Current models of synaptic vesicle trafficking implicate a core complex of proteins comprised of N-ethylmaleimide-sensitive factor (NSF), soluble NSF attachment proteins (SNAPs), and SNAREs in synaptic vesicle fusion and neurotransmitter release. Despite this progress, major challenges remain in establishing the in vivo functions of these proteins and their roles in determining the physiological properties of synapses. The present study employs glutamatergic adult neuromuscular synapses of Drosophila, which exhibit conserved properties of short-term synaptic plasticity with respect to mammalian glutamatergic synapses, to address these issues through genetic analysis. Our findings establish an in vivo role for SNAP-25 in synaptic vesicle priming, and support a zippering model of SNARE function in this process. Moreover, these studies define the contribution of SNAP-25-dependent vesicle priming to the detailed properties of short-term depression elicited by paired-pulse (PP) and train stimulation. In contrast, NSF is shown here not to be required for WT PP depression, but to be critical for maintaining neurotransmitter release during sustained stimulation. In keeping with this role, disruption of NSF function results in activity-dependent redistribution of the t-SNARE proteins, SYN-TAXIN and SNAP-25, away from neurotransmitter release sites (active zones). These findings support a role for NSF in replenishing active zone t-SNAREs for subsequent vesicle priming, and provide new insight into the spatial organization of SNARE protein cycling during synaptic activity. Together, the results reported here establish in vivo contributions of SNAP-25 and NSF to synaptic vesicle trafficking and define molecular mechanisms determining conserved functional properties of short-term depression.comatose ͉ Drosophila ͉ priming ͉ SNARE ͉ temperature-sensitive A wide variety of synapses exhibit short-term synaptic depression in response to repetitive stimulation. Although the underlying mechanisms remain a matter of intensive study and debate (1), it is generally agreed that many forms of short-term depression include activity-dependent changes in neurotransmitter release. In some cases, depression has been attributed to depletion of releaseready synaptic vesicle pools (1-6), and in depth analysis of certain mammalian synapses has defined the physiological factors underlying and accompanying depletion (7-10). Such studies have established the detailed functional properties of short-term synaptic depression. To further investigate the molecular determinants of these properties, the present study combines genetic approaches with detailed electrophysiological analysis of short-term depression at adult neuromuscular synapses of Drosophila. This system permits conserved properties of short-term synaptic depression to be examined in temperature-sensitive (TS) paralytic mutants allowing acute perturbation of specific gene products at native synapses.Studies of the neurotransmitter release apparatus have defined core protein interactions i...