Abstract
Synapse formation requires the accumulation of cytomatrix proteins and voltage-gated Ca(2+) channels (VGCCs) at presynaptic active zones (AZs). At Drosophila melanogaster larval neuromuscular junctions, a sequential process of AZ maturation is observed, with initial incorporation of early scaffolds followed by arrival of late scaffolds and VGCCs. To examine how AZ maturation regulates presynaptic output, serial imaging of AZ formation and function was performed at time-stamped synapses of male larvae expressing glutamate receptors linked to the photoconvertible protein mMaple. Quantal imaging demonstrated older synapses have higher synaptic efficacy and sustain greater release across development, while immature sites lacking VGCC accumulation supported spontaneous fusion. To examine how activity regulates AZ maturation, the effects of cell autonomous disruptions to neurotransmitter release were analyzed. Decreased synaptic transmission reduced AZ seeding and caused hyperaccumulation of material at existing AZs. Generation of an endogenous photoconvertible version of the AZ scaffold protein BRP revealed neuronal silencing decreased the protein's turnover. Although enlarged AZs are also observed in rab3 mutants, activity reduction acted through an independent mechanism that required postsynaptic glutamate receptor-dependent signaling. Endogenous tagging of the Unc13B early AZ scaffold and the Unc13A late AZ scaffold revealed activity reduction decreased seeding of both early and late scaffolds, in contrast to rab3 mutants. Together, these data indicate AZ maturation regulates presynaptic release mode and output strength, with neuronal activity shaping both AZ number and size across development.