Synaptic physiology and ultrastructure in comatose mutants define an in vivo role for NSF in neurotransmitter release

N-Ethylmaleimide-sensitive fusion protein (NSF) is a cytosolic protein thou ght to play a key role in vesicular transport in all eukaryotic cells. Alth ough NSF was proposed to function in the trafficking of synaptic vesicles r esponsible for neurotransmitter release, only recently have in vivo experim ents begun to reveal a specific function for NSF in this process. Our previ ous work showed that mutations in a Drosophila NSF gene, dNSF1, are respons ible for the temperature-sensitive paralytic phenotype in comatose (comt) m utants. In this study, we perform electrophysiological and ultrastructural analyses in three different comt alleles to investigate the function of dNS F1 at native synapses in vivo. Electrophysiological analysis of postsynapti c potentials and currents at adult neuromuscular synapses revealed that in the absence of repetitive stimulation, comt synapses exhibit wild-type neur otransmitter release at restrictive (paralytic) temperatures. In contrast, repetitive stimulation at restrictive temperatures revealed a progressive, activity-dependent reduction in neurotransmitter release in comt but not in wild type. These results indicate that dNSF1 does not participate directly in the fusion of vesicles with the target membrane but rather functions in maintaining the pool of readily releasable vesicles competent for fast cal cium-triggered fusion. To define dNSF1 function further, we used transmissi on electron microscopy to examine the distribution of vesicles within synap tic terminals, and observed a marked accumulation of docked vesicles at res trictive temperatures in comt. Together, the results reported here define a role for dNSF1 in the priming of docked synaptic vesicles for calcium-trig gered fusion.