Optogenetic approaches to neuromuscular synapse elimination

描述（由申请人提供）：神经元与其突触伴侣形成的突触在发育过程中具有可塑性，并且由于经验，在数量、强度和功能特性（如短期和长期可塑性）方面具有可塑性。一个充分研究的模型系统发育，活动依赖性的可塑性是小鼠神经肌肉突触，它经历活动依赖性的可塑性在发展中，这是一个标志，其较小的，较难接近的中枢神经系统的同行。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。一些证据表明，最活跃的轴突将拥有最强的突触，并成为赢家，维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，失去突触强度，并被淘汰。然而，虽然突触消除过程中事件的结构进展是已知的，并且功能进展的某些方面是已知的，但两者如何随着时间的推移相互关联是完全未知的。此外，以前的研究没有直接联系的时间信息活动模式/刺激的变化，突触强度的变化，突触面积的变化，或触发突触弱化-或突触消除-与差异活动在神经肌肉接头原位。 在这里，我们建议测试的假设，在双重神经支配的神经肌肉接头，一个输入异突触的活动削弱其他输入，突触损失，轴突萎缩和输入撤回之前。为了验证这一假设，我们将使用一系列转基因小鼠，其中小鼠Thy1.2启动子驱动所有胸锁乳突肌运动轴突及其神经末梢（Thy 1-ChR 2：：YFP 100）中的视紫红质：：YFP表达。对新生和成年小鼠的神经-肌肉制备物的初步研究表明，通过聚焦到ChR 2：：YFP+运动轴突或其末端上的488 nm激光的短暂脉冲，可以在许多小时内引发突触后肌纤维动作电位，所述脉冲以1至100 Hz的频率递送。当这些小鼠与在~50%的神经末梢（Thy 1-CFP50%）中表达CFP的小鼠杂交时，竞争性输入可以在空间上被区分并用光进行差异刺激。我们建议使用这些小鼠：1）确定刺激一个轴突引起未刺激轴突的突触的异突触弱化的时间参数和机制; 2）确定一个输入的异突触弱化如何导致突触丢失，轴突萎缩和输入撤回。这些研究将首次建立活动导致突触强度变化的机制的重要空间和时间方面，最终导致永久改变神经回路的突触消除。 公共卫生相关性：这项研究将为控制发育中突触命运的活动依赖性规则提供重要的新信息。发育中小鼠的神经肌肉突触，其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，通过该机制活动导致突触强度的异突触变化，最终导致突触消除，永久改变神经回路。当考虑脊髓损伤和神经肌肉轴疾病的治疗干预时，这些信息是重要的，这些疾病损害突触维持并最终导致运动神经元失活和/或死亡。

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.