A counterpart to pain: itch Adam P. Kardon and Sarah E. Ross Address: Departments of Neurobiology and Anesthesiology, The Center for Pain Research, 200 Lothrop St., W1465, Pittsburgh, PA 05213, USA. Phone: 617-624-9178; FAX: 412-648-1441

Pain is not the only defense mechanism that alerts us to a physical hazard — the other is itch, an unpleasant sensation that is distinct from pain because it provokes the desire to scratch. Although pain and itch feel different, these two sensations share many qualities. Both are aversive sensations that evolved to protect us. Moreover, both can become pathological, developing into chronic debilitating conditions that ruin one’s quality of life. Itch, in particular, represents the most common reason that a patient sees a dermatologist. It is estimated that ~10% of the general population and 30% of the elderly suffer from pruritis (the medical term for itch), which negatively affects sleep, mood, and quality of life, thereby presenting an enormous health burden (Weisshaar and Dalgard, 2009; Yosipovitch, 2008). Despite this prevalence, itch has received little attention, and the genetic underpinnings of itch sensitivity are virtually unknown. Here we discuss the limited number of genetic mutations in humans and in mice that have been found to affect itch sensitivity — either heightening or reducing it — and what such mutations tells us about how itch is detected and encoded in the nervous system. Why do we scratch? Scratching in response to a noxious agent is seen across vertebrates, who use this strategy to remove potential threats from the skin’s surface, thereby minimizing exposure to harm. Even fish scratch, typically using tail fins or by rubbing against abrasive rocks (Stein, 1983). The highly conserved nature of this scratching response suggests that itch, like pain, confers an important evolutionary advantage. The basics of itch The key difference between itch and pain is that itch is triggered by noxious stimuli at the very outermost aspect of the skin, whereas pain can be elicited from all cutaneous layers, ranging from superficial to deep, as well as most other regions of the body (Ross, 2011). Thus, the primary sensory neurons that detect pruritic stimuli are postulated to be a subset of dorsal root ganglia (DRG) and trigeminal neurons (perhaps 5%) that selectively innervate the dermis. Most itch fibers are likely to be a subset of unmyelinated, slowly conducting C-fibers, though recent studies suggest that some Aδ fibers may also be involved mediating itch (Ringkamp et al., 2011). What is the identity of these fibers? Remarkably, we do not yet know which sensory fibers mediate itch. Moreover, whether this subset of sensory neurons is specific for itch remains highly controversial. The best evidence that there exists a dedicated subset of peripheral sensory neurons that are selectively tuned to convey itch rather than nociception comes from experiments performing microneurography in humans. Using this technique, it is possible to isolate the activity of individual nerve fibers and identify those that respond to itch-inducing chemicals. Such experiments led to the discovery of C-fibers that are activated by pruritogens and whose activity corresponds to the sensation of itch in humans (Schmelz et al., 1997). Furthermore, there appear to be (at least) two subtypes of itch-sensitive afferents, those that respond to histamine, and those that respond to cowhage (Namer et al., 2008). Intriguingly, these afferents were found to have very slow conduction velocities and very large innervation territories, suggesting that pruriceptive C-fibers and nociceptive C-fibers may have distinct physical properties. However, as of yet there is no marker or genetic means to identify these elusive fibers. Hence the controversy about the coding of itch continues. Pruritogen receptors couple to Trp channels Part of the confusion about which sensory neurons are tuned to convey itch likely stems from the fact that many pruritogen receptors appear require Trp channels for activity—the very same Trp channels that we associated with

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pain: the capsaicin receptor, TrpV1 and the mustard oil receptor, TrpA1. In particular, several recent studies have revealed that MrgprA3 (a receptor for chloroquine), and MrgprC11 (a receptor for SLIGRL and Bam8-22) are coupled to TrpA1, whereas the histamine receptor is coupled to TrpV1 (Imamachi et al., 2009; Liu et al., 2011; Shim et al., 2007; Wilson et al., 2011). Since TrpV1 and TrpA1 are essential downstream mediators for a number of itch receptors, it is not surprising that neurons that respond to histamine and other pruritogens also respond to capsaicin and/or mustard oil (Ma et al., 2012). Noxious chemicals are not specific to pain or itch The recent finding that Trp channels are involved in mediating itch highlights an emerging concept — most noxious chemicals can elicit either pain or itch depending on the manner and concentration at which they are applied. For instance, although capsaicin is generally considered an algogen (pain producing), it can also elicit itch if it is applied to the skin in a punctate fashion (Sikand et al., 2009). Furthermore, the topical application of capsaicin can cause itch in addition to pain (Wang et al., 2010). Analogously, agents that are widely considered as pruritogens (itch-producing) can also elicit pain. For instance, histamine causes itch when applied to the surface of the skin, but it elicits pain when it is injected into underlying tissue (Rosenthal, 1977; Simone et al., 1987). In fact, many noxious agents have been found to cause both pain and itch, including SLIGRL, Bam8-22, serotonin, acetylcholine, bradykinin, endothelin-1, formalin and prostaglandin J2. Thus, classifying an aversive stimulus as either an algogen or a pruritogen is overly simplistic. Indeed, given that the function of both itch and pain is to warn us of noxious agents, it makes sense that the sensory neurons that are tuned to convey nociception and pruritoception would be broadly tuned to somewhat overlapping sets of irritating chemical stimuli (Figure 1).

So how is itch coded? Given this overlap, how can itch be distinguished from pain? This unanswered question remains at the center of the itch field. One possibility is that pain and itch are initially detected by sensory afferents that express common receptors but show distinct stratification within the skin (with fibers mediating itch being exclusively superficial). The

sensation of itch would require the activation itch-fibers alone — in other words, without the concurrent activation of nociceptors. This idea, which is the basis of the selectivity theory, also posits that the central nervous system plays a key role in helping to sharpen these two sensory modalities by providing a mechanism through which pain inhibits itch (Ross, 2011). Thus, if a small number of itch-selective sensory neurons are activated by a noxious agent at the outermost aspect of the skin, itch would would ensue. If, in contrast, if a larger number of both itch and nociceptive afferents are activated simultaneously, the nociceptive input would inhibit the itch signal and itch would be suppressed. The existence of such a neural circuit would provide the cellular basis for the everyday experience that scratching (which activates nociceptors) inhibits itch. Since counter-stimuli such as scratching applied many centimeters away from the site of itch (outside the receptive field of the primary stimulus) it is likely that central neurons are involved in mediating this effect. However, whether this inhibition of itch occurs at the level of the spinal cord, the brain, or both remains to be seen. In fact, we do not yet know which projection neurons convey information about itch from the spinal cord to the brain. Anterolateral cordotomy abolishes itch in humans suggesting that itch, like pain, is conveyed by spinothalamic pathways. Furthermore, the finding that the ablation of spinal neurons using a substance-P/saporin conjugate results in a significant reduction in itch sensitivity suggests that NK1R-expressing projection neurons may be involved (Carstens et al., 2010). However, since NK1R-expressing neurons are also known to convey information about pain, it is not clear what makes itch distinct. Moreover, a surprisingly large proportion of spinothalamic tract neurons respond to pruritogens, again raising questions about how itch is differentiated from pain (Davidson et al., 2012).

Figure 1. Many aversive chemicals can result in pain or itch depending on how they are applied.  

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Measuring itch in mice Although the receptors, neurons and neural circuits that underlie itch sensation are poorly understood relative to pain, there has lately been a surge in interest in understanding itch, and progress is now being made at a rapid pace. Part of this progress is due to fact that assays to quantify itch-associated behavior, which for years were only utilized in a handful of labs, are now widely accepted as a reasonable proxy for itch and are being implemented more broadly. Pruritus aptly lends itself to behavioral evaluation due to the nature of its associated physical response. Thus, in general, the same pruritogens that evoke itch sensation and scratching in humans appear to produce an analogous site-directed scratching response in mice, and this response is thought to be indicative of itch sensation. Behavioral tests to measure itch in rodents have been done using three types of assays. In the classic assay, a pruritogen is injected into the skin at the nape of the neck, and the resulting scratching behavior is quantified (Kuraishi et al., 1995). Although this assay has the advantage of being robust and easy to measure, it has some limitations. Specifically, biomechanical limitations prevent rodents from accessing the neck by any physical means besides scratching and thus it becomes unclear whether the response is due to itch, pain or any other cutaneous sensation. As an illustration, when injected into the skin at the nape of the neck, both histamine and capsaicin evoke a scratching response (Shimada and LaMotte, 2008). In response to the need for an itch assay that discriminates between itch- and pain-associated behaviors, Shamida and Lamotte developed an experimental model of itch in which chemicals are injected intradermally into the cheek. In this cheek model, algogens like capsaicin cause predominantly wiping with the forelimb, whereas pruritogens such as histamine elicit predominantly scratching with the hindlimb (Shimada and LaMotte, 2008). This assay has since been validated using a wide variety of chemical agents, including mustard oil, bradykinin, serotonin and agonists of PAR-2 and PAR-4 (Akiyama et al., 2010a). Table 1. Genetic loss-of-function models in mice that show elevated or reduced itch. * denotes conditional knockout; all others are constitutive loss-of-function models.

Recently, an alternative assay that allows the behavioral distinction between pain and itch has been developed based on the observation that algogens elicit licking whereas pruritogens evoke biting responses. For instance, mice with chemically-induced dry skin spend significantly more time biting (but no more time licking) the affected site compared to untreated controls (Akiyama et al., 2010b). Moreover, capsaicin applied to the calf results mainly in

Gene Knock-out

Pruritogen(s) Tested

Itch Response

Spontaneous Scratching

Acute Pain Response Reference(s)

GRPR 48/80, CQ, SLIGRL Reduced No No change (Sun and Chen, 2007)

HDC Induced contact dermatitis Reduced No Not done (Seike et al., 2005)

Mrgpr-cluster CQ, Bam8-22, SLIGRL Reduced No No change (Liu et al., 2009)

(Liu et al., 2011)

Pirt Histamine, CQ, 5-HT,α-Me-5-HT, SLIGRL, ET-1

Reduced No Reduced (Patel et al., 2011)

PLCβ3 Histamine, 48/80 5-HT, α-Me-5HT Reduced No No change (Han et al., 2006)

(Imamachi et al., 2009)

TLR3 Histamine, CQ, 5-HT, α-Me-5-HT, SLIGRL, ET-1

Reduced No No change (Liu et al., 2012a)

TLR7 SLIGRL, CQ, 5-HT, ET-1, imiquimod Reduced No No change (Liu et al., 2010a)

TP U-46619 Reduced No Not done (Andoh et al., 2007) TrpA1 CQ, Bam8-22, Reduced No Reduced (Wilson et al., 2011) TrpV1 Histamine Reduced No Reduced (Shim et al., 2007)

Bhlhb5* Histamine, α-Me-5-HT, SLIGRL, 48/80 Formalin, CQ

Elevated Yes No change (Ross et al., 2010)

PI3Kγ Histamine, SLIGRL Elevated No No change (Lee et al., 2011)

vGlut2* 48/80, SLIGRL, α-Me-5HT, capsaicin Elevated Yes Reduced (Liu et al., 2010b)

(Lagerstrom et al., 2010)

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licking behavior, whereas histamine results predominantly in biting behavior (LaMotte et al., 2011). Thus, licking vs. biting (which can be distinguished using a high speed video-camera) is an alternative behavioral readout to quantitatively distinguish between itch and pain.

Genetic Models of itch The genetic manipulation of molecules involved in the generation, propagation or perception of itch sensation give rise to one of two phenotypic outcomes — more or less scratching behavior. Overexpression of endogenous pruritogens leads to excessive scratching behavior, whereas mutations that lead to a reduction in pruritogens can protect mice from itch. Decreased scratching behavior is also observed when the sensory neurons that convey itch do not function properly. Conversely, genetic defects that disrupt the balance between pain and itch signaling can unmask neural circuits that are normally silenced, resulting in elevated itch. In this next section, we discuss the genetic mutations in mice and humans that result in abnormal itch, and how these mutations inform us about the signaling pathways and circuits that underlie itch. NGF signaling is required for the survival of pruritoceptors Nerve growth factor (NGF) is the principal neurotrophin responsible for the development of Aδ- and C-fibers (Petruska and Mendell, 2004). In these sensory neurons, activation of the tyrosine receptor kinase A (TrkA) by NGF is essential for both axonal outgrowth and neural survival. Consequently, mice deficient in either NGF or TrkA fail to develop the subset of primary afferents that normally function to detect and respond to noxious stimuli (Crowley et al., 1994; Smeyne et al., 1994). Similarly in humans, mutations that disrupt the function of NGF or TrkA cause the loss Aδ- and C-fibers which results in insensitivity to pain (Indo et al., 1996). Importantly, people with a total loss of unmyelinated and thinly myelinated fibers also show a complete absence of itch (Indo, 2010). This finding provides strong evidence that itch is mediated by TrkA-dependent fibers (Figure 2A) Particular itch receptors are required for specific types of itch Peripheral activation of sensory neurons by pruritic stimuli is the major mechanism underlying the transmission of cutaneous itch. In the classic model, histamine is released, mainly from mast cells in the dermis, and interacts with histamine receptors on nerve terminals in the skin (Shim and Oh, 2008). However (except for the treatment of hives) histamine receptor antagonists are usually ineffective at reducing pruritis, likely because histamine receptors represent just one of numerous itch receptors (Paus et al., 2006). Recently, genetic studies in mice have helped identify two new itch receptors — MrgprA3 and MrgprC11 (Figure 2B). These receptors belong to a family of approximately 30 Mas-related proteins that are selectively expressed in DRG neurons. When the entire cluster is deleted, the resulting mice show significantly reduced scratching behavior in response to the antimalarial drug chloroquine and the endogenous peptide Bam8-22, despite the fact that responses to histamine and acute nociception are normal (Liu et al., 2009). Furthermore, in contrast to those isolated from wildtype mice, DRG neurons from Mrgpr-cluster-/- mice do not respond to chloroquine or Bam8-22 in vitro, suggesting that Mrgrprs are essential for their activation. When neurons from Mrgpr-cluster-/- mice were transfected with individual Mrgpr genes, only two Mrgpr receptors appeared to be involved: expression of MrgprA3 conferred sensitivity to chloroquine, whereas expression of MrgprC11 conferred responsiveness to Bam8-22. Taken together, these findings provide genetic evidence that MrgprA3 is required for chloroquine-mediated itch and that MrgprC11 is required for Bam8-22-mediated itch. Protease-activated receptors (PARs) are a family of GPCRs that are cleaved at the N-terminal domain by various proteases including trypsin, revealing a tethered ligand that auto-activates the receptor (Liu et al., 2011). PAR2, one of four known PARs, is highly expressed on DRG neurons, and intradermal application of the endogenous serine protease trypsin or the PAR2 synthetic ligand SLIGRL leads to itch-associated responses in both humans and mice (Tsujii et al., 2008). Furthermore, PAR2 is activated by the active component in cowhage, the cysteine protease mucunain, which causes intense pruritus (Reddy et al., 2008). These findings initially suggested that PAR2 might be a receptor that mediates itch. However, recent genetic studies have called this idea into question. Thus, while SLIGRL clearly excites DRG neurons through activation of PAR2 in vitro, no difference is observed between PAR2-deficient and wildtype mice

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in the scratching response to SLIGRL (Liu et al., 2011). This finding implies that SLIGRL induces scratching behavior through a separate mechanism that is independent of PAR2. Indeed, recent evidence suggests SLIGRL-induced itch may instead be mediated exclusively by activation of MrgprC11 (Liu et al., 2011). Mrgpr-cluster-/- mice showed deficits in scratching behavior in response to SLIGRL. Additionally, DRG neurons from Mrgpr-cluster-/- mice responded to SLIGRL only following transfection with MrgprC11. These findings suggest that, although SLIGRL can activate PAR-2, it evokes scratching behavior via activation of MrgprC11, and not through PAR2 as was previously believed (Figure 2B).

Although PAR2 may not be an itch receptor per se, there is still circumstantial evidence that this receptor plays a role in itch. First, PAR2 and trypsin (which cleaves PAR2 to reveal its tethered ligand) are significantly elevated in patients with atopic dermatitis, and exogenous application of trypsin to itchy skin lesions enhances scratching behavior significantly in these patients (Steinhoff et al., 2003). In addition, PAR2-deficient mice exhibit milder symptoms of induced contact dermatitis when compared with wild-type mice (Kawagoe et al., 2002). Furthermore, Netherton syndrome, a severe genetic skin disease characterized by unrelenting atopic dermatitis-like skin lesions, results from a loss-of-function mutation in the gene encoding the serine protease inhibitor Kallikrein 5 that leads to significantly elevated levels of epidermal serine proteases (Briot et al., 2009) and activation of PAR2. These observations suggest PAR2 may play a role in pathological itch associated with atopic dermatitis. Perhaps the up-regulation of PAR2 and endogenous proteases in the skin leads to increased levels of SLIGRL-like peptides that activate nearby MrgprC11 to cause itch. An additional receptor that has been implicated in itch through genetic studies is the prostaniod receptor (TP), a receptor for the prostanoid thromboxane A2 (TXA2). In mice with atopic dermatitis, TXA2 is abnormally over-exexpressed in the skin, and acute intradermal injections of a stable analogue of TXA2 induce robust scratching behavior, suggesting that molecule may act as an endogenous pruritogen in dermatitis-related skin lesions (Andoh et al., 2007). TP is expressed on both small-diameter sensory neurons and keratinocytes, and TP-deficient mice show a complete attenuation of scratching behavior elicited by TXA2 analogues. Intriguingly, activation of the receptor causes a calcium influx in both DRG neurons and keratinocytes in vitro, raising the possibility that TXA2 acts on both cell types to mediate itch (Figures 2B and 3). Recent studies have also revealed an important role for classic components of the innate immune system in pruritic signaling. Toll-like receptors (TLRs) respond to infection by recognizing pathogen-associated molecular patterns

Figure 2. Genetic evidence implicating receptors, channels and signaling pathways in the detection of itch by sensory neurons. A. NGF and TrkA are required during development for the survival of neurons that mediate itch. B. MrgprC11 is required for Bam8-22- and SLIGRL-mediated itch, whereas MarprA3 is required for chloroquine-mediated itch; both of these Mrg receptors require TrpA1 for function. Histamine-mediated itch requires TrpV1, and PLCβ3 and PIRT. Itch mediated by the prostanoid thromboxane A2 requires the thromboxane receptor TP. Molecules implicated in itch through human mutations are indicated in green; those implicated by loss-of-functions studies in mouse are indicated in red. Note that new evidence also implicates Tlr3 and Tlr7 receptors in itch, though they were not included in this diagram since it is remains unclear how they are working mechanistically. It is likely that there are at least two populations of itch fibers, as suggested by this figure. However, it is not clear which subset(s) expresses TP.  

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and activating signaling cascades that lead to the production of cytokines, chemokines and inflammation (Liu et al., 2012b). Recent evidence suggests that two TLRs are expressed in small-diameter DRG neurons and are involved in mediating itch, though the underlying mechanism remains unclear. TLR7-deficient mice showed no deficits in pain-related behaviors, but exhibited reduced scratching behavior in response to a number of pruritogens including chloroquine, SLIGRL, endothelin-1 and 5-HT (Liu et al., 2010a). Furthermore, the TLR7 specific agonist imiquimod acts as a potent pruritogen in mice by directly activating DRG neurons. However, conflicting reports exist regarding whether imiquimod-evoked itch is TLR7-dependent (Kim et al., 2011). More recently, TLR3 has been implicated in itch, and TLR3-deficient mice exhibit significantly reduced scratching behavior in response to numerous pruritogens. Similar to TLR7-/- mice, TLR3-/- mice appear to have sensory defects that are specific to itch since these mutants respond normally to acute painful stimuli. Key signaling molecules are also required for itch Although TrpV1 and TrpA1 are not considered itch receptors, new evidence suggests that these two channels are key downstream mediators of a variety of pruritogens. The transient receptor potential (Trp) family of ion-permeable, trans-membrane receptors respond to a variety of sensory cues from the internal and external environment. One of the best understood members of this family, Transient receptor potential vanilloid receptor 1 (TrpV1) is expressed in a broad range of tissues including neurons and keratinocytes, and has been implicated in the detection of noxious stimuli. Apart from its classical agonist capsaicin, the TrpV1 receptor is activated by high temperatures, acidic pH, endocannibinoids and a variety of inflammatory mediators (Caterina et al., 2000). Now, new genetic evidence has also uncovered an unexpected role for TrpV1 in certain types of itch (Figure 2B). Mice lacking TrpV1 exhibit a significant reduction in scratching behavior compared to wildtype controls in response to histamine and compound 48/80, a mast cell activator that results in histamine release. Furthermore, DRG neurons cultured from TrpV1-/- mice fail to produce a calcium influx in response to histamine, indicating that TrpV1 is an essential downstream mediator of the histamine receptor. Moreover, pre-treatment with capsazepine, a TrpV1 antagonist inhibits calcium influx in response to histamine in cultured DRG neurons from wildtype mice. Finally, while 293T cells co-transfected with TrpV1 and H1R produce an inward current in response to histamine, those transfected with H1R alone failed to replicate this response (Shim et al., 2007). Together, these studies suggest that histamine-dependent itch is conveyed to the spinal cord by a subset of TrpV1-positive C-fibers that express the histamine receptor and that require TrpV1 for histamine responsiveness. Interestingly, while TrpV1 is required for histamine-mediated itch, it appears to be dispensable for itch mediated by 5-HT, chloroquine and SLIGRL. The notion that TrpV1 is more than a simple pain sensor is further evidenced by the role of TrpV1 modulators in itch. Pirt, a membrane protein expressed in sensory neurons, binds directly to TrpV1 and has been show to regulate its activity in a phosphatidylinositol-4,5-bisphoaphate (PIP2)-dependent manner. Furthermore, Pirt-deficient mice exhibit nearly identical sensory deficits compared to mice lacking TrpV1, though to a less severe extent (Kim et al., 2008). Consistent with this, histamine evoked itch is significantly reduced in Pirt-/- mice (Figure 2B) and, accordingly, the percentage of DRG neurons from these mice responding to histamine is decreased. Unexpectedly, Pirt-/- mice exhibit deficits in scratching behavior in response to chloroquine and 5-HT—two pruritogens that do not require the TrpV1 receptor—suggesting that Pirt may also play a role in mediating itch that is independent of TrpV1 (Patel et al., 2011). Whether Pirt also modulates TrpA1 activity remains to be seen, though this possibility is suggested by the finding that TrpA1, like TrpV1, is regulated by PIP2 (Patil et al., 2011).

A second molecule that may be involved in the coupling of the histamine receptor to TrpV1 is phospholipase Cβ (PLCβ) (Figure 2A). PLCβ3 is highly (90%) co-expressed with TrpV1 in DRG neurons and diacylglycerol, a product of PLCβ3, has been shown to directly activate TrpV1, providing a mechanism by which H1R could regulate TrpV1 activity (Woo et al., 2008). Consistent with this idea, mice lacking PLCβ3 mice show significantly less histamine-induced scratching, and DRG neurons isolated from these mice display a reduced calcium influx in response to histamine. Thus, PLCβ3 is a key molecular mediator that links activation of the histamine receptor to depolarization via TrpV1. However, not all of the effects of PLCβ3 on itch are dependent on TrpV1: PLCβ3-/- mice also show decreased 5-HT-dependent itch, despite the fact that 5-HT-dependent itch does not require TrpV1. One possibility is that is that PLCβ3 may also couple to other channels to mediate depolarization, and in this regard TrpA1 is a good candidate. TrpA1 is a polymodal cation channel that is largely co-expressed with TrpV1 in sensory neurons (Kobayashi et al., 2005) and is activated by a wide array of noxious, reactive compounds, including allyl isothiocyanate (mustard oil) and formalin (Macpherson et al., 2007). Like TrpV1, TrpA1 is activated downstream of GPCRs, such as the bradykinin receptor, making it an excellent candidate as a mediator of itch as well as pain

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(Bandell et al., 2004). In keeping with this idea, TrpA1-/- mice scratched significantly less in response to chloroquine and Bam8-22 compared to wildtype mice, implying TrpA1 acts downstream of both MrgprA3 and MrgprC11 to elicit an itch response (Wilson et al., 2011). This finding was confirmed using isolated DRG neurons from TrpA1-deficient mice, which failed to produce an inward current in response to chloroquine or Bam8-22. Further, GPCR-mediated activation of TrpA1 can occur in a PLC-dependent manner, making TrpA1 a promising candidate as the downstream target of PLCβ3 following 5-HT receptor activation. In addition, products of oxidative stress, a major component of many chronic itch conditions, can activate TrpA1, and, accordingly, TrpA1-deficient mice exhibited an almost complete attenuation of scratching behavior in response to oxidative stress following intradermal injections of various oxidants (Liu et al., 2012a). Together, these studies demonstrate that TrpA1 is the key signal transducer downstream of multiple histamine-independent itch pathways, raising the possibility that TrpA1 antagonists may help relieve the histamine-independent itch that is associated with a variety of skin disorders. A final signaling molecule that has been implicated itch through genetic loss-of-function mutations is phosphoinositide 3-kinase γ (PI3Kγ). However (unlike the PIRT, PLCβ3, TrpV1 and TrpA1) loss of PI3Kγ in mice gives rise to more, not less itch. Furthermore, though PI3Kγ-deficient mice show significantly elevated scratching behavior in response to PAR-2 and histamine, they show normal responses in assays for acute pain (Lee et al., 2011). Thus, loss of PI3Kγ appears to be relatively specific to itch. Importantly, while loss of PI3Kγ during development gives rise to mice with elevated itch, acute inhibition of PI3Kγ using PI3Kγ inhibitors does not increase itch; in fact, it appears to reduce it (Pereira et al., 2011). This finding raises the possibility that PI3Kγ is required developmentally for the proper formation of itch circuits rather than for the function of itch circuits per se in adult mice. Since PI3Kγ is broadly expressed, the cellular basis for this effect remains unknown. The Pruritogenic Soup New insights into the role of immune factors, such as the toll-like receptors, in itch highlights the emerging idea that neural and immune systems likely function together with the skin to protect the organism from invasion, using itch as a defense strategy. Indeed, though itch is temporarily relieved by scratching, this behavioral response ultimately results in the generation of more pruritogens by damaging the skin and recruiting the immune system. The influx of multiple pruritogenic mediators — a pruritogenic soup — is analogous to the inflammatory soup that contributes to nociception upon tissue injury (Figure 3).

The best-characterized component of this pruritogenic soup is histamine, and there is compelling genetic evidence that histamine is an important contributor to itch. Histamine is synthesized by histidine decarboxylase (HDC), and is stored for release in mast cells in the periphery. Mice unable to produce histamine due to a disruption of the HDC gene still respond to an intradermal histamine injection with vigorous scratching. However, in a model of chronic contact dermatitis,

 Figure 3. Itch as an integrated protective mechanism mediated by neurons, the immune system and the skin. Note that only signaling pathways for which there is genetic evidence are illustrated here.  

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HDC-/- mice fail to develop pruritus (Seike et al., 2005). Further genetic evidence for a role of histamine in itch comes from mice that lack mast cells, the major cell type that releases histamine in the skin. Mast cell deficiency is caused by a chromosomal inversion in the regulatory element of the c-kit gene (SASH mice). Importantly, SASH mice exhibit a low incidence of spontaneous idiopathic dermatitis (Grimbaldeston et al., 2005). Moreover, these mice are frequently used to validate models of allergy-associated itch and are an invaluable tool for assessing histamine-independence when characterizing a novel pruritogen. While providing immediate relief from itch, scratching inherently damages the skin, attracting cells that release a host of cytokines and proteases that serve as pruritic mediators that amplifying the itch signal. Understanding the genetic and molecular basis for this itch-scratch cycle is key for developing effective therapeutics for chronic itch conditions. Overexpression of Th2 versus Th1 cytokines leads to the development of atopic dermatitis-like skin lesions in mice, suggesting that these cytokines may act as endogenous pruritogens in pathological itch conditions (Jin et al., 2009). Several interleukins synthesized by CD4+ T-cells and other immune cells are concentrated in atopic dermatitis-like skin lesions, suggesting a role for this class of cytokines in itch signaling. IL-31 expression is upregulated in pruritic atopic dermatitis-related skin lesions, and mice overexpressing this cytokine spontaneously develop dermatitis by two months of age (Dillon et al., 2004). The pro-inflammatory cytokine IL-18 is also implicated in the development of atopic dermatitis. Furthermore, IL-18 stimulates the release of histamine and serotonin from mast cells, as well as promotes the production of IL-4 and IL-13, both of which, when overexpressed in mice, lead to the development of pruritic skin lesions (Nakanishi et al., 2001; Yoshimoto et al., 1999). Many additional genetic models of atopic dermatitis exist, and have recently been described in several excellent reviews (Inagaki and Nagai, 2009; Jin et al., 2009). A key role for the skin in itch In addition to providing a physical barrier between the internal and external environments, keratinocytes express a number of the same ion channels found in neurons. Interestingly, this includes many of the Trp channels that have been implicated in itch and pain signaling, including TrpV1, TrpA1 and TrpV3 (Denda and Tsutsumi, 2011). This raises the possibility that keratinocytes contribute to or modulate pruritic signaling; however the mechanism by which keratinocytes and neurons interact has yet to be fully elucidated (Figure 3). TrpV3 is a calcium-permeable cation channel activated by warm temperatures and a variety of chemical stimuli, including some endogenous mediators of atopic dermatitis (Moqrich et al., 2005) (Yoshida et al., 2006). In mice, TrpV3 is highly expressed in keratinocytes, but not DRG neurons in the epidermis(Peier et al., 2002). Interestingly, mice with a gain-of-function mutation in TrpV3 spontaneously develop dermatitis-like symptoms accompanied by intense pruritus (Steinhoff and Biro, 2009). Further histological examination of the skin of these mice revealed that a number of the pruritic mediators known to be involved in the pathogenesis of atopic dermatitis were significantly elevated. Accordingly, TrpV3-deficient mice fail to display increased scratching behavior following a model of induced allergic dermatitis (Yamamoto-Kasai et al., 2012). Furthermore, six human subjects suffering from Olmsted syndrome, a rare congenital disorder characterized by intense itching, were shown to possess gain-of-function mutations in TrpV3 at genetic loci comparable to TrpV3-mutant mice (Lin et al., 2012). These studies suggest itch-related skin disorders may result, in part, from increased signaling of TrpV3 in keratinocytes. How keratinocytes couple to itch-mediating sensory fibers remains unknown.

A shift in the balance of pain and itch One of the reasons that we scratch an itch is because scratching provides relief. While the neural basis for this phenomenon is not yet known, it is speculated that counter-stimuli such as scratching may inhibit itch through neural circuits in the spinal cord. This idea, which is called the selectivity theory, posits that nociceptive input such as scratching results in the inhibition of neurons that convey itch sensation (McMahon and Koltzenburg, 1992). What this means is that an aversive stimulus may only be able to elicit itch (rather than pain) if the input selectively activates itch fibers. If, in contrast, an aversive stimulus activates both nociceptors and pruritoceptors, the nociceptive input results in the inhibition of itch (Figure 4).

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If the selectivity model is correct, it implies that there is inhibition between pain and itch, and such inhibition would presumably be mediated by inhibitory interneurons. Recent work in our lab has identified spinal inhibitory interneurons that may fulfill this role. Specifically, we found that the Olig-related transcription factor, Bhlhb5 is required for the survival of a subset of inhibitory interneurons neurons in the superficial dorsal horn of the spinal cord that appear to function to inhibit itch (Ross et al., 2010). In Bhlhb5-/-

mice, specific populations of spinal interneurons die during embryonic development and the resulting mice show abnormally elevated itch and the development of self-inflicted skin lesions. Importantly, the selective ablation of Bhlhb5 in inhibitory neurons in the spinal cord (but not by loss of Bhlhb5 in sensory neurons or in the forebrain) recapitulated the elevated itch phenotype, strongly pointing to the idea that spinal inhibitory neurons are responsible. Whether these neurons mediate the inhibition of itch by nociceptive input remains to be determined, but the Bhlhb5-expressing neurons are certainly well positioned to mediate this role (Figure 4). Hints that nociceptive input is absolutely required for the inhibition of itch comes from a pair of recent studies that independently looked at the role of vesicular glutamate transporter 2 (VGLUT2) in sensory neurons. All sensory neurons are glutamatergic and, although there are three vesicular glutamate transporters in the genome, many sensory neurons only express one of them. In particular, a large number of C-fibers exclusively express VGLUT2, and so they cannot release glutamate if VGLUT2 is knocked out. As one might expect, the silencing of a population of C-fibers in this way results in mice that show reduced pain behaviors. Unexpectedly, however, this silencing also caused abnormally elevated itch (Lagerstrom et al., 2010; Liu et al., 2010b). This finding implies that the silenced neurons are normally involved in providing sensory input that inhibits itch. Exactly which neurons are mediating this effect is not completely clear, though it seems that neurons of the TrpV1 lineage are involved. Furthermore, it is speculated that nociceptive input from VGLUT2-expressing nociceptors might activate Bhlhb5-expressing neurons that inhibit itch. However, as of yet, there is no direct evidence for this idea (Figure 4). It is very likely that the spinal cord plays a key role in integrating various types of somatosensory input in order to interpret the nature of the stimulus. One spinal interneuron subtype that appears to be critical for itch in particular is a population that expresses gastrin-releasing peptide receptor (GRPR). Ablation of this population using a saporin-conjugate results in mice that respond normally to painful stimuli but are insensitive to pruritogens (Han et al., 2012; Sun et al., 2009). Furthermore, constitutive loss of GRPR during development likewise gives rise to mice that show reduced scratching in itch assays (Sun and Chen, 2007), though how loss of GRPR affects the function of GRPR-expressing neurons is not known. Indeed, the degree to which GRP itself is involved in itch has been somewhat controversial. Originally it was suggested that gastrin-releasing peptide (GRP) released from primary afferents might mediate itch (Sun et al., 2009). But subsequent studies have now revealed that evoked sensory responses between primary afferent C-fibers and GRPR-expressing neurons are mediated by glutamate rather than GRP (Koga et al., 2011). Furthermore, it appears that the majority of GRP in the spinal cord comes from spinal interneurons rather than sensory neurons (Fleming et al., 2012). Thus, although there is good evidence that GRPR-expressing

Figure 4. The inhibition of itch by counter-stimuli. This modified version of the selectivity theory incorporates data from several recent papers in a speculative manner. GRPR-expressing neurons appear mediate itch, whereas a subset of Bhlhb5-expressing interneurons appear to inhibit itch; specific inputs and outputs of these neurons are not yet known.  

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interneurons play a key role in the integration of itch in the spinal cord, the specific role of GRP within itch circuits is not clear. Genetic variation and itch in humans — challenges for the future Even slight genetic variation within species can have a strong effect on itch sensitivity. Chloroquine, a widely prescribed anti-malarial medication, causes systemic itch as a side effect in 8-20% of humans when taken orally. Furthermore, this chloroquine-induced scratching is far more prevalent in individuals with dark skin compared to those with lighter skin, a figure that becomes clinically relevant when one considers that chloroquine is prescribed most often in African countries. In fact, atopic dermatitis, HIV, diabetes, cirrhosis and cholestasis during pregnancy are all disorders with a pruritic component that varies greatly in intensity based on ethnicity (Tey and Yosipovitch, 2010). There are now a dozen or so genetic mutations in mouse that have been found to affect itch behavior. These mouse models are important because they are helping us understand how itch is encoded in the nervous system. However, the genetic underpinnings of itch sensitivity in humans is almost completely unknown and remains an important challenge for future research. Acknowledgments: We would like to thank Eric Burrage for assistance with the figures.

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