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Somatosensory neurons are highly heterogeneous with distinct types of neural cells responding to specific stimuli. However, the distribution and roles of cell-type-specific long intergenic noncoding RNAs (lincRNAs) in somatosensory neurons remain largely unexplored. Here, by utilizing droplet-based single-cell RNA-seq (scRNA-seq) and full-length Smart-seq2, we show that lincRNAs, but not coding mRNAs, are enriched in specific types of mouse somatosensory neurons. Profiling of lincRNAs from single neurons located in dorsal root ganglia (DRG) identifies 200 lincRNAs localized in specific types or subtypes of somatosensory neurons. Among them, the conserved cell-type-specific lincRNA CLAP associates with pruritus and is abundantly expressed in somatostatin (SST)-positive neurons. CLAP knockdown reduces histamine-induced Ca 2+ influx in cultured SST-positive neurons and in vivo reduces histamine-induced scratching in mice. In vivo knockdown of CLAP also decreases the expression of neuron-type-specific and itch-related genes in somatosensory neurons, and this partially depends on the RNA binding protein MSI2. Our data reveal a cell-type-specific landscape of lincRNAs and a function for CLAP in somatosensory neurons in sensory transmission., (© 2022 The Authors.)

Although histamine is a well-known itch mediator, histamine H 1 -receptor blockers often lack efficacy in chronic itch. Recent molecular and cellular based studies have shown that non-histaminergic mediators, such as proteases, neuropeptides and cytokines, along with their cognate receptors, are involved in evocation and modulation of itch sensation. Many of these molecules are produced and secreted by immune cells, which act on sensory nerve fibers distributed in the skin to cause itching and sensitization. This understanding of the connections between immune cell-derived mediators and sensory nerve fibers has led to the development of new treatments for itch. This review summarizes current knowledge of immune cell-derived itch mediators and neuronal response mechanisms, and discusses therapeutic agents that target these systems.

Two histamine receptor subtypes (HR), namely H1R and H4R, are involved in the transmission of histamine-induced itch as key components. Although exact downstream signaling mechanisms are still elusive, transient receptor potential (TRP) ion channels play important roles in the sensation of histaminergic and non-histaminergic itch. The aim of this study was to investigate the involvement of TRPV1 and TRPA1 channels in the transmission of histaminergic itch. The potential of TRPV1 and TRPA1 inhibitors to modulate H1R- and H4R-induced signal transmission was tested in a scratching assay in mice in vivo as well as via Ca 2+ imaging of murine sensory dorsal root ganglia (DRG) neurons in vitro. TRPV1 inhibition led to a reduction of H1R- and H4R- induced itch, whereas TRPA1 inhibition reduced H4R- but not H1R-induced itch. TRPV1 and TRPA1 inhibition resulted in a reduced Ca 2+ influx into sensory neurons in vitro. In conclusion, these results indicate that both channels, TRPV1 and TRPA1, are involved in the transmission of histamine-induced pruritus.

Background & Aims: Limited understanding of pruritus mechanisms in cholestatic liver diseases hinders development of antipruritic treatments. Previous studies implicated lysophosphatidic acid (LPA) as a potential mediator of cholestatic pruritus., Methods: Pruritogenicity of lysophosphatidylcholine (LPC), LPA's precursor, was examined in naïve mice, cholestatic mice, and nonhuman primates. LPC's pruritogenicity involving keratinocyte TRPV4 was studied using genetic and pharmacologic approaches, cultured keratinocytes, ion channel physiology, and structural computational modeling. Activation of pruriceptor sensory neurons by microRNA-146a (miR-146a), secreted from keratinocytes, was identified by in vitro and ex vivo Ca 2+ imaging assays. Sera from patients with primary biliary cholangitis were used for measuring the levels of LPC and miR-146a., Results: LPC was robustly pruritic in mice. TRPV4 in skin keratinocytes was essential for LPC-induced itch and itch in mice with cholestasis. Three-dimensional structural modeling, site-directed mutagenesis, and channel function analysis suggested a TRPV4 C-terminal motif for LPC binding and channel activation. In keratinocytes, TRPV4 activation by LPC induced extracellular release of miR-146a, which activated TRPV1 + sensory neurons to cause itch. LPC and miR-146a levels were both elevated in sera of patients with primary biliary cholangitis with itch and correlated with itch intensity. Moreover, LPC and miR-146a were also increased in sera of cholestatic mice and elicited itch in nonhuman primates., Conclusions: We identified LPC as a novel cholestatic pruritogen that induces itch through epithelia-sensory neuron cross talk, whereby it directly activates skin keratinocyte TRPV4, which rapidly releases miR-146a to activate skin-innervating TRPV1 + pruriceptor sensory neurons. Our findings support the new concept of the skin, as a sensory organ, playing a critical role in cholestatic itch, beyond liver, peripheral sensory neurons, and central neural pathways supporting pruriception., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Itch is a unique sensory experience that is encoded by genetically distinguishable neurons both in the peripheral nervous system (PNS) and central nervous system (CNS) to elicit a characteristic behavioral response (scratching). Itch interacts with the other sensory modalities at multiple locations, from its initiation in a particular dermatome to its transmission to the brain where it is finally perceived. In this review, we summarize the current understanding of the molecular and neural mechanisms of itch by starting in the periphery, where itch is initiated, and discussing the circuits involved in itch processing in the CNS., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Chronic, persistent itch is a devastating symptom that causes much suffering. In recent years, there has been great progress made in understanding the molecules, cells, and circuits underlying itch sensation. Once thought to be carried by pain-sensing neurons, itch is now believed to be capable of being transmitted by dedicated sensory labeled lines. Members of the Mas-related G protein-coupled receptor (Mrgpr) family demarcate an itch-specific labeled line in the peripheral nervous system. In the spinal cord, the expression of other proteins identifies additional populations of itch-dedicated sensory neurons. However, as evidence for labeled-line coding has mounted, studies promoting alternative itch-coding strategies have emerged, complicating our understanding of the neural basis of itch. In this review, we cover the molecules, cells, and circuits related to understanding the neural basis of itch, with a focus on the role of Mrgprs in mediating itch sensation.

Background: Pruritus is a cardinal symptom of atopic dermatitis, and an increased cutaneous sensory network is thought to contribute to pruritus. Although the immune cell-IL-31-neuron axis has been implicated in severe pruritus during atopic skin inflammation, IL-31's neuropoietic potential remains elusive., Objective: We sought to analyze the IL-31-related transcriptome in sensory neurons and to investigate whether IL-31 promotes sensory nerve fiber outgrowth., Methods: In vitro primary sensory neuron culture systems were subjected to whole-transcriptome sequencing, ingenuity pathway analysis, immunofluorescence, and nerve elongation, as well as branching assays after IL-31 stimulation. In vivo we investigated the cutaneous sensory neuronal network in wild-type, Il31-transgenic, and IL-31 pump-equipped mice., Results: Transgenic Il31 overexpression and subcutaneously delivered IL-31 induced an increase in the cutaneous nerve fiber density in lesional skin in vivo. Transcriptional profiling of IL-31-activated dorsal root ganglia neurons revealed enrichment for genes promoting nervous system development and neuronal outgrowth and negatively regulating cell death. Moreover, the growth cones of primary small-diameter dorsal root ganglia neurons showed abundant IL-31 receptor α expression. Indeed, IL-31 selectively promoted nerve fiber extension only in small-diameter neurons. Signal transducer and activator of transcription 3 phosphorylation mediated IL-31-induced neuronal outgrowth, and pharmacologic inhibition of signal transducer and activator of transcription 3 completely abolished this effect. In contrast, transient receptor potential cation channel vanilloid subtype 1 channels were dispensable for IL-31-induced neuronal sprouting., Conclusions: The pruritus- and TH2-associated novel cytokine IL-31 induces a distinct transcriptional program in sensory neurons, leading to nerve elongation and branching both in vitro and in vivo. This finding might help us understand the clinical observation that patients with atopic dermatitis experience increased sensitivity to minimal stimuli inducing sustained itch., (Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

The transient receptor potential channels (TRPs) respond to chemical irritants and temperature. TRPV1 responds to the itch-inducing endogenous signal histamine, and TRPA1 responds to the itch-inducing chemical chloroquine. We showed that, in sensory neurons, TRPV4 is important for both chloroquine- and histamine-induced itch and that TRPV1 has a role in chloroquine-induced itch. Chloroquine-induced scratching was reduced in mice in which TRPV1 was knocked down or pharmacologically inhibited. Both TRPV4 and TRPV1 were present in some sensory neurons. Pharmacological blockade of either TRPV4 or TRPV1 significantly attenuated the Ca(2+) response of sensory neurons exposed to histamine or chloroquine. Knockout of Trpv1 impaired Ca(2+) responses and reduced scratching behavior evoked by a TRPV4 agonist, whereas knockout of Trpv4 did not alter TRPV1-mediated capsaicin responses. Electrophysiological analysis of human embryonic kidney (HEK) 293 cells coexpressing TRPV4 and TRPV1 revealed that the presence of both channels enhanced the activation kinetics of TRPV4 but not of TRPV1. Biochemical and biophysical studies suggested a close proximity between TRPV4 and TRPV1 in dorsal root ganglion neurons and in cultured cells. Thus, our studies identified TRPV4 as a channel that contributes to both histamine- and chloroquine-induced itch and indicated that the function of TRPV4 in itch signaling involves TRPV1-mediated facilitation. TRP facilitation through the formation of heteromeric complexes could be a prevalent mechanism by which the vast array of somatosensory information is encoded in sensory neurons., (Copyright © 2016, American Association for the Advancement of Science.)

Itch, the unpleasant sensation that elicits a desire to scratch, is mediated by specific subtypes of cutaneous sensory neuron. Here, we identify a subpopulation of itch-sensing neurons based on their expression of the receptor tyrosine kinase Ret. We apply flow cytometry to isolate Ret-positive neurons from dorsal root ganglia and detected a distinct population marked by low levels of Ret and absence of isolectin B4 binding. We determine the transcriptional profile of these neurons and demonstrate that they express neuropeptides such as somatostatin (Sst), the NGF receptor TrkA, and multiple transcripts associated with itch. We validate the selective expression of Sst using an Sst-Cre driver line and ablated these neurons by generating mice in which the diphtheria toxin receptor is conditionally expressed from the sensory neuron-specific Avil locus. Sst-Cre::Avil(iDTR) mice display normal nociceptive responses to thermal and mechanical stimuli. However, scratching behavior evoked by interleukin-31 (IL-31) or agonist at the 5HT1F receptor is significantly reduced. Our data provide a molecular signature for a subpopulation of neurons activated by multiple pruritogens., (© 2016 The Authors.)

There have been many exciting recent advances in our understanding of the molecular and cellular basis of itch. These discoveries cover diverse aspects of itch sensation, from the identification of new receptors to the characterization of spinal cord itch circuits. A common thread of these studies is that they demonstrate that itch sensory signals are segregated from input for other somatosensory modalities, such as pain, touch, and thermosensation. This specificity is achieved by the expression of dedicated receptors and transmitters in a select population of sensory neurons which detect pruritogens. Further, recent studies show that itch specificity is maintained in a spinal cord circuit by the utilization of specific neurotransmitters and cognate receptors to convey input along a distinct cellular pathway., (Published by Elsevier Ltd.)

Chronic itch, or pruritus, is associated with a wide range of skin abnormalities. The mechanisms responsible for chronic itch induction and persistence remain unclear. We developed a mouse model in which a constitutively active form of the serine/threonine kinase BRAF was expressed in neurons gated by the sodium channel Nav1.8 (BRAF(Nav1.8) mice). We found that constitutive BRAF pathway activation in BRAF(Nav1.8) mice results in ectopic and enhanced expression of a cohort of itch-sensing genes, including gastrin-releasing peptide (GRP) and MAS-related GPCR member A3 (MRGPRA3), in nociceptors expressing transient receptor potential vanilloid 1 (TRPV1). BRAF(Nav1.8) mice showed de novo neuronal responsiveness to pruritogens, enhanced pruriceptor excitability, and heightened evoked and spontaneous scratching behavior. GRP receptor expression was increased in the spinal cord, indicating augmented coding capacity for itch subsequent to amplified pruriceptive inputs. Enhanced GRP expression and sustained ERK phosphorylation were observed in sensory neurons of mice with allergic contact dermatitis– or dry skin–elicited itch; however, spinal ERK activation was not required for maintaining central sensitization of itch. Inhibition of either BRAF or GRP signaling attenuated itch sensation in chronic itch mouse models. These data uncover RAF/MEK/ERK signaling as a key regulator that confers a subset of nociceptors with pruriceptive properties to initiate and maintain long-lasting itch sensation.

Itch-specific neurons have been sought for decades. The existence of such neurons has been doubted recently as a result of the observation that itch-mediating neurons also respond to painful stimuli. We genetically labeled and manipulated MrgprA3(+) neurons in the dorsal root ganglion (DRG) and found that they exclusively innervated the epidermis of the skin and responded to multiple pruritogens. Ablation of MrgprA3(+) neurons led to substantial reductions in scratching evoked by multiple pruritogens and occurring spontaneously under chronic itch conditions, whereas pain sensitivity remained intact. Notably, mice in which TRPV1 was exclusively expressed in MrgprA3(+) neurons exhibited itch, but not pain, behavior in response to capsaicin. Although MrgprA3(+) neurons were sensitive to noxious heat, activation of TRPV1 in these neurons by noxious heat did not alter pain behavior. These data suggest that MrgprA3 defines a specific subpopulation of DRG neurons mediating itch. Our study opens new avenues for studying itch and developing anti-pruritic therapies.

Background: Galanin is a key modulator of nociception, and it is also required for the developmental survival of a subset of C-fibre sensory neurons which are critical to the mediation of neuropathic and inflammatory pain. However, the potential modulatory roles played by galanin, or the galanin-dependent neurons, in pruritoceptive mechanisms underlying the sensation of itch have not been investigated., Findings: Here we report that mice carrying a loss-of-function mutation in the galanin gene (Gal-KO) show no differences in spontaneous behavioural itch responses compared to wild-type (WT) controls. Similarly, the responses to a range of pruritogens are not significantly different between the two genotypes., Conclusions: These results suggest that neither galanin expression, nor the galanin-dependent subpopulation of sensory neurons is required for itch-related behaviours.

Itch, also known as pruritus, is a common, intractable symptom of several skin diseases, such as atopic dermatitis and xerosis. TLRs mediate innate immunity and regulate neuropathic pain, but their roles in pruritus are elusive. Here, we report that scratching behaviors induced by histamine-dependent and -independent pruritogens are markedly reduced in mice lacking the Tlr3 gene. TLR3 is expressed mainly by small-sized primary sensory neurons in dorsal root ganglions (DRGs) that coexpress the itch signaling pathway components transient receptor potential subtype V1 and gastrin-releasing peptide. Notably, we found that treatment with a TLR3 agonist induces inward currents and action potentials in DRG neurons and elicited scratching in WT mice but not Tlr3(-/-) mice. Furthermore, excitatory synaptic transmission in spinal cord slices and long-term potentiation in the intact spinal cord were impaired in Tlr3(-/-) mice but not Tlr7(-/-) mice. Consequently, central sensitization-driven pain hypersensitivity, but not acute pain, was impaired in Tlr3(-/-) mice. In addition, TLR3 knockdown in DRGs also attenuated pruritus in WT mice. Finally, chronic itch in a dry skin condition was substantially reduced in Tlr3(-/-) mice. Our findings demonstrate a critical role of TLR3 in regulating sensory neuronal excitability, spinal cord synaptic transmission, and central sensitization. TLR3 may serve as a new target for developing anti-itch treatment.