SUMMARY The cellular and molecular mechanisms mediating histamine-independent itch in primary sensory neurons are largely unknown. Itch induced by chloroquine (CQ) is a common side-effect of this widely used anti-malarial drug. Here we show that Mrgprs, a family of G protein-coupled receptors expressed exclusively in peripheral sensory neurons, function as itch receptors. Mice lacking a cluster of Mrgpr genes display significant deficits in itch induced by CQ but not histamine. CQ directly excites sensory neurons in an Mrgpr-dependent manner. CQ specifically activates mouse MrgprA3 and human MrgprX1. Loss- and gain-of-function studies demonstrate that MrgprA3 is required for CQ responsiveness in mice. Furthermore, MrgprA3-expressing neurons respond to histamine and co-express Gastrin-Releasing Peptide, a peptide involved in itch sensation, and MrgprC11. Activation of these neurons with MrgprC11-specific agonist BAM8-22 induces itch in wild-type but not mutant mice. Therefore, Mrgprs may provide molecular access to itch-selective neurons and constitute novel targets for itch therapeutics.

An ex vivo, vagally innervated, lung preparation was used to address the hypothesis that vagal C-fibres comprise at least two distinct phenotypes. Histological and extracellular electrophysiological experiments revealed that vagal C-fibres innervating the pulmonary system are derived from cell bodies situated in two distinct vagal sensory ganglia. The jugular (superior) ganglion neurones project C-fibres to both the extrapulmonary airways (larynx, trachea and bronchus) and the lung parenchymal tissue. By contrast, C-fibres from nodose (inferior) neurones innervate primarily structures within the lungs. Histologically, nodose neurones projecting lung C-fibres were different from the jugular neurones in that they were significantly less likely to express neurokinins. The nerve terminals within the lungs of both nodose and jugular C-fibres responded with action potential discharge to capsaicin and bradykinin application, but only the nodose C-fibre population responded with action potential discharge to the P2X selective receptor agonist α,β-methylene-ATP. Whole cell patch clamp recording of capsaicin-sensitive nodose and jugular ganglion neurones retrogradely labelled from the lung tissue revealed that, like the nerve terminals, lung specific nodose C-fibre neurones express functional P2X receptors, whereas lung specific jugular C-fibres do not. The data support the hypothesis that both neural crest-derived neurones (jugular ganglia) and placode-derived neurones (nodose ganglia) project C-fibres in the vagus, and that these two C-fibre populations represent distinct phenotypes.

Transient receptor potential (TRP) A1 and TRPM8 are ion channels that have been localized to afferent nociceptive nerves. These TRP channels may be of particular relevance to respiratory nociceptors in that they can be activated by various inhaled irritants and/or cold air. We addressed the hypothesis that mouse vagal sensory nerves projecting to the airways express TRPA1 and TRPM8 and that they can be activated via these receptors. Single cell RT-PCR analysis revealed that TRPA1 mRNA, but not TRPM8, is uniformly expressed in lung-labelled TRPV1-expressing vagal sensory neurons. Neither TRPA1 nor TRPM8 mRNA was expressed in TRPV1-negative neurons. Capsaicin-sensitive, but not capsaicin-insensitive, lung-specific neurons responded to cinnamaldehyde, a TRPA1 agonist, with increases in intracellular calcium. Menthol, a TRPM8 agonist, was ineffective at increasing cellular calcium in lung-specific vagal sensory neurons. Cinnamaldehyde also induced TRPA1-like inward currents (as measured by means of whole cell patch clamp recordings) in capsaicin-sensitive neurons. In an ex vivo vagal innervated mouse lung preparation, cinnamaldehyde evoked action potential discharge in mouse vagal C-fibres with a peak frequency similar to that observed with capsaicin. Cinnamaldehyde inhalation in vivo mimicked capsaicin in eliciting strong central-reflex changes in breathing pattern. Taken together, our results support the hypothesis that TRPA1, but not TRPM8, is expressed in vagal sensory nerves innervating the airways. TRPA1 activation provides a mechanism by which certain environmental stimuli may elicit action potential discharge in airway afferent C-fibres and the consequent nocifensor reflexes.

Some vagal afferent nerves are thought to mediate autonomic responses evoked by noxious oesophageal stimuli and participate in the perception of pain originating in the oesophagus. However, the vagal nociceptive nerve phenotypes implicated in this function have yet to be identified. In this study, nociceptive fibres were defined by the capacity to discriminate noxious mechanical stimuli (wide range of oesophageal distension with pressure up to 100 mmHg) and detect noxious chemical stimuli (the activators of capsaicin receptor TRPV1). Using immunohistochemical techniques with retrogradely labelled oesophagus-specific neurones and performing extracellular recordings from the isolated vagally innervated oesophagus, we show that in the guinea-pig, the vagus nerves supply the oesophagus with a large population of nociceptive-like afferent nerve fibres. Vagal nociceptive-like fibres in the guinea-pig oesophagus are derived from two embryonically distinct sources: neurones situated in the nodose vagal ganglia and neurones situated in the jugular vagal ganglia. Nodose (placode-derived) nociceptive-like fibres are exclusively C-fibres sensitive to a P2X receptors agonist and rarely express the neuropeptide substance P. In contrast, jugular (neural crest-derived) nociceptive-like fibres include both A-fibres and C-fibres, are insensitive to P2X receptors agonist and mostly express substance P. The non-nociceptive vagal tension mechanoreceptors are distinguished from nociceptors by their saturable response to oesophageal distension and by the lack of TRPV1. These tension mechanoreceptors are exclusively A-fibres arising from the nodose ganglion. We conclude that the vagus nerves supply the guinea-pig oesophagus with nociceptors in addition to tension mechanoreceptors. The vagal nociceptive-like fibres in the oesophagus comprise two distinct subtypes dictated by the ganglionic location of their cell bodies.