Chloroquine (CQ) and histamine are pruritogens commonly used to study itch in the mouse. A novel skin-nerve preparation was used to evaluate chloroquine (CQ)- and histamine-induced activation of afferent nerves in the dorsal thoracic skin of the mouse. All CQ sensitive nerves were C-fibres, and were also sensitive to histamine. The response to CQ, but not histamine, was largely absent in mrgpr-cluster Δ mice, supporting the hypothesis that CQ evokes itch largely via stimulation of MrgprA3 receptors. The CQ-induced action potential discharge was largely absent in phospholipase Cβ3 knockout animals. The CQ and histamine responses were not influenced by removal of TRPA1, TRPV1, TRPC3 or TRPC6, nor by the TRP channel blocker Ruthenium Red. The bouts of scratching in response to CQ were not different between wild-type and TRPA1-deficient mice. A selective inhibitor of the calcium-activated chloride channel TMEM16A, N-((4-methoxy)-2-naphthyl)-5-nitroanthranilic acid (MONNA), inhibited CQ-induced action potential discharge at itch nerve terminals and bouts of scratching by about 50%. Although TRPA1 and TRPV1 channels may be involved in the scratching responses to intradermal pruritogens, this is unlikely to be due to an effect at the nerve terminals, where chloride channels may play a more important role.

We evaluated the effect of voltage-gated sodium channel 1 (Na1) blockers in three nonoverlapping C-fiber subtypes in the mouse skin: chloroquine (CQ)-sensitive C-fibers with high mechanical thresholds- second, CQ-insensitive, capsaicin-sensitive C-fibers with high mechanical thresholds- and CQ and capsaicin-insensitive C-fibers with a very low mechanical threshold-C-LTMs. Na1-blocking drugs were applied to the nerve terminal receptive fields using an innervated isolated dorsal mouse skin-nerve preparation where the drugs are delivered into the skin intra-arterially. We combined these studies with an analysis of the mRNA expression of the -subunits of Na1 in individual dorsal root ganglia neurons labeled from the same region of the skin. Our results show that virtually all nociceptors and itch C-fibers expressed the tetrodotoxin (TTX)-resistant channels Na1.8 and Na1.9. However, TTX applied selectively into the skin abolished the action potential firing in response to mechanical stimulation in 75% of the itch C-fibers, 100% of the nociceptors, and 100% of C-LTMs. Na1.7 was the most commonly expressed TTX-sensitive Na1 in all three C-fiber subtypes innervating the dorsal skin. Selectively blocking Na1.7 abolished responses in about 40% of itch C-fibers, 65% of nociceptors, but only 20% of C-LTMs. Blocking Na1.8 alone had no affect on the firing sensitivity of the C-fibers. However, in itch and nociceptive C-fibers where the activation was not inhibited with a Na1.7 blocker, adding the Na1.8 blocker silenced action potential discharge.