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Nearly all adults are infected with one or more herpes viruses. The most common are herpes simplex virus (HSV)-1 and HSV-2, which upon reactivation can cause painful skin and mucosal erosions. Patients who are immune compromised often experience frequent, atypical, or chronic lesions and thus a greatly diminished QOL. Pellino1 is a ubiquitin ligase involved in IL-1 and toll-like receptor signaling; however, the role of Pellino1 in skin immunity against HSV is unknown. In this study, using the mouse-flank HSV-1 skin infection model, we show that Pellino1 has several critical functions during active viral replication. Peli1

Some γ-glutamyl peptides including glutathione (γ-Glu-Cys-Gly) and γ-glutamyl-valyl-glycine (γ-Glu-Val-Gly=γ-EVG) are reported to increase the intensity of basic tastes, such as salty, sweet, and umami, although they have no taste themselves at tested concentrations. The mechanism of action of γ-glutamyl peptides is not clearly understood, but the calcium sensing receptor (CaSR) may be involved. Glutathione and γ-EVG enhance the pungency of some spices, and the present study investigated the effects of γ-EVG on the responses of trigeminal ganglion (TG) cells to thermosensitiveTRP channel agonists. Single-cell RT-PCR revealed that most CaSR-expressing cells co-expressed TRPV1 (sensitive to capsaicin) and TRPA1 (sensitive to allyl isothiocyanate=AITC). Intracellular Ca(2+) imaging showed that pretreatment with γ-EVG excited 7% of trigeminal ganglion (TG) cells and increased the amplitude of their responses to AITC, but not to capsaicin or menthol. The enhancing effect of γ-EVG was prevented by a CaSR inhibitor. The results indicate that γ-EVG increases AITC pungency by activating a subset of trigeminal ganglion cells that co-express CaSR and TRPA1.

Accumulating evidence has highlighted the essential roles of cytokines in itch processing. Although IL-23 and Th17 cytokines are elevated in inflammatory skin disorders, their role in itch is unknown. Here, we investigated the role of IL-23 and IL-17A in itch response using an in vitro calcium imaging of mouse dorsal root ganglion (DRG) neurons and an in vivo behaviour test. Calcium imaging studies revealed that a few DRG neurons (~5%) responded to either IL-23 or IL-17A. Pretreatment cells with IL-23 significantly reduced calcium responses to histamine and capsaicin but not chloroquine. Behaviour experiments showed neither IL-23 nor IL-17A evoked scratching. IL-23 significantly decreased histamine-evoked scratching without affecting chloroquine-evoked scratching. There was no difference in scratching between IL-17A- and vehicle-treated groups. These results indicate that IL-23 might play a role in regulating histaminergic itch via modulation of TRPV1 activity.

Gentle tactile stimuli, such as insects crawling on the skin, can cause itching sensation called mechanical itch. Recent studies have begun to shed light on the neural mechanisms of mechanical itch. Interestingly, the neural pathway for mechanical itch is apparently different from that for chemical itch triggered by the activation of pruriceptors with various mediators. Mechanical itch dysesthesia is frequently seen in patients with chronic itch. Mechanisms of this dysesthesia are plausibly involved in central sensitization. In this review, we summarize the current knowledge of mechanical itch under normal and pathological conditions.

The Janus kinase 1/3 inhibitor tofacitinib has demonstrated an antipruritic effect in two phase III studies in psoriasis. However, the mechanisms behind this antipruritic effect are still unknown. We presently investigated whether tofacitinib affects spontaneous itch as well as expression of itch-related cytokines and epidermal nerve fiber density (ENFD) in the imiquimod-induced mouse model of psoriasis. Psoriasis-like skin lesions were produced by daily topical application of imiquimod to the back skin. Imiquimod treatment resulted in spontaneous scratching, which was significantly inhibited by tofacitinib treatment. Imiquimod treatment significantly increased mRNA expression of Il22, Il23, and Il31, reduced peptidergic ENFD, and increased nonpeptidergic ENFD compared to naive mice. Tofacitinib significantly decreased the expression of those cytokines and increased peptidergic ENFD without a significant effect on nonpeptidergic ENFD. Tofacitinib may inhibit psoriatic itch through inhibition of cytokine expression as well as modulation of epidermal innervation.

Innocuous mechanical stimuli, such as rubbing or stroking the skin, relieve itch through the activation of low-threshold mechanoreceptors. However, the mechanisms behind this inhibition remain unknown. We presently investigated whether stroking the skin reduces the responses of superficial dorsal horn neurons to pruritogens in male C57BL/6J mice. Single-unit recordings revealed that neuronal responses to chloroquine were enhanced during skin stroking, and this was followed by suppression of firing below baseline levels after the termination of stroking. Most of these neurons additionally responded to capsaicin. Stroking did not suppress neuronal responses to capsaicin, indicating state-dependent inhibition. Vesicular glutamate transporter 3 (VGLUT3)-lineage sensory nerves compose a subset of low-threshold mechanoreceptors. Stroking-related inhibition of neuronal responses to chloroquine was diminished by optogenetic inhibition of VGLUT3-lineage sensory nerves in male and femaleVglut3-cre/NpHR-EYFPmice. Conversely, in male and femaleVglut3-cre/ChR2-EYFPmice, optogenetic stimulation of VGLUT3-lineage sensory nerves inhibited firing responses of spinal neurons to pruritogens after the termination of stimulation. This inhibition was nearly abolished by spinal delivery of the κ-opioid receptor antagonist nor-binaltorphimine dihydrochloride, but not the neuropeptide Y receptor Y1 antagonist BMS193885. Optogenetic stimulation of VGLUT3-lineage sensory nerves inhibited pruritogen-evoked scratching without affecting mechanical and thermal pain behaviors. Therefore, VGLUT3-lineage sensory nerves appear to mediate inhibition of itch by tactile stimuli.SIGNIFICANCE STATEMENTRubbing or stroking the skin is known to relieve itch. We investigated the mechanisms behind touch-evoked inhibition of itch in mice. Stroking the skin reduced the activity of itch-responsive spinal neurons. Optogenetic inhibition of VGLUT3-lineage sensory nerves diminished stroking-evoked inhibition, and optogenetic stimulation of VGLUT3-lineage nerves inhibited pruritogen-evoked firing. Together, our results provide a mechanistic understanding of touch-evoked inhibition of itch.

The antipruritic activity of gabapentin, an anticonvulsant, was studied in a mouse model of allergic itch. In mice sensitized by an extract of the salivary glands of the mosquito (ESGM), an intradermal injection of ESGM elicited scratching and increased peripheral nerve firing. Oral or intradermal administration of gabapentin at the ESGM injection site inhibited ESGM-induced scratching and peripheral nerve firing. However, gabapentin did not affect histamine-induced scratching. The distributions of immunoreactivity to the voltage-dependent calcium channel α2δ-1 subunit, a site of gabapentin action, and the histamine H1 receptor differed in the mouse dorsal root ganglia. The α2δ-1 subunit was mainly found in neurons that were 15–20 µm in diameter, whereas the H1 receptor was mainly in 20–30 µm neurons. In addition, α2δ-1 subunit immunoreactivity co-localized with that of transient receptor potential vanilloid 1 (TRPV1). These results suggest that gabapentin regulates allergic itch by acting on the calcium channel α2δ-1 subunit in peripheral TRPV1-positive neurons.

Skin thermal changes modulate itch sensitivity. However, the mechanisms of this modulation are still unclear. Using mouse models of acute and chronic itch, we investigated whether local innocuous thermal stimulation of the skin alters itch sensitivity and if blockade of thermosensitive transient receptor potential (TRP) channels can reduce these changes. Localized thermal changes were achieved by placing a thermal probe in contact with the back skin for 30 s. Warming the skin significantly increased serotonin-evoked scratching and spontaneous scratching in the ovalbumin model of atopic dermatitis but decreased histamine-evoked scratching. These changes were blocked by a TRPV4 antagonist. Cooling the skin significantly increased serotonin-evoked scratching but reduced histamine-evoked scratching. The increase in serotonin-evoked scratching, but not the reduction of histamine-evoked scratching, was blocked by TRPM8 antagonism. Chloroquine-evoked scratching was unaffected by either warming or cooling. Our data indicate that different itch signaling pathways are differentially modulated by skin thermal changes.

Chronic itch is the most troublesome symptom of psoriasis, a chronic autoimmune skin disease of scaling and inflammation. Epidermal hyperinnervation by itch-signaling fibers is considered to be one cause of chronic itch [32], and increased epidermal nerve density has been frequently reported in humans with chronic itch conditions, including psoriasis [12; 18; 29–32], with some exceptions [19; 24]. Nociceptive fibers are expressed throughout the epidermis and often categorized as peptidergic or nonpeptidergic fibers [7]. The former contain the neuropeptides calcitonin gene–related peptide (CGRP) and/or substance P, whereas the latter express the purinergic receptor P2X3 and isolectin B4. Ablation of the peptidergic or non-peptidergic subset of fibers produces modality-specific deficits in response to heat and mechanical stimuli, respectively [8; 40]. Enhanced intraepidermal nonpeptidergic nerve density has been reported previously in animal models of dry skin itch [25; 34]. The majority of nonpeptidergic sensory neurons express glial cell line-derived neurotrophic factor (GDNF) family receptor (GFR)α-1 and/or GFRα-2, which show preferential binding for GDNF and neurturin (NRTN), respectively [36]. These neurotrophic growth factors are important for the development and maintenance of nonpeptidergic sensory neurons [5; 9; 13; 15; 35; 41]. As such, overexpression of these growth factors could lead to increased nonpeptidergic nerve density, possibly leading to increased itch. We have reported that the imiquimod-induced psoriasis mouse model is useful for the investigation of chronic itch in psoriasis [23]. Utilizing this model, we presently investigated whether the epidermal density of nonpeptidergic nerves and the expression of their neurotrophic growth factors (GDNF and NRTN) are increased in psoriasis-like skin. Because this model exhibits spontaneous scratching that is histamine-dependent in the early phase (Day 2) and histamine-independent in the late phase (Day 7), we measured nerve fiber density at these time points. In addition, we tested whether inhibition of NRTN could affect the density of intraepidermal fibers and spontaneous scratching behavior.

Stress is the exacerbating factor of itch across patients with chronic itch due to different origins. However, the precise mechanisms behind stress-induced exacerbation of itch remain unknown. Chronic stress induces hyperexcitability of the amygdala, the centre of emotional processing. Recent findings on the itch neuronal pathways support a pivotal role of the amygdala for itch processing. We hypothesized that itch is enhanced by stress through hyperexcitation of the amygdala. Modulation of amygdala activity, therefore, may have therapeutic potential in the treatment of chronic itch.

Itch is a major indicator of psoriasis, but the underlying mechanisms behind this symptom are largely unknown. To investigate the neuronal mechanisms of psoriatic itch, we tested whether mice subjected to the imiquimod-induced psoriasis model exhibit itch-associated behaviors. Mice received daily topical applications of imiquimod to the rostral back skin for 7 days. Imiquimod-treated mice exhibited a significant increase in spontaneous scratching behavior directed to the treated area as well as touch-evoked scratching (alloknesis). To characterize this model, we measured the mRNA expression levels of pruritogens and itch-relevant receptors/channels using real-time reverse transcription PCR. The mRNA expression of MrgprA3, MrgprC11, and MrgprD decreased gradually over time in the dorsal root ganglion (DRG) cells. There was no significant change in the mRNA expression of TRPV1 or TRPA1 in DRG cells. TRPV4 mRNA expression was transiently increased in the DRG cells, whereas TRPM8 mRNA was significantly decreased. The mRNA expression levels of histidine decarboxylase and tryptophan hydroxylase 1, as well as the intensity of histamine and serotonin immunoreactivity, were transiently increased in the skin on day 2, returning to baseline by day 7. Histamine H1-receptor antagonists, chlorpheniramine and olopatadine, significantly inhibited spontaneous scratching on day 2, but not day 7. Neither chlorpheniramine nor olopatadine affected alloknesis on day 2 or day 7. These results may reflect the limited antipruritic effects of histamine H1-receptor antagonists on human psoriasis. The imiquimod-induced psoriasis model seems to be useful for the investigation of itch and its sensitization in psoriasis.

Itch consists of both sensory and affective components. For chronic itch patients, the affective component of itch affects both quality of life (leading to psychological comorbidities) and disease prognosis (by promoting scratching of itchy skin). We found that acute itch stimuli, such as histamine, induced anxiety-like behavior and increased activity (c-Fos expression) in the amygdala in adult male C57BL/6 mice. Itch stimuli also increased activity in projection areas to the amygdala, suggesting that these regions form a circuit for affective itch processing. Electrophysiological characterization of histamine-responsive amygdala neurons showed that this population was active on a behaviorally relevant timescale and partially overlapped with pain signaling. Selective optogenetic activation of histamine-responsive amygdala neurons in adult male and female Fos:CreERT2;R26Ai14mice using the Targeted Recombination in Active Populations system enhanced both scratching and anxiety-like behavior. These results highlight the importance of itch-responsive amygdala neurons in modulating itch-related affect and behavior.SIGNIFICANCE STATEMENTThe sensation of itch includes an affective component that leads to stress and anxiety in chronic itch patients. We investigated the neuronal basis of affective itch in mice, with a focus on the amygdala, the key brain region for the generation of anxiety. A subpopulation of amygdala neurons responded to itch stimuli such as histamine. Optogenetic activation of histamine-responsive amygdala neurons affected both scratching and anxiety-like behavior. Therefore, this population appears to be important for mediating the affective component of itch.

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Endothelin-1 (ET-1) has been implicated in nonhistaminergic itch. Here we used electrophysiological methods to investigate whether mouse superficial dorsal horn neurons respond to intradermal (id) injection of ET-1 and whether ET-1-sensitive neurons additionally respond to other pruritic and algesic stimuli or spinal superfusion of bombesin, a homolog of gastrin-releasing peptide (GRP) that excites spinal itch-signaling neurons. Single-unit recordings were made from lumbar dorsal horn neurons in pentobarbital-anesthetized C57BL/6 mice. We searched for units that exhibited elevated firing after id injection of ET-1 (1 μg/μl). Responsive units were further tested with mechanical stimuli, bombesin (spinal superfusion, 200 μg·ml−1·min−1), heating, cooling, and additional chemicals [histamine, chloroquine, allyl isothiocyanate (AITC), capsaicin]. Of 40 ET-1-responsive units, 48% responded to brush and pinch [wide dynamic range (WDR)] and 52% to pinch only [high threshold (HT)]. Ninety-three percent responded to noxious heat, 50% to cooling, and >70% to histamine, chloroquine, AITC, and capsaicin. Fifty-seven percent responded to bombesin, suggesting that they participate in spinal itch transmission. That most ET-1-sensitive spinal neurons also responded to pruritic and algesic stimuli is consistent with previous studies of pruritogen-responsive dorsal horn neurons. We previously hypothesized that pruritogen-sensitive neurons signal itch. The observation that ET-1 activates nociceptive neurons suggests that both itch and pain signals may be generated by ET-1 to result in simultaneous sensations of itch and pain, consistent with observations that ET-1 elicits both itch- and pain-related behaviors in animals and burning itch sensations in humans.

Itch is often defined as an ‘unpleasant sensation that provokes the desire to scratch’. Recent studies have provided novel insights into the molecular mechanisms underlying itch-selective signalling pathways from the periphery to the central nervous system. Chronic itch is often associated with inflammatory skin diseases such as atopic dermatitis, psoriasis or contact dermatitis, as well as systemic diseases such as renal, cholestatic, hematologic or endocrine pruritus or pruritus related to malignancy, and reduces quality of life. Chronic itch is thought to involve the sensitisation of itch-signalling pathways, the molecular and cellular processes of which we are just starting to uncover. This review article describes recent findings regarding the basic mechanisms of itch transmission from the skin to the brain. We further describe the neuronal changes that occur both peripherally and centrally under conditions of chronic itch. Key Concepts There are a variety of endogenous pruritogens and external itch stimuli. Itch transducing channels include TRPV1, TRPA1 and Nav1.7. Spinal excitatory and inhibitory interneurons process itch information. Descending noradrenergic and serotonergic systems modulate spinal itch transmission. Sensitisation occurs in both peripheral and central nervous system under conditions of chronic itch. NK1-expressing spinal neurons play a major role in itch sensitisation. Keywords: itch; pruritus; sodium channels; TRP channels; sensitisation; brain; spinal cord; pruriceptors; descending modulation

Itch is relayed to higher centers by projection neurons in the spinal and medullary dorsal horn. We employed a double-label method to map the ascending projections of pruriceptive and nociceptive trigeminal and spinal neurons. The retrograde tracer fluorogold (FG) was stereotaxically injected into the right thalamus or lateral parabrachial area (LPb) in mice. Seven days later, mice received intradermal (id) microinjection of histamine, chloroquine, capsaicin, or vehicle into the left cheek. Histamine, chloroquine, and capsaicin intradermally elicited similar distributions of Fos-positive neurons in the medial aspect of the superficial medullary and spinal dorsal horn from the trigeminal subnucleus caudalis to C2. Among neurons retrogradely labeled from the thalamus, 43%, 8%, and 22% were Fos-positive following id histamine, chloroquine, or capsaicin. Among the Fos-positive neurons following pruritic or capsaicin stimuli, ∼1-2% were retrogradely labeled with FG. Trigeminoparabrachial projection neurons exhibited a higher incidence of double labeling in the superficial dorsal horn. Among the neurons retrogradely labeled from LPb, 36%, 29%, and 33% were Fos positive following id injection of histamine, chloroquine, and capsaicin, respectively. Among Fos-positive neurons elicited by id histamine, chloroquine, and capsaicin, respectively, 3.7%, 4.3%, and 4.1% were retrogradely labeled from LPb. The present results indicate that, overall, relatively small subpopulations of pruriceptive and/or nociceptive neurons innervating the cheek project to thalamus or LPb. These results imply that the vast majority of pruritogen- and algogen-responsive spinal neurons are likely to function as interneurons relaying information to projection neurons and/or participating in segmental nocifensive circuits.

We investigated roles for spinal neurons expressing the neurokinin-1 receptor (NK1R) and/or gastrin-releasing peptide receptor (GRPR) in a mouse model of ovalbumin (OVA)-induced chronic atopic dermatitis. Mice receiving repeated topical application of OVA exhibited atopic-like skin lesions and behavioral signs of chronic itch including spontaneous scratching, touch-evoked scratching (alloknesis), and enhancement of chloroquine-evoked scratching (hyperknesis). Substance P-saporin (SP-SAP) and bombesin-saporin (BB-SAP) were intrathecally injected into OVA-sensitized mice to neurotoxically ablate NK1R- or GRPR-expressing spinal neurons, respectively. SP-SAP diminished the expression of NK1R in the superficial spinal dorsal horn and significantly attenuated all behavioral signs of chronic itch. BB-SAP reduced the spinal dorsal horn expression of GRPR and significantly attenuated hyperknesis, with no effect on spontaneous scratching or alloknesis. To investigate whether NK1R-expressing spinal neurons project in ascending somatosensory pathways, we performed a double-label study. The retrograde tracer, Fluorogold (FG), was injected into either the somatosensory thalamus or lateral parabrachial nucleus. In the upper cervical (C1-2) spinal cord, most neurons retrogradely labeled with FG were located in the dorsomedial aspect of the superficial dorsal horn. Of FG-labeled spinal neurons, 89% to 94% were double labeled for NK1R. These results indicate that NK1R-expressing spinal neurons play a major role in the expression of symptoms of chronic itch and give rise to ascending somatosensory projections. Gastrin-releasing peptide receptor-expressing spinal neurons contribute to hyperknesis but not to alloknesis or ongoing itch. NK1R-expressing spinal neurons represent a potential target to treat chronic itch.

We have further developed a behavioral model of itch and pain in the lower hindlimb (calf) originally reported by LaMotte et al. (2011) that allows comparisons with responses of lumbar dorsal horn neurons to pruritic and noxious stimuli. Intradermal (id) microinjection of the pruritogens histamine, SLIGRL-NH2 (agonist of PAR-2 and MrgprC11) and choloroquine (agonist of MrgprA3) into the calf of the lower limb elicited significant biting and a small amount of licking directed to the injection site, over a 30-min time course. Following id injection of histamine, low-threshold mechanical stimuli reliably elicited discrete episodes of biting (alloknesis) over a longer time course; significantly less alloknesis was observed following id injection of SLIGRL-NH2. Capsaicin injections elicited licking but little biting. Following id injection of capsaicin, low-threshold mechanical stimuli elicited discrete hindlimb flinches (allodynia) over a prolonged (>2 hr) time course. In single-unit recordings from superficial lumbar dorsal horn neurons, low-threshold mechanically-evoked responses were significantly enhanced, accompanied by receptive field expansion, following id injection of histamine in histamine-responsive neurons. This was not observed in histamine-insensitive neurons, or following id injection of saline or SLIGRL-NH2, regardless of whether the latter activated the neuron or not. These results suggest that itch-responsive neurons are selectively sensitized by histamine but not SLIGRL-NH2 to account for alloknesis. The presently-described “calf” model appears to distinguish between itch- and pain-related behavioral responses, and provides a basis to investigate lumbar spinal neural mechanisms underlying itch, alloknesis, pain and allodynia.

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.

Aims: Oxidative stress is thought to be involved in Friedreich's ataxia (FRDA), yet it has not been demonstrated in the target neurons that are first to degenerate. Using the YG8R mouse model of FRDA, microarray and neuritic growth experiments were carried out in the dorsal root ganglion (DRG), the primary site of neurodegeneration in this disease. Results: YG8R hemizygous mice exhibited defects in movement, and DRG neurites had growth defects. Microarray of DRG tissue identified decreased transcripts encoding the antioxidants, including peroxiredoxins, glutaredoxins, and glutathione S-transferase, and these were confirmed by immunoblots and quantitative real-time PCR. Because the decreased gene transcripts are the known targets of the antioxidant transcription factor nuclear factor-E2-related factor-2 (Nrf2), Nrf2 expression was measured; it was significantly decreased at the transcript and protein level in both the DRG and the cerebella of the YG8R hemizygous mouse; further, frataxin expression was significantly correlated with Nrf2 expression. Functionally, in YG8R hemizygous DRG, the total glutathione levels were reduced and explanted cells were more sensitive to the thioredoxin reductase (TxnRD) inhibitor auranofin, a thiol oxidant. In cell models of FRDA, including Schwann and the DRG, frataxin deficiency caused a decreased expression of the Nrf2 protein level in the nucleus, but not a defect in its translocation from the cytosol. Further, frataxin-deficient cells had decreased enzyme activity and expression of TxnRD, which is regulated by Nrf2, and were sensitive the TxnRD inhibitor auranofin. Innovation and Conclusion: These results support a mechanistic hypothesis in which frataxin deficiency decreases Nrf2 expression in vivo, causing the sensitivity to oxidative stress in target tissues the DRG and the cerebella, which contributes to the process of neurodegeneration. Antioxid. Redox Signal. 19, 1481–1493.

Scratching inhibits pruritogen-evoked responses of neurons in the superficial dorsal horn, implicating a spinal site for scratch inhibition of itch. We investigated if scratching differentially affects neurons depending on whether they are activated by itchy vs. painful stimuli, and if the degree of inhibition depends on the relative location of scratching. We recorded from rat lumbar dorsal horn neurons responsive to intradermal (id) microinjection of serotonin (5-hydroxytryptamine, 5-HT). During the response to 5-HT, scratch stimuli (3mm, 300 mN, 2 Hz, 20s) were delivered at the injection site within the mechanosensitive receptive field (on-site), or 4-30 mm away, outside of the receptive field (off-site). During off-site scratching, 5-HT-evoked firing was significantly attenuated followed by recovery. On-site scratching excited neurons, followed by a significant post-scratch decrease in 5-HT-evoked firing. Most neurons additionally responded to mustard oil (allyl isothiocyanate). Off-site scratching had no effect, while on-site scratching excited the neurons. These results indicate that scratching exerts a state-dependent inhibitory effect on responses of spinal neurons to pruritic but not algesic stimuli. Moreover, on-site scratching first excited neurons followed by inhibition, while off-site scratching immediately evoked the inhibition of pruritogen-evoked activity. This accounts for the suppression of itch by scratching at a distance from the site of the itchy stimulus.

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.

Itch is often triggered by warming the skin in patients with itchy dermatitis, but the underlying mechanism is largely unknown. We presently investigated if warming the skin enhances histamine- or serotonin (5-HT)-evoked itch behavior or responses of sensory dorsal root ganglion (DRG) cells, and if responses of superficial dorsal horn neurons to innocuous warming are enhanced by these pruritogens. In a temperature-controlled environmental chamber, mice exhibited greater scratching following intradermal injection of 5-HT, but not histamine, SLIGRL, or BAM8-22, when the skin surface temperature was above 36°C. Calcium imaging of DRG cells in a temperature-controlled bath revealed that responses to 5-HT, but not histamine, were significantly greater at a bath temperature of 35°C vs. lower temperatures. Single-unit recordings revealed a subpopulation of superficial dorsal horn neurons responsive to intradermal injection of 5-HT. Of these, 58% responded to innocuous skin warming (37°C) prior to intradermal injection of 5-HT, while 100% responded to warming following intradermal injection of 5-HT. Warming-evoked responses were superimposed on the 5-HT-evoked elevation in firing and were significantly larger compared with responses pre-5-HT, as long as 30 min after the intradermal injection of 5-HT. Five-HT-insensitive units, and units that either did or did not respond to intradermal histamine, did not exhibit any increase in the incidence of warmth sensitivity or in the mean response to warming following intradermal injection of the pruritogen. The results suggest that 5-HT-evoked responses of pruriceptors are enhanced during skin warming, leading to increased firing of 5-HT-sensitive dorsal horn neurons that signal nonhistaminergic itch. NEW & NOTEWORTHY Skin warming often exacerbates itch in patients with itchy dermatitis. We demonstrate that warming the skin enhanced serotonin-evoked, but not histamine-evoked, itch behavior and responses of sensory dorsal root ganglion cells. Moreover, serotonin, but not histamine, enhanced responses of superficial dorsal horn neurons to innocuous warming. The results suggest that skin warming selectively enhances the responses of serotonin-sensitive pruriceptors, leading to increased firing of serotonin-sensitive dorsal horn neurons that signal nonhistaminergic itch.

This chapter summarizes recent findings regarding the central transmission of acute and chronic itch. Itch is transduced by cutaneous pruriceptors that transmit signals to neurons in the superficial spinal cord. Spinal itch-signaling circuits utilize several neuropeptides whose receptors represent novel targets to block itch transmission. Itch is relieved by scratching, which activates spinal interneurons to inhibit itch-transmitting neurons. Spinal itch transmission is also thought to be modulated by descending pathways. Itch is transmitted rostrally via ascending pathways to activate a variety of brain regions involved in sensory discrimination of affective and motor responses to itch. The pathophysiological mechanisms of chronic itch are poorly understood but likely involve sensitization of itch-signaling pathways and/or dysfunction of itch-inhibitory circuits. Improved understanding of central itch mechanisms has identified a number of novel targets for the development of antipruritic treatment strategies.

Lightly touching normal skin near a site of itch can elicit itch sensation, a phenomenon known as alloknesis. To investigate the neural mechanisms of alloknesis, we have developed an animal model. Low-threshold mechanical stimulation of the skin normally does not elicit any response in naive C57/BL6 mice. Following acute intradermal (i.d.) injection of histamine in the rostral back, mechanical stimulation 7 mm from the injection site elicited discrete hindlimb scratch bouts directed toward the stimulus. This began at 10 minutes and peaked 20-40 minutes post histamine injection, declining over the next hour. Histamine itself elicited bouts of scratching not associated with the mechanical stimulus, which ceased after 30 minutes. Histamine- and touch-evoked scratching was inhibited by the μ-opiate antagonist naltrexone. Touch-evoked scratching was observed following i.d. 5-HT (5-hydroxytryptamine), a protease-activated receptor (PAR)-4 agonist, and an MrgprC11 agonist BAM8-22, but not chloroquine or a PAR-2 agonist. The histamine H1 receptor antagonist terfenadine prevented scratching and alloknesis evoked by histamine, but not 5-HT, a PAR-4 agonist or an MrgprC11 agonist. In mice with experimental dry skin, there was a time-dependent increase in spontaneous and touch-evoked scratching. This animal model appears to be useful to investigate neural mechanisms of itch and alloknesis.

The relief of itch by scratching is thought to involve inhibition of pruritogen-responsive neurons in the spinal cord. We recorded the responses of superficial dorsal horn neurons in mice to intradermal injection of the pruritogens chloroquine and histamine. Scratching within an area 5-17 mm distant from the injection site, outside of the units' mechanoreceptive fields (off-site), significantly inhibited chloroquine-evoked and histamine-evoked responses without affecting capsaicin-evoked firing. This is consistent with observations that scratching at a distance from a site of itch is antipruritic. In contrast, scratching directly at the injection site (within the receptive field; on-site) had no effect on chloroquine-evoked neuronal firing, but enhanced the same neurons' responses to intradermal injection of the algogen capsaicin. Moreover, neuronal responses to histamine were enhanced during on-site scratching, and this was followed by suppression of firing below baseline levels after termination of scratching. Scratching thus inhibits pruritogen-responsive neurons in a manner that depends on the input modality (i.e. pain vs. histamine-dependent or histamine-independent itch) and skin location.

Chronic itch is symptomatic of many skin conditions and systemic diseases. Little is known about pathophysiological alterations in itch-signaling neural pathways associated with chronic itch. We used a mouse model of hindpaw chronic dry skin itch to investigate properties of presumptive itch-signaling neurons. Neurons in the lumbar superficial dorsal horn ipsilateral to hindpaw dry skin treatment exhibited a high level of spontaneous activity that was inhibited by scratching the plantar surface. Most spontaneously active units exhibited further increases in firing rate following intradermal injection of an agonist of the protease-activated receptor PAR-2, or histamine. The large majority of pruritogen-responsive units also responded to capsaicin and allyl isothiocyanate. For neurons ipsilateral to dry skin treatment, responses elicited by the PAR-2 agonist, but not histamine or mechanical stimuli, were significantly larger compared with neurons ipsilateral to vehicle (water) treatment or neurons recorded in naïve (untreated) mice. The spontaneous activity may signal ongoing itch, while enhanced PAR-2 agonist-evoked responses may underlie hyperknesis (enhanced itch), both of which are symptomatic of many chronic itch conditions. The enhancement of neuronal responses evoked by the PAR-2 agonist, but not by histamine or mechanical stimuli, implies that the dry skin condition selectively sensitized PAR-2 agonist-sensitive primary afferent pruriceptors.

Chronic itch is a symptom of many skin conditions and systemic disease, and it has been hypothesized that the chronic itch may result from sensitization of itch-signaling pathways. We induced experimental chronic dry skin on the rostral back of mice, and observed a significant increase in spontaneous hindlimb scratches directed to the dry skin. Spontaneous scratching was significantly attenuated by a PAR-2 antibody and 5-HT2A receptor antagonist, indicating activation of these receptors by endogeneous mediators released under dry skin conditions. We also observed a significant increase in the number of scratch bouts evoked by acute intradermal injections of a protease-activated receptor (PAR)-2 agonist and serotonin (5-HT), but not histamine. We additionally investigated if pruritogen-evoked activity of dorsal root ganglion (DRG) neurons is enhanced in this model. DRG cells from dry skin mice exhibited significantly larger responses to the PAR-2 agonist and 5-HT, but not histamine. Spontaneous scratching may reflect ongoing itch, and enhanced pruritogen-evoked scratching may represent hyperknesis (enhanced itch), both potentially due to sensitization of itch-signaling neurons. The correspondence between enhanced behavioral scratching and DRG cell responses suggests that peripheral pruriceptors that respond to proteases and 5-HT, but not histamine, may be sensitized in dry skin itch.

Inactivation of descending pathways enhanced responses of spinal dorsal horn neurons to noxious stimuli, but little is known regarding tonic descending modulation of spinal itch transmission. To study effects of cervical spinal cold block on responses of dorsal horn neurons to itch-evoking and pain-evoking stimuli, single-unit recordings were made from superficial dorsal horn wide dynamic range and nociceptive-specific-type neurons in pentobarbital-anesthetized mice. Intradermal histamine excited 17 units. Cold block starting 1 minute after intradermal injection of histamine caused a marked decrease in firing. The histamine-evoked response during and following cold block was significantly lower compared with control histamine-evoked responses in the absence of cold block. A similar but weaker depressant effect of cold block was observed for dorsal horn unit responses to chloroquine. Twenty-six units responded to mustard oil allyl isothiocyanate (AITC), with a further significant increase in firing during the 1-minute period of cold block beginning 1 minute after AITC application. Activity during cold block was significantly greater compared with the same time period of control responses to AITC in the absence of cold block. Ten units' responses to noxious heat were significantly enhanced during cold block, while 6 units' responses were reduced and 18 unaffected. Cold block had no effect on mechanically evoked responses. These results indicate that spinal chemonociceptive transmission is under tonic descending inhibitory modulation, while spinal pruriceptive transmission is under an opposing, tonic descending facilitatory modulation.

Itch is relayed to higher centers by projection neurons in the spinal and medullary dorsal horn. We employed a double-label method to map the ascending projections of pruriceptive and nociceptive trigeminal and spinal neurons. The retrograde tracer fluorogold (FG) was stereotaxically injected into the right thalamus or lateral parabrachial area (LPb) in mice. Seven days later, mice received intradermal (id) microinjection of histamine, chloroquine, capsaicin, or vehicle into the left cheek. Histamine, chloroquine, and capsaicin intradermally elicited similar distributions of Fos-positive neurons in the medial aspect of the superficial medullary and spinal dorsal horn from the trigeminal subnucleus caudalis to C2. Among neurons retrogradely labeled from the thalamus, 43%, 8%, and 22% were Fos-positive following id histamine, chloroquine, or capsaicin. Among the Fos-positive neurons following pruritic or capsaicin stimuli, ∼1-2% were retrogradely labeled with FG. Trigeminoparabrachial projection neurons exhibited a higher incidence of double labeling in the superficial dorsal horn. Among the neurons retrogradely labeled from LPb, 36%, 29%, and 33% were Fos positive following id injection of histamine, chloroquine, and capsaicin, respectively. Among Fos-positive neurons elicited by id histamine, chloroquine, and capsaicin, respectively, 3.7%, 4.3%, and 4.1% were retrogradely labeled from LPb. The present results indicate that, overall, relatively small subpopulations of pruriceptive and/or nociceptive neurons innervating the cheek project to thalamus or LPb. These results imply that the vast majority of pruritogen- and algogen-responsive spinal neurons are likely to function as interneurons relaying information to projection neurons and/or participating in segmental nocifensive circuits.

Intrathecal administration of the neurotoxin bombesin-saporin reduces or abolishes pruritogen-evoked scratching behavior. We investigated whether spinal neurons that respond to intradermal (ID) injection of pruritogens also respond to spinal superfusion of bombesin and vice versa. Single-unit recordings were made from superficial lumbar spinal dorsal horn neurons in anesthetized mice. We identified neurons with three search strategies: 1) ID injection of the nonhistaminergic itch mediator chloroquine, 2) spinal superfusion of bombesin, and 3) noxious pinch. All units were tested with an array of itch mediators (chloroquine, histamine, SLIGRL, BAM8-22), algogens [capsaicin, allyl isothiocyanate (AITC)], and physical stimuli (brush, pinch, noxious heat, cooling) applied to the hindlimb receptive field. The vast majority of chloroquine-responsive units also responded to bombesin. Of 26 chloroquine-sensitive units tested, most responded to SLIGRL, half responded to histamine and/or BAM8-22, and most responded to capsaicin and/or AITC as well as noxious thermal and mechanical stimuli. Of 29 bombesin-responsive units, a large majority also responded to other itch mediators as well as AITC, capsaicin, and noxious thermal and mechanical stimuli. Responses to successive applications of bombesin exhibited tachyphylaxis. In contrast, of 36 units responsive to noxious pinch, the majority (67%) did not respond to ID chloroquine or spinal bombesin. It is suggested that chloroquine- and bombesin-sensitive spinal neurons signal itch from the skin.

The kappa-opioid agonist, nalfurafine, has been approved in Japan for treatment of itch in patients with chronic kidney disease. We presently investigated if systemic administration of nalfurafine inhibited ongoing or touch-evoked scratching behavior (alloknesis) following acute intradermal injection of histamine or the non-histaminergic itch mediator, chloroquine, in mice. We also investigated if nalfurafine suppressed spontaneous or touch-evoked scratching in an experimental model of chronic dry skin itch. Nalfurafine reduced scratching evoked by histamine and chloroquine. Following acute histamine, but not chloroquine, low-threshold mechanical stimuli reliably elicited directed hindlimb scratching behavior, which was significantly attenuated by nalfurafine. In mice with experimental dry skin, nalfurafine abolished spontaneous scratching but had no effect on alloknesis. Nalfurafine thus appears to be a promising treatment for acute itch as well as ongoing itch of dry skin.

In humans, pruritus (itch) is a common but poorly understood symptom in numerous skin and systemic diseases. Endothelin 1 (ET-1) evokes histamine-independent pruritus in mammals through activation of its cognate G protein-coupled receptor endothelin A receptor (ETAR). Here, we have identified neural endothelin-converting enzyme 1 (ECE-1) as a key regulator of ET-1-induced pruritus and neural signaling of itch. We show here that ETAR, ET-1, and ECE-1 are expressed and colocalize in murine dorsal root ganglia (DRG) neurons and human skin nerves. In murine DRG neurons, ET-1 induced internalization of ETAR within ECE-1-containing endosomes. ECE-1 inhibition slowed ETAR recycling yet prolonged ET-1-induced activation of ERK1/2, but not p38. In a murine itch model, ET-1-induced scratching behavior was substantially augmented by pharmacological ECE-1 inhibition and abrogated by treatment with an ERK1/2 inhibitor. Using iontophoresis, we demonstrated that ET-1 is a potent, partially histamine-independent pruritogen in humans. Immunohistochemical evaluation of skin from prurigo nodularis patients confirmed an upregulation of the ET-1/ETAR/ECE-1/ERK1/2 axis in patients with chronic itch. Together, our data identify the neural peptidase ECE-1 as a negative regulator of itch on sensory nerves by directly regulating ET-1-induced pruritus in humans and mice. Furthermore, these results implicate the ET-1/ECE-1/ERK1/2 pathway as a therapeutic target to treat pruritus in humans.