Fei Ru

Sensory neuron-specific GPCR Mrgprs are itch receptors mediating chloroquine-induced pruritus.

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 antimalarial 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 coexpress gastrin-releasing peptide, a peptide involved in itch sensation, and MrgprC11. Activation of these neurons with the 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.

P2X2 receptors differentiate placodal vs. neural crest C-fiber phenotypes innervating guinea pig lungs and esophagus

The lungs and esophagus are innervated by sensory neurons with somata in the nodose, jugular, and dorsal root ganglion. These sensory ganglia are derived from embryonic placode (nodose) and neural crest tissues (jugular and dorsal root ganglia; DRG). We addressed the hypothesis that the neurons embryonic origin (e.g., placode vs. neural crest) plays a greater role in determining particular aspects of its phenotype than the environment in which it innervates (e.g., lungs vs. esophagus). This hypothesis was tested using a combination of extracellular and patch-clamp electrophysiology and single-cell RT-PCR from guinea pig neurons. Nodose, but not jugular C-fibers innervating the lungs and esophagus, responded to alpha,beta-methylene ATP with action potential discharge that was sensitive to the P2X3 (P2X2/3) selective receptor antagonist A-317491. The somata of lung- and esophagus-specific sensory fibers were identified using retrograde tracing with a fluorescent dye. Esophageal- and lung-traced neurons from placodal tissue (nodose neurons) responded similarly to alpha,beta-methylene ATP (30 microM) with a large sustained inward current, whereas in neurons derived from neural crest tissue (jugular and DRG neurons), the same dose of alpha,beta-methylene ATP resulted in only a transient rapidly inactivating current or no detectable current. It has been shown previously that only activation of P2X2/3 heteromeric receptors produce sustained currents, whereas homomeric P2X3 receptor activation produces a rapidly inactivating current. Consistent with this, single-cell RT-PCR analysis revealed that the nodose ganglion neurons innervating the lungs and esophagus expressed mRNA for P2X2 and P2X3 subunits, whereas the vast majority of jugular and dorsal root ganglia innervating these tissues expressed only P2X3 mRNA with little to no P2X2 mRNA expression. We conclude that the responsiveness of C-fibers innervating the lungs and esophagus to ATP and other purinergic agonists is determined more by their embryonic origin than by the environment of the tissue they ultimately innervate.

Vagal afferent nerves with the properties of nociceptors.

Vagal afferent nerves are essential for optimal neural regulation of visceral organs, but are not often considered important for their defense. However, there are well-defined subsets of vagal afferent nerves that have activation properties indicative of specialization to detect potentially harmful stimuli (nociceptors). This is clearly exemplified by the vagal bronchopulmonary C-fibers that are quiescent in healthy lungs but are readily activated by noxious chemicals and inflammatory molecules. Vagal afferent nerves with similar activation properties have been also identified in the esophagus and probably exist in other visceral tissues. In addition, these putative vagal nociceptors often initiate defensive reflexes, can be sensitized, and have the capacity to induce central sensitization. This set of properties is a characteristic of nociceptors in somatic tissues.

Comparison of TRPA1-versus TRPV1-mediated cough in guinea pigs.

TRPA1 receptor is activated by endogenous inflammatory mediators and exogenous pollutant molecules relevant to respiratory diseases. Previous studies have implicated TRPA1 as a drug target for antitussive therapy. Here we evaluated the relative efficacy of TRPA1 activation to evoke cough. In conscious guinea pigs the TRPA1 agonist allyl-isothiocyanate (AITC) evoked cough with a maximally effective concentration of 10mM that was abolished by the selective TRPA1 antagonist AP-18. AITC (10mM) was approximately 3-times less effective in inducing cough than capsaicin (50 μM). Ex vivo single fiber extracellular recordings revealed that, similarly to capsaicin, AITC evoked activation in airway jugular C-fibers, but not in airway nodose Aδ-fibers. Consistent with the cough studies, AITC was approximately 3-times less effective than capsaicin in evoking sustained activation of the jugular C-fibers. Another TRPA1 agonist, cinnamaldehyde, was approximately twofold more effective than AITC in inducing cough. However, the cinnamaldehyde (10mM)-induced cough was only partially inhibited by the TRPA1 antagonist AP-18, and was abolished by combination of AP-18 and the TRPV1 antagonist I-RTX. We conclude that in naïve guinea pigs, TRPA1 activation initiates cough that is relatively modest compared to the cough initiated by TRPV1, likely due to lower efficacy of TRPA1 stimulation to induce sustained activation of airway C-fibers.

Selective silencing of NaV1.7 decreases excitability and conduction in vagal sensory neurons

Non‐technical summary  Sodium channels are obligatory for the conduction of action potentials along axons. There are several different sodium channel subtypes expressed in vagal sensory neurons, and it is difficult to pharmacologically block these subtypes selectively. We used virally delivered shRNA to selectively block the production of one of the sodium channel subtypes expressed in vagal sensory neurons, namely NaV1.7, and found that by selectively inhibiting the expression of this channel the conduction of action potentials was blocked in the majority of vagal sensory neurons. This study also shows that NaV1.7 is required for the elicitation of classical vagal reflexes such as cough.

Selective inhibition of vagal afferent nerve pathways regulating cough using Nav 1.7 shRNA silencing in guinea pig nodose ganglia.

Adeno-associated virus delivery systems and short hairpin RNA (shRNA) were used to selectively silence the voltage-gated sodium channel NaV 1.7 in the nodose ganglia of guinea pigs. The cough reflex in these animals was subsequently assessed. NaV 1.7 shRNA was delivered to the majority of nodose ganglia neurons [50-60% transfection rate determined by green fluorescent protein (GFP) gene cotransfection] and action potential conduction in the nodose vagal nerve fibers, as evaluated using an extracellular recording technique, was markedly and significantly reduced. By contrast, <5% of neurons in the jugular vagal ganglia neurons were transfected, and action potential conduction in the jugular vagal nerve fibers was unchanged. The control virus (with GFP expression) was without effect on action potential discharge and conduction in either ganglia. In vivo, NaV 1.7 silencing in the nodose ganglia nearly abolished cough evoked by mechanically probing the tracheal mucosa in anesthetized guinea pigs. Stimuli such as capsaicin and bradykinin that are known to stimulate both nodose and jugular C-fibers evoked coughing in conscious animals was unaffected by NaV 1.7 silencing in the nodose ganglia. Nodose C-fiber selective stimuli including adenosine, 2-methyl-5-HT, and ATP all failed to evoke coughing upon aerosol challenge. These results indicate that cough is independently regulated by two vagal afferent nerve subtypes in guinea pigs, with nodose Aδ fibers regulating cough evoked mechanically from the trachea and bradykinin- and capsaicin-evoked cough regulated by C-fibers arising from the jugular ganglia.

Adenosine-induced activation of esophageal nociceptors.

Clinical studies implicate adenosine acting on esophageal nociceptive pathways in the pathogenesis of noncardiac chest pain originating from the esophagus. However, the effect of adenosine on esophageal afferent nerve subtypes is incompletely understood. We addressed the hypothesis that adenosine selectively activates esophageal nociceptors. Whole cell perforated patch-clamp recordings and single-cell RT-PCR analysis were performed on the primary afferent neurons retrogradely labeled from the esophagus in the guinea pig. Extracellular recordings were made from the isolated innervated esophagus. In patch-clamp studies, adenosine evoked activation (inward current) in a majority of putative nociceptive (capsaicin-sensitive) vagal nodose, vagal jugular, and spinal dorsal root ganglia (DRG) neurons innervating the esophagus. Single-cell RT-PCR analysis indicated that the majority of the putative nociceptive (transient receptor potential V1-positive) neurons innervating the esophagus express the adenosine receptors. The neural crest-derived (spinal DRG and vagal jugular) esophageal nociceptors expressed predominantly the adenosine A(1) receptor while the placodes-derived vagal nodose nociceptors expressed the adenosine A(1) and/or A(2A) receptors. Consistent with the studies in the cell bodies, adenosine evoked activation (overt action potential discharge) in esophageal nociceptive nerve terminals. Furthermore, the neural crest-derived jugular nociceptors were activated by the selective A(1) receptor agonist CCPA, and the placodes-derived nodose nociceptors were activated by CCPA and/or the selective adenosine A(2A) receptor CGS-21680. In contrast to esophageal nociceptors, adenosine failed to stimulate the vagal esophageal low-threshold (tension) mechanosensors. We conclude that adenosine selectively activates esophageal nociceptors. Our data indicate that the esophageal neural crest-derived nociceptors can be activated via the adenosine A(1) receptor while the placodes-derived esophageal nociceptors can be activated via A(1) and/or A(2A) receptors. Direct activation of esophageal nociceptors via adenosine receptors may contribute to the symptoms in esophageal diseases.

5‐Hydroxytryptamine selectively activates the vagal nodose C‐fibre subtype in the guinea‐pig oesophagus

Abstract  The afferent neurons innervating the oesophagus originate from two embryonic sources: neurons located in vagal nodose ganglia originate from embryonic placodes and neurons located in vagal jugular and spinal dorsal root ganglia (DRG) originate from the neural crest. Here, we address the hypothesis that 5‐hydroxytryptamine (5‐HT) differentially stimulates afferent nerve subtypes in the oesophagus. Extracellular recordings of single unit activity originating from nerve terminals were made in the isolated innervated guinea‐pig oesophagus. Whole cell patch clamp recordings (35 °C) were made from the primary afferent neurons retrogradely labelled from the oesophagus. 5‐Hydroxytryptamine (10 μmol L−1) activated vagal nodose C‐fibres (70%) in the oesophagus but failed to activate overtly vagal jugular nerve fibres and oesophagus‐specific spinal DRG neurons. The response to 5‐HT in nodose C‐fibre nerve terminals was mimicked by the selective 5‐HT3 receptor agonist 2‐methyl‐5‐HT (10 μmol L−1) and nearly abolished by the 5‐HT3 receptor antagonists ondansetron (10 μmol L−1) and Y‐25130 (10 μmol L−1). In patch clamp studies, 2‐methyl‐5‐HT (10 μmol L−1) activated a proportion of isolated oesophagus‐specific nodose capsaicin‐sensitive neurons (putative cell bodies of nodose C‐fibres). We conclude that the responsiveness to 5‐HT discriminates placode‐derived (vagal nodose) C‐fibres from the neural crest‐derived (vagal jugular and spinal DRG) afferent nerves in the oesophagus. The response to 5‐HT in nodose C‐fibres is mediated by the 5‐HT3 receptor in their neuronal membrane.

Transgene expression and effective gene silencing in vagal afferent neurons in vivo using recombinant adeno-associated virus vectors

Vagal afferent fibres innervating thoracic structures such as the respiratory tract and oesophagus are diverse, comprising several subtypes of functionally distinct C‐fibres and A‐fibres. Both morphological and functional studies of these nerve subtypes would be advanced by selective, effective and long‐term transduction of vagal afferent neurons with viral vectors. Here we addressed the hypothesis that vagal sensory neurons can be transduced with adeno‐associated virus (AAV) vectors in vivo, in a manner that would be useful for morphological assessment of nerve terminals, using enhanced green fluorescent protein (eGFP), as well as for the selective knock‐down of specific genes of interest in a tissue‐selective manner. We found that a direct microinjection of AAV vectors into the vagal nodose ganglia in vivo leads to selective, effective and long‐lasting transduction of the vast majority of primary sensory vagal neurons without transduction of parasympathetic efferent neurons. The transduction of vagal neurons by pseudoserotype AAV2/8 vectors in vivo is sufficiently efficient such that it can be used to functionally silence TRPV1 gene expression using short hairpin RNA (shRNA). The eGFP encoded by AAV vectors is robustly transported to both the central and peripheral terminals of transduced vagal afferent neurons allowing for bright imaging of the nerve endings in living tissues and suitable for structure–function studies of vagal afferent nerve endings. Finally, the AAV2/8 vectors are efficiently taken up by the vagal nerve terminals in the visceral tissue and retrogradely transported to the cell body, allowing for tissue‐specific transduction.

The neural crest‐ and placodes‐derived afferent innervation of the mouse esophagus

Background  The mouse is an invaluable model for mechanistic studies of esophageal nerves, but the afferent innervation of the mouse esophagus is incompletely understood. Vagal afferent neurons are derived from two embryonic sources: neural crest and epibranchial placodes. We hypothesized that both neural crest and placodes contribute to the TRPV1‐positive (potentially nociceptive) vagal innervation of the mouse esophagus.