TRPV1-expressing primary afferents generate behavioral responses to pruritogens via multiple mechanisms

Abstract

The mechanisms that generate itch are poorly understood at both the molecular and cellular levels despite its clinical importance. To explore the peripheral neuronal mechanisms underlying itch, we assessed the behavioral responses (scratching) produced by s.c. injection of various pruritogens in PLCbeta3- or TRPV1-deficient mice. We provide evidence that at least 3 different molecular pathways contribute to the transduction of itch responses to different pruritogens: 1) histamine requires the function of both PLCbeta3 and the TRPV1 channel; 2) serotonin, or a selective agonist, alpha-methyl-serotonin (alpha-Me-5-HT), requires the presence of PLCbeta3 but not TRPV1, and 3) endothelin-1 (ET-1) does not require either PLCbeta3 or TRPV1. To determine whether the activity of these molecules is represented in a particular subpopulation of sensory neurons, we examined the behavioral consequences of selectively eliminating 2 nonoverlapping subsets of nociceptors. The genetic ablation of MrgprD(+) neurons that represent approximately 90% of cutaneous nonpeptidergic neurons did not affect the scratching responses to a number of pruritogens. In contrast, chemical ablation of the central branch of TRPV1(+) nociceptors led to a significant behavioral deficit for pruritogens, including alpha-Me-5-HT and ET-1, that is, the TRPV1-expressing nociceptor was required, whether or not TRPV1 itself was essential. Thus, TRPV1 neurons are equipped with multiple signaling mechanisms that respond to different pruritogens. Some of these require TRPV1 function; others use alternate signal transduction pathways.

Fig. 1.

Temperature, mechanical, and pain behavioral analysis in PLCβ3-deficient mice and their littermate controls. (A) Latency to exhibit evidence of discomfort (paw shaking and licking) on a hot plate (n = 8 per genotype). (B) Latency to tail withdrawal from a hot or cold water bath (n = 6 per genotype). (C) Percentage of time spent at 30 °C when given a choice between 30 °C and 40 °C (n = 4 per genotype). (D) Paw withdrawal frequency to von Frey filaments (n = 8 per genotype). (E) Latency to paw withdrawal from radiant heat before (“0”) and 2 days after CFA injection (n = 8 per genotype). (F) Paw thickness of the CFA-injected (ipsi) and uninjected (contra) hindpaw (n = 8 per genotype). (G) Time spent for paw licking and shaking after formalin injection into a hind paw during each 5-min interval of a 50-min test period (n = 8 per genotype). There were no significant differences at any point between 2 groups (P > 0.05, two-way ANOVA with Bonferroni posttests) for any of the behavioral tests. All data are presented as means ± SEM.

Fig. 2.

The scratching elicited by serotonin and α-Me-5-HT, but not other pruritogens, is eliminated in PLCβ3-deficient mice. (A) The total number of “bouts” of scratching per 30 min in response to s.c. injections into the nape of the neck of 8 different pruritic compounds, in a volume of 100 μL. Compounds tested: 5-HT (100 nmol), α-Me-5HT (30 μg), ET-1 (10 pmol), chloroquine (200 μg), formalin (100 μL of 0.6%), U-46619 (3.5 μg), SLIGRL-NH₂ (100 nmol), and agmantin (160 μg). At least 4 and up to 10 pairs of the mice were used for each compound. (B and C) Time course of scratching after injection of α-Me-5HT and ET-1 in wild-type (filled rectangle) versus PLCβ3−/− (filled circle) mice. Compared with wild-type mice (n = 8), PLCβ3-deficient mice (n = 8) show a complete abrogation for α-Me-5HT but no effect for ET-1. Data represent means ± SEM. Asterisks denote significant differences compared with littermate controls. *, P < 0.01, Mann–Whitney U test.

Fig. 3.

Fos-like immunoreactivity (FLI) in superficial dorsal horn of the rostral cervical spinal cord in wild-type and PLCβ3−/− mice after s.c. injection of α-Me-5HT or ET-1 into the nape of the neck. (A–D) There is a dense cluster of FLI neurons in the lateral part of laminae I and II, where primary afferents from the nape of the neck terminate. ET-1 induced FLI was not altered in the PCLβ3−/− mice compared with wild-type mice, but α-Me-5-HT-evoked FLI was greatly reduced in PCLβ3−/− mice (n = 4 to 8 per genotype for each compound). The graphs indicate number of FLI neurons in laminae I, II, V, and VI. The statistical difference between the 2 genotypes was significant for α-Me-5-HT-evoked FLI (*, P < 0.0001, Mann–Whitney U test), but not for ET-1. Error bars, means ± SEM.

Fig. 4.

PLCβ3-deficient mice show impaired licking behavioral responses to hindpaw injection of 5-HT (n = 4 per genotype) and α-Me-5-HT (n = 9 per genotype), but not to ET-1 (n = 8 per genotype). Note that the licking responses produced by thigh injection of α-Me-5-HT (n = 5 per genotype) are also significantly impaired in PLCβ3-deficient mice. *, P < 0.05, Student's t test for both 5-HT and α-Me-5-HT compared with wild-type mice. All data presented as means ± SEM.

Fig. 5.

TRPV1⁺ neurons are required for the behavioral responses to different pruritogens. (A) Scratching in response to histamine (n = 4 per genotypes), α-Me-5-HT (n = 5 per genotypes), and ET-1 (n = 4 per genotypes) in TRPV1-mutant mice. In contrast to histamine, scratching in responses to α-Me-5-HT and ET-1 was not altered in the mutant mice. *, P < 0.05, Student's t test for histamine comparing the 2 genotypes. (B) Licking in response to histamine, α-Me-5-HT, and ET-1, 1 d after intrathecal capsaicin or vehicle injection. Ablation of the central terminals of TRPV1-expressing neurons led to significant loss of the behavioral responses to both α-Me-5-HT and ET-1. *, P < 0.01, two-way rmANOVA followed by Sheffé's test; n = 3 for capsaicin treated or vehicle-treated). (C) Mice lacking MrgprD⁺ neurons do not show altered scratching behavior in response to 8 different itch-provoking compounds. Data represent means ± SEM; n = 4–8 for all tests.

Fig. 6.

This schematic illustrates 3 distinct and likely independent pathways through which itch can be induced by a histamine, α-Me-5-HT or ET-1 action upon TRPV1-expressing neurons.