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