Synaptic vesicle dynamics within neuromuscular junctions of synapto-phluorin expressing mice

The mammalian neuromuscular junction (NMJ) is a useful model synapse to study the relationship between synaptic structure and function, although these have rarely been studied together at the same synapses. At developing NMJs, different motor axon inputs to muscle fibers compete for maintenance of their synapses. Competition results in progressive changes in synaptic structure and strength that lead to the weakening and loss of some inputs, a process that has been called synapse elimination. Important questions that remain to be addressed include what is the temporal relationship between changes in synaptic strength and structure during this developmental period and how are synaptic structure and function related in the adult NMJ. To address these issues, we generated transgenic lines of mice in which the thy1.2 promoter drives expression of synapto-pHluorin (spH), as a means of optically measuring synaptic vesicle distribution and release. In both neonatal and adult NMJs spH is highly expressed in presynaptic terminals and is co-localized with other synaptic vesicle proteins. Experiments in adult NMJs reveal that spH does not alter normal synaptic function, that nerve stimulation leads to readily detectable and reproducible fluorescence changes that vary with stimulus frequency, and that these fluorescence changes are reliable indicators of neurotransmitter release. Measurements of activity induced fluorescence intensity changes in both adult and neonatal NMJs reveal a surprising amount of heterogeneity in synaptic vesicle release throughout individual motor axon terminals. The amount of release at a particular site, however, is highly correlated to the relative abundance of synaptic vesicles there, indicating that a relatively constant fraction of the total vesicular pool is released in response to neural activity. These studies reveal previously unknown relationships between synaptic structure and function at the mammalian NMJ and demonstrate the usefulness of spH expressing mice as a tool for studying neuromuscular synapses in adults as well as during development. These experiments should provide groundwork for future studies on the processes underlying activity dependent plasticity in the developing and adult nervous system