Synaptotagmin(syt), a synaptic vesicle-specific protein known to bind Ca2+ in the presence of phosphoiipids, has been proposed to mediate Ca2+-dependent neurotransmitter release. We have addressed the role of syt in neurotransmltter release in vivo by generating mutations in synapfofagmin(syf) in the fruitfly and assaying the subsequent effects on neurotransmission. Most embryos that lack syt fail to hatch and exhibit very reduced, uncoordinated muscle contractions. Larvae with partial lack-of-function mutations show almost no evoked excitatory junctional potentials (EJPs) in 0.4 mM Ca2+ and a 15-fold reduction in EJP amplitude in 1.0 mM ca2+ when compared with heterozygous controls. In contrast, we observe an increase in the frequency of spontaneous miniature EJPs in the mutants. These results provide in vivo evidence that syt plays a key role in Ca*+ activation of neurotransmitter release and indicate the existence of separate pathways for evoked and spontaneous neurotransmitter release. introduction

Communication between neurons and their targets at a synapse is initiated by release of neurotransmitter from presynaptic terminals (Del Castilio and Katz, 1956; Miledi, 1973). This release is thought to be based exclusively on the fusion of synaptic vesicles with the presynaptic cell membrane (Ceccarelli and Hurlbut, 1960; Heuser et al., 1979). Neurotransmission is initiated by depolarization of the presynaptic terminal that allows influx of extracellular Ca2+ via voltage-gated Ca*+ channels. The influx of Ca*+ is absolutely required for evoked neurotransmitter release (Mulkey and Zucker, 1991). In both squid giant axons and mammalian neurons, the lag between Ca2+ influx and postsynaptic effect is approximately 200 us (Llinas et al., 1961; Cope and Mendell, 1962). The requirement for Ca*+ and the speed of the process have led to the conclusion that Ca*+ must directly and rapidly activate the machinery involved with fusion of synaptic vesicles with the presynaptic membrane.