Chemosensory properties of murine nasal and cutaneous trigeminal neurons identified by viral tracing

BACKGROUND: Somatosensation of the mammalian head is mainly mediated by the trigeminal nerve that provides innervation of diverse tissues like the face skin, the conjunctiva of the eyes, blood vessels and the mucouse membranes of the oral and nasal cavities. Trigeminal perception encompasses thermosensation, touch, and pain. Trigeminal chemosensation from the nasal epithelia mainly evokes stinging, burning, or pungent sensations. In vitro characterization of trigeminal primary sensory neurons derives largely from analysis of complete neuronal populations prepared from sensory ganglia. Thus, functional properties of primary trigeminal afferents depending on the area of innervation remain largely unclear. RESULTS: We established a PrV based tracing technique to identify nasal and cutaneous trigeminal neurons in vitro. This approach allowed analysis and comparison of identified primary afferents by means of electrophysiological and imaging measurement techniques. Neurons were challenged with several agonists that were reported to exhibit specificity for known receptors, including TRP channels and purinergic receptors. In addition, TTX sensitivity of sodium currents and IB4 binding was investigated. Compared with cutaneous neurons, a larger fraction of nasal trigeminal neurons showed sensitivity for menthol and capsaicin. These findings pointed to TRPM8 and TRPV1 receptor protein expression largely in nasal neurons whereas for cutaneous neurons these receptors are present only in a smaller fraction. The majority of nasal neurons lacked P2X(3 )receptor-mediated currents but showed P2X(2)-mediated responses when stimulated with ATP. Interestingly, cutaneous neurons revealed largely TTX resistant sodium currents. A significantly higher fraction of nasal and cutaneous afferents showed IB4 binding when compared to randomly chosen trigeminal neurons. CONCLUSION: In conclusion, the usability of PrV mediated tracing of primary afferents was demonstrated. Using this technique it could be shown that compared with neurons innervating the skin nasal trigeminal neurons reveal pronounced chemosensitivity for TRPM8 and TRPV1 channel agonists and only partially meet properties typical for nociceptors. In contrast to P2X(3 )receptors, TRPM8 and TRPV1 receptors seem to be of pronounced physiological relevance for intranasal trigeminal sensation

Investigation of neuronal activity in the trigeminal system of mice

In dieser Dissertation wurden Fragestellungen zu zellulären Mechanismen trigeminaler Chemosensorik und Nozizeption am Modellorganismus Maus bearbeitet. Wesentlicher Bestandteil war die Etablierung einer viralen Tracingtechnik, die es erlaubt primäre trigeminale Neurone in Abhängigkeit des Innervationsgebietes zu identifizieren und $\textit {in vitro}$ mittels modernen elektrophysiologischen und bildgebenden Verfahren zu analysieren. In einem Doppeltracing-Ansatz konnten so Nasenschleimhaut-innervierende Afferenzen mit Neuronen verglichen werden, die die Gesichtshaut innervieren. Im Vergleich konnte gezeigt werden, dass ein großer Anteil nasaler trigeminaler Neurone Chemosensitivität für Menthol und Capsaizin besitzt. Haut innervierende Neurone wiesen weitgehend Charakteristika von Nozizeptoren auf. In weiterführenden Studien konnten im heterologen System neue Erkenntnisse zur Sensitivität der Rezeptorproteine TRPM8 und TRPA1 für trigeminale Agonisten wie Menthol und Linalool gewonnen werden.This dissertation deals with cellular mechanisms of trigeminal chemosensation and nociception in the mouse model. A key question was the establishment of a viral tracing technique allowing identification and analysis of primary trigeminal neurons in dependence of their peripheral innervation pattern. A double tracing approach was used in combination with $\textit {in vitro}$ measurements (Patch clamp, Calcium imaging) to characterise trigeminal neurons innervating the nasal mucosa or the face skin. It could be shown that a large fraction of nasal trigeminal afferents reveal chemosensitivity for the TRPM8 and TRPV1 receptor agonists menthol and capsaicin. Most skin innervating neurons showed features commonly attributed to nociceptors like P2X$_{3}$ receptor mediated response to ATP and binding of the isolectin B4 (IB4). Further, studies in a heterologous expression system revealed new findings concerning sensitivity of TRPM8 and TRPA1 receptors for trigeminal agonists like menthol and linalool

The calcium-conducting ion channel transient receptor potential canonical 6 is involved in macrophage inflammatory protein-2-induced migration of mouse neutrophils

The role of the calcium-conducting ion channel transient receptor potential canonical 6 (TRPC6) in macrophage inflammatory protein-2 (MIP-2) induced migration of mouse neutrophils was investigated.status: publishe

Bimodal action of menthol on the transient receptor potential channel TRPA1

TRPA1 is a calcium-permeable nonselective cation transient receptor potential (TRP) channel that functions as an excitatory ionotropic receptor in nociceptive neurons. TRPA1 is robustly activated by pungent substances in mustard oil, cinnamon, and garlic and mediates the inflammatory actions of environmental irritants and proalgesic agents. Here, we demonstrate a bimodal sensitivity of TRPA1 to menthol, a widely used cooling agent and known activator of the related cold receptor TRPM8. In whole-cell and single-channel recordings of heterologously expressed TRPA1, submicromolar to low-micromolar concentrations of menthol cause channel activation, whereas higher concentrations lead to a reversible channel block. In addition, we provide evidence for TRPA1-mediated menthol responses in mustard oil-sensitive trigeminal ganglion neurons. Our data indicate that TRPA1 is a highly sensitive menthol receptor that very likely contributes to the diverse psychophysical sensations after topical application of menthol to the skin or mucous membranes of the oral and nasal cavities.status: publishe

Nicotine activates the chemosensory cation channel TRPA1

Topical application of nicotine, as used in nicotine replacement therapies, causes irritation of the mucosa and skin. This reaction has been attributed to activation of nicotinic acetylcholine receptors (nAChRs) in chemosensory neurons. In contrast with this view, we found that the chemosensory cation channel transient receptor potential A1 (TRPA1) is crucially involved in nicotine-induced irritation. We found that micromolar concentrations of nicotine activated heterologously expressed mouse and human TRPA1. Nicotine acted in a membrane-delimited manner, stabilizing the open state(s) and destabilizing the closed state(s) of the channel. In the presence of the general nAChR blocker hexamethonium, nociceptive neurons showed nicotine-induced responses that were strongly reduced in TRPA1-deficient mice. Finally, TRPA1 mediated the mouse airway constriction reflex to nasal instillation of nicotine. The identification of TRPA1 as a nicotine target suggests that existing models of nicotine-induced irritation should be revised and may facilitate the development of smoking cessation therapies with less adverse effects.status: publishe

Nicotine activates the chemosensory cation channel TRPA1

8 pages, 7 figures.-- Supporting information available at: http://www.nature.com/neuro/journal/vaop/ncurrent/suppinfo/nn.2379_S1.htmlArticle in press.Topical application of nicotine, as used in nicotine replacement therapies, causes irritation of the mucosa and skin. This reaction has been attributed to activation of nicotinic acetylcholine receptors (nAChRs) in chemosensory neurons. In contrast with this view, we found that the chemosensory cation channel transient receptor potential A1 (TRPA1) is crucially involved in nicotine-induced irritation. We found that micromolar concentrations of nicotine activated heterologously expressed mouse and human TRPA1. Nicotine acted in a membrane-delimited manner, stabilizing the open state(s) and destabilizing the closed state(s) of the channel. In the presence of the general nAChR blocker hexamethonium, nociceptive neurons showed nicotine-induced responses that were strongly reduced in TRPA1-deficient mice. Finally, TRPA1 mediated the mouse airway constriction reflex to nasal instillation of nicotine. The identification of TRPA1 as a nicotine target suggests that existing models of nicotine-induced irritation should be revised and may facilitate the development of smoking cessation therapies with less adverse effects.K.T. and J.A.J.V. were supported by a postdoctoral mandate from KU Leuven and are currently postdoctoral fellows of the Research Foundation–Flanders (Fonds voor Wetenschappelijk Onderzoek, FWO). M.G. and W.E. are doctoral FWO fellows. V.M.M was supported by Spanish CONSOLIDER-INGENIO 2010 CSD2007-00023. This work was supported by grants from Inter-university Attraction Poles Programme (Belgian Science Policy, P6/28), FWO (G.0172.03 and G.0565.07), the Research Council of the KU Leuven (GOA 2004/07) and the Flemish Government (Excellentiefinanciering, EF/95/010).Peer reviewe