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Generation of the Mrgpra3GFP-Cre transgenic mouse line. (a) Diagram showing the mating strategy. (b–d) DRG sections from an Mrgpra3GFP-Cre; ROSA26tdTomato mouse stained with antibody to GFP. tdTomato fluorescence was visualized directly without staining. (e) Merged image of tdTomato fluorescence and bright field view of a DRG section from a Mrgpra3GFP-Cre; ROSA26tdTomato mouse. (f) In situ hybridization with an Mrgpra3 probe on the section shown in e. (g–j) Representative light view (g), fluorescent view (h) and Fura-2 ratiometric images of tdTomato+ DRG neurons labeled as 1, 2 and 3 (i,j). The color of the neurons switching from yellow to green indicates an increase of the intracellular calcium concentration. (k) Representative traces evoked by chloroquine (CQ, 1 mM) in the calcium imaging assay from the three tdTomato+ neurons labeled in j. (l) Representative traces of action potentials induced by chloroquine (1 mM) in neurons from Mrgpra3GFP-Cre mice. Chloroquine induced action potentials in GFP-Cre+ DRG neurons (10 of 11). In contrast, GFP-Cre− DRG neurons (12) did not show any response to chloroquine. (m) RT-PCR analysis of GFP-Cre expression in various tissues from Mrgpra3GFP-Cre mice. GFP-Cre was only detected in the DRG and trigeminal ganglia (TG). Actb, β-actin; WT, wild type. All scale bars represent 50 μm.
Source publication
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 o...
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... investigate the function of MrgprA3 + neurons, we generated a bacterial artificial chromosome (BAC) transgenic mouse line in which the GFP-Cre fusion protein was expressed under the control of the Mrgpra3 promoter (Fig. 1). The Mrgpra3 GFP-Cre transgenic line was fertile and showed no obvious phenotypic or behavioral abnormalities. We crossed Mrgpra3 GFP-Cre mice with Cre-dependent ROSA26 tdTomato reporter mice 15 in which Cre-active neurons are marked by the expression of tdTomato (Fig. 1a). Because of the strong fluorescence of tdTomato, these neurons ...
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... fusion protein was expressed under the control of the Mrgpra3 promoter (Fig. 1). The Mrgpra3 GFP-Cre transgenic line was fertile and showed no obvious phenotypic or behavioral abnormalities. We crossed Mrgpra3 GFP-Cre mice with Cre-dependent ROSA26 tdTomato reporter mice 15 in which Cre-active neurons are marked by the expression of tdTomato (Fig. 1a). Because of the strong fluorescence of tdTomato, these neurons can be visualized directly by epifluorescence without immunohistochemical staining (Fig. 1c,e,h). We used antibodies to GFP to stain adult DRG sections from Mrgpra3 GFP-Cre ; ROSA26 tdTomato mice. All GFP and tdTomato signals were colocalized in a subset of small-diameter ...
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... phenotypic or behavioral abnormalities. We crossed Mrgpra3 GFP-Cre mice with Cre-dependent ROSA26 tdTomato reporter mice 15 in which Cre-active neurons are marked by the expression of tdTomato (Fig. 1a). Because of the strong fluorescence of tdTomato, these neurons can be visualized directly by epifluorescence without immunohistochemical staining (Fig. 1c,e,h). We used antibodies to GFP to stain adult DRG sections from Mrgpra3 GFP-Cre ; ROSA26 tdTomato mice. All GFP and tdTomato signals were colocalized in a subset of small-diameter sensory neurons (312 double-labeled neurons from 3 mice; Fig. ...
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... these neurons can be visualized directly by epifluorescence without immunohistochemical staining (Fig. 1c,e,h). We used antibodies to GFP to stain adult DRG sections from Mrgpra3 GFP-Cre ; ROSA26 tdTomato mice. All GFP and tdTomato signals were colocalized in a subset of small-diameter sensory neurons (312 double-labeled neurons from 3 mice; Fig. ...
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... characterize Cre activity in the MrgprA3 + neurons, we performed Mrgpra3 in situ hybridization on DRG sections from Mrgpra3 GFP-Cre ; ROSA26 tdTomato mice. We collected tdTomato fluorescent images and bright field images of each DRG section before the in situ procedure and matched them to the same DRG section afterward (Fig. 1e,f). Of the tdTomato + neurons examined (389 neurons from 3 mice), 97.2 0.6% exhibited the MrgprA3 in situ signal and 95.5 0.5% of these MrgprA3 + in situ neurons (396 MrgprA3 + neurons from 3 mice) were also tdTomato + , suggesting that the expression of GFP-Cre is tightly controlled by the endogenous Mrgpra3 ...
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... characterize the Mrgpra3 GFP-Cre transgenic line, we used Ca 2+ imaging to examine the responses of dissociated GFP- Cre-labeled DRG neurons to chloroquine. Of the 112 tdTomato + neu- rons obtained from three Mrgpra3 GFP-Cre ; ROSA26 tdTomato mice, 90.8 4.6% showed robust increases in intracellular Ca 2+ following chloro- quine application ( Fig. 1g-k). In whole-cell patch-clamp recording, 10 of 11 GFP-Cre + neurons from Mrgpra3 GFP-Cre transgenic mice dis- played a train of action potentials following chloroquine ...
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... contrast, GFP-Cre -neurons failed to generate action potentials after a similar treatment (Fig. 1l). To examine the expression pattern of GFP-Cre, we performed reverse transcription PCR (RT-PCR) with intron-spanning primers on various adult mouse tissues. Among the tissues tested, GFP-Cre was found only in the DRG and trigeminal ganglia, which is consistent with the expression pattern of MrgprA3 (ref. 11; Fig. 1m). Taken together, ...
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... after a similar treatment (Fig. 1l). To examine the expression pattern of GFP-Cre, we performed reverse transcription PCR (RT-PCR) with intron-spanning primers on various adult mouse tissues. Among the tissues tested, GFP-Cre was found only in the DRG and trigeminal ganglia, which is consistent with the expression pattern of MrgprA3 (ref. 11; Fig. 1m). Taken together, these data indicate that we suc- cessfully generated a transgenic line in which GFP-Cre is specifically expressed in MrgprA3 + neurons and that acts as a powerful tool to examine the function of MrgprA3 + ...
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... CGRP and IB4, 63.1% of the MrgprA3 + neurons expressed both CGRP and IB4 (106 tdTomato + neurons; Supplementary Fig. 1a-d). Thus, MrgprA3 + neurons repre- sent a unique population of DRG neurons that are labeled by both the nonpeptidergic marker IB4 and the peptidergic marker CGRP. ...
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... addition, we performed double-labeling experiments using antibodies to TRPV1 and tdTomato in DRG and found that 19% of TRPV1 + DRG neurons coexpressed MrgprA3, whereas 88.3% of MrgprA3 + neurons coexpressed TRPV1 (Supplementary Fig. 1e-g). Furthermore, 93.3% of MrgprA3 + neurons expressed MrgprC11 (Supplementary Fig. 1h-k). ...
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... addition, we performed double-labeling experiments using antibodies to TRPV1 and tdTomato in DRG and found that 19% of TRPV1 + DRG neurons coexpressed MrgprA3, whereas 88.3% of MrgprA3 + neurons coexpressed TRPV1 (Supplementary Fig. 1e-g). Furthermore, 93.3% of MrgprA3 + neurons expressed MrgprC11 (Supplementary Fig. 1h-k). ...
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... central projections of MrgprA3 + fibers co-terminated with IB4 + fibers in spinal lamina II middle (Supplementary Fig. 1l) 17 . This portion of lamina II is ventral to the CGRP + lamina and dorsal to the PKC lam- ina ( Supplementary Fig. 1m-o). ...
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... central projections of MrgprA3 + fibers co-terminated with IB4 + fibers in spinal lamina II middle (Supplementary Fig. 1l) 17 . This portion of lamina II is ventral to the CGRP + lamina and dorsal to the PKC lam- ina ( Supplementary Fig. 1m-o). Although we found that MrgprA3 + neurons expressed CGRP in cell bodies in the DRG (Fig. 2j-l), only minimal overlap between the MrgprA3 + central projections and the terminals of other CGRP + neurons was observed ( Supplementary Fig. 1m). ...
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... portion of lamina II is ventral to the CGRP + lamina and dorsal to the PKC lam- ina ( Supplementary Fig. 1m-o). Although we found that MrgprA3 + neurons expressed CGRP in cell bodies in the DRG (Fig. 2j-l), only minimal overlap between the MrgprA3 + central projections and the terminals of other CGRP + neurons was observed ( Supplementary Fig. 1m). This could be explained by the fact that MrgprA3 expression is restricted to cell bodies with low CGRP expression. ...
Similar publications
Many primary sensory neurons are polymodal, responding to multiple stimulus modalities (chemical, thermal, or mechanical), yet each modality is recognized differently. Although polymodality implies that stimulus encoding occurs in higher centers, such as the spinal cord or brain, recent sensory neuron ablation studies find that behavioral responses...
Citations
... Prior research using electrophysiological recording and in vitro preparations suggests that most somatosensory neurons respond to multiple stimulus modalities, rendering them polymodal (Crain, 1956;Dichter and Fischbach, 1977;Djouhri and Lawson, 2004). Contradicting this perspective, recent studies propose that the majority of somatosensory neurons are modality-specific (i.e., respond exclusively to specific stimuli, such as noxious mechanical forces, heat or cold) (Abrahamsen et al., 2008;Lagerström et al., 2011;Han et al., 2013;Vrontou et al., 2013;Emery et al., 2016). The cause of these discrepancies remains unclear and may be attributed to differences in experimental models (e.g., in vitro, ex vivo, or in vivo) and recording methods. ...
Introduction
Peripheral sensory neurons serve as the initial responders to the external environment. How these neurons react to different sensory stimuli, such as mechanical or thermal forces applied to the skin, remains unclear.
Methods
Using in vivo two-photon Ca²⁺ imaging in the lumbar 4 dorsal root ganglion (DRG) of awake Thy1.2-GCaMP6s mice, we assessed neuronal responses to various mechanical (punctate or dynamic) and thermal forces (heat or cold) sequentially applied to the paw plantar surface.
Results
Our data indicate that in normal awake male mice, approximately 14 and 38% of DRG neurons respond to either single or multiple modalities of stimulation. Anesthesia substantially reduces the number of responsive neurons but does not alter the ratio of cells exhibiting single-modal responses versus multi-modal responses. Following peripheral nerve injury, DRG cells exhibit a more than 5.1-fold increase in spontaneous neuronal activity and a 1.5-fold increase in sensory stimulus-evoked activity. As neuropathic pain resulting from nerve injury progresses, the polymodal nature of sensory neurons intensifies. The polymodal population increases from 39.1 to 56.9%, while the modality-specific population decreases from 14.7 to 5.0% within a period of 5 days.
Discussion
Our study underscores polymodality as a significant characteristic of primary sensory neurons, which becomes more pronounced during the development of neuropathic pain.
... The underlying neural mechanisms of these pain-itch interactions have been explored, with significant findings revealing their operation at the spinal cord level [3][4][5][6][7][8] . However, despite the progress made at the spinal cord level, minimal research exists thus far regarding the involvement of supraspinal regions in the neural processing of these interactions 2 . ...
... Furthermore, using optogenetic terminal inhibition techniques, we discovered that the neural circuit of ORX neurons from LH to the PAG region plays a crucial part in this bidirectional modulation of itch and pain processing (Figs. 3,5). We also examined the involvement of ORX neurons in the processing of chronic itch, utilizing a chronic contact dermatitis model, which suggests its potential as a novel therapeutic target for chronic pruritus (Fig. 4). ...
Pain and itch are recognized as antagonistically regulated sensations; pain suppresses itch, whilst pain inhibition enhances itch. The neural mechanisms at the central nervous system (CNS) underlying these pain-itch interactions still need to be explored. Here, we revealed the contrasting role of orexin-producing neurons (ORX neurons) in the lateral hypothalamus (LH), which suppresses pain while enhancing itch neural processing, by applying optogenetics to the acute pruritus and pain model. We also revealed that the circuit of ORX neurons from LH to periaqueductal gray regions served in the contrasting modulation of itch and pain processing using optogenetic terminal inhibition techniques. Additionally, by using an atopic dermatitis model, we confirmed the involvement of ORX neurons in regulating chronic itch processing, which could lead to a novel therapeutic target for persistent pruritus in clinical settings. Our findings provide new insight into the mechanism of antagonistic regulation between pain and itch in the CNS.
... Together, these results indicate that MrgprA3 + neurons are itch selective primary sensory neurons [8,11]. Indeed, a recent study demonstrated that selective activation of these pruriceptive neurons using capsaicin, in the absence of nociceptive neurons activation, elicits itch [13]. ...
... Although TRPV1 was reported to contribute to MrgprA3 + mediated itch sensations [13], the involvement Mrgprs (including MrgprA3) in capsaicin induced itch and pain in CD disorder has not been tested. To examine this possibility, we generated the CD model using Mrgprs -/mice. ...
... Primary sensory neurons that express MrgprA3 were recently reported to mediate both itch and pain -metabotropic activation of MrgprA3 + neurons evoked itch in mice, while ionotropic activation these neurons using optogenetics or ATP induced aversive responses distinct from scratching [28]. While broad activation of TRPV1, an ion channel, by capsaicin results in burning pain, selective activation of MrgprA3 + neurons using capsaicin, in the absence of nociceptive neuron activation, evokes itch [13]. The contribution of MrgprA3 + neurons and TRPV1 channels to CD-associated chronic itch and pain, however, is still unclear. ...
Rationale: Noxious stimuli are often perceived as itchy in patients with chronic dermatitis (CD); however, itch and pain mechanisms of CD are not known.
Methods: TRPV1 involvement in CD was analyzed using a SADBE induced CD-like mouse model, and several loss- and gain-of-function mouse models. Trigeminal TRPV1 channel and MrgprA3⁺ neuron functions were analyzed by calcium imaging and whole-cell patch-clamp recordings. Lesional CD-like skin from mice were analyzed by unbiased metabolomic analysis. 20-HETE availability in human and mouse skin were determined by LC/MS and ELISA. And finally, HET0016, a selective 20-HETE synthase inhibitor, was used to evaluate if blocking skin TRPV1 activation alleviates CD-associated chronic itch or pain.
Results: While normally a pain inducing chemical, capsaicin induced both itch and pain in mice with CD condition. DREADD silencing of MrgprA3⁺ primary sensory neurons in these mice selectively decreased capsaicin induced scratching, but not pain-related wiping behavior. In the mice with CD condition, MrgprA3⁺ neurons showed elevated ERK phosphorylation. Further experiments showed that MrgprA3⁺ neurons from MrgprA3;Braf mice, which have constitutively active BRAF in MrgprA3⁺ neurons, were significantly more excitable and responded more strongly to capsaicin. Importantly, capsaicin induced both itch and pain in MrgprA3;Braf mice in an MrgprA3⁺ neuron dependent manner. Finally, the arachidonic acid metabolite 20-HETE, which can activate TRPV1, was significantly elevated in the lesional skin of mice and patients with CD. Treatment with the selective 20-HETE synthase inhibitor HET0016 alleviated itch in mice with CD condition.
Conclusion: Our results demonstrate that 20-HETE activates TRPV1 channels on sensitized MrgprA3⁺ neurons, and induces allokinesis in lesional CD skin. Blockade of 20-HETE synthesis or silencing of TRPV1-MrgprA3⁺ neuron signaling offers promising therapeutic strategies for alleviating CD-associated chronic itch.
... NP2 expressing MrgprA3 and MrgprC11, activated by chloroquine and BAM8-22, respectively; and NP3 expressing histamine and serotonin receptors (5)(6)(7)(8)(9). Other itch receptors such as PAR2, interleukins (IL-4, -13, -31 and -33) or thymic stromal lymphopoietin (TSLP) are also expressed in these neurons (3,(9)(10)(11)(12). ...
... Deletion of the Mrgpr-cluster or specific ablation of MrgprA3 + neurons has been shown to decrease itch in dry skin, contact dermatitis, and allergic dermatitis mouse models (5,64). Additionally, induction of chronic dermatitis through skin treatment with SADBE increases the excitability of MrgprA3 + neurons (39). ...
TRESK (K2P18.1) is a background K+ channel expressed in sensory neurons, where it modulates the resting membrane potential, action potential firing and neuronal excitability. A subset of these sensory neurons, which express specific TRPs and Mas-related G protein-coupled receptors (Mrgprs), are activated by pruritogens and mediate itch sensations. Because TRESK is involved in somatosensation and pain transduction, we evaluated the contribution of this channel to pruritic sensitivity and its potential as a target for the treatment of chronic itch pathologies. By combining RNA in situ hybridization, calcium imaging, electrophysiological and behavioral approaches, we found that TRESK is involved in the modulation of non-histaminergic itch. TRESK is coexpressed with MrgprD+ and MrgprA3+ in sensory neurons and MrgprA3+ neurons from TRESK-/- animals display an enhanced firing compared to WT counterparts. Interestingly, acute itch to intradermal injection of chloroquine is significantly enhanced in the absence of TRESK but not the response to histamine, BAM8-22 or LTC4. TRESK deletion also enhanced chronic itch in mice models of Allergic Contact Dermatitis and Dry Skin. In the mouse model imiquimod-induced psoriasiform dermatitis, the absence of TRESK produced a significantly enhanced scratching behavior, which developed earlier and was more robust. Finally, enhancing TRESK function with the channel activator cloxyquin diminished both acute and chronic itch in WT mice but not in KO animals. In summary, our data indicates that TRESK is involved in regulating the excitability of a subset of sensory neurons that mediate histaminergic-independent itch. Enhancing the channel function with specific activators constitutes a novel anti-pruritic therapeutic method that can be combined with other compounds for the treatment of non-histaminergic itch, for which appropriate treatments are lacking.
... TRP channels are non-selective ion channels primarily located on the plasma membrane in various cells and are divided into six families: TRPV (vanilloid), TRPA (ankyrin), TRPM (melastatin), TRPP (polycystin), TRPC (canonical), and TRPML (mucolipin) [16]. Among the members of the TRP channel family, TRPV1, TRPA1, TRPV3, and TRPV4 are especially known to be involved in itch transduction [17][18][19]. ...
... Similar to C-fibers, an evaluation of the sensory nerves that express MrgprA3-expressing DRG neurons also express receptors for histamine, gastrin-releasing peptide (GRP) [56], and capsaicin (TRPV1) [57]. Han et al. found that the ablation of MrgprA3-expressing neurons in the DRG reduced itch behavior without disturbing pain [17]. Moreover, the expression of TLR7 in primary sensory neurons is required specifically for inducing itch, excluding pain. ...
Chronic itch is a debilitating condition with limited treatment options, severely affecting quality of life. The identification of pruriceptors has sparked a growing interest in the therapeutic potential of TRP channels in the context of itch. In this regard, we provided a comprehensive overview of the site-specific expression of TRP channels and their associated functions in response to a range of pruritogens. Although several potent antipruritic compounds that target specific TRP channels have been developed and have demonstrated efficacy in various chronic itch conditions through experimental means, a more thorough understanding of the potential for adverse effects or interactions with other TRP channels or GPCRs is necessary to develop novel and selective therapeutics that target TRP channels for treating chronic itch. This review focuses on the mechanism of itch associated with TRP channels at specific sites, from the skin to the sensory neuron, with the aim of suggesting specific therapeutic targets for treating this condition.
... Nonhistamine receptors of importance in AD include cytokine receptors and various G protein-coupled receptors other than histamine receptors, such as protease-activated receptors (PARs), toll-like receptors (TLRs), and Mas-related G protein-coupled receptors (Mrgprs) [20,30,32]. Genetic and functional analyses initially supported the existence of itchspecific neurons that express Mrgprs [33]. MrgprX2 is upregulated in patients with AD and correlates with itch intensity [34]. ...
Atopic dermatitis (AD) is a chronic, relapsing immunoinflammatory skin condition characterized by sensations such as pruritis, pain, and neuronal hypersensitivity. The mechanisms underlying these sensations are multifactorial and involve complex crosstalk among several cutaneous components. This review explores the role these components play in the pathophysiology of atopic dermatitis. In the skin intercellular spaces, sensory nerves interact with keratinocytes and immune cells via myriad mediators and receptors. These interactions generate action potentials that transmit pruritis and pain signals from the peripheral nervous system to the brain. Keratinocytes, the most abundant cell type in the epidermis, are key effector cells, triggering crosstalk with immune cells and sensory neurons to elicit pruritis, pain, and inflammation. Filaggrin expression by keratinocytes is reduced in atopic dermatitis, causing a weakened skin barrier and elevated skin pH. Fibroblasts are the main cell type in the dermis and, in atopic dermatitis, appear to reduce keratinocyte differentiation, further weakening the skin barrier. Fibroblasts and mast cells promote inflammation while dermal dendritic cells appear to attenuate inflammation. Inflammatory cytokines and chemokines play a major role in AD pathogenesis. Type 2 immune responses typically generate pruritis, and the type 1 and type 3 responses generate pain. Type 2 responses and increased skin pH contribute to barrier dysfunction and promote dysbiosis of the skin microbiome, causing the proliferation of Staphyloccocus aureus. In conclusion, understanding the dynamic interactions between cutaneous components in AD could drive the development of therapies to improve the quality of life for patients with AD.
... However, these fibers can also respond to noxious heat [4,12]. In mice, different pruritogens activate specific subtypes of G protein-coupled receptors (GPCRs) involved in itch signaling: chloroquine activates MrgprA3, bovine adrenal medulla peptide [8][9][10][11][12][13][14][15][16][17][18][19][20][21][22] activates MrgprC11, β-alanine activates MrgprD, and cowhage (mucunain) activates protease-activated receptors PAR2 and PAR4 [13][14][15]. These receptors presumably label neuron populations that signal itch sensations in mice. ...
... However, these fibers can also respond to noxious heat [4,12]. In mice, different pruritogens activate specific subtypes of G protein-coupled receptors (GPCRs) involved in itch signaling: chloroquine activates MrgprA3, bovine adrenal medulla peptide [8][9][10][11][12][13][14][15][16][17][18][19][20][21][22] activates MrgprC11, β-alanine activates MrgprD, and cowhage (mucunain) activates protease-activated receptors PAR2 and PAR4 [13][14][15]. These receptors presumably label neuron populations that signal itch sensations in mice. ...
... These receptors presumably label neuron populations that signal itch sensations in mice. The selective activation of a specific neuron class carrying the Mrg-prA3 receptor by the classical algogen capsaicin results in pure itch (scratching) behavior in mice [13]. ...
Background
Chronic itch (chronic pruritus) is a major therapeutic challenge that remains poorly understood despite the extensive recent analysis of human pruriceptors. It is unclear how the peripheral nervous system differentiates the signaling of non-histaminergic itch and pain.
Methods
Here we used psychophysical analysis and microneurography (single nerve fiber recordings) in healthy human volunteers to explore the distinct signaling mechanisms of itch, using the pruritogens β-alanine, BAM 8-22 and cowhage extract.
Results
The mode of application (injection or focal application using inactivated cowhage spicules) influenced the itch/pain ratio in sensations induced by BAM 8-22 and cowhage but not β-alanine. We found that sensitizing pre-injections of prostaglandin E2 increased the pain component of BAM 8-22 but not the other pruritogens. A-fibers contributed only to itch induced by β-alanine. TRPV1 and TRPA1 were necessary for itch signaling induced by all three pruritogens. In single-fiber recordings, we found that BAM 8-22 and β-alanine injection activated nearly all CM-fibers (to different extents) but not CMi-fibers, whereas cowhage extract injection activated only 56% of CM-fibers but also 25% of CMi-fibers. A “slow bursting discharge pattern” was evoked in 25% of CM-fibers by β-alanine, in 35% by BAM 8-22, but in only 10% by cowhage extract.
Conclusion
Our results indicate that no labeled line exists for these pruritogens in humans. A combination of different mechanisms, specific for each pruritogen, leads to itching sensations rather than pain. Notably, non-receptor-based mechanisms such as spatial contrast or discharge pattern coding seem to be important processes. These findings will facilitate the discovery of therapeutic targets for chronic pruritus, which are unlikely to be treated effectively by single receptor blockade.
... 26,35 Some evidence has suggested the existence of itch-specific neurons that express Mas-related G protein-coupled receptors (Mrgprs), at least in mice. 36 Alternatively, the differentiation between itch and pain may be based on spinal processing. ...
... 54 Genetic and functional analyses initially supported the existence of itch-specific neurons that express MrgprA3, at least in mice. 36 Mrgprs are expressed exclusively on peripheral sensory neurons and mast cells, and the dorsal root ganglia of Mrgprpositive neurons also express gastrin-releasing peptide, which is an itch-selective neurotransmitter in the spinal cord. 14,56,57,96 Human MrgprX1 and/or MrgprX2 can be activated to induce itch by various substances (eg, substance P, BAM8-22, and chloroquine), is required for downstream itch signaling. ...
... Previous studies have identified several highly restricted populations of primary sensory neurons detecting a particular sensory modality (e.g., TRPM8 + neurons for cold, and MrgprA3 + neurons for itch). 4,5 However, most nociceptive afferent neurons are polymodal and can be involved in both pain and itch. How polymodal sensory neurons differentiate and encode pain and itch signals remains unclear. ...
... These results reveal differential mechanisms in coding and transmission of pain and itch signaling in MrgprD + polymodal sensory neurons, complementing previous studies of itch-selective populations. [2][3][4]17 ...
... Pain behavior experiments-Pain behavioral tests were conducted as previously described. 4,24 In the von Frey assays, mice were placed into acrylic behavioral chambers on a metal mesh. Von Frey filaments, each applying a specific force, were applied to the underside of the paw until bending. ...
Pain and itch coding mechanisms in polymodal sensory neurons remain elusive. MrgprD⁺ neurons represent a major polymodal population and mediate both mechanical pain and nonhistaminergic itch. Here, we show that chemogenetic activation of MrgprD⁺ neurons elicited both pain- and itch-related behavior in a dose-dependent manner, revealing an unanticipated compatibility between pain and itch in polymodal neurons. While VGlut2-dependent glutamate release is required for both pain and itch transmission from MrgprD⁺ neurons, the neuropeptide neuromedin B (NMB) is selectively required for itch signaling. Electrophysiological recordings further demonstrated that glutamate synergizes with NMB to excite NMB-sensitive postsynaptic neurons. Ablation of these spinal neurons selectively abolished itch signals from MrgprD⁺ neurons, without affecting pain signals, suggesting a dedicated itch-processing central circuit. These findings reveal distinct neurotransmitters and neural circuit requirements for pain and itch signaling from MrgprD⁺ polymodal sensory neurons, providing new insights on coding and processing of pain and itch.
... Although our understanding remains incomplete, sensory receptors expressed on peripheral axon terminals are thought to determine the nociceptive or pruriceptive functionality of these primary sensory neurons 18 . These neurons were also shown, however, to transmit the same pain-or itch-speci c signal regardless of the type of sensory receptor they express, indicating that they act as labeled lines 19,20 . Pain and itch signals are transmitted from the spinal cord to the thalamus through the spinothalamic tract (STT) as well as parabrachial nucleus (PBN) and then to several brain regions including the ACC 21,22 . ...
Pain and itch perception both evoke aversive but qualitatively different feelings. The transmission pathways and brain areas that process pain and itch are related, with the anterior cingulate cortex (ACC) being important for the affective dimension of both sensations. The cellular mechanisms by which these two somatosensory stimuli are processed in the same brain area, however, remain largely unknown. Here we identified distinct neuronal populations related to pain and itch processing in layer II/III of the ACC. These include neurons activated by both itch and pain stimuli separated by a short time interval and modality-specific neurons activated only by either itch or pain stimuli regardless of the interval between them. Using the dual-eGRASP (enhanced green fluorescent protein reconstitution across synaptic partners) technique, we found that pain- and itch-specific neurons preferentially receive synaptic connections from mediodorsal thalamic neurons activated by pain and itch stimuli, respectively. Using an inhibitory designer receptor exclusively activated by a designer drug (DREADD), we found that although suppressing itch- or pain-specific neurons reduced pruriception or nociception, respectively, neither type of inhibition affected the opposite modality. Together, these results indicate that the processing of itch and pain information in the ACC involves activity-dependent and modality-specific neuronal populations, and that pain and itch are processed by functionally distinct ACC neuronal subsets.
