In vivo calcium signaling during synaptic refinement at the Drosophila neuromuscular junction

Abstract

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 Drosophila neuromuscular junctions (NMJs), where low-frequency Ca2+ oscillations are required for synaptic refinement and the response to the muscle-derived chemorepellant Sema2a. We examined embryonic growth cone filopodia in vivo to directly observe their exploration and to analyze the episodic Ca2+ oscillations involved in refinement. Motoneuron filopodia repeatedly contacted off-target muscle fibers over several hours during late embryogenesis, with episodic Ca2+ signals present in both motile filopodia as well as in later-stabilized synaptic boutons. The Ca2+ transients matured over several hours into regular low-frequency (0.03Hz) oscillations. In vivo imaging of intact embryos of both sexes revealed that the formation of ectopic filopodia is increased in Sema2a heterozygotes. We provide genetic evidence suggesting a complex presynaptic Ca2+-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.