gastrin-releasing-peptide and Pruritus

Itch is one of the most primal sensations, being both ubiquitous and important for the well-being of animals. For more than a century, a desire to understand how itch is encoded by the nervous system has prompted the advancement of many theories. Within the past 15 years, our understanding of the molecular and neural mechanisms of itch has undergone a major transformation, and this remarkable progress continues today without any sign of abating. Here I describe accumulating evidence that indicates that itch is distinguished from pain through the actions of itch-specific neuropeptides that relay itch information to the spinal cord. According to this model, classical neurotransmitters transmit, inhibit and modulate itch information in a context-, space- and time-dependent manner but do not encode itch specificity. Gastrin-releasing peptide (GRP) is proposed to be a key itch-specific neuropeptide, with spinal neurons expressing GRP receptor (GRPR) functioning as a key part of a convergent circuit for the conveyance of peripheral itch information to the brain.

Topics: Animals; Gastrin-Releasing Peptide; Humans; Neurons; Neuropeptides; Pruritus; Spinal Cord

Itch, in particular chronic forms, has been widely recognized as an important clinical problem, but much less is known about the mechanisms of itch in comparison with other sensory modalities such as pain. Recently, considerable progress has been made in dissecting the circuit mechanisms of itch at both the spinal and supraspinal levels. Major components of the spinal neural circuit underlying both chemical and mechanical itch have now been identified, along with the circuits relaying ascending transmission and the descending modulation of itch. In this review, we summarize the progress in elucidating the neural circuit mechanism of itch at spinal and supraspinal levels.

Topics: Animals; Brain; Gastrin-Releasing Peptide; Humans; Neurons; Pruritus; Receptors, Bombesin; Signal Transduction; Spinal Cord

Astrocytes are the most abundant glial cells in the central nervous system (CNS), including the spinal cord. Neuronal damage induces astrocytes to become reactive and contribute to various CNS pathologies. Recent studies have demonstrated that astrocytes in the spinal dorsal horn (SDH) become reactive in a transcription factor signal transducer and activator of transcription 3-dependent manner without neuronal damage under chronic itch conditions, causing release of the factor lipocalin-2, leading to induction of sensitization of gastrin releasing peptide-induced chemical itch signaling in the SDH. In this review, we describe recent advances in our understanding of SDH neuronal pathways for itch transmission, the mechanisms of SDH astrocytic activation and its contribution to abnormal itch processing and discuss the role of reactive astrocytes in the SDH in abnormal sensory processing under chronic itch conditions.

Topics: Astrocytes; Chronic Disease; Gastrin-Releasing Peptide; Humans; Lipocalin-2; Neural Pathways; Pruritus; Signal Transduction; Spinal Cord Dorsal Horn; STAT3 Transcription Factor

Neuropathic itch is clinically important but has received much less attention as compared to neuropathic pain. In the past decade, itch-specific pathways have been characterized on a cellular and molecular level, but their exact role in the pathophysiology of neuropathic itch is still unclear. Traditionally, mutually exclusive theories for itch such as labeled line, temporal/spatial pattern, or intensity theory have been proposed, and experimental studies in mice mainly favor the specificity theory of itch. By contrast, results in humans also suggest a role for spatial and temporal patterns in neuropathic itch. Rarefication of skin innervation in neuropathy could provide a "spatial contrast" discharge pattern, and axotomy could induce de novo expression of the itch-specific spinal neuropeptide, gastrin-releasing peptide, in primary afferent nociceptors, thereby modulating itch processing in the dorsal horn. Thus, clinical neuropathy may generate itch by changes in the spatial and temporal discharge patterns of nociceptors, hijacking the labeled line processing of itch and abandoning the canonical scheme of mutual exclusive itch theories. Moreover, the overlap between itch and pain symptoms in neuropathy patients complicates direct translation from animal experiments and, on a clinical level, necessitates collaboration between medical specialities, such as dermatologists, anesthesiologists, and neurologists.

Topics: Animals; Gastrin-Releasing Peptide; Humans; Neuralgia; Nociceptors; Pruritus

The current review highlights recent advances in physiological and pharmacological researches in biology of mammalian bombesin-like peptides (BLPs).. BLPs and their receptors were found to have regulatory roles in many biological processes in central nervous system. Two BLPs, neuromedin B and gastrin-releasing peptide (GRP), and their receptors are required for regulation of basal and induced sighing activity in rodents. This is the first study demonstrating central pathways involved in regulation of sighing activity. GRP receptor (GRPR) expressing neurons are excitatory glutamatergic interneurons located in the dorsal lamina without projections outside the spinal cord and mediate itch signals via vesicular glutamate transporter 2. Those neurons receive itch signals and make synapses with the parabrachial nucleus projecting spinal neurons to transmit itch signals to parabrachial nucleus. GRP expressing interneurons function in a proposed 'leaky gate model' to interpret the mechanism of both pain and itch transmission. In addition to recent advances of biology in nervous system, BLPs and their receptors were found to play potential regulatory roles in innate and adaptive immune responses and tissue development.. Several important biological roles of BLPs and their receptors in nervous system were identified. Together with researches regarding central roles of BLPs, studies revealing the regulatory roles of BLPs and their receptors in immunology and tissue development provide us with novel insights into understanding of the biology of BLPs and their receptors.

Topics: Animals; Biological Phenomena; Bombesin; Gastrin-Releasing Peptide; Glucose; Humans; Neurokinin B; Organogenesis; Pain Perception; Pruritus; Receptors, Bombesin

To highlight the most recent advances regarding gastrointestinal peptides and their relation to chronic itch, with focus on gastrin-releasing peptide (GRP), substance P, and their respective receptors.. GRP and its high-affinity GRP receptor (GRPR) have been identified as key regulators in the spinal cord itch pathway and may be involved in the maintenance of chronic itch sensation. Several neuropeptides including GRP, neuromedin B, and substance P regulate itch signals in a cooperative or inhibitory manner on the spinal level. Small clinical studies show that neurokinin 1 receptor antagonists might be of benefit in the treatment of chronic itch.. Chronic itch is a burdensome clinical problem, for which no specific treatment is available. Studies on the mechanisms of pruriceptive sensation and its signaling to the central nervous system (CNS) via the spinal cord have elucidated a number of peptides that are implicated in the regulation of itch-specific signaling pathways. Among those, GRP and its high-affinity GRP receptor have been proposed as key elements in the itch-specific neuronal pathways.

Topics: Gastrin-Releasing Peptide; Humans; Neurokinin-1 Receptor Antagonists; Neurotransmitter Agents; Pruritus; Signal Transduction; Spinal Cord; Substance P

Pruritus is commonly associated with cholestatic disorders and shows wide interindividual variability. The presence of skin lesions due to scratching and the application of a visual analogue scale are useful for clinical evaluation. Although the pathophysiology of this entity is not well understood, advances have recently been made in understanding of the pruritoceptive neural pathway, which shares certain similarities with the nociceptive pathway, although there are other distinguishing characteristics such as the action of a specific neurotransmitter, GPR, on the first synapsis at the posterior horn of the spinal cord. Amongst the modulator systems of the pruritoceptive pathway is the action of the endogenous opioids. An increase of these opioids in cholestatic situations is the most widely accepted hypothesis for pruritus in these patients. Some treatments have proven efficacy in randomized clinical trials in patients with cholestatic disorders, such as anion exchange resins, rifampicin, opioid antagonists and ursodeoxycholic acid; the latter is especially useful in intrahepatic cholestasis of pregnancy.

Topics: Animals; Anion Exchange Resins; Cholestasis; Evidence-Based Medicine; Female; Gastrin-Releasing Peptide; Haplorhini; Humans; Male; Models, Neurological; Narcotic Antagonists; Neural Pathways; Opioid Peptides; Posterior Horn Cells; Pregnancy; Pregnancy Complications; Pruritus; Randomized Controlled Trials as Topic; Rifampin; Ursodeoxycholic Acid

Neurons in the superficial dorsal horn that express the gastrin-releasing peptide receptor (GRPR) are strongly implicated in spinal itch pathways. However, a recent study reported that many of these correspond to vertical cells, a population of interneurons that are believed to transmit nociceptive information. In this study, we have used a GRPR CreERT2 mouse line to identify and target cells that possess Grpr mRNA. We find that the GRPR cells are highly concentrated in lamina I and the outer part of lamina II, that they are all glutamatergic, and that they account for ∼15% of the excitatory neurons in the superficial dorsal horn. We had previously identified 6 neurochemically distinct excitatory interneuron populations in this region based on neuropeptide expression and the GRPR cells are largely separate from these, although they show some overlap with cells that express substance P. Anatomical analysis revealed that the GRPR neurons are indeed vertical cells, and that their axons target each other, as well as arborising in regions that contain projection neurons: lamina I, the lateral spinal nucleus, and the lateral part of lamina V. Surprisingly, given the proposed role of GRPR cells in itch, we found that most of the cells received monosynaptic input from Trpv1-expressing (nociceptive) afferents, that the majority responded to noxious and pruritic stimuli, and that chemogenetically activating them resulted in pain-related and itch-related behaviours. Together, these findings suggest that the GRPR cells are involved in spinal cord circuits that underlie both pain and itch.

Topics: Animals; Gastrin-Releasing Peptide; Interneurons; Mice; Pain; Posterior Horn Cells; Pruritus; Receptors, Bombesin; Spinal Cord; Spinal Cord Dorsal Horn

Gastrin releasing peptide receptor (GRPR), a member of the bombesin (BBN) G protein-coupled receptors, is aberrantly overexpressed in several malignant tumors, including those of the breast, prostate, pancreas, lung, and central nervous system. Additionally, it also mediates non-histaminergic itch and pathological itch conditions in mice. Thus, GRPR could be an attractive target for cancer and itch therapy. Here, we report the inactive state crystal structure of human GRPR in complex with the non-peptide antagonist PD176252, as well as two active state cryo-electron microscopy (cryo-EM) structures of GRPR bound to the endogenous peptide agonist gastrin-releasing peptide and the synthetic BBN analog [D-Phe

Topics: Animals; Bombesin; Cryoelectron Microscopy; Gastrin-Releasing Peptide; Humans; Male; Mice; Neoplasms; Pruritus; Receptors, Bombesin

In today's society, people are subjected to many social stressors, and excessive chronic stress causes functional disruption of the neuroendocrine system and many diseases. Although the exacerbation of atopic dermatitis with symptoms of itching and erectile dysfunction is induced by chronic stress, the details of the mechanisms are unknown. Here, we examined the effects of chronic stress on itch sensation and male sexual function at the behavioral and molecular levels, focusing on two distinct gastrin-releasing peptide (GRP) systems that independently regulate itch transmission, i.e., the somatosensory GRP system, and male sexual function, i.e., the lumbosacral autonomic GRP system, in the spinal cord. In a rat model of chronic stress induced by chronic corticosterone (CORT) administration, we observed increased plasma CORT concentrations, decreased body weight, and increased anxiety-like behavior, similar to that observed in humans. Chronic CORT exposure induced hypersensitivity to itch and increased the Grp mRNA level in the spinal somatosensory system, but there was no change in pain or tactile sensitivity. Antagonists of the somatosensory GRP receptor, an itch-specific mediator, suppressed itch hypersensitivity induced by chronic CORT exposure. In contrast, chronic CORT exposure decreased male sexual behavior, ejaculated semen volume, vesicular gland weight, and plasma testosterone levels. However, there were no effects on the expression of Grp mRNA or protein in the lumbosacral autonomic GRP system, which regulates male sexual function. In summary, chronic stress model rats showed itch hypersensitivity and impaired sexual function in males, and the involvement of the spinal GRP systems was apparent in itch hypersensitivity.

Topics: Animals; Corticosterone; Gastrin-Releasing Peptide; Humans; Male; Pruritus; Rats; RNA, Messenger; Spinal Cord

Itch (pruritus) is a common unpleasant feeling, often accompanied by the urge of scratching the skin. It is the main symptom of many systemic and skin diseases, which can seriously affect the patient's quality of life. Geraniol (GE; trans-3,7-dimethyl-2,6-octadien-1-ol) is a natural monoterpene with diverse effects, including anti-inflammatory, antioxidant, neuroprotective, anti-nociceptive, and anticancer properties. The study aims to examine the effects of GE on acute and chronic itch, and explore the underlying mechanisms.. Acute itch was investigated by using Chloroquine and compound 48/80 induced model, followed by manifestation of diphenylcyclopropenone (DCP)-induced allergic contact dermatitis and the acetone-ether-water (AEW)-induced dry skin model in mice. The scratching behavior, skin thickness, c-Fos expression, and GRPR protein expression in the spinal cord were subsequently monitored and evaluated by behavioral tests as well as pharmacological and pharmacogenetic technologies.. GE alleviates both acute and chronic itch via modulating the spinal GABA/GRPR signaling in mice. Findings of this study reveal that GE may provide promising therapeutic options for itch management. Also, considering the pivotal role of essential oils in aromatherapy, GE has great application potential in aromatherapy for treating skin diseases, and especially the skin with severe pruritus.

Topics: Animals; Antipruritics; Bicuculline; Chloroquine; gamma-Aminobutyric Acid; Gastrin-Releasing Peptide; Mice; Pruritus; Quality of Life; Spinal Cord

A11 dopaminergic neurons regulate somatosensory transduction by projecting from the diencephalon to the spinal cord, but the function of this descending projection in itch remained elusive. Here, we report that dopaminergic projection neurons from the A11 nucleus to the spinal dorsal horn (dopaminergic

Topics: Dopamine; Dopaminergic Neurons; Gastrin-Releasing Peptide; Glutamic Acid; Humans; Pruritus; Receptors, AMPA; Receptors, Bombesin; Spinal Cord

Although pruritus, commonly known as itch, is a common and debilitating symptom associated with various skin conditions, there is a lack of effective therapies available. Xanthotoxol (XAN), a biologically active linear furocoumarin, shows potential in the treatment of various neurological disorders. In this study, we discovered that administering XAN either through intraperitoneal or intrathecal injections effectively reduced scratching behavior induced by compound 48/80 or chloroquine. Importantly, XAN also substantially alleviates chronic itch in dry skin and allergic contact dermatitis mice. Substantial progress has highlighted the crucial role of gastrin-releasing peptide (GRP)-gastrin-releasing peptide receptor (GRPR) signaling in the dorsal spinal cord in transmitting various types of itch. Our behavior tests revealed that XAN significantly alleviated scratching behaviors induced by intrathecal administration of GRP or GRPR agonist bombesin. Furthermore, XAN reduced the activation of neurons in the spinal cord caused by intrathecal administration of GRP in mice. Moreover, XAN attenuates the activation of spinal GRPR-positive neurons in itchy mice. These findings suggest that XAN mitigates itch in mice by suppressing spinal GRP/GRPR signaling, thereby establishing XAN as a promising therapeutic option for treating pruritus.

Topics: Animals; Furocoumarins; Gastrin-Releasing Peptide; Mice; Mice, Inbred C57BL; Pruritus; Receptors, Bombesin; Spinal Cord

Despite accumulating evidence in rodents, the functional role of neuromedin B (NMB) in regulating somatosensory systems in primate spinal cord is unknown. We aimed to compare the expression patterns of NMB and its receptor (NMBR) and the behavioral effects of intrathecal (i.t.) NMB with gastrin-releasing peptide (GRP) on itch or pain in non-human primates (NHPs). We used six adult rhesus monkeys. The mRNA or protein expressions of NMB, GRP, and their receptors were evaluated by quantitative reverse transcription polymerase chain reaction, immunohistochemistry, or in situ hybridization. We determined the behavioral effects of NMB or GRP via acute thermal nociception, capsaicin-induced thermal allodynia, and itch scratching response assays. NMB expression levels were greater than those of GRP in the dorsal root ganglia and spinal dorsal horn. Conversely, NMBR expression was significantly lower than GRP receptor (GRPR). I.t. NMB elicited only mild scratching responses, whereas GRP caused robust scratching responses. GRP- and NMB-elicited scratching responses were attenuated by GRPR (RC-3095) and NMBR (PD168368) antagonists, respectively. Moreover, i.t. NMB and GRP did not induce thermal hypersensitivity and GRPR and NMBR antagonists did not affect peripherally elicited thermal allodynia. Consistently, NMBR expression was low in both itch- and pain-responsive neurons in the spinal dorsal horn. Spinal NMB-NMBR system plays a minimal functional role in the neurotransmission of itch and pain in primates. Unlike the functional significance of the GRP-GRPR system in itch, drugs targeting the spinal NMB-NMBR system may not effectively alleviate non-NMBR-mediated itch.

Topics: Animals; Gastrin-Releasing Peptide; Hyperalgesia; Neurokinin B; Pain; Primates; Pruritus; Receptors, Bombesin; Spinal Cord; Spinal Cord Dorsal Horn

Gastrin-releasing peptide (GRP) and its receptor (GRPR) have been identified as itch mediators in the spinal and trigeminal somatosensory systems in rodents. In primates, there are few reports of GRP/GRPR expression or function in the spinal sensory system and virtually nothing is known in the trigeminal system. The aim of the present study was to characterize GRP and GRPR in the trigeminal and spinal somatosensory system of Japanese macaque monkeys (Macaca fuscata). cDNA encoding GRP was isolated from the macaque dorsal root ganglion (DRG) and exhibited an amino acid sequence that was highly conserved among mammals and especially in primates. Immunohistochemical analysis demonstrated that GRP was expressed mainly in the small-sized trigeminal ganglion and DRG in adult macaque monkeys. Densely stained GRP-immunoreactive (ir) fibers were observed in superficial layers of the spinal trigeminal nucleus caudalis (Sp5C) and the spinal cord. In contrast, GRP-ir fibers were rarely observed in the principal sensory trigeminal nucleus and oral and interpolar divisions of the spinal trigeminal nucleus. cDNA cloning, in situ hybridization, and Western blot revealed substantial expression of GRPR mRNA and GRPR protein in the macaque spinal dorsal horn and Sp5C. Our Western ligand blot and ligand derivative stain for GRPR revealed that GRP directly bound in the macaque Sp5C and spinal dorsal horn as reported in rodents. Finally, GRP-ir fibers were also detected in the human spinal dorsal horn. The spinal and trigeminal itch neural circuits labeled with GRP and GRPR appear to function also in primates.

Topics: Animals; DNA, Complementary; Gastrin-Releasing Peptide; Humans; Ligands; Macaca fuscata; Pruritus; Receptors, Bombesin; Sense Organs

Itch and pain are both unpleasant, but they are discrete sensations. Both of these sensations are transmitted by C-fibers and processed in laminae I-II of the dorsal horn. To examine whether pruriception modulates pain, we first confirmed the activation of cells in the itch-related circuits that were positive for gastrin-releasing peptide (GRP) and GRP receptor (GRPR) using a paw formalin injection model. This pain model with typical biphasic pain behavior increased c-Fos but did not affect the expressions of

Topics: Analgesics; Formaldehyde; Gastrin-Releasing Peptide; Humans; Nerve Fibers, Unmyelinated; Pain; Posterior Horn Cells; Pruritus; Receptors, Bombesin; Spinal Cord; Spinal Cord Dorsal Horn

Contagious itch behavior informs conspecifics of adverse environment and is crucial for the survival of social animals. Gastrin-releasing peptide (GRP) and its receptor (GRPR) in the suprachiasmatic nucleus (SCN) of the hypothalamus mediates contagious itch behavior in mice. Here, we show that intrinsically photosensitive retina ganglion cells (ipRGCs) convey visual itch information, independently of melanopsin, from the retina to GRP neurons via PACAP-PAC1R signaling. Moreover, GRPR neurons relay itch information to the paraventricular nucleus of the thalamus (PVT). Surprisingly, neither the visual cortex nor superior colliculus is involved in contagious itch. In vivo calcium imaging and extracellular recordings reveal contagious itch-specific neural dynamics of GRPR neurons. Thus, we propose that the retina-ipRGC-SCN-PVT pathway constitutes a previously unknown visual pathway that probably evolved for motion vision that encodes salient environmental cues and enables animals to imitate behaviors of conspecifics as an anticipatory mechanism to cope with adverse conditions.

Topics: Animals; Calcium; Gastrin-Releasing Peptide; Mice; Pituitary Adenylate Cyclase-Activating Polypeptide; Pruritus; Retina; Retinal Ganglion Cells; Suprachiasmatic Nucleus; Visual Pathways

Several lines of evidence have clarified that the key transmission pathways of itching sensation travel from the periphery to the central nervous system (CNS). Despite the functional significance of gastrin-releasing peptide (GRP) and its cognate receptor in the itch processing mechanism in the spinal dorsal horn (SDH), the roles of GRP-expressing (GRP

Topics: Animals; Behavior, Animal; Clozapine; Cyclopropanes; Dermatitis, Contact; Female; Gastrin-Releasing Peptide; Haptens; Male; Mice, Inbred C57BL; Mice, Transgenic; Neurons; Pruritus

Chronic itch is a highly debilitating symptom among patients with inflammatory skin diseases. Recent studies have revealed that gastrin-releasing peptide (GRP) and its receptor (gastrin-releasing peptide receptor [GRPR]) in the spinal dorsal horn (SDH) play a central role in itch transmission.. We aimed to investigate whether GRP-GRPR signaling is altered in SDH neurons in a mouse model of chronic itch and to determine the potential mechanisms underlying these alterations.. Patch-clamp recordings from enhanced green fluorescent protein (EGFP)-expressing (GRPR. We observed potentiation of GRP-evoked excitation in the GRPR. Our findings indicate that, under chronic itch conditions, the GRP-induced excitability of GRPR

Topics: Animals; Astrocytes; Chronic Disease; Disease Models, Animal; Gastrin-Releasing Peptide; Male; Mice; Mice, Transgenic; Posterior Horn Cells; Pruritus; Receptors, Bombesin; Signal Transduction

Gastrin-releasing peptide (GRP) functions as a neurotransmitter for non-histaminergic itch, but its site of action (sensory neurons vs spinal cord) remains controversial. To determine the role of GRP in sensory neurons, we generated a floxed Grp mouse line. We found that conditional knockout of Grp in sensory neurons results in attenuated non-histaminergic itch, without impairing histamine-induced itch. Using a Grp-Cre knock-in mouse line, we show that the upper epidermis of the skin is exclusively innervated by GRP fibers, whose activation via optogeneics and chemogenetics in the skin evokes itch- but not pain-related scratching or wiping behaviors. In contrast, intersectional genetic ablation of spinal Grp neurons does not affect itch nor pain transmission, demonstrating that spinal Grp neurons are dispensable for itch transmission. These data indicate that GRP is a neuropeptide in sensory neurons for non-histaminergic itch, and GRP sensory neurons are dedicated to itch transmission.

Topics: Animals; Disease Models, Animal; Gastrin-Releasing Peptide; Gene Knockout Techniques; Histamine; Mice; Mice, Inbred C57BL; Mice, Knockout; Neurotransmitter Agents; Pain; Pruritus; Sensory Receptor Cells; Skin; Spinal Cord; Transcriptome

Itch and pain are distinct unpleasant sensations that can be triggered from the same receptive fields in the skin, raising the question of how pruriception and nociception are coded and discriminated. Here, we tested the multimodal capacity of peripheral first-order neurons, focusing on the genetically defined subpopulation of mouse C-fibers that express the chloroquine receptor MrgprA3. Using optogenetics, chemogenetics, and pharmacology, we assessed the behavioral effects of their selective stimulation in a wide variety of conditions. We show that metabotropic Gq-linked stimulation of these C-afferents, through activation of native MrgprA3 receptors or DREADDs, evokes stereotypical pruriceptive rather than nocifensive behaviors. In contrast, fast ionotropic stimulation of these same neurons through light-gated cation channels or native ATP-gated P2X3 channels predominantly evokes nocifensive rather than pruriceptive responses. We conclude that C-afferents display intrinsic multimodality, and we provide evidence that optogenetic and chemogenetic interventions on the same neuronal populations can drive distinct behavioral outputs.

Topics: Adenosine Triphosphate; Animals; Channelrhodopsins; Chloroquine; Ganglia, Spinal; Gastrin-Releasing Peptide; Light; Mice; Nerve Fibers, Unmyelinated; Neurons, Afferent; Nociception; Optogenetics; Pain; Pruritus; Receptors, G-Protein-Coupled; Receptors, Opioid; Receptors, Purinergic P2X3; Transient Receptor Potential Channels

In the past 10 years specific pathways for pruritus have been characterized on a cellular and molecular level but their exact role in the pathophysiology of neuropathic pruritus remains unclear. This also applies to the question which of the competing theories for pruritus, e.g. specificity, temporal/spatial pattern or intensity, would best apply. While experimental trials on mice have mostly confirmed the theory of specificity, the results on humans indicate a role of spatial and temporal patterns. The skin innervation is greatly reduced by the neuropathy and could provide a "spatial contrast pattern" and the axotomy could induce a de novo expression of gastrin-releasing peptide (GRP) in primarily afferent nociceptors and thus modulate spinal pruritus processing. In addition, the overlap of pruritus and pain in neuropathy patients complicates the direct translation from animal experiments and requires collaboration at the clinical level between pain medicine and dermatology.

Topics: Animals; Gastrin-Releasing Peptide; Humans; Mice; Nociceptors; Pain; Peripheral Nervous System Diseases; Pruritus; Skin

Gastrin-releasing peptide (GRP) is a spinal itch transmitter expressed by a small population of dorsal horn interneurons (GRP neurons). The contribution of these neurons to spinal itch relay is still only incompletely understood, and their potential contribution to pain-related behaviors remains controversial. Here, we have addressed this question in a series of experiments performed in

Topics: Animals; Disease Models, Animal; Gastrin-Releasing Peptide; Interneurons; Male; Mice, Transgenic; Nociception; Pain; Posterior Horn Cells; Pruritus

Topics: Animals; Astrocytes; Dermatitis, Allergic Contact; Disease Models, Animal; Gastrin-Releasing Peptide; Interleukin-1 Receptor-Like 1 Protein; Interleukin-33; Janus Kinase 2; Male; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Oligodendroglia; Pruritus; Signal Transduction; Spinal Cord; STAT3 Transcription Factor

Spinal transmission of pruritoceptive (itch) signals requires transneuronal signaling by gastrin-releasing peptide (GRP) produced by a subpopulation of dorsal horn excitatory interneurons. These neurons also express the glutamatergic marker vGluT2, raising the question of why glutamate alone is insufficient for spinal itch relay. Using optogenetics together with slice electrophysiology and mouse behavior, we demonstrate that baseline synaptic coupling between GRP and GRP receptor (GRPR) neurons is too weak for suprathreshold excitation. Only when we mimicked the endogenous firing of GRP neurons and stimulated them repetitively to fire bursts of action potentials did GRPR neurons depolarize progressively and become excitable by GRP neurons. GRPR but not glutamate receptor antagonism prevented this action. Provoking itch-like behavior by optogenetic activation of spinal GRP neurons required similar stimulation paradigms. These results establish a spinal gating mechanism for itch that requires sustained repetitive activity of presynaptic GRP neurons and postsynaptic GRP signaling to drive GRPR neuron output.

Topics: Action Potentials; Animals; Behavior, Animal; Female; Gastrin-Releasing Peptide; Immunohistochemistry; Male; Mice; Mice, Transgenic; Neurons; Optogenetics; Potassium Channel Blockers; Pruritus; Receptors, Bombesin; Receptors, Glutamate; Receptors, Presynaptic; Spinal Cord

In this issue of Neuron, Pagani et al. (2019) find that itch signaling occurs only when GRP neurons fire action potentials in bursts. This enables GRP release and the activation of GRPR neurons, which help carry the itch signal to the brain.

Topics: Action Potentials; Gastrin-Releasing Peptide; Humans; Neurons; Pruritus; Spinal Cord

Itch sensation consists of both sensory and emotional components. The molecular and cellular mechanisms underlying the transduction and transmission of itch sensation have been studied extensively in rodents. However, whether itch induces emotional responses in mice still remains unknown. We found that pruritogens induced conditioned place aversion (CPA) in mice, and that the CPA lasted for at least two weeks. Disruption of itch signal transmission by depletion of peripheral sensory fibers expressing TRPV1 (transient receptor potential vanilloid subfamily, member 1) attenuated chloroquine-induced CPA. Consistently, ablation of itch-specific neurons that express gastrin-releasing peptide receptor in the spinal cord also abolished itch-induced CPA, confirming that itch-induced CPA is dependent on the spinal itch circuit. Thus, these results demonstrate that itch can induce CPA in mice, which requires peripheral itch signal inputs.

Topics: Animals; Ganglia, Spinal; Gastrin-Releasing Peptide; Male; Mice, Inbred C57BL; Pruritus; Receptors, Bombesin; Sensation; Sensory Receptor Cells; Spinal Cord

A key question in our understanding of itch coding mechanisms is whether itch is relayed by dedicated molecular and neuronal pathways. Previous studies suggested that gastrin-releasing peptide (GRP) is an itch-specific neurotransmitter. Neuromedin B (NMB) is a mammalian member of the bombesin family of peptides closely related to GRP, but its role in itch is unclear. Here, we show that itch deficits in mice lacking NMB or GRP are non-redundant and Nmb/Grp double KO (DKO) mice displayed additive deficits. Furthermore, both Nmb/Grp and Nmbr/Grpr DKO mice responded normally to a wide array of noxious stimuli. Ablation of NMBR neurons partially attenuated peripherally induced itch without compromising nociceptive processing. Importantly, electrophysiological studies suggested that GRPR neurons receive glutamatergic input from NMBR neurons. Thus, we propose that NMB and GRP may transmit discrete itch information and NMBR neurons are an integral part of neural circuits for itch in the spinal cord.

Topics: Animals; Antipruritics; Behavior, Animal; Capsaicin; Disease Models, Animal; Gastrin-Releasing Peptide; Histamine; Humans; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Neurokinin B; Nociception; Nociceptors; Pruritus; Receptors, Bombesin; Signal Transduction; Skin; Spinal Cord

Itch is a protective sensation producing a desire to scratch. Pathologic itch can be a chronic symptom of illnesses such as uremia, cholestatic liver disease, neuropathies and dermatitis, however current therapeutic options are limited. Many types of cell surface receptors, including those present on cells in the skin, on sensory neurons and on neurons in the spinal cord, have been implicated in itch signaling. The role of G protein signaling in the regulation of pruriception is poorly understood. We identify here 2 G protein signaling components whose mutation impairs itch sensation. R7bp (a.k.a. Rgs7bp) is a palmitoylated membrane anchoring protein expressed in neurons that facilitates Gαi/o -directed GTPase activating protein activity mediated by the Gβ5/R7-RGS complex. Knockout of R7bp diminishes scratching responses to multiple cutaneously applied and intrathecally-administered pruritogens in mice. Knock-in to mice of a GTPase activating protein-insensitive mutant of Gαo (Gnao1 G184S/+) produces a similar pruriceptive phenotype. The pruriceptive defect in R7bp knockout mice was rescued in double knockout mice also lacking Oprk1, encoding the G protein-coupled kappa-opioid receptor whose activation is known to inhibit itch sensation. In a model of atopic dermatitis (eczema), R7bp knockout mice showed diminished scratching behavior and enhanced sensitivity to kappa opioid agonists. Taken together, our results indicate that R7bp is a key regulator of itch sensation and suggest the potential targeting of R7bp-dependent GTPase activating protein activity as a novel therapeutic strategy for pathological itch.

Topics: Animals; Camphor; Cells, Cultured; Chromones; Endothelin-1; Female; Ganglia, Spinal; Gastrin-Releasing Peptide; Gene Expression Regulation; GTP-Binding Protein alpha Subunits, Gi-Go; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mutation; Natriuretic Peptide, Brain; Nociception; Pain Threshold; Pruritus; Psychomotor Performance; Receptors, Opioid, kappa; RGS Proteins; Sensation; Sensory Receptor Cells

Topics: Animals; Blotting, Western; Dermatitis, Atopic; Disease Models, Animal; Gastrin-Releasing Peptide; Humans; Immunohistochemistry; Interleukin-22; Interleukins; Keratinocytes; Mice; Mice, Inbred C57BL; Mice, Transgenic; Pruritus; Real-Time Polymerase Chain Reaction; Signal Transduction; Skin

Coding of itch versus pain has been heatedly debated for decades. However, the current coding theories (labeled line, intensity, and selectivity theory) cannot accommodate all experimental observations. Here we identified a subset of spinal interneurons, labeled by gastrin-releasing peptide (Grp), that receive direct synaptic input from both pain and itch primary sensory neurons. When activated, these Grp

Topics: Animals; Ganglia, Spinal; Gastrin-Releasing Peptide; Interneurons; Mice; Models, Animal; Neurons, Afferent; Pain; Pruritus; Receptors, Bombesin; Spinal Cord

B-type natriuretic peptide (BNP)-natriuretic peptide receptor A (NPRA) and gastrin-releasing peptide (GRP)-GRP receptor (GRPR) systems contribute to spinal processing of itch. However, pharmacological and anatomic evidence of these two spinal ligand-receptor systems are still not clear. The aim of this study was to determine the spinal functions of BNP-NPRA and GRP-GRPR systems for regulating scratching activities in mice by using pharmacological and immunohistochemical approaches. Our results showed that intrathecal administration of BNP (0.3-3 nmol) dose dependently elicited scratching responses, which could be blocked by the NPRA antagonist (Arg6,β-cyclohexyl-Ala8,D-Tic16,Arg17,Cys18)-atrial natriuretic factor(6-18) amide (A71915). However, A71915 had no effect on intrathecal GRP-induced scratching. In contrast, pretreatment with a GRPR antagonist (D-Tpi6,Leu13ψ(CH2-NH)-Leu14)bombesin(6-14) (RC-3095) inhibited BNP-induced scratching. Immunostaining revealed that NPRA proteins colocalize with GRP, but not GRPR, in the superficial area of dorsal horn, whereas BNP proteins do not colocalize with either GRP or GRPR in the dorsal horn. Intradermal administration of ligands including endothelin-1, U-46619, bovine adrenal medulla 8-22, and Ser-Leu-Ile-Gly-Arg-Leu-NH2 (SLIGRL) increased scratching bouts at different levels of magnitude. Pretreatment with intrathecal A71915 did not affect scratching responses elicited by all four pruritogens, whereas pretreatment with RC-3095 only inhibited SLIGRL-induced scratching. Interestingly, immunostaining showed that RC-3095, but not A71915, inhibited SLIGRL-elicited c-Fos activation in the spinal dorsal horn, which was in line with behavioral outcomes. These findings demonstrate that: 1) BNP-NPRA system may function upstream of the GRP-GRPR system to regulate itch in the mouse spinal cord, and 2) both NPRA and GRPR antagonists may have antipruritic efficacy against centrally, but not peripherally, elicited itch.

Topics: Animals; Atrial Natriuretic Factor; Bombesin; Gastrin-Releasing Peptide; Male; Mice; Natriuretic Peptide, Brain; Peptide Fragments; Pruritus; Receptors, Atrial Natriuretic Factor; Receptors, Bombesin; Spinal Cord; Tetrahydroisoquinolines

There are substantial disagreements about the expression of gastrin-releasing peptide (GRP) in sensory neurons and whether GRP antibody cross-reacts with substance P (SP). These concerns necessitate a critical revaluation of GRP expression using additional approaches. Here, we show that a widely used GRP antibody specifically recognizes GRP but not SP. In the spinal cord of mice lacking SP (Tac1KO), the expression of not only GRP but also other peptides, notably neuropeptide Y (NPY), is significantly diminished. We detectedGrpmRNA in dorsal root ganglias using reverse transcription polymerase chain reaction, in situ hybridization and RNA-seq. We demonstrated thatGrpmRNA and protein are upregulated in dorsal root ganglias, but not in the spinal cord, of mice with chronic itch. Few GRP(+)immunostaining signals were detected in spinal sections following dorsal rhizotomy and GRP(+)cell bodies were not detected in dissociated dorsal horn neurons. Ultrastructural analysis further shows that substantially more GRPergic fibers form synaptic contacts with gastrin releasing peptide receptor-positive (GRPR(+)) neurons than SPergic fibers. Our comprehensive study demonstrates that a majority of GRPergic fibers are of primary afferent origin. A number of factors such as low copy number ofGrptranscripts, small percentage of cells expressingGrp, and the use of an eGFP GENSAT transgenic as a surrogate for GRP protein have contributed to the controversy. Optimization of experimental procedures facilitates the specific detection of GRP expression in dorsal root ganglia neurons.

Topics: Amino Acid Sequence; Animals; Antibody Specificity; Cell Count; Cells, Cultured; Chronic Disease; Ganglia, Spinal; Gastrin-Releasing Peptide; Gene Deletion; Gene Expression Regulation; Male; Mice, Inbred C57BL; Mice, Knockout; Posterior Horn Cells; Pruritus; Receptors, Bombesin; RNA, Messenger; Spinal Cord; Substance P

Spinal itch transmission has been reported to be mediated by at least two neuronal populations in spinal dorsal horn, neurons expressing brain-natriuretic peptide (BNP) receptor (Npra) and gastrin-releasing peptide (GRP) receptor (GRPR). Although Npra-expressing neurons were shown to be upstream of GRPR- expressing neurons in spinal itch transmission, the roles of BNP and GRP in the spinal neurotransmission of histamine-dependent and -independent itch remains unclear. Using in vivo electrophysiology and behavior analysis, this study examined the responses of chloroquine (histamine-independent pruritogen)-responsive and histamine-responsive dorsal horn neurons to spinal applications of BNP and GRP. Electrophysiologically, 9.5% of chloroquine-responsive neurons responded to BNP, 33.3% to GRP, and 4.8% to both, indicating that almost half of chloroquine-responsive neurons were unresponsive to both BNP and GRP. In contrast, histamine-responsive neurons did not respond to spinal BNP application, whereas 30% responded to spinal GRP application, indicating that 70% of histamine-responsive neurons were unresponsive to both BNP and GRP. Behavioral analyses showed differences in the time-course and frequency of scratching responses evoked by intrathecal BNP and GRP. These findings provide evidence that most BNP-Npra and GRP-GRPR signaling involve different pathways of spinal itch transmission, and that multiple neurotransmitters, in addition to BNP and GRP, are involved in spinal itch transmission. The electrophysiological results also suggest that spinal BNP contributes little to histaminergic itch directly.

Topics: Action Potentials; Animals; Chloroquine; Gastrin-Releasing Peptide; Histamine; Male; Mice; Mice, Inbred C57BL; Natriuretic Peptide, Brain; Posterior Horn Cells; Pruritus

Itch and pain are intimately related and may share similar peripheral and central mechanisms and pathways. However, it has been believed that synaptic glutamate release from a group of peripheral nociceptors is required to sense pain and suppress itch. Although we previously demonstrated that phosphorylation of GluN2B subunits of the NMDA receptor at Tyr1472 is important for central sensitization in a neuropathic pain model of mice with a knock-in mutation of the Tyr1472 site to phenylalanine of GluN2B (Y1472F-KI), the role of NMDA receptors in itch transmission remains unknown. Here, we demonstrated that the scratching behaviors elicited by various pruritogens applied to the cheek and c-fos expression in the region innervated by the trigeminal nerve were markedly attenuated in the Y1472F-KI mice. The c-fos immunoreactivity was co-localized with the receptor of gastrin-releasing peptide (GRP). Scratching behaviors evoked by chloroquine were inhibited by the NMDA receptor antagonists D-AP5 and CP101,606 and by the Src kinase inhibitor PP2. Direct activation of the trigeminal region by intracisternal administration of NMDA and GRP induced robust scratching behaviors, both of which were reduced by the GRP receptor antagonist RC-3095. Taken together, the data obtained in this present study are the first to demonstrate that phosphorylation of GluN2B subunit at Tyr1472 is important for trigeminal transmission of itch and suggest that the NMDA receptor activation occurs upstream of the GRP-GRP receptor pathway.

Topics: Animals; Behavior, Animal; Gastrin-Releasing Peptide; Glutamic Acid; Mice; Neuralgia; Phosphorylation; Pruritus; Receptors, Bombesin; Receptors, N-Methyl-D-Aspartate; Signal Transduction; Spinal Cord

Atopic dermatitis (AD) is a multifactorial inflammatory skin disease characterized by skin barrier dysfunction, allergic inflammation and intractable pruritus resistant to conventional antipruritic treatments, including H. This study assessed the correlation between plasma GzmB levels and severity of pruritus and dermatitis, in AD patients.. Plasma was collected from 46 patients with AD, 24 patients with psoriasis, and 30 healthy controls. AD severity was assessed with the scoring atopic dermatitis (SCORAD) index, psoriasis severity with the psoriasis area and severity index (PASI), and degree of pruritus by visual analogue scale (VAS) score. GzmA, GzmB and gastrin releasing peptide (GRP) levels were measured by enzyme-linked immunosorbent assays.. Plasma GzmB concentrations were significantly higher in patients with AD and psoriasis than in healthy controls. Correlation analyses showed that plasma GzmB concentrations positively correlated with SCORAD and serum levels of severity markers such as thymus and activation-regulated chemokine, and lactate dehydrogenase in AD patients. Moreover, plasma levels of GRP, an itch-related peptide, were higher in patients with AD, positively correlating with VAS score and plasma GzmB level. In addition, plasma GzmB concentration was significantly lower in the treatment group than the untreated group with AD. Meanwhile, there were no correlations among GzmB levels, VAS score and PASI score in patients with psoriasis. In contrast to the results of plasma GzmB, plasma GzmA levels were unchanged among AD, psoriasis and healthy groups, and showed no correlations with VAS score and SCORAD index in patients with AD.. Plasma GzmB levels may reflect the degree of pruritus and dermatitis in patients with AD.

Topics: Adult; Case-Control Studies; Dermatitis; Dermatitis, Atopic; Enzyme-Linked Immunosorbent Assay; Female; Gastrin-Releasing Peptide; Gene Expression Regulation; Granzymes; Humans; Inflammation; Killer Cells, Natural; Male; Middle Aged; Pruritus; Psoriasis; T-Lymphocytes, Cytotoxic

Topics: Adolescent; Adult; Dermatitis, Atopic; Eczema; Female; Gastrin-Releasing Peptide; Humans; Male; Middle Aged; Pruritus; Young Adult

Activated mammalian target of rapamycin (P-mTOR) has been shown to maintain the sensitivity of subsets of small-diameter primary afferent A-nociceptors. Local or systemic inhibition of the mTOR complex 1 (mTORC1) pathway reduced punctate mechanical and cold sensitivity in neuropathic pain and therefore offered a new approach to chronic pain control. In this study, we have investigated the effects of the rapamycin analog temsirolimus (CCI-779) on itch. Bouts of scratching induced by the histamine-dependent pruritogenic compound 48/80 and histamine-independent pruritogens, chloroquine and SLIGRL-NH2, injected intradermally were significantly reduced by local (intradermal) or systemic (intraperitoneal, i.p.) pretreatment with CCI-779. We also investigated the action of metformin, a drug taken to control type 2 diabetes and recently shown to inhibit mTORC1 in vivo. Although the response to nonhistaminergic stimuli was reduced at all of the time points tested, scratching to compound 48/80 was modified by metformin only when the drug was injected 24 hours before this pruritogen. We also examined the colocalization of P-mTOR with gastrin-releasing peptide, a putative marker for some itch-sensitive primary afferents, and found that P-mTOR was coexpressed in less than 5% of gastrin-releasing peptide-positive fibers in the mouse skin. Taken together, the data highlight the role that P-mTOR-positive A-fibers play in itch signaling and underline the importance of the mTORC1 pathway in the regulation of homeostatic primary afferent functions such as pain and itch. The actions of the antidiabetic drug metformin in ameliorating nonhistamine-mediated itch also suggest a new therapeutic route for the control of this category of pruritus.

Topics: Animals; Disease Models, Animal; Gastrin-Releasing Peptide; Histamine; Hypoglycemic Agents; Male; Mechanistic Target of Rapamycin Complex 1; Metformin; Mice; Mice, Inbred C57BL; Multiprotein Complexes; Neuralgia; Phosphoproteins; Protein Kinase Inhibitors; Pruritus; Signal Transduction; Sirolimus; Skin; TOR Serine-Threonine Kinases; Treatment Outcome

How neuropeptides in the primate spinal cord regulate itch and pain is largely unknown. Here we elucidate the sensory functions of spinal opioid-related peptides and gastrin-releasing peptide (GRP) in awake, behaving monkeys. Following intrathecal administration, β-endorphin (10-100 nmol) and GRP (1-10 nmol) dose-dependently elicit the same degree of robust itch scratching, which can be inhibited by mu-opioid peptide (MOP) receptor and GRP receptor (BB2) antagonists, respectively. Unlike β-endorphin, which produces itch and attenuates inflammatory pain, GRP only elicits itch without affecting pain. In contrast, enkephalins (100-1000 nmol) and nociceptin-orphanin FQ (3-30 nmol) only inhibit pain without eliciting itch. More intriguingly, dynorphin A(1-17) (10-100 nmol) dose-dependently attenuates both β-endorphin- and GRP-elicited robust scratching without affecting pain processing. The anti-itch effects of dynorphin A can be reversed by a kappa-opioid peptide (KOP) receptor antagonist nor-binaltorphimine. These nonhuman primate behavioral models with spinal delivery of ligands advance our understanding of distinct functions of neuropeptides for modulating itch and pain. In particular, we demonstrate causal links for itch-eliciting effects by β-endorphin-MOP receptor and GRP-BB2 receptor systems and itch-inhibiting effects by the dynorphin A-KOP receptor system. These studies will facilitate transforming discoveries of novel ligand-receptor systems into future therapies as antipruritics and/or analgesics in humans.

Topics: Analgesics, Opioid; Animals; Behavior, Animal; beta-Endorphin; Dynorphins; Female; Gastrin-Releasing Peptide; Hyperalgesia; Injections, Spinal; Macaca mulatta; Male; Neuropeptides; Pain; Pruritus; Spinal Cord

Chronic itch is an intractable symptom of inflammatory skin diseases, such as atopic and contact dermatitis. Recent studies have revealed neuronal pathways selective for itch, but the mechanisms by which itch turns into a pathological chronic state are poorly understood. Using mouse models of atopic and contact dermatitis, we demonstrate a long-term reactive state of astrocytes in the dorsal horn of the spinal segments that corresponds to lesioned, itchy skin. We found that reactive astrogliosis depended on the activation of signal transducer and activator of transcription 3 (STAT3). Conditional disruption of astrocytic STAT3 suppressed chronic itch, and pharmacological inhibition of spinal STAT3 ameliorated the fully developed chronic itch. Mice with atopic dermatitis exhibited an increase in scratching elicited by intrathecal administration of the itch-inducer gastrin-releasing peptide (GRP), and this enhancement was normalized by suppressing STAT3-mediated reactive astrogliosis. Moreover, we identified lipocalin-2 (LCN2) as an astrocytic STAT3-dependent upregulated factor that was crucial for chronic itch, and we demonstrated that intrathecal administration of LCN2 to normal mice increased spinal GRP-evoked scratching. Our findings indicate that STAT3-dependent reactive astrocytes act as critical amplifiers of itching through a mechanism involving the enhancement of spinal itch signals by LCN2, thereby providing a previously unrecognized target for treating chronic itch.

Topics: Acute-Phase Proteins; Animals; Astrocytes; Chronic Disease; Gastrin-Releasing Peptide; Lipocalin-2; Lipocalins; Male; Mice; Mice, Inbred C57BL; Oncogene Proteins; Pruritus; Spinal Cord Dorsal Horn; STAT3 Transcription Factor

The gastrin-releasing peptide (GRP) and its receptor (GRPR) are important components of itch transmission. Upstream, but not downstream, aspects of GRPR signaling have been investigated extensively. We hypothesize that GRPR signals in part through the PI3Kγ/Akt pathway. We used pharmacological, electrophysiological, and behavioral approaches to further evaluate GRPR downstream signaling pathways. Our data show that GRP directly activates small-size capsaicin-sensitive DRG neurons, an effect that translates into transient calcium flux and membrane depolarization (∼ 20 mV). GRPR activation also induces Akt phosphorylation, a proxy for PI3Kγ activity, in ex vivo naive mouse spinal cords and in GRPR transiently expressing HEK293 cells. The intrathecal injection of GRP led to intense scratching, an effect largely reduced by either GRPR antagonists or PI3Kγ inhibitor. Scratching behavior was also induced by the intrathecal injection of an Akt activator. In a dry skin model of itch, we show that GRPR blockade or PI3Kγ inhibition reversed the scratching behavior. Altogether, these findings are highly suggestive that GRPR is expressed by the central terminals of DRG nociceptive afferents, which transmit itch via the PI3Kγ/Akt pathway.. Itch is the most common symptom of the skin and is related to noncutaneous diseases. It severely impairs patients' quality of life when it becomes chronic and there is no specific or effective available therapy, mainly because itch pathophysiology is not completely elucidated. Our findings indicate that the enzyme PI3Kγ is a key central mediator of itch transmission. Therefore, we suggest PI3Kγ as an attractive target for the development of new anti-pruritic drugs. With this study, we take a step forward in our understanding of the mechanisms underlying the central transmission of itch sensation.

Topics: Action Potentials; Animals; Anticarcinogenic Agents; Bombesin; Capsaicin; Central Nervous System; Disease Models, Animal; Enzyme Inhibitors; Ganglia, Spinal; Gastrin-Releasing Peptide; Indoles; Male; Mice; Neurons; p-Methoxy-N-methylphenethylamine; Pain Threshold; Peptide Fragments; Phosphatidylinositol 3-Kinase; Pruritus; Quinoxalines; Reaction Time; Receptors, Bombesin; Synaptic Transmission; Thiazolidinediones

We investigated roles for substance P (SP), gastrin-releasing peptide (GRP), and glutamate in the spinal neurotransmission of histamine-dependent and -independent itch. In anesthetized mice, responses of single superficial dorsal horn neurons to intradermal (i.d.) injection of chloroquine were partially reduced by spinal application of the α-amino-3-hydroxy-5-methyl-4-isoxazole proprionate acid (AMPA)/kainate antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Co-application of CNQX plus a neurokinin-1 (NK-1) antagonist produced stronger inhibition, while co-application of CNQX, NK-1, and GRP receptor (GRPR) antagonists completely inhibited firing. Nociceptive-specific and wide dynamic range-type neurons exhibited differential suppression by CNQX plus either the GRPR or NK-1 antagonist, respectively. Neuronal responses elicited by i.d. histamine were abolished by CNQX alone. In behavioral studies, individual intrathecal administration of a GRPR, NK-1, or AMPA antagonist each significantly attenuated chloroquine-evoked scratching behavior. Co-administration of the NK-1 and AMPA antagonists was more effective, and administration of all 3 antagonists abolished scratching. Intrathecal CNQX alone prevented histamine-evoked scratching behavior. We additionally employed a double-label strategy to investigate molecular markers of pruritogen-sensitive dorsal root ganglion (DRG) cells. DRG cells responsive to histamine and/or chloroquine, identified by calcium imaging, were then processed for co-expression of SP, GRP, or vesicular glutamate transporter type 2 (VGLUT2) immunofluorescence. Subpopulations of chloroquine- and/or histamine-sensitive DRG cells were immunopositive for SP and/or GRP, with >80% immunopositive for VGLUT2. These results indicate that SP, GRP, and glutamate each partially contribute to histamine-independent itch. Histamine-evoked itch is mediated primarily by glutamate, with GRP playing a lesser role. Co-application of NK-1, GRP, and AMPA receptor antagonists may prove beneficial in treating chronic itch.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Animals; Antirheumatic Agents; Bombesin; Chloroquine; Drug Combinations; Excitatory Amino Acid Antagonists; Ganglia, Spinal; Gastrin-Releasing Peptide; Glutamic Acid; Male; Mice; Mice, Inbred C57BL; Neurokinin-1 Receptor Antagonists; Neurons; Peptide Fragments; Piperidines; Pruritus; Substance P; Vesicular Glutamate Transport Protein 2

A recent study by Mishra and Hoon identified B-type natriuretic peptide (BNP) as an important peptide for itch transmission and proposed that BNP activates spinal natriuretic peptide receptor-A (NPRA) expressing neurons, which release gastrin releasing peptide (GRP) to activate GRP receptor (GRPR) expressing neurons to relay itch information from the periphery to the brain (Science 340:968-971, 2013). A central premise for the validity of this novel pathway is the absence of GRP in the dorsal root ganglion (DRG) neurons. To this end, they showed that Grp mRNA in DRG neurons is either absent or barely detectable and claimed that BNP but not GRP is a major neurotransmitter for itch in pruriceptors. They showed that NPRA immunostaining is perfectly co-localized with Grp-eGFP in the spinal cord, and a few acute pain behaviors in Nppb-/- mice were tested. They claimed that BNP is an itch-selective peptide that acts as the first station of a dedicated neuronal pathway comprising a GRP-GRPR cascade for itch. However, our studies, along with the others, do not support their claims.. We were unable to reproduce the immunostaining of BNP and NPRA as shown by Mishra and Hoon. By contrast, we were able to detect Grp mRNA in DRGs using in situ hybridization and real time RT-PCR. We show that the expression pattern of Grp mRNA is comparable to that of GRP protein in DRGs. Pharmacological and genetic blockade of GRP-GRPR signaling does not significantly affect intrathecal BNP-induced scratching behavior. We show that BNP inhibits inflammatory pain and morphine analgesia.. Accumulating evidence demonstrates that GRP is a key neurotransmitter in pruriceptors for mediating histamine-independent itch. BNP-NPRA signaling is involved in both itch and pain and does not function upstream of the GRP-GRPR dedicated neuronal pathway. The site of BNP action in itch and pain and its relationship with GRP remain to be clarified.

Topics: Animals; Ganglia, Spinal; Gastrin-Releasing Peptide; Gene Expression Regulation; Inflammation; Male; Mice; Mice, Inbred C57BL; Natriuretic Peptide, Brain; Pain; Pruritus; Receptors, Atrial Natriuretic Factor; Receptors, Bombesin; RNA, Messenger; Signal Transduction; Spinal Cord

Patients with cholestatic disease have increased systemic concentrations of bile acids (BAs) and profound pruritus. The G-protein-coupled BA receptor 1 TGR5 (encoded by GPBAR1) is expressed by primary sensory neurons; its activation induces neuronal hyperexcitability and scratching by unknown mechanisms. We investigated whether the transient receptor potential ankyrin 1 (TRPA1) is involved in BA-evoked, TGR5-dependent pruritus in mice.. Co-expression of TGR5 and TRPA1 in cutaneous afferent neurons isolated from mice was analyzed by immunofluorescence, in situ hybridization, and single-cell polymerase chain reaction. TGR5-induced activation of TRPA1 was studied in in HEK293 cells, Xenopus laevis oocytes, and primary sensory neurons by measuring Ca(2+) signals. The contribution of TRPA1 to TGR5-induced release of pruritogenic neuropeptides, activation of spinal neurons, and scratching behavior were studied using TRPA1 antagonists or Trpa1(-/-) mice.. TGR5 and TRPA1 protein and messenger RNA were expressed by cutaneous afferent neurons. In HEK cells, oocytes, and neurons co-expressing TGR5 and TRPA1, BAs caused TGR5-dependent activation and sensitization of TRPA1 by mechanisms that required Gβγ, protein kinase C, and Ca(2+). Antagonists or deletion of TRPA1 prevented BA-stimulated release of the pruritogenic neuropeptides gastrin-releasing peptide and atrial natriuretic peptide B in the spinal cord. Disruption of Trpa1 in mice blocked BA-induced expression of Fos in spinal neurons and prevented BA-stimulated scratching. Spontaneous scratching was exacerbated in transgenic mice that overexpressed TRG5. Administration of a TRPA1 antagonist or the BA sequestrant colestipol, which lowered circulating levels of BAs, prevented exacerbated spontaneous scratching in TGR5 overexpressing mice.. BAs induce pruritus in mice by co-activation of TGR5 and TRPA1. Antagonists of TGR5 and TRPA1, or inhibitors of the signaling mechanism by which TGR5 activates TRPA1, might be developed for treatment of cholestatic pruritus.

Topics: Animals; Bile Acids and Salts; Cholestasis; Disease Models, Animal; Ganglia, Spinal; Gastrin-Releasing Peptide; HEK293 Cells; Humans; Mice, Knockout; Natriuretic Peptides; Neurons, Afferent; Nociceptors; Oocytes; Primary Cell Culture; Pruritus; Receptors, G-Protein-Coupled; Transient Receptor Potential Channels; TRPA1 Cation Channel; Xenopus laevis

We previously showed that gastrin-releasing peptide receptor (GRPR) in the spinal cord is important for mediating nonhistaminergic itch. Neuromedin B receptor (NMBR), the second member of the mammalian bombesin receptor family, is expressed in a largely nonoverlapping pattern with GRPR in the superficial spinal cord, and its role in itch transmission remains unclear. Here, we report that Nmbr knock-out (KO) mice exhibited normal scratching behavior in response to intradermal injection of pruritogens. However, mice lacking both Nmbr and Grpr (DKO mice) showed significant deficits in histaminergic itch. In contrast, the chloroquine (CQ)-evoked scratching behavior of DKO mice is not further reduced compared with Grpr KO mice. These results suggest that NMBR and GRPR could compensate for the loss of each other to maintain normal histamine-evoked itch, whereas GRPR is exclusively required for CQ-evoked scratching behavior. Interestingly, GRPR activity is enhanced in Nmbr KO mice despite the lack of upregulation of Grpr expression; so is NMBR in Grpr KO mice. We found that NMB acts exclusively through NMBR for itch transmission, whereas GRP can signal through both receptors, albeit to NMBR to a much lesser extent. Although NMBR and NMBR(+) neurons are dispensable for histaminergic itch, GRPR(+) neurons are likely to act downstream of NMBR(+) neurons to integrate NMB-NMBR-encoded histaminergic itch information in normal physiological conditions. Together, we define the respective function of NMBR and GRPR in itch transmission, and reveal an unexpected relationship not only between the two receptors but also between the two populations of interneurons in itch signaling.

Topics: Animals; Gastrin-Releasing Peptide; Histamine; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Neural Inhibition; Nociception; Posterior Horn Cells; Pruritus; Receptors, Bombesin; Signal Transduction

Central serotonin (5-hydroxytryptophan, 5-HT) modulates somatosensory transduction, but how it achieves sensory modality-specific modulation remains unclear. Here we report that enhancing serotonergic tone via administration of 5-HT potentiates itch sensation, whereas mice lacking 5-HT or serotonergic neurons in the brainstem exhibit markedly reduced scratching behavior. Through pharmacological and behavioral screening, we identified 5-HT1A as a key receptor in facilitating gastrin-releasing peptide (GRP)-dependent scratching behavior. Coactivation of 5-HT1A and GRP receptors (GRPR) greatly potentiates subthreshold, GRP-induced Ca(2+) transients, and action potential firing of GRPR(+) neurons. Immunostaining, biochemical, and biophysical studies suggest that 5-HT1A and GRPR may function as receptor heteromeric complexes. Furthermore, 5-HT1A blockade significantly attenuates, whereas its activation contributes to, long-lasting itch transmission. Thus, our studies demonstrate that the descending 5-HT system facilitates GRP-GRPR signaling via 5-HT1A to augment itch-specific outputs, and a disruption of crosstalk between 5-HT1A and GRPR may be a useful antipruritic strategy.

Topics: Action Potentials; Animals; Calcium Signaling; Gastrin-Releasing Peptide; Male; Mice; Neurons; Pruritus; Receptor, Serotonin, 5-HT1A; Receptors, Bombesin; Signal Transduction; Spinal Cord

Patients with cholestatic disease exhibit pruritus and analgesia, but the mechanisms underlying these symptoms are unknown. We report that bile acids, which are elevated in the circulation and tissues during cholestasis, cause itch and analgesia by activating the GPCR TGR5. TGR5 was detected in peptidergic neurons of mouse dorsal root ganglia and spinal cord that transmit itch and pain, and in dermal macrophages that contain opioids. Bile acids and a TGR5-selective agonist induced hyperexcitability of dorsal root ganglia neurons and stimulated the release of the itch and analgesia transmitters gastrin-releasing peptide and leucine-enkephalin. Intradermal injection of bile acids and a TGR5-selective agonist stimulated scratching behavior by gastrin-releasing peptide- and opioid-dependent mechanisms in mice. Scratching was attenuated in Tgr5-KO mice but exacerbated in Tgr5-Tg mice (overexpressing mouse TGR5), which exhibited spontaneous pruritus. Intraplantar and intrathecal injection of bile acids caused analgesia to mechanical stimulation of the paw by an opioid-dependent mechanism. Both peripheral and central mechanisms of analgesia were absent from Tgr5-KO mice. Thus, bile acids activate TGR5 on sensory nerves, stimulating the release of neuropeptides in the spinal cord that transmit itch and analgesia. These mechanisms could contribute to pruritus and painless jaundice that occur during cholestatic liver diseases.

Topics: Action Potentials; Animals; Bile Acids and Salts; Capsaicin; Cells, Cultured; Cholestasis; Dermis; Enkephalin, Leucine; Female; Ganglia, Spinal; Gastrin-Releasing Peptide; Gene Expression; Macrophages; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Neurons; Opioid Peptides; Organ Specificity; Pain; Pain Perception; Patch-Clamp Techniques; Pruritus; Rats; Rats, Sprague-Dawley; Receptors, G-Protein-Coupled; Single-Cell Analysis; Spinal Cord

Topics: Animals; Chronic Disease; Disease Models, Animal; Female; Gastrin-Releasing Peptide; Macaca fascicularis; Nerve Fibers; Pruritus; Receptors, Bombesin; Skin; Spinal Cord

Topics: Aged; Aged, 80 and over; Female; Gastrin-Releasing Peptide; Humans; Male; Middle Aged; Nerve Fibers, Myelinated; Nerve Fibers, Unmyelinated; Pruritus; Skin

Itch is triggered by somatosensory neurons expressing the ion channel TRPV1 (transient receptor potential cation channel subfamily V member 1), but the mechanisms underlying this nociceptive response remain poorly understood. Here, we show that the neuropeptide natriuretic polypeptide b (Nppb) is expressed in a subset of TRPV1 neurons and found that Nppb(-/-) mice selectively lose almost all behavioral responses to itch-inducing agents. 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, gastrin-releasing peptide, 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.

Topics: Animals; Chloroquine; Endothelin-1; Gastrin-Releasing Peptide; Histamine; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Natriuretic Peptide, Brain; Nociception; Phospholipase C beta; Pruritus; Receptors, Atrial Natriuretic Factor; Sensory Receptor Cells; Spinal Cord; TRPV Cation Channels

Pruritus (itch) is a severe side effect associated with the use of drugs as well as hepatic and hematological disorders. Previous studies in rodents suggest that bombesin receptor subtypes i.e. receptors for gastrin-releasing peptide (GRPr) and neuromedin B (NMBr) differentially regulate itch scratching. However, to what degree spinal GRPr and NMBr regulate scratching evoked by intrathecally administered bombesin-related peptides is not known. The first aim of this study was to pharmacologically compare the dose-response curves for scratching induced by intrathecally administered bombesin-related peptides versus morphine, which is known to elicit itch in humans. The second aim was to determine if spinal GRPr and NMBr selectively or generally mediate scratching behavior. Mice received intrathecal injection of bombesin (0.01-0.3 nmol), GRP (0.01-0.3 nmol), NMB (0.1-1 nmol) or morphine (0.3-3 nmol) and were observed for one hour for scratching activity. Bombesin elicited most profound scratching over one hour followed by GRP and NMB, whereas morphine failed to evoke scratching response indicating the insensitivity of mouse models to intrathecal opioid-induced itch. Intrathecal pretreatment with GRPr antagonist RC-3095 (0.03-0.1 nmol) produced a parallel rightward shift in the dose response curve of GRP-induced scratching but not NMB-induced scratching. Similarly, PD168368 (1-3 nmol) only attenuated NMB but not GRP-induced scratching. Individual or co-administration of RC-3095 and PD168368 failed to alter bombesin-evoked scratching. A higher dose of RC-3095 (0.3 nmol) generally suppressed scratching induced by all three peptides but also compromised motor function in the rotarod test. Together, these data indicate that spinal GRPr and NMBr independently drive itch neurotransmission in mice and may not mediate bombesin-induced scratching. GRPr antagonists at functionally receptor-selective doses only block spinal GRP-elicited scratching but the suppression of scratching at higher doses is confounded by motor impairment.

Topics: Animals; Behavior, Animal; Bombesin; Gastrin-Releasing Peptide; Indoles; Injections, Spinal; Male; Mice; Morphine; Motor Activity; Peptide Fragments; Pruritus; Pyridines; Receptors, Bombesin; Spinal Cord

Spatial and temporal cues govern the genesis of a diverse array of neurons located in the dorsal spinal cord, including dI1-dI6, dIL(A), and dIL(B) subtypes, but their physiological functions are poorly understood. Here we generated a new line of conditional knock-out (CKO) mice, in which the homeobox gene Tlx3 was removed in dI5 and dIL(B) cells. In these CKO mice, development of a subset of excitatory neurons located in laminae I and II was impaired, including itch-related GRPR-expressing neurons, PKCγ-expressing neurons, and neurons expressing three neuropeptide genes: somatostatin, preprotachykinin 1, and the gastrin-releasing peptide. These CKO mice displayed marked deficits in generating nocifensive motor behaviors evoked by a range of pain-related or itch-related stimuli. The mutants also failed to exhibit escape response evoked by dynamic mechanical stimuli but retained the ability to sense innocuous cooling and/or warm. Thus, our studies provide new insight into the ontogeny of spinal neurons processing distinct sensory modalities.

Topics: Animals; Animals, Newborn; Capsaicin; Cell Count; Chloroquine; Embryo, Mammalian; Ganglia, Spinal; Gastrin-Releasing Peptide; Gene Expression Regulation, Developmental; Homeodomain Proteins; Mice; Mice, Transgenic; Muscle Proteins; Neurons; Oligopeptides; Pain; Physical Stimulation; Protein Kinase C; Protein Precursors; Pruritus; Psychomotor Performance; Sensation; Somatostatin; Tachykinins; Vesicular Glutamate Transport Protein 1

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.

Topics: Animals; Chronic Disease; Disease Models, Animal; Gastrin-Releasing Peptide; Gene Expression; Humans; MAP Kinase Signaling System; Mice; Mice, Knockout; Mice, Transgenic; NAV1.8 Voltage-Gated Sodium Channel; Nociceptors; Proto-Oncogene Proteins B-raf; Pruritus; Receptors, Bombesin; Receptors, G-Protein-Coupled; Sensory Receptor Cells; Spinal Cord; TRPV Cation Channels

Recent studies support roles for neurokinin-1 (NK-1) and gastrin-releasing peptide (GRP) receptor-expressing spinal neurons in itch. We presently investigated expression of substance P (SP) and GRP in pruritogen-responsive primary sensory neurons and roles for these neuropeptides in itch signaling. Responses of dorsal root ganglion (DRG) cells to various pruritogens were observed by calcium imaging. DRG cells were then processed for SP, GRP, and isolectin B-4 (IB4; a marker for nonpeptidergic neurons) immunofluorescence. Of pruritogen-responsive DRG cells, 11.8-26.8%, 21.8-40.0%, and 21.4-26.8% were immunopositive for SP, GRP, and IB4, respectively. In behavioral studies, both systemic and intrathecal administration of a NK-1 receptor antagonist significantly attenuated scratching evoked by chloroquine and a protease-activated receptor 2 agonist, SLIGRL, but not histamine, bovine adrenal medulla peptide 8-22 (BAM8-22), or serotonin. Systemic or intrathecal administration of a GRP receptor antagonist attenuated scratching evoked by chloroquine and SLIGRL but not BAM8-22 or histamine. The GRP receptor antagonist enhanced scratching evoked by serotonin. These results indicate that SP and GRP expressed in primary sensory neurons are partially involved as neurotransmitters in histamine-independent itch signaling from the skin to the spinal cord.

Topics: Animals; Calcium; Chloroquine; Ganglia, Spinal; Gastrin-Releasing Peptide; Histamine; Male; Mice; Mice, Inbred C57BL; Neurokinin-1 Receptor Antagonists; Oligopeptides; Peptide Fragments; Pruritus; Receptors, Bombesin; Sensory Receptor Cells; Serotonin; Signal Transduction; Substance P

Topics: Adult; Dermatitis, Atopic; Female; Gastrin-Releasing Peptide; Humans; Male; Middle Aged; Pruritus; Young Adult

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.

Topics: Action Potentials; Animals; Ganglia, Spinal; Gastrin-Releasing Peptide; Gene Expression Regulation; Gene Knockdown Techniques; Membrane Glycoproteins; Mice; Mice, Knockout; Pain; Pruritus; Sensory Receptor Cells; Spinal Cord; Synaptic Transmission; Toll-Like Receptor 3; Toll-Like Receptor 7; TRPV Cation Channels

Itch is one of the major somatosensory modalities. Some recent findings have proposed that gastrin releasing peptide (Grp) is expressed in a subset of dorsal root ganglion (DRG) neurons and functions as a selective neurotransmitter for transferring itch information to spinal cord interneurons. However, expression data from public databases and earlier literatures indicate that Grp mRNA is only detected in dorsal spinal cord (dSC) whereas its family member neuromedin B (Nmb) is highly expressed in DRG neurons. These contradictory results argue that a thorough characterization of the expression of Grp and Nmb is warranted.. Grp mRNA is highly expressed in dSC but is barely detectable in DRGs of juvenile and adult mice. Anti-bombesin serum specifically recognizes Grp but not Nmb. Grp is present in a small number of small-diameter DRG neurons and in abundance in layers I and II of the spinal cord. The reduction of dSC Grp after dorsal root rhizotomy is significantly different from those of DRG derived markers but similar to that of a spinal cord neuronal marker. Double fluorescent in situ of Nmb and other molecular markers indicate that Nmb is highly and selectively expressed in nociceptive and itch-sensitive DRG neurons.. The majority of dSC Grp is synthesized locally in dorsal spinal cord neurons. On the other hand, Nmb is highly expressed in pain- and itch-sensing DRG neurons. Our findings provide direct anatomic evidence that Grp could function locally in the dorsal spinal cord in addition to its roles in DRG neurons and that Nmb has potential roles in nociceptive and itch-sensitive neurons. These results will improve our understanding about roles of Grp and Nmb in mediating itch sensation.

Topics: Aging; Amino Acid Sequence; Animals; Antibody Specificity; Bombesin; Cold Temperature; Ganglia, Spinal; Gastrin-Releasing Peptide; Gene Expression Regulation, Developmental; Humans; Immune Sera; Mechanotransduction, Cellular; Mice; Molecular Sequence Data; Neurokinin B; Nociceptors; Pain; Pain Threshold; Physical Stimulation; Protein Transport; Pruritus; Receptors, Bombesin; Rhizotomy; RNA, Messenger; Sensory Receptor Cells; Spinal Cord

Gastrin-releasing peptide (GRP) has been implicated in the itch-scratch cycle. We investigated if this gut-brain-skin peptide plays a role in the compulsive, hindleg scratching of the neck of mice by 5'-guanidinonaltrindole (GNTI), the kappa opioid receptor antagonist, and in the antipruritic activity of nalfurafine, the kappa opioid agonist. Previously, we showed that GNTI (0.03-1mg/kg, s.c.) elicits dose-related scratching and that nalfurafine (0.001-0.02mg/kg, s.c.) inhibits this behavior in mice. Utilizing immunohistochemistry, GRP positive nerve fibers were detected in mouse skin and superficial layer of the dorsal horn of the spinal cord as well as GRP positive cells in the dorsal root ganglion. Pretreating mice with either a pseudopeptide GRP receptor antagonist, RC-3095 (10-30mg/kg, s.c. at -15min), or a peptide GRP receptor antagonist, [d-Phe(6)]bombesin(6-13) methyl ester (2-100nmol, i.t. at -10min), did not suppress GNTI-induced scratching. However, pretreating mice with either antagonist inhibited scratching precipitated by the GRP receptor agonist, GRP(18-27) (2nmol, i.t.). Pretreating mice with a muscarinic M(1) receptor agonist, McN-A-343 (1.5-15μg/5μl, i.t. at -10min) antagonized GNTI-induced scratching. Norbinaltorphimine (20mg/kg, i.p. at -18 to -20h), a kappa opioid antagonist, countered the antiscratch activity of nalfurafine. We conclude that (a) the GRP receptor system does not mediate GNTI-induced scratching and (b) the kappa opioid system is involved, at least in part, in the scratch suppressing activity of nalfurafine.

Topics: (4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethylammonium Chloride; Animals; Bombesin; Compulsive Behavior; Ganglia, Spinal; Gastrin-Releasing Peptide; Grooming; Guanidines; Locomotion; Male; Mice; Morphinans; Naltrexone; Nerve Fibers; Peptide Fragments; Pirenzepine; Pruritus; Receptor, Muscarinic M1; Receptors, Bombesin; Receptors, Opioid, kappa; Skin; Spinal Cord; Spiro Compounds

Bombesin is a pruritogenic agent that causes intense itch-scratching activity in rodents. Bombesin has high affinity for the gastrin-releasing peptide (GRP) receptor (GRPr) and the neuromedin B (NMB) receptor (NMBr). The aim of this study was to investigate pharmacologically the ability of GRPr and NMBr to elicit scratching behavior in rats. The intracerebroventricular route was selected for drug delivery because the study focused on supraspinal sites of action. The magnitude and duration of scratching produced by the naturally occurring peptides GRP and NMB were characterized. Antagonists selective for GRPr [(d-Tpi6, Leu13Ψ(CH2-NH)-Leu14)Bombesin(6-14) (RC-3095)] and NMBr [(S)-α-methyl-α-[[[(4-nitrophenyl)amino]carbonyl]amino]-N-[[1-(2-pyridinyl)cyclohexyl]methyl]-1H-indole-3-propanamide (PD168368)] were used to define the role of GRPr and NMBr in the scratching response. After intracerebroventricular administration, GRP (0.03-0.3 nmol) and NMB (0.1-1 nmol) dose-dependently elicited marked scratching. There was a tolerance to scratching elicited by daily repeated administration of bombesin, GRP, or NMB. Presession administration of RC-3095 (0.1-1 nmol) and PD168368 (0.3-3 nmol) dose-dependently antagonized scratching elicited by GRP and NMB, respectively. More importantly, 1 nmol of RC-3095 failed to block NMB-elicited scratching, and 3 nmol of PD168368 failed to block GRP-elicited scratching. In addition, pretreatment with effective doses of RC-3095 or PD168368 alone or in combination did not block bombesin-elicited scratching. Through the use of the selective antagonists RC-3095 and PD168368, this study demonstrates that central GRPr and NMBr act independently to elicit scratching behavior and there is an additional, unidentified receptor mechanism underlying bombesin-elicited scratching.

Topics: Animals; Bombesin; Dose-Response Relationship, Drug; Gastrin-Releasing Peptide; Indoles; Infusions, Intraventricular; Male; Neurokinin B; Neurotransmitter Agents; Peptide Fragments; Pruritus; Pyridines; Rats; Rats, Wistar; Receptors, Bombesin

Itch sensation is one of the major sensory experiences of human and animals. Recent studies have proposed that gastrin releasing peptide (GRP) is a key neurotransmitter for itch in spinal cord. However, no direct evidence is available to indicate that GRP actually mediate responses between primary afferent fibers and dorsal horn neurons. Here we performed integrative neurobiological experiments to test this question. We found that a small population of rat dorsal horn neurons responded to GRP application with increases in calcium signaling. Whole-cell patch-clamp recordings revealed that a part of superficial dorsal horn neurons responded to GRP application with the increase of action potential firing in adult rats and mice, and these dorsal horn neurons received exclusively primary afferent C-fiber inputs. On the other hands, few A(δ) inputs receiving cells were found to be GRP positive. Finally, we found that evoked sensory responses between primary afferent C fibers and GRP positive superficial dorsal horn neurons are mediated by glutamate but not GRP. CNQX, a blocker of AMPA and kainate (KA) receptors, completely inhibited evoked EPSCs, including in those Fos-GFP positive dorsal horn cells activated by itching. Our findings provide the direct evidence that glutamate is the principal excitatory transmitter between C fibers and GRP positive dorsal horn neurons. Our results will help to understand the neuronal mechanism of itch and aid future treatment for patients with pruritic disease.

Topics: Action Potentials; Animals; Evoked Potentials; Excitatory Postsynaptic Potentials; Gastrin-Releasing Peptide; Glutamic Acid; Green Fluorescent Proteins; Histamine; Injections, Intradermal; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Nerve Fibers, Unmyelinated; Neurotransmitter Agents; Posterior Horn Cells; Pruritus; Rats; Rats, Sprague-Dawley; Spinal Cord; Synaptic Transmission

Topics: Animals; Dermatitis, Atopic; Disease Models, Animal; Gastrin-Releasing Peptide; Male; Mice; Mice, Mutant Strains; Nerve Fibers, Unmyelinated; Pruritus; Skin

Itching, or pruritus, is defined as an unpleasant cutaneous sensation that serves as a physiological self-protective mechanism to prevent the body from being hurt by harmful external agents. Chronic itch represents a significant clinical problem resulting from renal diseases and liver diseases, as well as several serious skin diseases such as atopic dermatitis. The identity of the itch-specific mediator in the central nervous system, however, remains elusive. Here we describe that the gastrin-releasing peptide receptor (GRPR) plays an important part in mediating itch sensation in the dorsal spinal cord. We found that gastrin-releasing peptide is specifically expressed in a small subset of peptidergic dorsal root ganglion neurons, whereas expression of its receptor GRPR is restricted to lamina I of the dorsal spinal cord. GRPR mutant mice showed comparable thermal, mechanical, inflammatory and neuropathic pain responses relative to wild-type mice. In contrast, induction of scratching behaviour was significantly reduced in GRPR mutant mice in response to pruritogenic stimuli, whereas normal responses were evoked by painful stimuli. Moreover, direct spinal cerebrospinal fluid injection of a GRPR antagonist significantly inhibited scratching behaviour in three independent itch models. These data demonstrate that GRPR is required for mediating the itch sensation rather than pain, at the spinal level. Our results thus indicate that GRPR may represent the first molecule that is dedicated to mediating the itch sensation in the dorsal horn of the spinal cord, and thus may provide a central therapeutic target for antipruritic drug development.

Topics: Animals; Ganglia, Spinal; Gastrin-Releasing Peptide; Mice; Motor Activity; Mutation; Pain; Posterior Horn Cells; Pruritus; Receptors, Bombesin; Spinal Cord