Perturbation of the synaptic release machinery in hippocampal neurons by overexpression of SNAP-25 with the Semliki Forest virus vector

We have examined whether the Semliki Forest virus (SFV) expression vector c an be used to manipulate the exocytotic machinery in cultured hippocampal n eurons. Autaptic responses were recorded in individually identified neurons which overexpressed either a non-synaptic protein, the transferrin recepto r, or the synaptic SNARE protein SNAP-25 (synaptosomal-associated protein o f 25 kDA), In neurons overexpressing the transferrin receptor, autaptic res ponses occurred in a similar proportion and had similar amplitudes (12-18 h postinfection) as in uninfected control neurons. With increasing time afte r the infection, an increasing proportion of the transferrin receptor-overe xpressing neurons showed changes in the shape of the cell body, but the aut aptic responses appeared normal as long as recordings could be performed (u p to 30 h postinfection), In contrast, in SNAP-25-overexpressing neurons, t he proportion of responding cells was reduced 12-18 h after the infection, and the amplitude of the autaptic current in responding neurons was also re duced. The sensitivity to exogenously applied glutamate was, however, uncha nged. Biochemical analysis showed that 50% of the overexpressed SNAP-25 was palmitoylated. The levels of two other SNAREs, syntaxin and synaptobrevin (also called vesicle-associated membrane protein), were not affected. Our r esults indicate that the SFV vector can provide an effective tool to study the function of proteins participating in neurotransmitter release.