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 Ca2+ transients in a corresponding fraction of TG neurons. Ca2+ transients were sensitive to inhibition of NKCC1 and voltage-gated Ca2+ channels (VGCCs). Ca2+ 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 Ca2+-activated Cl− channels (CaCCs) in TGs of mice. Pharmacological inhibition of CaCCs reduced the amplitude of capsaicin-induced responses of TG neurons in Ca2+ 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 Ca2+-activated Cl−-dependent signal amplification mechanism in TG neurons that requires intracellular Cl− accumulation by NKCC1 and the activation of CaCCs.

感觉神经元的细胞内氯离子浓度（[Cl−]i）可调控信号传递与信号放大过程。在背根神经节（DRG）与嗅觉感觉神经元（OSNs）中，氯离子可通过钠-钾-2氯协同转运蛋白1（NKCC1）完成蓄积，使细胞内氯离子浓度高于电化学平衡水平，并在氯离子通道开放时引发去极化性氯离子外流。本研究借助药理学与成像学方法、膜片钳技术以及行为学测试，探究了野生型（WT）与NKCC1基因敲除（NKCC1−/−）小鼠三叉神经节（TG）初级感觉神经元内的[Cl−]i及氯离子的功能。野生型TG神经元的[Cl−]i显示存在依赖NKCC1的主动氯离子蓄积过程。γ-氨基丁酸A型受体（GABAA受体）的激活可在对应比例的TG神经元中引发[Cl−]i下降与钙瞬变。该钙瞬变对NKCC1与电压门控钙通道（VGCCs）的抑制剂敏感。辣椒素——瞬时受体电位香草酸亚型1（TRPV1）的经典激动剂——所诱导的钙响应在NKCC1−/−小鼠的TG神经元中被削弱，但在细胞外氯离子浓度降低的条件下则会升高，这提示辣椒素诱导的响应存在氯离子依赖性放大机制。通过下一代测序技术（NGS），我们在小鼠TG组织中检测到多种钙激活氯离子通道（CaCCs）的表达。对CaCCs进行药理学抑制后，TG神经元在钙成像与电生理记录中辣椒素诱导的响应幅度均出现下降。在行为学范式中，NKCC1−/−小鼠对伤害性刺激辣椒素的回避行为减弱。综上，本研究结果强烈支持TG神经元中存在钙激活氯离子依赖性信号放大机制，该机制依赖NKCC1介导的细胞内氯离子蓄积与钙激活氯离子通道的激活。