Synaptic & intrinsic motoneuron excitability in ALS excitotoxicity

描述（由申请人提供）：肌萎缩侧索硬化症（ALS）是一种成人发病的神经退行性疾病，其特异性靶向运动神经元。在20%的家族性ALS（fALS）病例中，超氧化物歧化酶（SOD 1）存在特定突变。具有突变的人SOD1的小鼠显示出与人ALS患者相同的病理学和表型症状，因此被广泛用作fALS的动物模型。然而，SOD 1在决定运动神经元死亡中的作用仍不清楚。一个主要的假设是，由于运动神经元过度兴奋导致细胞内钙超载引起的兴奋性毒性导致选择性运动神经元死亡，因为运动神经元缓冲钙的能力较低。该建议旨在确定野生型和SOD 1运动神经元之间内在和突触运动神经元兴奋性变化的相对贡献。此外，还将评估唯一FDA批准的ALS药物阿舒唑对内在和突触运动神经元兴奋性的影响。这项建议的目的将通过使用胚胎培养的运动神经元，新生儿脊髓切片，和一种新的体外骶髓制备完成。这些制剂的使用将允许野生型和SOD 1小鼠之间的运动神经元兴奋性的有效和完整的表征。在体外骶髓准备允许内在和突触运动神经元兴奋性进行评估，通过细胞内记录在整个发展和成年的时间点。它还使我们能够在治疗水平和多个时间过程中给予阿曲唑，以广泛表征阿曲唑随时间的影响。新生儿脊髓切片制备允许使用全细胞膜片钳配置使用药物的直接浴应用以抑制特定离子通道来评估内在与突触运动神经元兴奋性。此外，可以确定阿曲唑对运动神经元的直接和即时影响。胚胎培养的运动神经元提供了一个了解出生前运动神经元异常的窗口，也为我们提供了一个没有突触输入的准备。在该制剂中，可以分离出在多个时间过程中施用的阿曲唑对运动神经元的内在性质的影响。ALS运动神经元变性的原因需要确定，以开发新的药物治疗和了解运动神经元功能的差异或变化将导致这些未来药物的具体目标。此外，运动神经元功能的早期可检测的变化可能使使用先发制人的治疗，可以显着延迟甚至阻止疾病的发展。

DESCRIPTION (provided by applicant): Amyotrophic lateral sclerosis (ALS) is an adult onset neurodegenerative disease which specifically targets motoneurons. In 20% of familial ALS (fALS) cases, there is a specific mutation in superoxide dismutase (SOD1). Mice with mutated human SOD1, display the same pathological and phenotypical symptoms in human ALS patients and are therefore widely used as an animal model of fALS. However, the role SOD1 plays in determining motoneuron death remains unclear. A leading hypothesis is that excitotoxicity caused by an overload of calcium into the cell due to motoneuron hyperexcitability causes selective motoneuron death because motoneurons have a low capacity to buffer calcium. This proposal aims to determine the relative contribution of changes in intrinsic and synaptic motoneuron excitability between wild type and SOD1 motoneurons. Furthermore, the effect of Riluzole, the only FDA approved drug for ALS, on both intrinsic and synaptic motoneuron excitability will also be assessed. The aims of this proposal will be accomplished through the use of embryonic cultured motoneurons, neonatal spinal cord slices, and a new in vitro sacral cord preparation. The use of these preparations will allow an efficient and complete characterization of motoneuron excitability between wild type and SOD1 mice. The in vitro sacral cord preparation allows intrinsic and synaptic motoneuron excitability to be assessed through intracellular recordings at time points throughout development and adulthood. It also enables us to administer Riluzole at therapeutic levels and in multiple time courses to extensively characterize the effects of Riluzole over time. The neonatal spinal cord slice preparation allows intrinsic versus synaptic motoneuron excitability to be assessed using direct bath application of drugs to inhibit specific ion channels using a whole cell patch clamp configuration. Also, the direct and immediate effects of Riluzole on motoneurons can be determined. The embryonic cultured motoneurons provide a window into motoneuron abnormalities present before birth and also give us a preparation where no synaptic inputs are present. In this preparation the effects of Riluzole administered over multiple time courses on the intrinsic properties of motoneurons can be isolated. The cause(s) of motoneuron degeneration in ALS needs to be determined in order to develop novel drugs treatments and understanding differences or changes in motoneuron function will lead to specific targets for these future drugs. Furthermore, early detectable changes in motoneuron function may enable the use of pre-emptive therapies that can significantly delay or even arrest the development of the disease.