Optogenetic approaches to neuromuscular synapse elimination

消除神经肌肉突触的光遗传学方法

• 批准号: 8465923
• 负责人: RITA J. BALICE-GORDON
• 金额: $ 19.3万
• 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 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8465923
• 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.

描述（由申请人提供）：神经元与其突触伙伴的突触在开发过程中具有延展性，并且由于经验，在数量，强度和功能特性方面，例如短期和长期可塑性。研究良好的发展模型系统是小鼠神经肌肉突触，它在发育中经历了活动依赖性可塑性，这是其较小，较不访问的CNS对应物的标志。在胚胎晚期和早期生命中，神经肌肉突触被消除，其中一个轴突的突触与其他轴突的突触相匹配，这些轴突支配了相同的肌肉纤维。几条证据表明，最活跃的轴突将具有最强的突触，并成为赢家，并保持肌肉纤维的单个神经支配，而其他较低的活性轴突将衰弱，失去突触强度并消除。但是，尽管已知突触消除过程中事件的结构进程，并且已知功能进展的某些方面，但随着时间的推移，两者如何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区域的变化联系起来，或者触发突触弱化 - 或突触消除 - 与神经肌肉接头的不同活性。 在这里，我们建议测试以下假设：在双性支配的神经肌肉连接处，一个输入异突触的活性会削弱另一个输入，先前的突触损失，轴突萎缩和输入戒断。为了检验这一假设，我们将使用一系列转基因小鼠，其中小鼠Thy1.2启动子在所有胸骨类肌肉运动轴突及其神经末端驱动crunnerrhodopsin :: YFP的表达（Thy1-Chr2 :: YFP100）。在新生儿和成年小鼠的神经肌肉制剂中进行的初步研究表明，突触后肌肉纤维动作电位可以通过488 nm激光的简短脉冲聚焦于CHR2 :: YFP+运动轴突或其末端从1到100 Hz传递。当这些小鼠越过约50％的神经末端表达CFP的小鼠（THY1-CFP50％）时，可以在空间区分竞争的输入并用光差异刺激竞争的输入。我们建议将这些小鼠用于：1）确定一个时间参数和机制，通过这些参数刺激一个轴突会导致异突触弱化的突触突触的突触； 2）确定一个输入的异突触削弱如何导致突触丧失，轴突萎缩和输入戒断。这些研究将首次确定活性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，从而永久改变神经回路。