The cells and circuitry for itch responses in mice

Abstract

Itch is triggered by somatosensory neurons expressing the ion channel TRPV1 (transient receptor potential cation channel subfamily V member 1), but the mechanisms underlying this nociceptive response remain poorly understood. Here, we show that the neuropeptide natriuretic polypeptide b (Nppb) is expressed in a subset of TRPV1 neurons and found that Nppb(-/-) mice selectively lose almost all behavioral responses to itch-inducing agents. Nppb triggered potent scratching when injected intrathecally in wild-type and Nppb(-/-) mice, showing that this neuropeptide evokes itch when released from somatosensory neurons. Itch responses were blocked by toxin-mediated ablation of Nppb-receptor-expressing cells, but a second neuropeptide, gastrin-releasing peptide, still induced strong responses in the toxin-treated animals. Thus, our results define the primary pruriceptive neurons, characterize Nppb as an itch-selective neuropeptide, and reveal the next two stages of this dedicated neuronal pathway.

Fig. 1. Nppb is always co-expressed with itch related signaling molecules TRPV1 and PLCβ3

TRPV1-DTA mice exhibit dramatically reduced scratch responses following intradermal injection of pruritic agents; histamine (100 µg in 10 µl); chloroquine (100 µg); endothelin 1 (1µM); methyl-serotonin (30 µg); PAR2 agonist SLIGRL-NH2 (100 µg) and compound 48/80 (100 µg). Itch inducing substances were injected intradermally into the shoulder of mice and numbers of scratching bouts were assessed over 30 minutes; data are mean ± s.e.m (n ≥ 7 animals) normalized to wild-type litter controls. (B) In situ hybridization (ISH) of sections through DRG illustrating loss of Nppb-expression in TRPV1-DTA animals; quantitation of Nppb expressing neurons revealed that 7 ± 0.6 % of NeuN-positive C4 DRG neurons express the neuropeptide in wild type mice (n=6). (C) Double label ISH of DRG demonstrates that Nppb (green) and TRPV1 (red) are co-expressed in the same sensory neurons; note that only a subset of TRPV1 expressing neurons contain Nppb. (D) All Nppb-positive neurons also express PLCβ3 (red) but many PLCβ3 neurons are Nppb negative. (E) Double label ISH also shows that Nppb-positive neurons (green) all express MrgprA receptors (including MrgprA3, the receptor for chloroquine; red), with more than 70% of MrgprA expressing-neurons also containing the neuropeptide. (F) Double label immunostaining demonstrates complete overlap between expression of MrgprC11 (red), the receptor for the pruritogen SLIGL-NH₂ and Nppb (green) in somatosensory neurons. Also see Fig. S2 for additional characterization of neurons expressing Nppb.

Fig. 2. Generation and characterization of Nppb ^−/− mice

(A) Schematic representation showing the disruption of the Nppb gene by insertion of a splice acceptor-lacZ cassette into the second exon used to generate Nppb^−/− mice. (B) In situ hybridization of sections through DRG reveals that Nppb expression is lost in Nppb^−/− animals. (C) Nppb ^−/− mice retained normal reactions to thermal, touch and proprioceptive stimuli, (D) but exhibit greatly attenuated responses to a range of pruritic agents (Table S2); data are mean ± s.e.m (n≥ 7 animals) normalized to wild-type litter controls. Significant differences between genotypes were determined using Student’s t-test with * indicating P < 0.001. (E) Intrathecal injection of Nppb (5µg in 10µl) into the lumbar 4–5 segment of control and Nppb ^−/− mice induced repeated bouts of scratching; injection of GRP (1nM in 10µl) also triggered itch responses in both mutant and control animals. Data represent mean values ± s.e.m. (n≥ 5 animals); no significant differences in response between genotypes were found (Student’s t-test). Typical behavioral responses of control and Nppb ^−/− mice to histamine, Nppb and the nociceptive neuropeptide substance P are illustrated in Videos S1 – 3.

Fig. 3. Selective ablation of Npra receptor neurons in the spinal cord impairs pruriception

(A) Expression of Npra in the dorsal spinal cord was assessed using ISH. In normal mice (upper panel), a significant subset of interneurons in the outer layer express Npra, however after intrathecal administration of Nppb-saporin (lower panel) few Npra neurons remained. In contrast, the number of GRP-receptor positive cells (right panels) were unaffected by Nppb-saporin. (B) Nppb-saporin treated mice retain normal reactions to thermal, touch and proprioceptive stimuli, but (C) exhibit greatly attenuated responses to intradermal injection of histamine (100 µg in 10 µl) or intrathecal administration of Nppb. Data represent means normalized against untreated controls ± s.e.m. (n≥ 5 animals). Significant differences between genotypes were determined using Student’s t-test with * indicating P < 0.01.

Fig. 4. GRP acts directly downstream of Nppb in the rodent pruriceptive circuit

(A) qPCR was used to quantitate expression of GRP and Nppb relative to GAPDH in the DRG and spinal cord. GRP is robustly expressed in the spinal cord (at a level comparable with Tac1) but is almost undetectable in the DRG. Nppb is prominently expressed in DRG, but is not present in the spinal cord. Data represent mean ± s.e.m for triplicate cDNA preparations each analyzed in 2 separate PCR reactions. (B) ISH was used to analyze GRP expression in the dorsal horn of normal (left panel) and Nppb-saporin treated mice (right panel); note that many GRP expressing interneurons are lost on ablation of Npra-expressing cells. (C) A significant number of GRP-neurons was eliminated following Nppb-saporin (Nppb-sap) treatment. GRP-saporin (GRP-sap), which targets GRP-receptor neurons has no effect on the number of GRP-interneurons; data represent mean ± s.e.m. (n≥ 4 animals). Significant differences between groups were determined using Student’s t-test with * indicating P < 0.001. (D) Ablation of Npra-neurons had no effect on intrathecally administered GRP induced scratch responses. (E) Scratching induced by lumbar injection of Nppb was strongly attenuated by pretreatment with a selective GRP antagonist or by the ablation of GRPR-expressing neurons with GRP-saporin. Data (D, E) are mean ± s.e.m. (n≥ 6 animals) and * indicates P < 0.001 (Student’s t-test). (F) Double label immunohistochemistry was used to localize interneurons expressing Npra (green) and GRP-driven EGFP (red) in sections through the dorsal horn of Tg(GRP-EGFP) mice. Left panels show typical individual staining patterns while the right panel shows merged Npra and GRP images with the cell nuclei counterstained with DAPI (grey); the outline of the dorsal horn is dotted. (G) Model of the first three stages of the pruriceptive circuit with the critical neuropeptide used at each stage indicated.