. The human DTR binds diphtheria toxin (DTX) with 10 5 -fold higher affinity than does the endogenous mouse receptor. Heterozygous Mrgprd DTR/⫹ mice (hereafter referred to as Mrgprd DTR mice) expressed DTR in an identical pattern as a GFP reporter expressed from a second targeted Mrgprd allele (Fig. 1 B–D). Injection of DTX into adult Mrg- prd DTR mice produced a virtually complete (⬎98%) loss of Mrgprd ⫹ cell bodies in the DRG (Fig. 1 E and F) and in their central and peripheral fibers (Fig. 1 I–N). Consistent with Mrgprd expression in the nonpeptidergic afferents, DTX treat- ment caused an 82.4% reduction in labeling for IB4 but no change in labeling for calcitonin gene-related peptide (CGRP), a marker of peptidergic afferents [Fig. 1 E–N and supporting information (SI) Table S1]. The overall reduction in neuron number is commensurate with the size of the Mrgprd ⫹ popula- Author contributions: D.J.C., H.L., S.D.S., M.J.Z., A.I.B., and D.J.A. designed research; D.J.C., H.L., L.L., S.D.S., and M.J.Z. performed research; D.J.C., H.L., L.L., S.D.S., M.J.Z., A.I.B., and D.J.A. analyzed data; and D.J.C., A.I.B., and D.J.A. wrote the paper. The authors declare no conflict of interest. Freely available online through the PNAS open access option. 1 D.J.C., H.L., L.L., and S.D.S. contributed equally to this work. 2 To whom correspondence may be addressed. E-mail: allan.basbaum@ucsf.edu or wuwei@caltech.edu. This article contains supporting information online at www.pnas.org/cgi/content/full/ 0901507106/DCSupplemental. PNAS 兩 June 2, 2009 兩 vol. 106 兩 no. 22 兩 9075–9080 NEUROSCIENCE Behavioral responses to painful stimuli require peripheral sensory neurons called nociceptors. Electrophysiological studies show that most C-fiber nociceptors are polymodal (i.e., respond to multiple noxious stimulus modalities, such as mechanical and thermal); nevertheless, these stimuli are perceived as distinct. Therefore, it is believed that discrimination among these modalities only occurs at spinal or supraspinal levels of processing. Here, we provide evidence to the contrary. Genetic ablation in adulthood of unmy- elinated sensory neurons expressing the G protein-coupled recep- tor Mrgprd reduces behavioral sensitivity to noxious mechanical stimuli but not to heat or cold stimuli. Conversely, pharmacological ablation of the central branches of TRPV1 ⴙ nociceptors, which constitute a nonoverlapping population, selectively abolishes nox- ious heat pain sensitivity. Combined elimination of both popula- tions yielded an additive phenotype with no additional behavioral deficits, ruling out a redundant contribution of these populations to heat and mechanical pain sensitivity. This double-dissociation suggests that the brain can distinguish different noxious stimulus modalities from the earliest stages of sensory processing.