Calcium Signaling during Synaptic Refinement at the Neuromuscular Junction.

Neural activity plays a key role in pruning aberrant synapses in various neural systems, including the mammalian cortex, where low-frequency (0.01 Hz) calcium oscillations refine topographic maps. However, the activity-dependent molecular mechanisms remain incompletely understood. Activity-dependent pruning also occurs at embryonic neuromuscular junctions (NMJs), where low-frequency Ca oscillations are required for synaptic refinement and the response to the muscle-derived chemorepellant Sema2a. We examined embryonic growth cone filopodia to directly observe their exploration and to analyze the episodic Ca oscillations involved in refinement. Motoneuron filopodia repeatedly contacted off-target muscle fibers over several hours during late embryogenesis, with episodic Ca signals present in both motile filopodia as well as in later-stabilized synaptic boutons. The Ca transients matured over several hours into regular low-frequency (0.03 Hz) oscillations. imaging of intact embryos of both sexes revealed that the formation of ectopic filopodia is increased in heterozygotes. We provide genetic evidence suggesting a complex presynaptic Ca-dependent signaling network underlying refinement that involves the phosphatases calcineurin and protein phosphatase-1, as well the serine/threonine kinases CaMKII and PKA. Significantly, this network influenced the neuron's response to the muscle's Sema2a chemorepellant, critical for the removal of off-target contacts. To address the question of how synaptic connectivity is established during development, we examined the behavior of growth cone filopodia during the exploration of both correct and off-target muscle fibers in embryos. We demonstrate that filopodia repeatedly contact off-target muscles over several hours, until they ultimately retract. We show that intracellular signals are observed in motile and stabilized "ectopic" contacts. Several genetic experiments provide insight in the molecular pathway underlying network refinement, which includes oscillatory calcium signals via voltage-gated calcium channels as a key component. Calcium orchestrates the activity of several kinases and phosphatases, which interact in a coordinated fashion to regulate chemorepulsion exerted by the muscle.