Cinnamaldehyde elicits itch behavior via TRPV1and TRPV4 but not TRPA1

Domocos, Dan PhD^a,b; Follansbee, Taylor PhD^a; Nguyen, Amanda BS^a; Nguyen, Tony BS^a; Carstens, Mirela I. BS^a; Carstens, Earl PhD^a,Author Information Itch: July-September 2020 – Volume 5 – Issue 3 – p e36doi: 10.1097/itx.0000000000000036

Abstract

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 TRPV1and 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.

A spinal neural circuitry for converting touch to itch sensation

Sihan Chen, Xiao-Fei Gao, Yuxi Zhou, Ben-Long Liu, Xian-Yu Liu, Yufen Zhang, Devin M Barry, Kun Liu, Yingfu Jiao, Rita Bardoni, Weifeng Yu , Zhou-Feng Chen

Abstract

Touch and itch sensations are crucial for evoking defensive and emotional responses, and light tactile touch may induce unpleasant itch sensations (mechanical itch or alloknesis). The neural substrate for touch-to-itch conversion in the spinal cord remains elusive. We report that spinal interneurons expressing Tachykinin 2-Cre (Tac2^Cre) receive direct Aβ low threshold mechanoreceptor (LTMR) input and form monosynaptic connections with GRPR neurons. Ablation or inhibition markedly reduces mechanical but not acute chemical itch nor noxious touch information. Chemogenetic inhibition of Tac2^Cre neurons also displays pronounced deficit in chronic dry skin itch, a type of chemical itch in mice. Consistently, ablation of gastrin-releasing peptide receptor (GRPR) neurons, which are essential for transmitting chemical itch, also abolishes mechanical itch. Together, these results suggest that innocuous touch and chemical itch information converge on GRPR neurons and thus map an exquisite spinal circuitry hard-wired for converting innocuous touch to irritating itch.

Abstract

Background: Chronic itch is a highly debilitating symptom among patients with inflammatory skin diseases. Recent studies have revealed that gastrin-releasing peptide (GRP) and its receptor (gastrin-releasing peptide receptor [GRPR]) in the spinal dorsal horn (SDH) play a central role in itch transmission.

Objective: We aimed to investigate whether GRP-GRPR signaling is altered in SDH neurons in a mouse model of chronic itch and to determine the potential mechanisms underlying these alterations.

Methods: Patch-clamp recordings from enhanced green fluorescent protein (EGFP)–expressing (GRPR1) SDH neurons were used to examine GRP-GRPR signaling in spinal cord slices obtained from Grpr-EGFP mice. Immunohistochemical, genetic (gene expression and editing through adeno-associated virus vectors), and behavioral approaches were also used for in vivo experiments.

Results: We observed potentiation of GRP-evoked excitation in the GRPR1 SDH neurons of mice with contact dermatitis, without concomitant changes in GRPR expression. Interestingly, increases in excitation were attenuated by suppressing the reactive state of SDH astrocytes, which are known to be reactive in patients with chronic itch conditions. Furthermore, CRISPR-Cas9–mediated astrocyte-selective in vivo editing of a gene encoding lipocalin-2 (LCN2), an astrocytic factor implicated in chronic itch, suppressed increases in GRP-induced excitation of GRPR1 neurons, repetitive scratching, and skin damage in mice with contact dermatitis. Moreover, LCN2 potentiated GRP-induced excitation of GRPR1 neurons in normal mice.

Conclusion: Our findings indicate that, under chronic itch conditions, the GRP-induced excitability of GRPR1 SDH neurons is enhanced through a non–cell-autonomous mechanism involving LCN2 derived from reactive astrocytes.

Key words: Contact dermatitis, chronic itch, gastrin-releasing peptide, gastrin-releasing peptide receptor, spinal dorsal horn neurons, astrocytes, lipocalin 2, CRISPR-Cas9, patch-clamp recordings