Elimination of the excess synaptic contacts established in the early stages of neuronal development is required to refine the function of neuronal circuits. Here we investigate whether secreted protein acidic and rich in cysteine (SPARC), a molecule produced by glial cells, is involved in synapse removal. SPARC production peaks when innervation of the rat superior cervical ganglion and the tail of Xenopus tropicalis tadpoles are remodeled. The formation of new cholinergic synapses in autaptic single-cell microcultures is inhibited by SPARC. The effect resides in the C-terminal domain, which is also responsible for triggering a concentration-and time-dependent disassembly of stable cholinergic synapses. The loss of synaptic contacts is associated with the formation of retracted axon terminals containing multivesicular bodies and secondary lysosomes. The biological relevance of in vitro results was supported by injecting the tail of Xenopus tropicalis tadpoles with peptide 4.2, a 20-aa sequence derived from SPARC that mimics full-length protein effects. Swimming was severely impaired at ∼5 h after peptide application, caused by the massive elimination of neuromuscular junctions and pruning of axonal branches. Effects revert by 6 d after injection, as motor innervation reforms. In conclusion, SPARC triggers a cell-autonomous program of synapse elimination in cholinergic neurons that likely occurs when protein production peaks during normal development.neuron-glia interaction | synapse elimination | SPARC | Xenopus tropicalis | cholinergic synapse

Highlights d High-pressure freezing of cultured brain tissue without cryoprotectants for EM d Flash-and-freeze functional EM of identified synapses in complex neural circuits d Direct correlation of activity-induced functional states and ultrastructure d Dissection of the spatiotemporal organization of endocytosis in identified synapses

Highlights d The Munc13-1 C 2 B domain controls synaptic vesicle replenishment rates d Blocking Ca 2+ -phospholipid-C 2 B signaling attenuates vesicle replenishment d Enhancing Ca 2+ -phospholipid-C 2 B signaling accelerates vesicle replenishment d This process determines short-term plasticity and fidelity of synaptic transmission

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