Role of thermosensitive transient receptor potential (TRP) channels in thermal preference of male and female mice.

Transient Receptor Potential (TRP) ion channels are important for sensing environmental temperature. In rodents, TRPV4 senses warmth (25-34 °C), TRPV1 senses heat (>42 °C), TRPA1 putatively senses cold (<17 °C), and TRPM8 senses cool-cold (18-26 °C). We investigated if knockout (KO) mice lacking these TRP channels exhibited changes in thermal preference. Thermal preference was tested using a dual hot-cold plate with one thermoelectric surface set at 30 °C and the adjacent surface at a temperature of 15-45 °C in 5 °C increments. Blinded observers counted the number of times mice crossed through an opening between plates and the percentage of time spent on the 30 °C plate. In a separate experiment, observers blinded as to genotype also assessed the temperature at the location on a thermal gradient (1.83 m, 4-50 °C) occupied by the mouse at 5- or 10-min intervals over 2 h. Male and female wildtype mice preferred 30 °C and significantly avoided colder (15-20 °C) and hotter (40-45 °C) temperatures. Male TRPV1KOs and TRPA1KOs, and TRPV4KOs of both sexes, were similar, while female WTs, TRPV1KOs, TRPA1KOs and TRPM8KOs did not show significant thermal preferences across the temperature range. Male and female TRPM8KOs did not significantly avoid the coldest temperatures. Male mice (except for TRPM8KOs) exhibited significantly fewer plate crossings at hot and cold temperatures and more crossings at thermoneutral temperatures, while females exhibited a similar but non-significant trend. Occupancy temperatures along the thermal gradient exhibited a broad distribution that shrank somewhat over time. Mean occupancy temperatures (recorded at 90-120 min) were significantly higher for females (30-34 °C) compared to males (26-27 °C) of all genotypes, except for TRPA1KOs which exhibited no sex difference. The results indicate (1) sex differences with females (except TRPA1KOs) preferring warmer temperatures, (2) reduced thermosensitivity in female TRPV1KOs, and (3) reduced sensitivity to cold and innocuous warmth in male and female TRPM8KOs consistent with previous studies.

TRPM8-dependent shaking in mammals and birds.

Removing water from wet fur or feathers is important for thermoregulation in warm-blooded animals. The "wet dog shake" (WDS) behavior has been largely characterized in mammals but to a much lesser extent in birds. Although it is known that TRPM8 is the main molecular transducer of low temperature in mammals, it is not clear if wetness-induced shaking in furred and feathered animals is dependent on TRPM8. Here, we show that a novel TRPM8 agonist induces WDS in rodents and, importantly, in birds, similar to the shaking behavior evoked by water-spraying. Furthermore, the WDS onset depends on TRPM8, as we show in water-sprayed mice. Overall, our results provide multiple evidence for a TRPM8 dependence of WDS behaviors in all tested species. These suggest that a convergent evolution selected similar shaking behaviors to expel water from fur and feathers, with TRPM8 being involved in wetness sensing in both mammals and birds.

Targeting Transient Receptor Potential (TRP) Channels, Mas-Related G-Protein-Coupled Receptors (Mrgprs), and Protease-Activated Receptors (PARs) to Relieve Itch.

Itch (pruritus) is a sensation in the skin that provokes the desire to scratch. The sensation of itch is mediated through a subclass of primary afferent sensory neurons, termed pruriceptors, which express molecular receptors that are activated by itch-evoking ligands. Also expressed in pruriceptors are several types of Transient Receptor Potential (TRP) channels. TRP channels are a diverse class of cation channels that are responsive to various somatosensory stimuli like touch, pain, itch, and temperature. In pruriceptors, TRP channels can be activated through intracellular signaling cascades initiated by pruritogen receptors and underly neuronal activation. In this review, we discuss the role of TRP channels TRPA1, TRPV1, TRPV2, TRPV3, TRPV4, TRPM8, and TRPC3/4 in acute and chronic pruritus. Since these channels often mediate itch in association with pruritogen receptors, we also discuss Mas-related G-protein-coupled receptors (Mrgprs) and protease-activated receptors (PARs). Additionally, we cover the exciting therapeutic targets amongst the TRP family, as well as Mrgprs and PARs for the treatment of pruritus.

Inhibition of itch by neurokinin 1 receptor (Tacr1) -expressing ON cells in the rostral ventromedial medulla in mice.

The rostral ventromedial medulla (RVM) is important in descending modulation of spinal nociceptive transmission, but it is unclear if the RVM also modulates spinal pruriceptive transmission. RVM ON cells are activated by noxious algesic and pruritic stimuli and are pronociceptive. Many RVM-spinal projection neurons express the neurokinin-1 receptor (Tacr1), and ON-cells are excited by local administration of substance P (SP). We hypothesized that Tacr1-expressing RVM ON cells exert an inhibitory effect on itch opposite to their pronociceptive action. Intramedullary microinjection of SP significantly potentiated RVM ON cells and reduced pruritogen-evoked scratching while producing mild mechanical sensitization. Chemogenetic activation of RVM Tacr1-expressing RVM neurons also reduced acute pruritogen-evoked scratching. Optotagging experiments confirmed RVM Tacr1-expressing neurons to be ON cells. We conclude that Tacr1-expressing ON cells in RVM play a significant role in the modulation of pruriceptive transmission.

Thermal Hyperalgesia and Mechanical Allodynia Elicited by Histamine and Non-histaminergic Itch Mediators: Respective Involvement of TRPV1 and TRPA1.

Acute itch is elicited by histamine, as well as non-histaminergic itch mediators including chloroquine, BAM8-22 and Ser-Leu-Ile-Gly-Arg-Leu (SLIGRL). When injected intradermally, histamine binds to histamine H1 and H4 receptors that activate transient receptor potential vanilloid 1 (TRPV1) to depolarize pruriceptors. Chloroquine, BAM8-22, and SLIGRL, respectively, bind to Mas-related G-protein-coupled receptors MrgprA3, MrgprC11, and MrgprC11/PAR2 that in turn activate transient receptor potential ankyrin 1 (TRPA1). In this study we tested if histamine, chloroquine, BAM8-22 and SLIGRL elicit thermal hyperalgesia and mechanical allodynia in adult male mice. We measured the latency of hindpaw withdrawal from a noxious heat stimulus, and the threshold for hindpaw withdrawal from a von Frey mechanical stimulus. Intraplantar injection of histamine resulted in significant thermal hyperalgesia (p < 0.001) and mechanical allodynia (p < 0.001) ipsilaterally that persisted for 1 h. Pretreatment with the TRPV1 antagonist AMG-517 (10 or 20 µg), but not the TRPA1 antagonist HC-030031 (50 or 100 µg), significantly attenuated the magnitude and time course of thermal hyperalgesia and mechanical allodynia elicited by histamine (p < 0.001 for both), indicating that these effects are mediated by TRPV1. In contrast, pretreatment with the TRPA1 antagonist significantly reduced thermal hyperalgesia and mechanical allodynia elicited by chloroquine (p < 0.001 for both ), BAM-822 (p < 0.01, p < 0.001, respectively) and SLGRL (p < 0.05, p < 0.001, respectively), indicating that effects elicited by these non-histaminergic itch mediators require TRPA1. TRPV1 and TRPA1 channel inhibitors thus may have potential use in reducing hyperalgesia and allodynia associated with histaminergic and non-histaminergic itch, respectively.

Role of 5-HT1A and 5-HT3 receptors in serotonergic activation of sensory neurons in relation to itch and pain behavior in the rat.

Serotonin (5-hydroxytryptamine, 5-HT) released by platelets, mast cells, and immunocytes is a potent inflammatory mediator which modulates pain and itch sensing in the peripheral nervous system. The serotonergic receptors expressed by primary afferent neurons involved in these sensory functions are not fully identified and appear to be to a large extent species dependent. Moreover, the mechanisms through which 5-HT receptor activation is coupled to changes in neuronal excitability have not been completely revealed. Using a combination of in vitro (calcium and voltage imaging and patch-clamp) and in vivo behavioral methods, we used both male and female Wistar rats to provide evidence for the involvement of two 5-HT receptor subtypes, 5-HT1A and 5-HT3, in mediating the sustained and transient effects, respectively, of 5-HT on rat primary afferent neurons involved in pain and itch processing. In addition, our results are consistent with a model in which sustained serotonergic responses triggered via the 5-HT1A receptor are due to closure of background potassium channels, followed by membrane depolarization and action potentials, during which the activation of voltage-gated calcium channels leads to calcium entry. Our results may provide a better understanding of mammalian serotonergic itch signaling.

The Challenge of Basic Itch Research.

Basic mechanisms and pathways of itch signaling are reviewed, with an emphasis on the progress to date as well as remaining challenges in translating current knowledge to the clinical treatment of chronic itch. Recent studies reveal 3 subsets of pruriceptive sensory neurons highly expressing itch-related genes. Their fibers project into the spinal cord to activate neurons expressing gastrin releasing peptide (GRP) and its receptor (GRPR), which connect to neurons that express the substance P (NK-1) receptor and project to the parabrachial nucleus and thalamus. Spinal inhibitory interneurons release GABA, glycine and dynorphin to modulate segmental itch transmission. However, near-ly all pruriceptive neurons also respond to algogens such as capsaicin. Alternative theories of itch-pain discrimination, such as intensity or spatial contrast, are based on the observation that focal stimulation of nociceptive nerve endings elicits itch while more wide-spread stimulation elicits pain. These findings cloud the issue of a labeled line for itch- a long-debated but currently unresolved challenge. In higher primates there is a dichotomy of histaminergic and non-histaminergic itch-signaling pathways which is less demarcated in rodents, suggesting species differences. A cardinal symptom of chronic itch is alloknesis, i.e., mechanical or touch-evoked itch. Recent evidence indicates that low-threshold mechanosensory afferents can access the spinal itch pathway, but are normally kept in check by inhibitory interneurons expressing neuropeptide Y (NPY). In chronic itch, NPY-mediated inhibition is reduced, allowing touch to excite itch-signaling pathways. These recent advances provide novel targets for development of therapeutic strategies to relieve chronic itch.

Cinnamaldehyde elicits itch behavior via TRPV1 and TRPV4 but not TRPA1.

INTRODUCTION: Cinnamaldehyde (CA) elicits itch sensation in humans. We investigated if CA elicits scratching behavior in mice and determined the roles for TRPV1, TRPA1, and TRPV4. MATERIALS AND METHODS: Scratching behavior elicited by intradermal injection of CA was assessed in wildtype (WT) mice and knockout (KO) mice lacking TRPV1, TRPA1, TRPV4, or deficient in mast cells. We also assessed scratching and wet dog shakes elicited by low-threshold mechanical stimulation of skin treated topically with CA or vehicle. Using calcium imaging we tested if CA activates dorsal root ganglion (DRG) neurons of each genotype. RESULTS: Intradermal cheek injection of CA elicited dose-dependent hindlimb scratch bouts, with fewer forelimb wipes and facial groom bouts that were not dose-dependent. CA elicited significantly fewer scratch bouts in TRPV1 and TRPV4 KO mice, but not TRPA1KOs, compared with WTs. There were no sex differences across genotypes. The histamine H1 antagonist cetirizine did not affect CA-evoked scratching, which was normal in mast cell deficient mice, indicating lack of histamine involvement. Scores for alloknesis were significantly greater following topical application of CA compared with vehicle. Post-CA alloknesis scores were significantly higher in TRPV4KOs of both sexes and in female TRPV1 and TRPA1KOs, compared with WTs. Low threshold mechanical stimuli also elicited significantly more wet dog shakes in mice treated topically with 20% CA, with significantly fewer in TRPV1, TRPA1, and TRPV4KOs compared with WTs. In calcium imaging studies, CA excited 24% of WT DRG cells, significantly fewer (11.5%) in cells from TRPV4KOs, and none in TRPA1KOs. Responses of cells of all genotypes exhibited significant sensitization to repeated CA stimulation. Sensitization was significantly enhanced by IL-4, which itself excited 16% of WT DRG cells and none from TRPA1KOs. DISCUSSION: The results indicate that TRPA1 is dispensable for CA-evoked scratching, which depends partly on TRPV1 and TRPV4.

Transient receptor potential ankyrin 1 (TRPA1) positively regulates imiquimod-induced, psoriasiform dermal inflammation in mice.

Transient receptor potential ankyrin 1 (TRPA1), a membrane protein ion channel, is known to mediate itch and pain in skin. The function of TRPA1, however, in psoriasiform dermatitis (PsD) is uncertain. Herein, we found that expression of TRPA1 is highly up-regulated in human psoriatic lesional skin. To study the role of TRPA1 in PsD, we assessed Psoriasis Severity Index (PSI) scores, transepidermal water loss (TEWL), skin thickness and pathology, and examined dermal inflammatory infiltrates, Th17-related genes and itch-related genes in c57BL/6 as wild-type (WT) and TRPA1 gene knockout (KO) mice following daily application of topical IMQ cream for 5 days. Compared with WT mice, clinical scores, skin thickness change and TEWL scores were similar on day 3, but were significantly decreased on day 5 in IMQ-treated TRPA1 KO mice (vs WT mice), suggesting reduced inflammation and skin barrier defects. Additionally, the relative area of epidermal Munro's microabscesses and mRNA levels of neutrophil inducible chemokines (S100A8, S100A9 and CXCL1) were decreased in the treated skin of TRPA1 KO mice, suggesting that neutrophil recruitment was impaired in the KO mice. Furthermore, mast cells, CD31+ blood vascular cells, CD45+ leukocytes and CD3+ T cells were all reduced in the treated skin of TRPA1 KO mice. Lastly, mRNA expression levels of IL-1ß, IL-6, IL-23, IL-17A, IL-17F and IL-22 were decreased in TRPA1 KO mice. In summary, these results suggest a key role for TRPA1 in psoriasiform inflammation and raising its potential as a target for therapeutic intervention.

Signs of chronic itch in the mouse imiquimod model of psoriasiform dermatitis: sex differences and roles of TRPV1 and TRPA1.

Plaque psoriasis is a chronic inflammatory skin disease that affects a substantial proportion of the world population. This disorder is characterized by scaly, thick skin, intense ongoing itch, and itch from light touch (such as clothing contacting skin, called "alloknesis"). Imiquimod is a topical treatment for basal cell carcinomas and warts that has been used to create a mouse model of plaque psoriasis. Imiquimod-treated male, but not female, wildtype B6 mice showed significant increases in spontaneous scratching, while both sexes exhibited increased alloknesis, indicative of chronic itch. TRPV1 and TRPA1 knockout (KO) mice all exhibited numeric increases in spontaneous scratching which were significant for TRPV1KO mice and TRPA1KO males. Female TRPV1KO and TRPA1KO mice exhibited imiquimod-induced increases in alloknesis scores that did not significantly differ from wildtypes, while alloknesis scores in imiquimod-treated male TRPV1KO and TRPA1KO mice were significantly lower compared with wildtypes, suggesting that these ion channels are necessary for the development of alloknesis in males but not females in this model. Curiously, none of the groups exhibited any significant overall change in chloroquine-evoked scratching following imiquimod treatment, indicating that hyperknesis does not develop in this mouse model. Overall, the data indicate that there are sex differences in this mouse model of psoriasis, and that TRPV1 and TRPA1 ion channels have a small role in promoting the development of itch sensitization. This contrasts with the far greater role these channels play in the manifestation of skin changes in psoriatic dermatitis.

TRPV1 mediates inflammation and hyperplasia in imiquimod (IMQ)-induced psoriasiform dermatitis (PsD) in mice.

BACKGROUND: Transient Receptor Potential Vanilloid 1 (TRPV1) is known to mediate itch and neurogenic inflammation, but the role of TRPV1 in psoriasiform dermal inflammation is poorly understood. OBJECTIVE: To investigate the function of TRPV1 in imiquimod (IMQ)-induced psoriasiform dermatitis (PsD) in mice. METHODS: Following daily treatment of topical IMQ cream for consecutive 5 days in C57BL/6 wide-type (WT) and TRPV1 gene knockout (KO) mice, we assessed the psoriasis severity index (PSI) scores, transepidermal water loss (TEWL), dermal inflammatory infiltrates, as well as gene expression levels for psoriasis related genes in mouse skin lesions. RESULTS: Compared with WT mice, the clinical and TEWL scores, the extent of skin hyperplasia, the area of Munro microabscesses (MM) and angiogenesis of psoriasis were all significantly decreased in TRPV1 KO mice triggered with IMQ, suggesting a reduction in skin inflammation and barrier defects. In addition, the infiltration of CD45+ leukocytes, mast cells as well as CD3+ T cells was all reduced in the IMQ-treated skin of TRPV1 KO mice. Quantitative Real-time PCR (RT-qPCR) revealed that expression levels of IL-1ß, IL-6, IL-23, S100A8 were decreased while IL-10 was increased in TRPV1 KO mice. CONCLUSIONS: In summary, key markers of psoriatic inflammation and epidermal hyperplasia are reduced in TRPV1 KO mice, indicating the involvement of TRPV1 in the psoriasiform inflammation and suggesting its potential as a therapeutic target.

Opposing effects of cervical spinal cold block on spinal itch and pain transmission.

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.

Involvement of TRPV4 in Serotonin-Evoked Scratching.

Several thermosensitive transient receptor potential channels (transient receptor potential vanilloid type-1, -3; transient receptor potential cation channel, subfamily A, member 1) have been implicated in itch. In contrast, the role of transient receptor potential vanilloid type-4 (TRPV4) in itch is unknown. Therefore, we investigated if TRPV4, a temperature-sensitive cation channel, plays an important role in acute itch in mice. Four different pruritogens, including serotonin (5-hydroxytryptamine [5-HT]), histamine, SLIGRL (protease-activated receptors 2/mas-related G-protein-coupled receptor C11 agonist), and chloroquine (mas-related G-protein-coupled receptor A3 agonist), were intradermally injected into mice and itch-related scratching behavior was assessed. TRPV4 knockout mice exhibited significantly fewer 5-HT-evoked scratching bouts compared with wild-type mice. Notably, no differences between TRPV4 knockout and wild-type mice were observed in the number of scratch bouts elicited by SLIGRL and histamine. Pretreatment with a TRPV4 antagonist significantly attenuated 5-HT-evoked scratching in vivo. Using calcium imaging in cultured primary murine dorsal root ganglion neurons, the response of neurons after 5-HT application, but not other pruritogens, was significantly lower in TRPV4 knockout compared with wild-type mice. A TRPV4 antagonist significantly suppressed 5-HT-evoked responses in dorsal root ganglion cells from wild-type mice. Approximately 90% of 5-HT-sensitive dorsal root ganglion neurons were immunoreactive for an antibody to TRPV4, as assessed by calcium imaging. These results indicate that 5-HT-induced itch is linked to TRPV4.

Anatomical evidence of pruriceptive trigeminothalamic and trigeminoparabrachial projection neurons in mice.

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.

A central role for spinal dorsal horn neurons that express neurokinin-1 receptors in chronic itch.

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.

Nalfurafine suppresses pruritogen- and touch-evoked scratching behavior in models of acute and chronic itch in mice.

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.

Role of spinal bombesin-responsive neurons in nonhistaminergic itch.

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.

Effects of gustatory nerve transection and/or ovariectomy on oral capsaicin avoidance in rats.

The incidence of chronic oral pain such as burning mouth syndrome is greater in peri-menopausal females, and was postulated to be associated with gustatory nerve damage. We investigated whether bilateral transection of the chorda tympani, with or without accompanying ovariectomy, affected oral capsaicin avoidance in rats. Female rats had restricted access to 2 bottles, 1 bottle containing capsaicin (concentration range: 0.33-33 µM/L) and the other vehicle. Percent volume of capsaicin consumption and lick counts were measured. The concentration series was tested before and 0.5, 3, 6, 9, and 12 months after the following surgical procedures: (a) bilateral transection of the chorda tympani (CTx); (b) ovariectomy (OVx); (3) CTx plus OVx; or (4) sham CT surgery. Before surgery there was a concentration-dependent decrease in licks and volume of capsaicin consumed, with a threshold between 0.1 and 0.3 ppm. The majority of drink licks occurred during the first 9 minutes of access. Over the 12-month test period, the CTx group did not exhibit reduced capsaicin consumption, and consumed significantly more capsaicin at 6 and 9 months postsurgery. Rats in the OVx group consistently consumed significantly less capsaicin and exhibited significantly higher counts of capsaicin-evoked Fos-like immunoreactivity in the dorsomedial trigeminal subnucleus caudalis (Vc) compared to all other treatment groups. That CTx, with or without OVx, did not enhance capsaicin avoidance indicates that damage to the gustatory system does not disinhibit trigeminal nociceptive transmission.

Roles of glutamate, substance P, and gastrin-releasing peptide as spinal neurotransmitters of histaminergic and nonhistaminergic itch.

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.

Eugenol and carvacrol induce temporally desensitizing patterns of oral irritation and enhance innocuous warmth and noxious heat sensation on the tongue.

Eugenol and carvacrol, from the spices clove and oregano, respectively, are agonists of TRPV3, which is implicated in transduction of warmth and possibly heat pain. We investigated the temporal dynamics of lingual irritation elicited by these agents, and their effects on innocuous warmth and heat pain, using a half-tongue method in human subjects. The irritant sensation elicited by both eugenol and carvacrol decreased across repeated applications at a 1-minute interstimulus interval (self-desensitization) which persisted for at least 10 minutes. Both agents also cross-desensitized capsaicin-evoked irritation. Eugenol and carvacrol significantly increased the magnitude of perceived innocuous warmth (44 °C) for >10 minutes, and briefly (<5 minutes) enhanced heat pain elicited by a 49 °C stimulus. Similar albeit weaker effects were observed when thermal stimuli were applied after the tongue had been desensitized by repeated application of eugenol or carvacrol, indicating that the effect is not due solely to summation of chemoirritant and thermal sensations. Neither chemical affected sensations of innocuous cool or cold pain. A separate group of subjects was asked to subdivide eugenol and carvacrol irritancy into subqualities, the most frequently reported being numbing and warmth, with brief burning, stinging/pricking, and tingle, confirming an earlier study. Eugenol, but not carvacrol, reduced detection of low-threshold mechanical stimuli. Eugenol and carvacrol enhancement of innocuous warmth may involve sensitization of thermal gating of TRPV3 expressed in peripheral warm fibers. The brief heat hyperalgesia following eugenol may involve a TRPV3-mediated enhancement of thermal gating of TRPV1 expressed in lingual polymodal nociceptors.