Electrical and mechanical properties of motor units in a mouse model of ALS

描述（由申请人提供）： 该提案的重点是识别 ALS 小鼠模型中运动神经元退化时的功能缺陷，基于最近发表的初步结果表明，通常指定其激活模式的运动神经元特性可能在其退化中发挥关键作用。我们特别关注运动神经元的大小。运动神经元通常充当运动单元的中心组件，运动单元由运动神经元、其轴突和受神经支配的肌纤维组成。因此，大小不仅涉及细胞体，还涉及树突（反映输入的数量）和轴突末端分支（与受神经支配的肌纤维的数量成正比）。正常情况下，运动神经元被从小到大激活：S型运动单位在运动神经元解剖结构和肌纤维数量方面都很小，所有这些都很慢。接下来是更大、更快的运动单位（FR 和 FF 型）。然而，对 ALS 标准动物模型（突变型 SOD1 小鼠）外周肌纤维去神经支配的研究表明，最初无法产生力的顺序相反：FF > FR >S，即从大到小。这种相反的序列表明尺寸过大是一种导致退化的缺陷，事实上，我们最近惊讶地发现突变的 SOD1 运动神经元在很小的时候（出生前 10 天）就开始过度生长。这是在第一个 FF 运动单位开始无法产生力量之前很久（大约 50 天），甚至在典型症状出现之前更长时间（90 天）。值得注意的是，这些较大细胞的固有电特性也被扭曲，可能导致代谢和兴奋性毒性应激的结合。此外，输入结构可能会发生变化。为了研究大小、内在兴奋性和突触输入之间的关系，需要对成年状态的小鼠运动神经元进行细胞内研究。我们开发了 3 种新制剂，可首次对成人状态的骶腰椎和脑干运动神经元进行细胞内研究。两种是体外的，可以进行系统的药物研究，而另一种是在原位的，可以直接比较运动神经元的电特性与其机械特性。因此，原位准备研究将确定运动神经元在受到力破坏时的特性。目标 1 使用原位制备来检验过大尺寸可预测力失效模式的假设。目标 2 使用体外骶索制剂来评估内在电特性和输入是否存在平行上调，以匹配尺寸扭曲，而目标 3 使用脑干切片来查看这些较小的运动神经元是否经历相同的模式。在目标 4 中，长期给药用于确定电特性的改变是否会导致尺寸的变化。总体而言，这项工作构成了研究 ALS 机制的新方法。

DESCRIPTION (provided by applicant): This proposal focuses on identifying functional deficits in motoneurons as they degenerate in a mouse model of ALS, based on the recently published and preliminary results indicating that motoneuron properties that normally specify their activation patterns may play a key role in their degeneration. We focus especially on motoneuron size. The motoneuron normally functions as the central component of a motor unit, which consists of the motoneuron, its axon and the muscle fibers innervated. Thus size involves not just the cell body but also the dendrites (which reflect number of inputs) and axon terminal branches (which is proportional to number of innervated muscle fibers). Normally, motoneurons are activated from small to large: type S motor units are small in terms of motoneuron anatomy and number of muscle fibers, all of which are slow. Progressive larger and faster motor units follow (type FR and FFs). Yet studies of the denervation of muscle fibers in the periphery in a standard animal model of ALS, the mutant SOD1 mouse, indicate that initial failure to generate force occurs in the opposite sequence: FF > FR >S, i.e. from large to small. This reverse sequence suggests excess size is a deficit that contributes to degeneration and indeed we have recently been surprised to find that mutant SOD1 motoneurons began to grow excessively at a very young age, before 10 days of birth. This is long before the first FF motor units begin to fail in force generation (about 50 days) and even longer before classic symptom onset (90 days). Remarkably, the intrinsic electrical properties of these larger cells are also distorted, potentially leading to a combination of metabolic and excitotoxic stress. In addition, changes in the structure of input could occur. To investigate the relations between size, intrinsic excitability and synaptic input requires intracellular study of mouse motoneurons in the adult state. We have developed 3 new preparations that allow the first intracellular studies of motoneuron in the adult state for sacral lumbar and brainstem motoneurons. Two are in vitro, allowing systematic drug studies while one is in situ, allowing direct comparison of motoneuron electrical properties to its mechanical properties. Thus the in situ prep studies will identify the properties of the motoneuron as it undergoes force failure. Aim 1 uses the in situ preparation to test the hypothesis that excess size predicts the pattern of force failure. Aim 2 uses an in vitro sacral cord preparation to asses whether there is parallel upregulation in intrinsic electrical properties and inputs to match the distortion in size, while Aim 3 uses brainstem slice to see if these smaller motoneurons undergo the same pattern. In Aim 4, chronic drug administration is used to determine if alterations in electrical properties cause changes in size. Overall, this work constitutes a new approach to study of mechanisms of ALS.