Chronic itch, or pruritus, is associated with a wide range of skin abnormalities. The mechanisms responsible for chronic itch induction and persistence remain unclear. We developed a mouse model in which a constitutively active form of the serine/threonine kinase BRAF was expressed in neurons gated by the sodium channel Nav1.8 (BRAF Nav1.8 mice). We found that constitutive BRAF pathway activation in BRAF Nav1.8 mice results in ectopic and enhanced expression of a cohort of itch-sensing genes, including gastrin-releasing peptide (GRP) and MAS-related GPCR member A3 (MRGPRA3), in nociceptors expressing transient receptor potential vanilloid 1 (TRPV1). BRAF Nav1.8 mice showed de novo neuronal responsiveness to pruritogens, enhanced pruriceptor excitability, and heightened evoked and spontaneous scratching behavior. GRP receptor expression was increased in the spinal cord, indicating augmented coding capacity for itch subsequent to amplified pruriceptive inputs. Enhanced GRP expression and sustained ERK phosphorylation were observed in sensory neurons of mice with allergic contact dermatitis-or dry skin-elicited itch; however, spinal ERK activation was not required for maintaining central sensitization of itch. Inhibition of either BRAF or GRP signaling attenuated itch sensation in chronic itch mouse models. These data uncover RAF/MEK/ERK signaling as a key regulator that confers a subset of nociceptors with pruriceptive properties to initiate and maintain long-lasting itch sensation. IntroductionThe ability of the brain to discriminate pain from itch in order to make binary decisions -eliciting either withdrawal or scratching behavior -is critically dependent on the functional connectivity of the somatosensory system. Itch information, along with pain, is conveyed by primary afferents of the dorsal root ganglion (DRG) to the spinal cord and of the trigeminal ganglion neurons to the trigeminal subnucleus caudalis of the brainstem, respectively, which in turn supplies input to the somatosensory cortex through spinothalamic tract or trigeminothalamic tract neurons (1-4). At the molecular level, emerging evidence suggests that activation of GPCRs in sensory neurons is likely responsible for relaying distinct types of acute stimulus-evoked itch (5-7). In addition, several transient receptor potential (TRP) channels, including TRP vanilloid 1 (TRPV1) and TRPA1, have been implicated in mediating histaminergic and nonhistaminergic itch, respectively (8-10). In the spinal cord, gastrin-releasing peptide (GRP) receptor (GRPR) and neurons expressing GRPR are key mediators dedicated to the coding of itch sensation (11-13). In contrast to acute itch, chronic itch may arise from an altered or diseased state of the

SUMMARY Central serotonin (5-HT) modulates somatosensory transduction, but how it achieves sensory modality-specific modulation remains unclear. Here we report that enhancing serotonergic tone via administration of 5-hydroxytryptophan potentiates itch sensation, whereas mice lacking 5-HT or serotonergic neurons in the brainstem exhibit markedly reduced scratching behavior. Through pharmacological and behavioral screening, we identified 5-HT1A as a key receptor in facilitating gastrin-releasing peptide (GRP)-dependent scratching behavior. Co-activation of 5-HT1A and GRP receptors (GRPR) greatly potentiates subthreshold, GRP-induced Ca2+ transients and action potential firing of GRPR+ neurons. Immunostaining, biochemical and biophysical studies suggest that 5-HT1A and GRPR may function as receptor heteromeric complexes. Furthermore, 5-HT1A blockade significantly attenuates, whereas its activation contributes to, long-lasting itch transmission. Thus, our studies demonstrate that the descending 5-HT system facilitates GRP-GRPR signaling via 5-HT1A to augment itch-specific outputs and a disruption of crosstalk between 5-HT1A and GRPR may be a useful anti-pruritic strategy.

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

Highlights d Neurons in l/vlPAG exhibit scratching behavior related to neural activities d Ablation of Tac1 + but not SST + neurons decreases itchinduced scratching behavior d Activation of Tac1 + neurons induces spontaneous scratching and grooming behaviors d l/vlPAG Tac1 + neurons modulate spinal itch processing via a RVM-dependent pathway