Optogenetic approaches to neuromuscular synapse elimination

描述（由申请人提供）：由神经元及其突触伙伴组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。小鼠神经肌肉突触在发育过程中经历了活动依赖的可塑性，这是它们更小、更难以接近的中枢神经系统的一个标志。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。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，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，功能进展的某些方面也是已知的，但两者如何随着时间的推移而相互关联是完全未知的。此外，以前的研究没有直接将活动模式/刺激的时间信息与突触强度的变化和突触区域的变化联系起来，也没有在神经肌肉连接处原位的不同活动触发突触减弱或突触消除。

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.