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Itch is triggered by somatosensory neurons expressing the ion-channel TRPV1 but the mechanisms underlying this nociceptive response remain poorly understood. Here we showed that the neuropeptide, Nppb, is expressed in a subset of TRPV1-neurons and found that Nppb−/− mice selectively lose almost all behavioral responses to itch-inducing agents. Importantly, Nppb triggered potent scratching when injected intrathecally in wild type and Nppb−/− mice showing that this neuropeptide evokes itch when released from somatosensory neurons. Itch responses were blocked by toxin-mediated ablation of Nppb-receptor expressing cells but a second neuropeptide, GRP, still induced strong responses in the toxin treated animals. Thus our results define the primary pruriceptive neurons, characterize Nppb as an itch-selective neuropeptide and reveal the next two stages of this dedicated neuronal pathway.
Pruritic responses are triggered by somatosensory neurons, with several itch-inducing agents acting through pathway involving the ion-channel TRPV1 (1, 2) and the effector enzyme PLCβ3 (3). Recently, agonist induced silencing of TRPV1-expressing neurons was shown to result in a profound loss of all itch responses (2) but also affects thermosensation and pain (4). In contrast, a much more specific loss of pruriception was observed in mice lacking the gastrin releasing peptide (GRP) receptor (5) and in animals where the spinal interneurons expressing this receptor had been specifically targeted with a GRP-conjugated toxin (GRP-saporin) (6). Thus it was hypothesized that GRP is the neurotransmitter that initiates a labeled line for itch (5, 6).
Previously, we generated mice that had lost all TRPV1-lineage neurons (7). These mice have major pruritic deficits (Fig. 1A) as well as a complete loss of thermosensory input (7) in keeping with previous reports using capsaicin-induced lesions (2, 4). To identify candidate molecules that might mediate itch signaling, we used a differential microarray based screen that identified many TRPV1-enriched transcripts (Table S1). Amongst these, the natriuretic polypeptide b (Nppb) is prominently expressed in a small subset of DRG neurons but is dramatically decreased in sensory ganglia from TRPV1-DTA animals (Fig. 1B). Double label ISH explicitly demonstrates that all Nppb-expressing neurons containTRPV1 (Fig. 1C) and PLCβ3 (Fig. 1D), which are critically required for histamine induced scratching in mice (2, 3). Moreover, double labeling (Fig. 1E, F) shows almost complete overlap between the expression of Nppb and two Mas related G protein coupled receptors that have recently been shown to detect specific pruritogens (8–10).
We generated Nppb−/− animals (Fig. 2A) and showed that these mutants displayed no detectable expression of Nppb (Fig. 2B). The mice were healthy, had normal numbers of nociceptive, touch, and proprioceptive neurons, and the distribution and number of dorsal horn interneurons was unaffected by gene disruption (Fig. S1). Nppb−/− mice retained normal responses to thermal, nocieptive, touch, and proprioceptive stimulation when tested using standard assays (Fig. 2C). We performed intradermal injections and recorded numbers of scratching bouts for substances that directly activate pruriceptors and with compound 48/80 that causes itch via an indirect route (11). All these agents (Table S2) reliably triggered multiple bouts of scratching in control animals (Fig. 2D, grey bars) but Nppb−/− mice were almost completely insensitive to the full range of pruritic substances tested (Fig. 2D, red bars).
How does Nppb induce this stereotyped scratch response? We reasoned that because this peptide is prominently expressed in somatosensory neurons the most plausible explanation for its role would be if it acted as a specific itch related neuromodulator (or neurotransmitter) in the spinal cord. Indeed intrathecal injection of Nppb induced profound scratching behavior in wild type animals (Fig. 2E, grey bar) demonstrating that spinal-Nppb is sufficient to induce itch even without activation of the peripheral neurons that express it. Intrathecal injection of Nppb into Nppb−/− mice also led to an equivalent phenotype (Fig. 2E, red bar). Loss of Nppb in sensory afferents was thus responsible for the pruriceptive deficits seen in mutant mice. Therefore we suggest that Nppb-expression delineates the subset of somatosensory neurons that detect pruritic agents and that central release of Nppb from these neurons is necessary for the itch response.
Because Nppb is responsible for transmitting the peripheral signals that trigger pruritic responses, its receptor Npra (12) should be expressed at the site of afferent fiber synaptic connections in the spinal dorsal horn and mark the secondary neurons in the itch response circuit. Therefore we used ISH to localize Npra expression in the dorsal horn and found that it is indeed expressed in a limited subset of neurons (most likely interneurons, see below), primarily in the outer layer i.e. lamina I (Fig. 3A left panel) corresponding to the terminal field of TRPV1 expressing sensory neurons (13).
To examine whether the Npra neurons in the spinal cord function in the itch circuit and if they are selectively required for pruritogen induced scratching (rather than other somatosensory responses) we used a targeted-toxin cell ablation strategy (14). We injected an Nppb-saporin conjugate intrathecally into wild type mice to target their Npra expressing cells and assessed the effectiveness, specificity and behavioral consequences of administering this toxin. Approximately 70 % of Npra-positive neurons were eliminated after administration of toxin (Figs. 3A, S3). This targeting was highly selective, with neither cells expressing the GRP-receptor nor other dorsal horn interneurons affected by Nppb-saporin treatment (Figs. 3A, S3). Toxin injected mice displayed normal responses to thermal, touch, and painful stimulation (Fig. 3B). However, we observed a dramatic reduction in scratching evoked by histamine (Fig. 3C) indicating that these neurons are required for itch responses but not for other somatosensory pathways.
How do these findings fit with current models of itch behavior? The GRP-receptor has been shown to be a key element in the pruritic pathway (5, 6) with the suggestion that GRP might be the primary neurotransmitter for itch. However, this view has also been questioned (11, 15). We were unable to detect more than trace quantities of GRP expression in the DRG using a sensitive qPCR assay (Fig. 4A). Similarly, somatosensory neurons from GRP-reporter mice Tg(GRP-EGFP) were negative for EGFP expression (Figs. S4A). In contrast, ISH for GRP and analysis of EGFP expression in the GRP-reporter mice revealed that this neuropeptide is present in a population of neurons in the dorsal horn (Figs. 4B, S4A) as reported previously (15, 16). Therefore we concluded that GRP cannot act at the level of pruriception but must function downstream of Nppb and applied three complementary functional strategies to substantiate this hypothesis and dissect the itch response circuit.
First we demonstrated that GRP induced scratching was unaltered either by Nppb-knockout (Fig. 2E) or by the ablation of Npra-expressing neurons (Fig. 4D). Second, we showed that pharmacological inhibition of the GRP-receptor not only attenuated behavioral responses to the pruritic agent histamine or GRP injection (Fig. S4B), but also inhibited scratching after intrathecal administration of Nppb (Fig. 4E). Lastly, we tested mice with selective ablation of GRP-receptor expressing neurons and again found significantly reduced itch responses to Nppb (Fig. 4E). These data place GRP downstream of Nppb in the itch response circuit, suggest that the secondary pruriceptors are targets for one neuropeptide, Nppb and in turn signal through a second peptide, GRP. Indeed, just as this model predicts, all Npra-expressing neurons in the dorsal horn contain GRP (Fig. 4F) and Nppb-saporin treatment significantly reduces the number of GRP expressing cells (Fig. 4B, right panel).
Our results molecularly characterize the first three stations of an itch response pathway in mice (Fig. 4G), demonstrate that Nppb marks the primary sensory neurons and show that this peptide is both necessary and sufficient for transmission of peripheral signals that induce stereotypic itch responses. Unlike previously characterized receptors (8, 9) and signaling molecules (1–3) that affect the detection of particular itch-inducing agents, Nppb is necessary for responses to a wide range of pruritogens (i.e., compounds classed as inducing histamine and non-histamine related itch, Table S2). Our data also refine the role GRP and GRP-receptor cells play in the itch response pathway by placing them at later stages than had been hypothesized previously (5, 6). Many questions about itch remain unanswered, including its close relationship to sensing pain (17), its slow kinetics and long duration, as well as reports from human and non-human primate studies that different central pathways mediate histamine and non-hitamine itch (18–21). Because Nppb is critically required for pruriception in mice, future studies involving ablation and/or activation of the Nppb-expressing somatosensory neurons together with circuit tracing may reveal whether these cells directly trigger a spinal scratch reflex, are selective detectors for the sensation of itch or are in fact more broadly-tuned nociceptors. Such experiments will help reveal the central mechanism(s) for itch, explain the interactions between pruriception and other somatosensory signals and ultimately provide a powerful stimulus for the rational design of novel therapies to alleviate chronic itch.
We are grateful to Nick Ryba for encouragement and helpful advice and for valuable suggestions. The Nppb−/− mice used for this research project were generated ES cells obtained from the NCRR-NIH supported KOMP repository (www.komp.org) and engineered by the Wellcome Trust Sanger Institute and the Mouse Biology Program (www.mousebiology.org). We thank NIDCR GTF for help generating Nppb mutant mice. The mouse strain STOCK Tg(GRPEGFP) was obtained from the MMRRC, a NCRR-NIH funded strain repository, and was donated to the MMRRC by the NINDS funded GENSAT BAC transgenic project. This research was supported by the intramural research program of the NIH, NIDCR.
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