Department of Physiology and Cellular Biophysics.
Neurobiology Course, Marine Biological Laboratory, Woods Hole, Massachusetts 02543.
J Neurosci. 2019 Sep 4;39(36):7086-7101. doi: 10.1523/JNEUROSCI.2817-18.2019. Epub 2019 Jul 12.
Small-diameter vesicular glutamate transporter 3-lineage (Vglut3) dorsal root ganglion (DRG) neurons play an important role in mechanosensation and thermal hypersensitivity; however, little is known about their intrinsic electrical properties. We therefore set out to investigate mechanisms of excitability within this population. Calcium microfluorimetry analysis of male and female mouse DRG neurons demonstrated that the cooling compound menthol selectively activates a subset of Vglut3 neurons. Whole-cell recordings showed that small-diameter Vglut3 DRG neurons fire menthol-evoked action potentials and exhibited robust, transient receptor potential melastatin 8 (TRPM8)-dependent discharges at room temperature. This heightened excitability was confirmed by current-clamp and action potential phase-plot analyses, which showed menthol-sensitive Vglut3 neurons to have more depolarized membrane potentials, lower firing thresholds, and higher evoked firing frequencies compared with menthol-insensitive Vglut3 neurons. A biophysical analysis revealed voltage-gated sodium channel (Na) currents in menthol-sensitive Vglut3 neurons were resistant to entry into slow inactivation compared with menthol-insensitive neurons. Multiplex hybridization showed similar distributions of tetrodotoxin (TTX)-sensitive Na transcripts between TRPM8-positive and -negative Vglut3 neurons; however, Na1.8 transcripts, which encode TTX-resistant channels, were more prevalent in TRPM8-negative neurons. Conversely, pharmacological analyses identified distinct functional contributions of Na subunits, with Na1.1 driving firing in menthol-sensitive neurons, whereas other small-diameter Vglut3 neurons rely primarily on TTX-resistant Na channels. Additionally, when Na1.1 channels were blocked, the remaining Na current readily entered into slow inactivation in menthol-sensitive Vglut3 neurons. Thus, these data demonstrate that TTX-sensitive Nas drive action potential firing in menthol-sensitive sensory neurons and contribute to their heightened excitability. Somatosensory neurons encode various sensory modalities including thermoreception, mechanoreception, nociception, and itch. This report identifies a previously unknown requirement for tetrodotoxin-sensitive sodium channels in action potential firing in a discrete subpopulation of small-diameter sensory neurons that are activated by the cooling agent menthol. Together, our results provide a mechanistic understanding of factors that control intrinsic excitability in functionally distinct subsets of peripheral neurons. Furthermore, as menthol has been used for centuries as an analgesic and anti-pruritic, these findings support the viability of Na1.1 as a therapeutic target for sensory disorders.
小直径囊泡谷氨酸转运体 3 谱系（Vglut3）背根神经节（DRG）神经元在机械感觉和热超敏反应中发挥重要作用；然而，它们的内在电特性知之甚少。因此，我们着手研究该群体中兴奋性的机制。对雄性和雌性小鼠 DRG 神经元的钙微荧光分析表明，冷却化合物薄荷醇选择性激活 Vglut3 神经元的一个亚群。全细胞记录显示，小直径 Vglut3 DRG 神经元发射薄荷醇诱发的动作电位，并在室温下表现出强烈的、瞬时受体电位 melastatin 8（TRPM8）依赖性放电。电流箝位和动作电位相图分析证实了这种兴奋性的增强，结果表明，与薄荷醇不敏感的 Vglut3 神经元相比，薄荷醇敏感的 Vglut3 神经元具有更去极化的膜电位、更低的放电阈值和更高的诱发放电频率。生物物理分析表明，与薄荷醇不敏感的神经元相比，薄荷醇敏感的 Vglut3 神经元中的电压门控钠通道（Na）电流不易进入缓慢失活。多重杂交显示，TRPM8 阳性和阴性 Vglut3 神经元之间的河豚毒素（TTX）敏感 Na 转录本分布相似；然而，编码 TTX 抗性通道的 Na1.8 转录本在 TRPM8 阴性神经元中更为普遍。相反，药理学分析确定了 Na 亚基的不同功能贡献，Na1.1 驱动薄荷醇敏感神经元的放电，而其他小直径 Vglut3 神经元主要依赖于 TTX 抗性 Na 通道。此外，当 Na1.1 通道被阻断时，剩余的 Na 电流很容易在薄荷醇敏感的 Vglut3 神经元中进入缓慢失活。因此，这些数据表明，TTX 敏感的 Na 驱动薄荷醇敏感感觉神经元中的动作电位放电，并有助于提高其兴奋性。感觉神经元编码各种感觉模式，包括温度觉、机械觉、痛觉和瘙痒。本报告确定了一个以前未知的要求，即在薄荷醇激活的离散小直径感觉神经元亚群中，TTX 敏感的钠离子通道在动作电位放电中起作用。总之，我们的结果提供了对不同功能亚群外周神经元内在兴奋性的控制因素的机制理解。此外，由于薄荷醇作为一种镇痛药和抗瘙痒药已经使用了几个世纪，这些发现支持 Na1.1 作为感觉障碍治疗靶点的可行性。
