Cellular and molecular mechanisms of cold-adapted mechanosensation in the hibernating squirrel

项目摘要 十三线地松鼠是一种专性冬眠动物，季节性地降低体温， 37°在活跃状态下到2-4°C在麻木。虽然这样的温度通常会抑制外周信号传导， 作为哺乳动物，迟钝的松鼠对触觉线索保持敏感，并能被触觉唤醒。 （机械感觉）。然而，涉及的神经生物学机制仍然未知。长期 本研究的目的是确定提供抗寒性的细胞和分子特征， 机械感觉来支持哺乳动物的极端热耐受性。 压力刺激由背根神经节（DRG）中的一个专门的神经元子集感知，称为压力刺激。 机械感受器机械门控离子通道如Piezo 2将压力转化为离子电流， 激活电压门控离子通道，通过动作电位（AP）将信号传递到下游。 Piezo 2和电压门控离子通道具有进化成熟的温度依赖性动力学 允许热阻的操作。本研究将在Dr. 耶鲁大学的Elena Gracheva和Slav Bagriantsev评估了离子通道的核心假设， 机械感受器的改变是十三行地面冷适应机械感觉的基础 松鼠 Gracheva和Bagriantsev实验室过去的工作表明，该物种的组成性低， 温度敏感性和麻木期间伤害性AP机械功能降低。我们的初步工作还 表明机械敏感电流在麻痹期间被保留。因此，我们结合使用 原位杂交，RNA测序和电生理学来评估松鼠DRG神经元是否具有 选择性增强机械敏感性，以其他感觉方式为代价，相对于小鼠 DRG神经元（Aim 1）。此外，冷暴露使AP变宽，并破坏典型细胞中的电压梯度。 哺乳动物神经元由于Na+/K+ ATP酶泵的动力学减慢。相比之下，我们的初步工作 显示冷暴露的迟钝神经元显示AP增宽减少和完整的静息膜电位。 根据这些数据，我们使用全细胞和离体电生理学来评估松鼠是否 机械感受器具有耐寒的生电机制（Aim 2）。 据我们所知，这项研究代表了第一次尝试解剖冷的细胞机制， 哺乳动物的适应性机械感觉阐明极端寒冷条件下的神经功能有可能 为临床低温手术后对抗神经元损伤提供了新的靶点。

PROJECT SUMMARY The thirteen-lined ground squirrel is an obligate hibernator that seasonally drops its body temperature from 37° in the active state to 2-4°C during torpor. While such temperatures typically inhibit peripheral signaling in mammals, torpid squirrels remain sensitive to tactile cues and can be awakened by the sense of touch (mechanosensation). However, the neurobiological mechanisms involved remain unknown. The long-term goal of this study is to identify the cellular and molecular signatures that afford cold-resistant mechanosensation to support extreme thermal tolerance in mammals. Pressure stimuli are sensed by a specialized subset of neurons in the dorsal root ganglion (DRG) known as mechanoreceptors. Mechanically gated ion channels such as Piezo2 convert pressure into ionic current that activates voltage-gated ion channels, which relay the signal downstream through action potentials (APs). Piezo2 and voltage-gated ion channels have temperature-dependent dynamics ripe for evolutionary manipulations permitting thermal resistance. This study, which will be conducted at the laboratories of Drs. Elena Gracheva and Slav Bagriantsev at Yale University, assesses the central hypothesis that ion channel modifications in mechanoreceptors underlie cold-adapted mechanosensation in the thirteen-lined ground squirrel. Past work from the Gracheva and Bagriantsev labs has shown that this species shows constitutively low thermosensitivity and decreased function of nociceptive AP machinery during torpor. Our preliminary work also shows that mechanosensitive currents are preserved during torpor. Accordingly, we use a combination of in situ hybridization, RNA sequencing and electrophysiology to assess whether squirrel DRG neurons have selectively potentiated mechanosensitivity at the cost of other sensory modalities, relative to mouse DRG neurons (Aim 1). Moreover, cold exposure widens APs and disrupts voltage gradients in typical mammalian neurons due to slowed kinetics of the Na+/K+ ATPase pump. In contrast, our preliminary work shows that cold-exposed torpid neurons show decreased AP widening and intact resting membrane potential. In light of these data, we use whole-cell and ex vivo electrophysiology to assess whether squirrel mechanoreceptors have cold-resistant electrogenic machinery (Aim 2). To the best of our knowledge, this study represents the first attempt to dissect cellular mechanisms of cold- adapted mechanosensation in mammals. Elucidating neural function in extreme cold has the potential to provide novel targets for combatting neuronal damage following clinical hypothermic procedures.