Die Wirkung von Nikotin auf trigeminal exprimierte Ionenkanäle

Der $\textit {Nervus Trigeminus}$ leistet unter anderem die sensorische Innervation von Epithelien des Kopfes. Sensorische Neurone dieses Nervs reagieren auf eine Vielzahl verschiedener Stimuli. Das weit verbreitete Suchtmittel Nikotin wirkt als chemischer Stimulus auf trigeminale Neurone. Die vorliegende Arbeit erweitert Kenntnisse über die Mechanismen der chemosensorischen Detektion von Nikotin auf der Ebene trigeminaler Neurone der Maus und humaner einzelner Rezeptoren mit dem Schwerpunkt auf ionotropen Rezeptoren. Für humane nikotinische Acetylcholinrezeptoren sowie TRPA1-Kanäle wurde hier mittels elektrophysiologischer Methoden erstmals die Stereoselektivität für die Nikotinenantiomere (+)- und (-)-Nikotin untersucht. Zudem wurde mittels Transkriptomanalysen einzelner Nikotin-sensitiver Neurone eine Anzahl von Rezeptoren gefunden, die als Kandidaten für unbekannte Nikotinrezeptoren weitere Ansätze bieten, das Wissen über die Wahrnehmung von Nikotin zu vervollständigen

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

Deep sequencing of the murine olfactory receptor neuron transcriptome

The ability of animals to sense and differentiate among thousands of odorants relies on a large set of olfactory receptors (OR) and a multitude of accessory proteins within the olfactory epithelium (OE). ORs and related signaling mechanisms have been the subject of intensive studies over the past years, but our knowledge regarding olfactory processing remains limited. The recent development of next generation sequencing (NGS) techniques encouraged us to assess the transcriptome of the murine OE. We analyzed RNA from OEs of female and male adult mice and from fluorescence-activated cell sorting (FACS)-sorted olfactory receptor neurons (ORNs) obtained from transgenic OMP-GFP mice. The Illumina RNA-Seq protocol was utilized to generate up to 86 million reads per transcriptome. In OE samples, nearly all OR and trace amine-associated receptor (TAAR) genes involved in the perception of volatile amines were detectably expressed. Other genes known to participate in olfactory signaling pathways were among the 200 genes with the highest expression levels in the OE. To identify OE-specific genes, we compared olfactory neuron expression profiles with RNA-Seq transcriptome data from different murine tissues. By analyzing different transcript classes, we detected the expression of non-olfactory GPCRs in ORNs and established an expression ranking for GPCRs detected in the OE. We also identified other previously undescribed membrane proteins as potential new players in olfaction. The quantitative and comprehensive transcriptome data provide a virtually complete catalogue of genes expressed in the OE and present a useful tool to uncover candidate genes involved in, for example, olfactory signaling, OR trafficking and recycling, and proliferation

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

Characterization of the olfactory receptors expressed in human spermatozoa

The detection of external cues is fundamental for human spermatozoa to locate the oocyte in the female reproductive tract. This task requires a specific chemoreceptor repertoire that is expressed on the surface of human spermatozoa, which is not fully identified to date. Olfactory receptors (ORs) are candidate molecules and have been attributed to be involved in sperm chemotaxis and chemokinesis, indicating an important role in mammalian spermatozoa. An increasing importance has been suggested for spermatozoal RNA, which led us to investigate the expression of all 387 OR genes. This study provides the first comprehensive analysis of OR transcripts in human spermatozoa of several individuals by RNA-Seq. We detected 91 different transcripts in the spermatozoa samples that could be aligned to annotated OR genes. Using stranded mRNA-Seq, we detected a class of these putative OR transcripts in an antisense orientation, indicating a different function, rather than coding for a functional OR protein. Nevertheless, we were able to detect OR proteins in various compartments of human spermatozoa, indicating distinct functions in human sperm. A panel of various OR ligands induced Ca$^{2+}$ signals in human spermatozoa, which could be inhibited by mibefradil. This study indicates that a variety of ORs are expressed at the mRNA and protein level in human spermatozoa