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

描述(由申请人提供)： 根据最近发表的初步结果表明，运动神经元的特性通常指定它们的激活模式，可能在它们的退化中起关键作用。我们特别关注运动神经元的大小。运动神经元通常是运动单位的中枢组成部分，运动单位由运动神经元、轴突和支配的肌纤维组成。因此，大小不仅涉及细胞体，还包括树突(反映输入的数量)和轴突终末分支(与神经支配的肌肉纤维的数量成正比)。正常情况下，运动神经元是由小到大被激活的：S型运动神经元在运动神经元解剖和肌纤维数量方面都很小，都很慢。紧随其后的是渐进式更大、更快的电机单元(FR和FF型)。然而，对肌萎缩侧索硬化症的标准动物模型--突变的SOD1鼠--的外周肌肉纤维失神经的研究表明，最初无法产生力量的顺序相反：FF&GT；FR&GT；S，即从大到小。这个相反的序列表明，过大的尺寸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运动神经元在非常小的年龄，在出生10天之前就开始过度生长。这是在第一批FF马达开始发生故障之前很久(大约50天)，甚至在典型症状出现之前(90天)。值得注意的是，这些较大细胞的固有电特性也被扭曲，潜在地导致新陈代谢和兴奋毒性应激的组合。此外，投入的结构也可能发生变化。为了研究大小、内在兴奋性和突触输入之间的关系，需要对成年状态下的小鼠运动神经元进行细胞内研究。我们已经开发了3种新的制剂，使首次在成年状态下对骶骨、腰椎和脑干运动神经元进行细胞内研究。两个是在体外，允许系统的药物研究，而一个是在原位，允许直接比较运动神经元的电特性和它的机械特性。因此，原位PREP研究将确定运动神经元在经历力故障时的特性。目的1使用原位准备来检验尺寸过大预示力量失效模式的假设。Aim 2使用体外骶髓准备来评估是否存在与大小扭曲相匹配的内在电特性和输入的平行上调，而Aim 3使用脑干切片来观察这些较小的运动神经元是否经历了相同的模式。在目标4中，长期给药被用来确定电特性的改变是否会导致大小的变化。总之，这项工作构成了研究肌萎缩侧索硬化发病机制的新途径。

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.