Characterization of developmental and molecular factors underlying release heterogeneity at Drosophila synapses

Neurons communicate through neurotransmitter release at specialized synaptic regions known as active zones (AZs). Using biosensors to visualize single synaptic vesicle fusion events at Drosophila neuromuscular junctions, we analyzed the developmental and molecular determinants of release probability (P) for a defined connection with ~300 AZs. P was heterogeneous but represented a stable feature of each AZ. P remained stable during high frequency stimulation and retained heterogeneity in mutants lacking the Ca²⁺ sensor Synaptotagmin 1. P correlated with both presynaptic Ca²⁺ channel abundance and Ca²⁺ influx at individual release sites. P heterogeneity also correlated with glutamate receptor abundance, with high P connections developing receptor subtype segregation. Intravital imaging throughout development revealed that AZs acquire high P during a multi-day maturation period, with P heterogeneity largely reflecting AZ age. The rate of synapse maturation was activity-dependent, as both increases and decreases in neuronal activity modulated glutamate receptor field size and segregation.