Abstract

Itch, initiated by the activation of sensory neurons, is associated frequently with dermatological diseases. MrgprA3+ sensory neurons have been identified as one of the major itch-sensing neuronal populations. Mounting evidence has demonstrated that peripheral pathological conditions induce physiological regulation of sensory neurons, which is critical for the maintenance of chronic itch sensation. However, the underlying molecular mechanisms are not clear. Here, we performed RNA sequencing of genetically labeled MrgprA3+ neurons under both naïve and allergic contact dermatitis conditions. Our results revealed the unique molecular signature of itch-sensing neurons and the distinct transcriptional profile changes that result in response to dermatitis. We found enrichment of nine Mrgpr family members and two histamine receptors in MrgprA3+ neurons, suggesting that MrgprA3+ neurons are a direct neuronal target for histamine and Mrgpr agonists. In addition, PTPN6 and PCDH12 were identified as highly selective markers of MrgprA3+ neurons. We also discovered that MrgprA3+ neurons respond to skin dermatitis in a way that is unique from other sensory neurons by regulating a combination of transcriptional factors, ion channels, and key molecules involved in synaptic transmission. These results significantly increase our knowledge of itch transmission and uncover potential targets for combating itch. Itch, initiated by the activation of sensory neurons, is associated frequently with dermatological diseases. MrgprA3+ sensory neurons have been identified as one of the major itch-sensing neuronal populations. Mounting evidence has demonstrated that peripheral pathological conditions induce physiological regulation of sensory neurons, which is critical for the maintenance of chronic itch sensation. However, the underlying molecular mechanisms are not clear. Here, we performed RNA sequencing of genetically labeled MrgprA3+ neurons under both naïve and allergic contact dermatitis conditions. Our results revealed the unique molecular signature of itch-sensing neurons and the distinct transcriptional profile changes that result in response to dermatitis. We found enrichment of nine Mrgpr family members and two histamine receptors in MrgprA3+ neurons, suggesting that MrgprA3+ neurons are a direct neuronal target for histamine and Mrgpr agonists. In addition, PTPN6 and PCDH12 were identified as highly selective markers of MrgprA3+ neurons. We also discovered that MrgprA3+ neurons respond to skin dermatitis in a way that is unique from other sensory neurons by regulating a combination of transcriptional factors, ion channels, and key molecules involved in synaptic transmission. These results significantly increase our knowledge of itch transmission and uncover potential targets for combating itch. Chronic itch is a devastating symptom frequently associated with dermatological diseases such as atopic dermatitis, psoriasis, and lichen planus (Yosipovitch and Bernhard, 2013Yosipovitch G. Bernhard J.D. Clinical practice. Chronic pruritus.N Engl J Med. 2013; 368: 1625-1634Crossref PubMed Scopus (239) Google Scholar). Itch sensation is initiated by the activation of sensory neurons whose cell bodies reside in the dorsal root ganglion (DRG) or trigeminal ganglion. Skin-innervating sensory neurons project their peripheral and central axons to the skin and spinal cord, respectively, providing a bridge for information flow from the peripheral skin to the central nervous system. Cutaneous stimuli or insults in the inflamed skin, such as pruritogens, are transduced into neuronal activities in the skin nerves to activate itch neural circuits and convey the signals to the brain (Ikoma et al., 2006Ikoma A. Steinhoff M. Ständer S. Yosipovitch G. Schmelz M. The neurobiology of itch.Nat Rev Neurosci. 2006; 7: 535-547Crossref PubMed Scopus (653) Google Scholar). Three pruriceptive sensory neuron subtypes have been identified by both functional studies and single-cell RNA sequencing (RNA-seq) analysis and named NP1–3, each of which expresses distinct itch receptor combinations (Chiu et al., 2014Chiu I.M. Barrett L.B. Williams E.K. Strochlic D.E. Lee S. Weyer A.D. et al.Transcriptional profiling at whole population and single cell levels reveals somatosensory neuron molecular diversity.eLife. 2014; 3e04660Crossref Scopus (123) Google Scholar, Hu et al., 2016Hu G. Huang K. Hu Y. Du G. Xue Z. Zhu X. et al.Single-cell RNA-seq reveals distinct injury responses in different types of DRG sensory neurons.Sci Rep. 2016; 6: 31851Crossref PubMed Scopus (68) Google Scholar, Kupari et al., 2019Kupari J. Häring M. Agirre E. Castelo-Branco G. Ernfors P. 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Memic F. van der Zwan J. et al.Molecular architecture of the mouse nervous system.Cell. 2018; 174: 999-1014.e22Abstract Full Text Full Text PDF PubMed Scopus (646) Google Scholar). NP1 neurons are classified by the expression of MrgprD (Liu et al., 2012Liu Q. Sikand P. Ma C. Tang Z. Han L. Li Z. et al.Mechanisms of itch evoked by beta-alanine.J Neurosci. 2012; 32: 14532-14537Crossref PubMed Scopus (161) Google Scholar), whereas NP2 neurons are classified by the expression of MrgprA3 and MrgprC11 (Han et al., 2013Han L. Ma C. Liu Q. Weng H.J. Cui Y. Tang Z. et al.A subpopulation of nociceptors specifically linked to itch.Nat Neurosci. 2013; 16: 174-182Crossref PubMed Scopus (311) Google Scholar, Liu et al., 2009Liu Q. Tang Z. Surdenikova L. Kim S. Patel K.N. Kim A. et al.Sensory neuron-specific GPCR Mrgprs are itch receptors mediating chloroquine-induced pruritus.Cell. 2009; 139: 1353-1365Abstract Full Text Full Text PDF PubMed Scopus (479) Google Scholar). NP3 neurons express the most itch receptors, including two coreceptors for IL-31 (IL31RA and OSMR), CYSLTR2, HTR1F, and S1PR1 (Huang et al., 2018Huang J. Polgár E. Solinski H.J. Mishra S.K. Tseng P.Y. Iwagaki N. et al.Circuit dissection of the role of somatostatin in itch and pain.Nat Neurosci. 2018; 21: 707-716Crossref PubMed Scopus (90) Google Scholar, Mishra and Hoon, 2013Mishra S.K. Hoon M.A. The cells and circuitry for itch responses in mice.Science. 2013; 340: 968-971Crossref PubMed Scopus (276) Google Scholar, Solinski et al., 2019Solinski H.J. Kriegbaum M.C. Tseng P.Y. Earnest T.W. Gu X. Barik A. et al.Nppb neurons are sensors of mast cell-induced itch.Cell Rep. 2019; 26 (3561–73.e4)Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, Storan et al., 2015Storan E.R. O'Gorman S.M. McDonald I.D. Steinhoff M. Role of cytokines and chemokines in itch.Handb Exp Pharmacol. 2015; 226: 163-176Crossref PubMed Scopus (31) Google Scholar, Usoskin et al., 2015Usoskin D. Furlan A. Islam S. Abdo H. Lönnerberg P. Lou D. et al.Unbiased classification of sensory neuron types by large-scale single-cell RNA sequencing.Nat Neurosci. 2015; 18: 145-153Crossref PubMed Scopus (864) Google Scholar). Peripheral pathological conditions can change the physiological properties of the innervating sensory neurons profoundly and modulate the communication of sensory neurons with second-order spinal neurons by utilizing both transcriptional and translational modifications (Basbaum et al., 2009Basbaum A.I. Bautista D.M. Scherrer G. Julius D. Cellular and molecular mechanisms of pain.Cell. 2009; 139: 267-284Abstract Full Text Full Text PDF PubMed Scopus (2063) Google Scholar, Piomelli and Sasso, 2014Piomelli D. Sasso O. Peripheral gating of pain signals by endogenous lipid mediators.Nat Neurosci. 2014; 17: 164-174Crossref PubMed Scopus (149) Google Scholar, Waxman and Zamponi, 2014Waxman S.G. Zamponi G.W. Regulating excitability of peripheral afferents: emerging ion channel targets.Nat Neurosci. 2014; 17: 153-163Crossref PubMed Scopus (207) Google Scholar). Indeed, our recent studies demonstrated that NP2 MrgprA3+ neurons exhibit elevated itch receptor expression and hyperinnervation in the skin in a mouse dry skin model (Zhu et al., 2017Zhu Y. Hanson C.E. Liu Q. Han L. Mrgprs activation is required for chronic itch conditions in mice.Itch (Phila). 2017; 2: e9Crossref PubMed Google Scholar) and enhanced excitability and spontaneous activities in a contact dermatitis model (Qu et al., 2014Qu L. Fan N. Ma C. Wang T. Han L. Fu K. et al.Enhanced excitability of MRGPRA3- and MRGPRD-positive nociceptors in a model of inflammatory itch and pain.Brain. 2014; 137: 1039-1050Crossref PubMed Scopus (58) Google Scholar). However, the molecular mechanisms underlying those changes are not clear. Our study presents extensive transcriptional profiling of pruriceptive MrgprA3+ and demonstrate distinct transcriptional profile changes in both MrgprA3+ and MrgprA3− neurons following allergic contact dermatitis (ACD). In our previous study, we generated an MrgprA3GFP-Cre line in which the expression of GFP-Cre is controlled by the MrgprA3 promoter (Han et al., 2013Han L. Ma C. Liu Q. Weng H.J. Cui Y. Tang Z. et al.A subpopulation of nociceptors specifically linked to itch.Nat Neurosci. 2013; 16: 174-182Crossref PubMed Scopus (311) Google Scholar). We treated the lower back skin of MrgprA3GFP-Cre mice to induce ACD. We selected the lower back skin to minimize possible scratching-induced skin injury because mice are not able to scratch their lower back skin effectively. The treatment induced parakeratosis, dense inflammatory cell infiltration in the skin (Figure 1a), and spontaneous scratching behavior (Figure 1b). Thoracic and lumbar DRGs (T11–L6) innervating the treated skin area were isolated after treatment and enzymatically dissociated. MrgprA3+ neurons and MrgprA3− neurons were FACS-purified based on GFP fluorescence, and the total RNA was extracted for RNA-seq analysis. Principal component analysis showed clear segregation of MrgprA3+ and MrgprA3− neurons, both before and after the ACD treatment (Figure 1c), demonstrating that MrgprA3+ neurons are a distinct subpopulation of DRG sensory neurons. To quantitatively evaluate the difference in the transcriptome profiles of different groups, we used the support vector machine–based classification model to classify control and ACD treatment samples (Cortes and Vapnik, 1995Cortes C. Vapnik V. Support-vector networks.Mach Learn. 1995; 20: 273-297Crossref Scopus (0) Google Scholar). The area under the receiver operating characteristic curve (Fawcett, 2006Fawcett T. An introduction to ROC analysis.Pattern Recogn Lett. 2006; 27: 861-874Crossref Scopus (10922) Google Scholar) is clearly higher for the MrgprA3+ neuron samples (Figure 1d), suggesting that the MrgprA3+ neurons exhibited a larger transcriptome profile change compared with the MrgprA3− neurons after ACD treatment. This data is consistent with the key role MrgprA3+ neurons play in mediating ACD itch. We first compared the data from MrgprA3+ and MrgprA3− populations in the control group. A total of 1,845 differentially expressed genes (DEGs), including 584 upregulated genes and 1,261 downregulated genes, were identified in MrgprA3+ neurons (Supplementary Table S1). Significant Gene Ontology (GO) Biological Process terms of the genes enriched in MrgprA3+ neurons include itch (custom GO term), membrane hyperpolarization, and response to ATP, consistent with MrgprA3+ neurons function as itch-sensing neurons (Figure 1e). Conversely, GO terms associated with genes enriched in MrgprA3− neurons include pain, thermal sensation, mechanical sensation, and locomotion, which is unsurprising because MrgprA3− neurons include pain sensors, touch sensors, and proprioceptors (Figure 1f). GO terms such as neurogenesis, neuron differentiation, and ion transport are associated with genes enriched in both groups, suggesting that the two neuronal populations engage different gene sets to perform the same biological process. Similar results were also demonstrated by the GO Molecular Function analysis (transmembrane transporter activity and cation channel activity). Many GO categories associated with MrgprA3+ neurons are related to ion channels in GO Molecular Function, suggesting that MrgprA3+ neurons utilize distinct groups of ion channels to perform neuronal functions. We next examined the gene expression pattern of known genes that are important for sensory neuron functions. The first group we analyzed were Mrgpr family members (Figure 2a) (Meixiong and Dong, 2017Meixiong J. Dong X. Mas-related G protein-coupled receptors and the biology of itch sensation.Annu Rev Genet. 2017; 51: 103-121Crossref PubMed Scopus (35) Google Scholar). Consistent with previous studies from our group and others, Mrgprb4, Mrgprc11, and Mrgpra1 are enriched in MrgprA3+ neurons (Han et al., 2013Han L. Ma C. Liu Q. Weng H.J. Cui Y. Tang Z. et al.A subpopulation of nociceptors specifically linked to itch.Nat Neurosci. 2013; 16: 174-182Crossref PubMed Scopus (311) Google Scholar, Li et al., 2016Li C.L. Li K.C. Wu D. Chen Y. Luo H. Zhao J.R. et al.Somatosensory neuron types identified by high-coverage single-cell RNA-sequencing and functional heterogeneity.Cell Res. 2016; 26: 83-102Crossref PubMed Scopus (195) Google Scholar, Lou et al., 2015Lou S. Pan X. Huang T. Duan B. Yang F.C. Yang J. et al.Incoherent feed-forward regulatory loops control segregation of C-mechanoreceptors, nociceptors, and pruriceptors.J Neurosci. 2015; 35: 5317-5329Crossref PubMed Scopus (16) Google Scholar, Meixiong et al., 2019aMeixiong J. Vasavda C. Green D. Zheng Q. Qi L. Kwatra S.G. et al.Identification of a bilirubin receptor that may mediate a component of cholestatic itch.eLife. 2019; 8Crossref PubMed Scopus (32) Google Scholar). Five other Mrgprs with unknown functions, including Mrgpra2b, Mrgpra4, Mrgpra7, Mrgpra8, and Mrgprb5, are also highly enriched in MrgprA3+ neurons. It is interesting to see nine members of the same family enriched in MrgprA3+ neurons, which only constitute 8% of the DRG sensory neurons. We then analyzed 19 known genes that play an important role in itch sensation (Figure 2b). Two histamine receptors (Hrh1 and Hrh2) and Plcβ3, an isozyme mediating histamine-induced sensory neuron activation, are enriched in MrgprA3+ neurons (Han and Dong, 2014Han L. Dong X. Itch mechanisms and circuits.Annu Rev Biophys. 2014; 43: 331-355Crossref PubMed Scopus (92) Google Scholar, Han et al., 2006Han S.K. Mancino V. Simon M.I. Phospholipase Cbeta 3 mediates the scratching response activated by the histamine H1 receptor on C-fiber nociceptive neurons.Neuron. 2006; 52: 691-703Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Neuromedian B, a neuropeptide required for scratching behaviors induced by histamine; compound 48/80; and 5-HT (Wan et al., 2017Wan L. Jin H. Liu X.Y. Jeffry J. Barry D.M. Shen K.F. et al.Distinct roles of NMB and GRP in itch transmission.Sci Rep. 2017; 7: 15466Crossref PubMed Scopus (22) Google Scholar) are also enriched in MrgprA3+ neurons. The enrichment of these genes and Mrgprs suggests that MrgprA3+ neurons are important direct mediators of the Mrgpr agonists and histamine-induced itch.Figure 4In situ hybridization validation of identified markers in MrgprA3+ neurons. FISH and Venn diagrams showing the expression overlapping of (a) Hrh1, (b) Hrh2, (a and b) PLCβ3, (c) Ptpn6, and (d) Pcdh12 with Mrgpra3. Bar = 20 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) As MrgprA3− neurons include NP1 and NP3 populations (Usoskin et al., 2015Usoskin D. Furlan A. Islam S. Abdo H. Lönnerberg P. Lou D. et al.Unbiased classification of sensory neuron types by large-scale single-cell RNA sequencing.Nat Neurosci. 2015; 18: 145-153Crossref PubMed Scopus (864) Google Scholar), they are similarly enriched for many itch receptors and itch-related molecules including Nppb and Sst. Both coreceptors for IL-31 (Il31ra and Osmr) are highly expressed in both MrgprA3+ and MrgprA3− neurons, although MrgprA3− neurons are more enriched for Il31ra, suggesting that MrgprA3+ neurons are part of the neuronal population mediating IL-31–induced itch. Lpar1 and Lpar3 are downregulated in MrgprA3+ neurons (Kremer et al., 2010Kremer A.E. Martens J.J. Kulik W. Ruëff F. Kuiper E.M. van Buuren H.R. et al.Lysophosphatidic acid is a potential mediator of cholestatic pruritus.Gastroenterology. 2010; 139 (1018.e1): 1008-1018Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar). The expression of both endothelin receptors and Ece1 (Kido-Nakahara et al., 2014Kido-Nakahara M. Buddenkotte J. Kempkes C. Ikoma A. Cevikbas F. Akiyama T. et al.Neural peptidase endothelin-converting enzyme 1 regulates endothelin 1-induced pruritus.J Clin Invest. 2014; 124: 2683-2695Crossref PubMed Scopus (52) Google Scholar) is relatively higher in the MrgprA3− population. Htr7 (Morita et al., 2015Morita T. McClain S.P. Batia L.M. Pellegrino M. Wilson S.R. Kienzler M.A. et al.HTR7 mediates serotonergic acute and chronic itch.Neuron. 2015; 87: 124-138Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) is preferentially expressed in MrgprA3− neurons. We next examined the expression patterns of several families that are key players in nociception, which include TRP channels, purinergic receptors, prostaglandin receptors, and acid-sensing ion channels (Figure 2c and d) (Basbaum et al., 2009Basbaum A.I. Bautista D.M. Scherrer G. Julius D. Cellular and molecular mechanisms of pain.Cell. 2009; 139: 267-284Abstract Full Text Full Text PDF PubMed Scopus (2063) Google Scholar). The only TRP channel enriched in MrgprA3+ neurons is Trpc3, whose function in itch sensation is not yet clear (Figure 2c). Three other genes are observed to be specifically enriched in MrgprA3+ neurons including P2rx2, P2ry2, and Ptger4 (EP4). The enrichments of P2rx2 and P2ry2 raise the question if ATP plays a role in itch induction, which has not been intensively investigated yet. Previous studies have shown an increased level of prostaglandin E2, an agonist for EP4, in the skin of patients with atopic dermatitis (Fogh et al., 1989Fogh K. Herlin T. Kragballe K. Eicosanoids in skin of patients with atopic dermatitis: prostaglandin E2 and leukotriene B4 are present in biologically active concentrations.J Allergy Clin Immunol. 1989; 83: 450-455Abstract Full Text PDF PubMed Scopus (125) Google Scholar). Consistently, blockade of PGE2 signaling reduced spontaneous scratching behavior in a mouse model of dermatitis (Emrick et al., 2018Emrick J.J. Mathur A. Wei J. Gracheva E.O. Gronert K. Rosenblum M.D. et al.Tissue-specific contributions of Tmem79 to atopic dermatitis and mast cell-mediated histaminergic itch.Proc Natl Acad Sci USA. 2018; 115: E12091-E12100Crossref PubMed Scopus (15) Google Scholar). Our analysis suggests that PGE2 might activate MrgprA3+ neurons via the activation of EP4. Many markers for nonpruriceptive neurons are downregulated in MrgprA3+ neurons (Figure 2f), including markers for large-diameter neurons (Vglut1, Ldhb, Cavna1h, Spp1, and Fam19a1), markers for proprioceptors (Pvalb), and a marker for C-low-threshold mechanoreceptors (Th and Vglut3) (Usoskin et al., 2015Usoskin D. Furlan A. Islam S. Abdo H. Lönnerberg P. Lou D. et al.Unbiased classification of sensory neuron types by large-scale single-cell RNA sequencing.Nat Neurosci. 2015; 18: 145-153Crossref PubMed Scopus (864) Google Scholar). Nefm and Nefh are enriched in MrgprA3− neurons. By contrast, Ina, an internexin neuronal intermediate filament, is enriched in MrgprA3+ neurons. Expression of neurotrophin receptors has long been one of the criteria to classify somatosensory neurons (Lallemend and Ernfors, 2012Lallemend F. Ernfors P. Molecular interactions underlying the specification of sensory neurons.Trends Neurosci. 2012; 35: 373-381Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). MrgprA3+ neurons are enriched for Gfra1 and Ntrk1 (Trka). We also observed the enrichment of Runx2, the only RUNX family member that has not been linked to somatosensory neurons differentiation and function, in MrgprA3+ neurons (Figure 2e). Similar to the previous single-cell RNA-seq analysis, we found that MrgprA3+ neurons are enriched for neuromedin B, CGRP (Calca and Calcb), and Agrp. In addition, we found that MrgprA3+ neurons are enriched for Pcsk2 and Pcsk1n, both of which are involved in the processing of neuropeptide and have never been linked to sensory neuron function (Figure 2g). MrgprA3+ neurons employ unique combinations of ion channels to maintain the resting membrane potential and initiate the neuronal firing. For sodium channels (Figure 3a), Nav1.9 (Scn11a) is enriched in MrgprA3+ neurons, which is in line with its known role in itch sensation (Salvatierra et al., 2018Salvatierra J. Diaz-Bustamante M. Meixiong J. Tierney E. Dong X. Bosmans F. A disease mutation reveals a role for NaV1.9 in acute itch.J Clin Invest. 2018; 128: 5434-5447Crossref PubMed Scopus (16) Google Scholar). The other two enriched members in MrgprA3+ neurons are the β subunits of voltage-gated sodium channels Scn2b and Scn3b. Cacng2 is the only enriched voltage-gated calcium channel in the MrgprA3+ neurons (Figure 3b). Ttyh2, a calcium-activated chloride channel (Suzuki, 2006Suzuki M. The Drosophila tweety family: molecular candidates for large-conductance Ca2+-activated Cl- channels.Exp Physiol. 2006; 91: 141-147Crossref PubMed Scopus (48) Google Scholar), is highly enriched in MrgprA3+ neurons (Figure 3c). We observed seven potassium channels enriched in MrgprA3+ neurons with Kcnk18 as the only enriched leaky potassium channel (TRESK) (Figure 3d) (Sano et al., 2003Sano Y. Inamura K. Miyake A. Mochizuki S. Kitada C. Yokoi H. et al.A novel two-pore domain K+ channel, TRESK, is localized in the spinal cord.J Biol Chem. 2003; 278: 27406-27412Crossref PubMed Scopus (120) Google Scholar). We perfomed RNAscope in situ hybridization analysis to validate the enrichment of histamine receptors in vivo. We found that approximately 14% of DRG neurons express both Hrh1 and Plcb3 (Figure 4a and b), suggesting that they are histamine-sensing neurons. Most MrgprA3+ neurons (82.9%) express both Hrh1 and Plcb3. Similar results were observed for Hrh2. We did not observe the expression of Hrh4 in DRG sensory neurons. We also validated two genes (Ptpn6 and Pcdh12) identified to be highly enriched in MrgprA3+ neurons (Figure 4). A total of 83.3% (80/96) of Ptpn6+ neurons express MrgprA3 (Figure 4c), and most Pcdh12+ neurons (69.9%, 93/133) coexpress MrgprA3 (Figure 4d). These results show that the enriched genes identified by our RNA-seq analysis also exist in vivo. ACD treatment significantly changed the expression profile of both MrgprA3+ neurons and MrgprA3− neurons, demonstrating the common neuro-immuno crosstalk between inflamed skin and innervating sensory neurons. We have identified 408 upregulated genes and 425 downregulated genes in MrgprA3+ neurons (Figure 5a, Supplementary Table S2). Similarly, 292 upregulated genes and 420 downregulated genes were identified in MrgprA3− neurons (Figure 5a, Supplementary Table S3). Inflamed skin caused significant damage to all of the innervating sensory neurons, as demonstrated by the enrichment of GO terms such as programmed cell death and cellular response to stress associated with both neuron types. However, the genes affected in the two neuronal populations are largely nonoverlapping (Figure 5a), which indicates that MrgprA3+ neurons respond to the dermatitis condition in a very different way from other sensory neuron subtypes. Indeed, unique GO terms are identified to be associated with the two neuronal populations (Figure 5b). A small percentage of affected genes (Figure 5a, Supplementary Table S4) were changed in both neuronal populations. Among them, we identified several interesting genes including those related to inflammatory responses such as Il17ra, Tnfrsf1a, and Ptgfr; those related to neuronal function such as Syt2, Syt7, and Slc12a2; and those related to intracellular signaling transduction in sensory neurons such as Pla2r1 and Dgkz. Distinct sets of transcription factors were identified within the two neuronal populations (Figure 6a), most of which lack known sensory neuron function. Etv5, an essential molecule regulating sensory neuron differentiation and axonal growth during development (Fontanet et al., 2013Fontanet P. Irala D. Alsina F.C. Paratcha G. Ledda F. Pea 3 transcription factor family members Etv4 and Etv5 mediate retrograde signaling and axonal growth of DRG sensory neurons in response to NGF.J Neurosci. 2013; 33: 15940-15951Crossref PubMed Scopus (24) Google Scholar), is upregulated in MrgprA3+ neurons. Six4, a transcriptional factor required for the survival of sensory neurons, is downregulated in MrgprA3+ neurons (Konishi et al., 2006Konishi Y. Ikeda K. Iwakura Y. Kawakami K. Six1 and Six4 promote survival of sensory neurons during early trigeminal gangliogenesis.Brain Res. 2006; 1116: 93-102Crossref PubMed Scopus (63) Google Scholar, Moody and LaMantia, 2015Moody S.A. LaMantia A.S. 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Chen C.L. et al.Mechanisms of compartmentalized expression of Mrg class G-protein-coupled sensory receptors.J Neurosci. 2008; 28: 125-132Crossref PubMed Scopus (58) Google Scholar). We then focused our analysis on the genes related to sensory neuron function (Figure 6b). Both MrgprA3+ and MrgprA3− neurons showed a distinct gene change pattern after ACD treatment. Three itch receptors (Hrh2, Il31ra, and Lpar3) were changed in MrgprA3− neurons, whereas we did not observe any significant change of itch receptors in MrgprA3+ neurons. Asic3, P2rx5, and Ptges were changed in MrgprA3+ neurons, whereas Bdkrb2, P2rx6, and Ptges2 were changed in MrgprA3− neurons. A TNF receptor (Tnfrsf1a) and several IL receptors (Il1r1, Il1ra, and Il17ra) were changed, indicating their involvement in sensory neurons in response to skin inflammation. We observed a significant increase of Ngfr (P75) only in MrgprA3+ neurons, consistent with the role of NGF in promoting chronic itch and sensory nerve sensitization (Ikoma et al., 2006Ikoma A. Steinhoff M. Ständer S. Yosipovitch G. Schmelz M. The neurobiology of itch.Nat Rev Neurosci. 2006; 7: 535-547Crossref PubMed Scopus (653) Google Scholar, Mollanazar et al., 2016Mollanazar N.K. Smith P.K. Yosipovitch G. Mediators of chronic pruritus in atopic dermatitis: getting the itch out?.Clin Rev Allergy Immunol. 2016; 51: 263-292Crossref PubMed Scopus (140) Google Scholar). Many ion channels have been identified as well, indicating the change of electrophysiological properties of neurons (Figure 6c). Among them, Cacng5 shows the greatest changes in MrgprA3+ neurons, whereas Kcne3 shows the greatest change in MrgprA3− neurons. Studies have shown that peripheral insults can lead to changes in the sensory signal transmission to spinal cord neurons and presynaptic regulation by the local spinal circuits or descending fibers. We found significant changes in many neurotransmitter receptors (Figure 6d). Among them, GRIA2 (GLUR2) was increased in both MrgprA3+ and MrgprA3− neurons. Many genes related to synaptic transmission were also identified (Figure 6e and f) including Syt2, which was increased in both neuron types; Syt7, which was decreased in both neuron types; and Ca