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

描述（由申请人提供）： 这项建议的重点是确定运动神经元的功能缺陷，因为他们在ALS小鼠模型中退化，根据最近发表的初步结果表明，运动神经元的属性，通常指定其激活模式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。运动单位由运动神经元、轴突和受神经支配的肌纤维组成，运动神经元是运动单位的中枢。因此，大小不仅涉及细胞体，还涉及树突（反映输入的数量）和轴突终末分支（与受神经支配的肌纤维的数量成比例）。正常情况下，运动神经元从小到大被激活：S型运动单元在运动神经元解剖和肌纤维数量方面都很小，所有这些都很慢。运动单位逐渐变大和变快（FR型和FF型）。然而，在ALS的标准动物模型（突变型SOD1小鼠）中对外周肌纤维去神经支配的研究表明，最初产生力的失败以相反的顺序发生：FF > FR >S，即从大到小。这种相反的序列表明，过大的尺寸是导致退化的缺陷，事实上，我们最近惊讶地发现，突变的SOD 1运动神经元在出生前10天就开始过度生长。这是在第一FF运动单元开始不能产生力之前很久（约50天），甚至在典型症状发作之前更久（90天）。值得注意的是，这些较大细胞的内在电特性也被扭曲，可能导致代谢和兴奋性毒性应激的结合。此外，投入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，允许在成人状态下对骶腰椎和脑干运动神经元进行第一次细胞内研究。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性能，其机械性能。因此，原位预备研究将确定运动神经元的性质，因为它经历了力的失败。目的1使用原位制备来检验过量尺寸预测力失效模式的假设。目标2使用体外骶髓制备来评估内在电特性和输入是否存在平行上调以匹配大小的失真，而目标3使用脑干切片来观察这些较小的运动神经元是否经历相同的模式。在目标4中，慢性药物施用用于确定电特性的改变是否引起尺寸的变化。总之，这项工作构成了一个新的方法来研究ALS的机制。

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.