Inhibition of neurotransmitter release in the lamprey reticulospinal synapse by antibody-mediated disruption of SNAP-25 function

The lamprey giant reticulospinal synapse can be used to manipulate the mole cular machinery of synaptic vesicle exocytosis by presynaptic microinjectio n. Here we test the effect of disrupting the function of the SNARE protein SNAP-25, Polyclonal SNAP-25 antibodies were shown in an in vitro assay to i nhibit the binding between syntaxin and SNAP-25, When microinjected presyna ptically, these antibodies produced a potent inhibition of the synaptic res ponse, Ba2+ spikes recorded in the presynaptic axon were not altered, indic ating that the effect was not due to a reduced presynaptic Ca2+ entry, Elec tron microscopic analysis showed that synaptic vesicle clusters had a simil ar organization in synapses of antibody-injected axons as in control axons, and the number of synaptic vesicles in apparent contact with the presynapt ic plasma membrane was also similar. Clathrin-coated pits, which normally o ccur at the plasma membrane around stimulated synapses, were not detected a fter injection of SNAP-25 antibodies, consistent with a blockade of vesicle cycling. Thus, SNAP-25 antibodies, which disrupt the interaction with synt axin, inhibit neurotransmitter release without affecting the number of syna ptic vesicles at the plasma membrane. These results provide further support to the view that the formation of SNARE complexes is critical for membrane fusion, but not for the targeting of synaptic vesicles to the presynaptic membrane.