From molecules to behavior

Die sensorischen Neurone des Trigeminusnervs (5. Hirnnerv) detektieren physikalische und chemische Stimuli durch freie Nervenendigungen in den Epithelien des Kopfes und Gesichts. Die vorliegende Arbeit befasst sich mit verschiedenen Aspekten der trigeminalen Wahrnehmung chemischer Stimuli auf Ebene des Verhaltens und in Einzelzellen. Unter Anwendung psychophysischer Techniken wurden Wechselwirkungen des trigeminalen mit anderen chemosensorischen Sinnen beim Menschen untersucht, insbesondere der Einfluss des Schmeckens auf die Schmerzwahrnehmung. Polyphenole aus Tee und Rotwein, die beim Verzehr ein trockenes Mundgefühl (Adstringenz) erzeugen, wurden als adäquate trigeminale Stimuli identifiziert und die Transduktionsmechanismen untersucht. Die Arbeit befasst sich auch mit der kaum bekannten Funktion des $Na^{+}-K^{+}-2Cl^{-}-Kotransporters$ 1 im trigeminalen Sinnessystem. Hierzu wurden Experimente an trigeminalen Ganglienneuronen und Verhaltenstests an gendeletierten Mäusen durchgeführt

RNA-Seq analysis of human trigeminal and dorsal root ganglia with a focus on chemoreceptors

The chemosensory capacity of the somatosensory system relies on the appropriate expression of chemoreceptors, which detect chemical stimuli and transduce sensory information into cellular signals. Knowledge of the complete repertoire of the chemoreceptors expressed in human sensory ganglia is lacking. This study employed the next-generation sequencing technique (RNA-Seq) to conduct the first expression analysis of human trigeminal ganglia (TG) and dorsal root ganglia (DRG). We analyzed the data with a focus on G-protein coupled receptors (GPCRs) and ion channels, which are (potentially) involved in chemosensation by somatosensory neurons in the human TG and DRG. For years, transient receptor potential (TRP) channels have been considered the main group of receptors for chemosensation in the trigeminal system. Interestingly, we could show that sensory ganglia also express a panel of different olfactory receptors (ORs) with putative chemosensory function. To characterize OR expression in more detail, we performed microarray, semi-quantitative RT-PCR experiments, and immunohistochemical staining. Additionally, we analyzed the expression data to identify further known or putative classes of chemoreceptors in the human TG and DRG. Our results give an overview of the major classes of chemoreceptors expressed in the human TG and DRG and provide the basis for a broader understanding of the reception of chemical cues

Transient receptor potential channels encode volatile chemicals sensed by rat trigeminal ganglion neurons

Primary sensory afferents of the dorsal root and trigeminal ganglia constantly transmit sensory information depicting the individual’s physical and chemical environment to higher brain regions. Beyond the typical trigeminal stimuli (e.g. irritants), environmental stimuli comprise a plethora of volatile chemicals with olfactory components (odorants). In spite of a complete loss of their sense of smell, anosmic patients may retain the ability to roughly discriminate between different volatile compounds. While the detailed mechanisms remain elusive, sensory structures belonging to the trigeminal system seem to be responsible for this phenomenon. In order to gain a better understanding of the mechanisms underlying the activation of the trigeminal system by volatile chemicals, we investigated odorant-induced membrane potential changes in cultured rat trigeminal neurons induced by the odorants vanillin, heliotropyl acetone, helional, and geraniol. We observed the dose-dependent depolarization of trigeminal neurons upon application of these substances occurring in a stimulus-specific manner and could show that distinct neuronal populations respond to different odorants. Using specific antagonists, we found evidence that TRPA1, TRPM8, and/or TRPV1 contribute to the activation. In order to further test this hypothesis, we used recombinantly expressed rat and human variants of these channels to investigate whether they are indeed activated by the odorants tested. We additionally found that the odorants dose-dependently inhibit two-pore potassium channels TASK1 and TASK3 heterologously expressed In $\textit {Xenopus laevis}$ oocytes. We suggest that the capability of various odorants to activate different TRP channels and to inhibit potassium channels causes neuronal depolarization and activation of distinct subpopulations of trigeminal sensory neurons, forming the basis for a specific representation of volatile chemicals in the trigeminal ganglia

Comprehensive RNA-seq expression analysis of sensory ganglia with a focus on ion channels and GPCRs in trigeminal ganglia

The specific functions of sensory systems depend on the tissue-specific expression of genes that code for molecular sensor proteins that are necessary for stimulus detection and membrane signaling. Using the Next Generation Sequencing technique (RNA-Seq), we analyzed the complete transcriptome of the trigeminal ganglia (TG) and dorsal root ganglia (DRG) of adult mice. Focusing on genes with an expression level higher than 1 FPKM (fragments per kilobase of transcript per million mapped reads), we detected the expression of 12984 genes in the TG and 13195 in the DRG. To analyze the specific gene expression patterns of the peripheral neuronal tissues, we compared their gene expression profiles with that of the liver, brain, olfactory epithelium, and skeletal muscle. The transcriptome data of the TG and DRG were scanned for virtually all known G-protein-coupled receptors (GPCRs) as well as for ion channels. The expression profile was ranked with regard to the level and specificity for the TG. In total, we detected 106 non-olfactory GPCRs and 33 ion channels that had not been previously described as expressed in the TG. To validate the RNA-Seq data, $\textit {in situ}$ hybridization experiments were performed for several of the newly detected transcripts. To identify differences in expression profiles between the sensory ganglia, the RNA-Seq data of the TG and DRG were compared. Among the differentially expressed genes (> 1 FPKM), 65 and 117 were expressed at least 10-fold higher in the TG and DRG, respectively. Our transcriptome analysis allows a comprehensive overview of all ion channels and G protein-coupled receptors that are expressed in trigeminal ganglia and provides additional approaches for the investigation of trigeminal sensing as well as for the physiological and pathophysiological mechanisms of pain

Trigeminal ganglion neurons of mice show intracellular chloride accumulation and chloride-dependent amplification of capsaicin-induced responses

Intracellular $Cl^{−}$ concentrations $([Cl^{−}]_{i}$) of sensory neurons regulate signal transmission and signal amplification. In dorsal root ganglion (DRG) and olfactory sensory neurons (OSNs), $Cl^{−}$ is accumulated by the $Na^{+}-K^{+}-2Cl^{−}$ cotransporter 1 (NKCC1), resulting in a $[Cl^{−}]_{i}$ above electrochemical equilibrium and a depolarizing $Cl^{−}$ efflux upon $Cl^{−}$ channel opening. Here, we investigate the $[Cl^{−}]_{i}$ and function of $Cl^{−}$ in primary sensory neurons of trigeminal ganglia (TG) of wild type (WT) and $NKCC1^{−/−}$ mice using pharmacological and imaging approaches, patch-clamping, as well as behavioral testing. The $[Cl^{−}]_{i}$ of WT TG neurons indicated active NKCC1-dependent $Cl^{−}$ accumulation. Gamma-aminobutyric acid $(GABA)_{A}$ receptor activation induced a reduction of $[Cl^{−}]_{i}$ as well as $Ca^{2+}$ transients in a corresponding fraction of TG neurons. $Ca^{2+}$ transients were sensitive to inhibition of NKCC1 and voltage-gated $Ca^{2+}$ channels (VGCCs). $Ca^{2+}$ responses induced by capsaicin, a prototypical stimulus of transient receptor potential vanilloid subfamily member-1 (TRPV1) were diminished in $NKCC1^{−/−}$ TG neurons, but elevated under conditions of a lowered $[Cl^{−}]_{o}$ suggesting a $Cl^{−}$-dependent amplification of capsaicin-induced responses. Using next generation sequencing (NGS), we found expression of different $Ca^{2+}$-activated $Cl^{−}$ channels (CaCCs) in TGs of mice. Pharmacological inhibition of CaCCs reduced the amplitude of capsaicin-induced responses of TG neurons in $Ca^{2+}$ imaging and electrophysiological recordings. In a behavioral paradigm, $NKCC1^{−/−}$ mice showed less avoidance of the aversive stimulus capsaicin. In summary, our results strongly argue for a $Ca^{2+}$-activated $Cl^{−}$-dependent signal amplification mechanism in TG neurons that requires intracellular $Cl^{−}$ accumulation by NKCC1 and the activation of CaCCs

Comprehensive RNA-Seq Expression Analysis of Sensory Ganglia with a Focus on Ion Channels and GPCRs in Trigeminal Ganglia

The specific functions of sensory systems depend on the tissue-specific expression of genes that code for molecular sensor proteins that are necessary for stimulus detection and membrane signaling. Using the Next Generation Sequencing technique (RNA-Seq), we analyzed the complete transcriptome of the trigeminal ganglia (TG) and dorsal root ganglia (DRG) of adult mice. Focusing on genes with an expression level higher than 1 FPKM (fragments per kilobase of transcript per million mapped reads), we detected the expression of 12984 genes in the TG and 13195 in the DRG. To analyze the specific gene expression patterns of the peripheral neuronal tissues, we compared their gene expression profiles with that of the liver, brain, olfactory epithelium, and skeletal muscle. The transcriptome data of the TG and DRG were scanned for virtually all known G-protein-coupled receptors (GPCRs) as well as for ion channels. The expression profile was ranked with regard to the level and specificity for the TG. In total, we detected 106 non-olfactory GPCRs and 33 ion channels that had not been previously described as expressed in the TG. To validate the RNA-Seq data, in situ hybridization experiments were performed for several of the newly detected transcripts. To identify differences in expression profiles between the sensory ganglia, the RNA-Seq data of the TG and DRG were compared. Among the differentially expressed genes (> 1 FPKM), 65 and 117 were expressed at least 10-fold higher in the TG and DRG, respectively. Our transcriptome analysis allows a comprehensive overview of all ion channels and G protein-coupled receptors that are expressed in trigeminal ganglia and provides additional approaches for the investigation of trigeminal sensing as well as for the physiological and pathophysiological mechanisms of pain