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.