Optogenetic approaches to neuromuscular synapse elimination

消除神经肌肉突触的光遗传学方法

• 批准号: 8302585
• 负责人: RITA J. BALICE-GORDON
• 金额: $ 22.8万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-15 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8302585
• 关键词: Action Potentials; Adult; Area; Atrophic; Axon; Biological Models; Caliber; Cessation of life; Development; Embryo; Event; Hour; Image; In Situ; Lasers; Life; Light; Link; Maintenance; Motor; Motor Neurons; Mus; Muscle Fibers; Myoblasts; Neonatal; Nerve; Nerve-Muscle Preparations; Neuromuscular Diseases; Neuromuscular Junction; Neurons; Pattern; Physiologic pulse; Property; Spinal; Spinal cord injury; Sternocleidomastoid Muscle; Study models; Synapses; Testing; Therapeutic Intervention; Time; Transgenic Mice; Withdrawal; Work; Xenopus; experience; functional outcomes; in vivo; nerve supply; neural circuit; neuromuscular; optogenetics; postnatal; postsynaptic; promoter; relating to nervous system; research study

DESCRIPTION (provided by applicant): Synapses made by a neuron with its synaptic partners are malleable during development, and as a consequence of experience, with respect to number, strength, and functional properties such as short and long term plasticity. A well studied model system for developmental, activity-dependent plasticity is mouse neuromuscular synapses, which undergo activity-dependent plasticity in development that is a hallmark of their smaller, less accessible CNS counterparts. During late embryonic and early postnatal life, neuromuscular synapses undergo elimination, in which the synapses of one axon are pitted in competition against the synapses of other axons innervating the same muscle fiber. Several lines of evidence suggest that the most active axon will have the strongest synapses and emerge as the winner, maintaining single innervation of a muscle fiber, while other, less active axons will wither, lose synaptic strength, and become eliminated. However, while the structural progression of events during synapse elimination is known, and some aspects of the functional progression are known, how the two are interrelated over time is entirely unknown. Furthermore, no previous studies have directly linked temporal information about activity patterns/stimulation to changes in synaptic strength to changes in synaptic area, or have triggered synapse weakening - or synapse elimination - with differential activity at neuromuscular junctions in situ. Here we propose to test the hypothesis that at dually innervated neuromuscular junctions, the activity of one input heterosynaptically weakens the other input, preceding synapse loss, axon atrophy and input withdrawal. To test this hypothesis, we will use a line of transgenic mice in which the mouse Thy1.2 promoter drives expression of Channelrhodopsin::YFP in all sternomastoid muscle motor axons and their nerve terminals (Thy1-ChR2::YFP100). Preliminary studies in nerve- muscle preparations from neonatal and adult mice show that postsynaptic muscle fiber action potentials can be elicited for many hours by brief pulses of 488 nm laser light focused onto ChR2::YFP+ motor axons or their terminals delivered from 1 to 100 Hz. When these mice are crossed to mice that express CFP in ~50% of nerve terminals (Thy1-CFP50%), competing inputs can be spatially discriminated and differentially stimulated with light. We propose to use these mice to: 1) to determine the temporal parameters and mechanism by which stimulation of one axon causes heterosynaptic weakening of the synapses of the unstimulated axon; and 2) determine how heterosynaptic weakening of one input results in synapse loss, axon atrophy and input withdrawal. These studies will establish, for the first time, important spatial and temporal aspects of the mechanism by which activity leads to changes in synaptic strength, culminating in synapse elimination that permanently alters neural circuitry. PUBLIC HEALTH RELEVANCE: The proposed studies will provide important, new information on the activity-dependent rules that govern the fate of developing synapses. Neuromuscular synapses in developing mice, whose maintenance is modulated by neural activity, will be used as a model system. These studies will establish important and previously unknown spatial and temporal features of the mechanism by which activity leads to heterosynaptic changes in synaptic strength, culminating in synapse elimination that permanently alters neural circuitry. This information is important when considering therapeutic interventions for spinal cord injury and diseases of the neuromuscular axis that compromise synaptic maintenance and culminate in motor neuron inactivity and/or death.

描述(由申请人提供)：神经元与其突触伙伴形成的突触在发育过程中是可塑性的，作为经验的结果，在数量、强度和功能特性(如短期和长期可塑性)方面是可塑性的。小鼠神经肌肉突触是一个研究得很好的发育、活动依赖性可塑性的模型系统，它在发育过程中经历活动依赖性可塑性，这是较小、较难获得的中枢神经系统突触的一个标志。在胚胎晚期和出生后早期，神经肌肉突触经历消除，其中一个轴突的突触与支配同一肌肉纤维的其他轴突的突触竞争。几条证据表明，最活跃的轴突将拥有最强的突触，并成为赢家，保持肌肉纤维的单一神经支配，而其他不太活跃的轴突将萎缩，失去突触力量，并被淘汰。然而，虽然突触消除过程中事件的结构进程是已知的，功能进程的某些方面也是已知的，但这两者如何随着时间的推移相互关联是完全未知的。此外，以前没有研究将有关活动模式/刺激的时间信息与突触强度的变化与突触面积的变化直接联系起来，也没有研究直接将突触减弱或突触消除与原位神经肌肉接头的不同活动联系起来。 在此，我们提出了一个假设，即在双重神经支配的神经肌肉接头上，一个输入的活动异向地削弱了另一个输入，先于突触丢失、轴突萎缩和输入撤退。为了验证这一假设，我们将使用一组转基因小鼠，其中小鼠Thy1.2启动子驱动通道视紫红质：：YFP在所有胸乳突肌运动轴突及其神经末梢(Thy1-ChR2：：YFP 100)中的表达。对新生和成年小鼠神经肌肉标本的初步研究表明，488 nm激光聚焦于ChR2：：YFP+运动轴突或其终末，以1~100赫兹传递，可诱发突触后肌肉纤维动作电位数小时。当这些小鼠与在~50%的神经末梢(Thy1-CFP50%)表达CFP的小鼠杂交时，竞争的输入可以在空间上被区分，并用光进行不同的刺激。我们建议使用这些小鼠来：1)确定刺激一个轴突导致未刺激轴突的突触异突触减弱的时间参数和机制；以及2)确定一个输入的异突触减弱如何导致突触丢失、轴突萎缩和输入撤退。这些研究将首次确定活动导致突触强度变化的机制的重要空间和时间方面，最终导致突触消除，永久改变神经回路。 与公共健康相关：拟议中的研究将提供重要的、新的信息，这些信息涉及支配突触发育命运的活动依赖规则。发育中的小鼠的神经肌肉突触的维持由神经活动调节，将被用作模型系统。这些研究将建立重要的和以前未知的空间和时间特征的机制，通过这种机制，活动导致突触强度的异构性变化，最终导致突触消除，永久改变神经回路。当考虑对脊髓损伤和神经肌肉轴疾病进行治疗干预时，这些信息是重要的，这些疾病损害了突触的维持，最终导致运动神经元不活动和/或死亡。