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.

The high incidence of treatment-resistant pain calls for the urgent preclinical translation of new analgesics. Understanding the behavioral readout of pain in animals is crucial for efficacy evaluation when developing novel analgesics. Mas-related G protein-coupled receptor D-positive (Mrgprd+) and transient receptor potential vanilloid 1-positive (TRPV1+) sensory neurons are two major non-overlapping subpopulations of C-fiber nociceptors. Their activation has been reported to provoke diverse nocifensive behaviors. However, what kind of behavior reliably represents subjectively conscious pain perception needs to be revisited. Here, we generated transgenic mice in which Mrgprd+ or TRPV1+ sensory neurons specifically express channelrhodopsin-2 (ChR2). Under physiological conditions, optogenetic activation of hindpaw Mrgprd+ afferents evoked reflexive behaviors (lifting, etc.), but failed to produce aversion. In contrast, TRPV1+ afferents activation evoked marked reflexive behaviors and affective responses (licking, etc.), as well as robust aversion. Under neuropathic pain conditions induced by spared nerve injury (SNI), affective behaviors and avoidance can be elicited by Mrgprd+ afferents excitation. Mechanistically, spinal cord-lateral parabrachial nucleus (lPBN) projecting neurons in superficial layers (lamina I-II o ) were activated by TRPV1+ nociceptors in naïve conditions or by Mrgprd+ nociceptors after SNI, whereas only deep spinal cord neurons were activated by Mrgprd+ nociceptors in naïve conditions. Moreover, the excitatory inputs from Mrgprd+ afferents to neurons within inner lamina II (II i ) are partially gated under normal conditions. Altogether, we conclude that optogenetic activation of the adult Mrgprd+ nociceptors drives non-pain-like reflexive behaviors via the deep spinal cord pathway under physiological conditions and drives pain-like affective behaviors via superficial spinal cord pathway under pathological conditions. The distinct spinal pathway transmitting different forms of nocifensive behaviors provides different therapeutic targets. Moreover, this study appeals to the rational evaluation of preclinical analgesic efficacy by using comprehensive and suitable behavioral assays, as well as by assessing neural activity in the two distinct pathways.

Cutaneous mast cells mediate numerous skin inflammatory processes and have anatomical and functional associations with sensory afferent neurons. We reveal that epidermal nerve endings from a subset of sensory nonpeptidergic neurons expressing MrgprD are reduced by the absence of Langerhans cells. Loss of epidermal innervation or ablation of MrgprD-expressing neurons increased expression of a mast cell gene module, including the activating receptor, Mrgprb2, resulting in increased mast cell degranulation and cutaneous inflammation in multiple disease models. Agonism of MrgprD-expressing neurons reduced expression of module genes and suppressed mast cell responses. MrgprD-expressing neurons released glutamate which was increased by MrgprD agonism. Inhibiting glutamate release or glutamate receptor binding yielded hyperresponsive mast cells with a genomic state similar to that in mice lacking MrgprD-expressing neurons. These data demonstrate that MrgprD-expressing neurons suppress mast cell hyperresponsiveness and skin inflammation via glutamate release, thereby revealing an unexpected neuroimmune mechanism maintaining cutaneous immune homeostasis.

Interplay between physiological systems in the body plays a prominent role in health and disease. At the cellular level, such interplay is orchestrated through the binding of specific ligands to their receptors expressed on cell surface. G protein-coupled receptors (GPCR) are seven-transmembrane domain receptors that initiate various cellular responses and regulate homeostasis. In this review, we focus on particular GPCRs named Mas-related G protein-coupled receptors (Mrgprs) mainly expressed by sensory neurons and specialized immune cells. We describe the different subfamilies of Mrgprs and their specific ligands, as well as recent advances in the field that illustrate the role played by these receptors in neuro-immune biological processes, including itch, pain and inflammation in diverse organs.

Due to their pivotal role in nociception and mast cell biology, the family of Mas-related G protein-coupled receptors (Mrgprs) has recently gained attention for their possible expression and role(s) in the gastrointestinal tract. In this context, based on immunocytochemical stainings using a commercial antibody, a recent study by Zhou et al. reported that the murine Mrgprd member is expressed in mouse gut lamina propria immune cells and in the outer smooth muscle layers pointing to a potential role for MrgprD in inflammatory responses and intestinal immunity. Immunohistochemical staining for G protein-coupled receptors (GPCRs), however, remains challenging and should be cautiously interpreted using appropriate specificity controls. Using the same antibody with an identical dilution, we did observe a similar staining in the same wild-type mouse strain, but an identical staining pattern was also found in mice lacking the MrgprD receptor, indicating that this antibody recognizes epitopes other than those of MrgprD. Moreover, in situ hybridization for MrgprD further indicated the absence of receptor mRNA expression in lamina propria immune cells and in the outer smooth muscle layers. Therefore, the results and conclusions regarding the presence of MrgprD at protein level within the GI wall as described in the study of Zhou and collaborators should be interpreted with strong caution and should be reconsidered in the light of the emerging possible roles of MrgprD and therapeutic perspectives in gastrointestinal pathophysiology.

MrgprD, a Mas-related G protein-coupled receptor, is initially identified in sensory neurons of mouse dorsal root ganglia (DRG) and has been suggested to participate in somatosensation. However, MrgprD has recently been found to be expressed outside the nervous system such as in aortic endothelia cells and neutrophils. In this study, we used immunohistochemistry to detect the expression and localization of MrgprD in mouse intestinal tract. The immunoreactivity (IR) of MrgprD was found in the smooth muscle layers of small intestine, colon and rectum. In addition, MrgprD IR was colocalized with F4/80-positive macrophages and CD3-positive T lymphocytes resident in the lamina propria of intestinal mucosa. MrgprD was also found to be expressed in primary peritoneal macrophages and splenic T lymphocytes. Furthermore, the presence of MrgprD mRNA and its protein was detected in murine macrophage-like RAW 264.7 and human T lymphocyte Jurkat cell lines. Our study shows, for the first time, the expression and localization of MrgprD in the intestinal tract and in macrophages and T lymphocytes, indicating the potential roles of MrgprD in intestinal mobility and immunity.

Itch is a common symptom of diseases of the skin but can also accompany diseases of other tissues including the nervous system. Acute itch from chemicals experimentally applied to the skin is initiated and maintained by action potential activity in a subset of nociceptive neurons. But whether these pruriceptive neurons are active or might become intrinsically more excitable under the pathological conditions that produce persistent itch and nociceptive sensations in humans is largely unexplored. Recently, two distinct types of cutaneous nociceptive dorsal root ganglion neurons were identified as responding to pruritic chemicals and playing a role in itch sensation. One expressed the mas-related G-coupled protein receptor MRGPRA3 and the other MRGPRD (MRGPRA3+ and MRGPRD+ neurons, respectively). Here we tested whether these two distinct pruriceptive nociceptors exhibited an enhanced excitability after the development of contact hypersensitivity, an animal model of allergic contact dermatitis, a common pruritic disorder in humans. The characteristics of increased excitability of pruriceptive neurons during this disorder may also pertain to the same types of neurons active in other pruritic diseases or pathologies that affect the nervous system and other tissues or organs. We found that challenging the skin of the calf of the hind paw or the cheek of previously sensitized mice with the hapten, squaric acid dibutyl ester, produced symptoms of contact hypersensitivity including an increase in skin thickness and site-directed spontaneous pain-like (licking or wiping) and itch-like (biting or scratching) behaviours. Ablation of MRGPRA3+ neurons led to a significant reduction in spontaneous scratching of the hapten-challenged nape of the neck of previously sensitized mice. In vivo, electrophysiological recordings revealed that MRGPRA3+ and MRGPRD+ neurons innervating the hapten-challenged skin exhibited a greater incidence of spontaneous activity and/or abnormal after-discharges in response to mechanical and heat stimuli applied to their receptive fields compared with neurons from the vehicle-treated control animals. Whole-cell recordings in vitro showed that both MRGPRA3+ and MRGPRD+ neurons from hapten-challenged mice displayed a significantly more depolarized resting membrane potential, decreased rheobase, and greater number of action potentials at twice rheobase compared with neurons from vehicle controls. These signs of neuronal hyperexcitability were associated with a significant increase in the peak amplitude of tetrodotoxin-sensitive and resistant sodium currents. Thus, the hyperexcitability of MRGPRA3+ and MRGPRD+ neurons, brought about in part by enhanced sodium currents, may contribute to the spontaneous itch- and pain-related behaviours accompanying contact hypersensitivity and/or other inflammatory diseases in humans.

Previous behavioral studies have revealed that CaV3.2 T-type calcium channels support peripheral nociceptive transmission and electrophysiological studies have established the presence of T-currents in putative nociceptive sensory neurons of dorsal root ganglion (DRG). To date, however, the localization pattern of this key nociceptive channel in the soma and peripheral axons of these cells has not been demonstrated due to lack of isoform-selective anti-CaV3.2 antibodies. In the present study a new polyclonal CaV3.2 antibody is used to localize CaV3.2 expression in rodent DRG neurons using different staining techniques including confocal and electron microscopy (EM). Confocal microscopy of both acutely dissociated cells and short-term cultures demonstrated strong immunofluorescence of anti-CaV3.2 antibody that was largely confined to smaller diameter DRG neurons where it co-localized with established immuno-markers of unmyelinated nociceptors, such as, CGRP, IB4 and peripherin. In contrast, a smaller proportion of these CaV3.2-labeled DRG cells also co-expressed neurofilament 200 (NF200), a marker of myelinated sensory neurons. In the rat sciatic nerve preparation, confocal microscopy demonstrated anti-CaV3.2 immunofluorescence which was co-localized with both peripherin and NF200. Further, EM revealed immuno-gold labeling of CaV3.2 preferentially in association with unmyelinated sensory fibers from mouse sciatic nerve. Finally, we demonstrated the expression of CaV3.2 channels in peripheral nerve endings of mouse hindpaw skin as shown by co-localization with Mrgpd-GFP-positive fibers. The CaV3.2 expression within the soma and peripheral axons of nociceptive sensory neurons further demonstrates the importance of this channel in peripheral pain transmission.