Summary Recent studies suggest that synaptic vesicles (SVs) giving rise to spontaneous neurotransmission are distinct from those that carry out evoked release. However, the molecular basis of this dichotomy remains unclear. Here, we focused on two non-canonical SNARE molecules, Vps10p-tail-interactor-1a (vti1a) and VAMP7, previously shown to reside on SVs. Using simultaneous multicolor imaging at individual synapses, we could show that compared to the more abundant vesicular SNARE synaptobrevin, both vti1a and VAMP7 were reluctantly mobilized during activity. Vti1a, but not VAMP7, showed robust trafficking under resting conditions which could be partly matched by synaptobrevin. Furthermore, loss of vti1a function selectively reduced high frequency spontaneous neurotransmitter release detected postsynaptically. Expression of a truncated version of vti1a augmented spontaneous release more than full-length vti1a, suggesting an autoinhibitory process regulates vti1a function. Taken together, these results support the premise that in its native form vti1a selectively maintains spontaneous neurotransmitter release.

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Recent studies suggest that stimulus-evoked and spontaneous neurotransmitter release processes are mechanistically distinct. Here we targeted the non-canonical synaptic vesicle SNAREs Vps10p-tail-interactor-1a (vti1a) and vesicle-associated membrane protein 7 (VAMP7) to specifically inhibit spontaneous release events and probe whether these events signal independently of evoked release to the postsynaptic neuron. We found that loss of vti1a and VAMP7 impairs spontaneous high-frequency glutamate release and augments unitary event amplitudes by reducing postsynaptic eukaryotic elongation factor 2 kinase (eEF2K) activity subsequent to the reduction in N-methyl-D-aspartate receptor (NMDAR) activity. Presynaptic, but not postsynaptic, loss of vti1a and VAMP7 occludes NMDAR antagonist-induced synaptic potentiation in an intact circuit, confirming the role of these vesicular SNAREs in setting synaptic strength. Collectively, these results demonstrate that spontaneous neurotransmission signals independently of stimulus-evoked release and highlight its role as a key regulator of postsynaptic efficacy.

Action potential-evoked vesicle fusion comprises the majority of neurotransmission within chemical synapses, but action potentialindependent spontaneous neurotransmission also contributes to the collection of signals sent to the postsynaptic cell. Previous work has implicated spontaneous neurotransmission in homeostatic synaptic scaling, but few studies have selectively manipulated spontaneous neurotransmission without substantial changes in evoked neurotransmission to study this function in detail. Here we used a quadruple knockdown strategy to reduce levels of proteins within the soluble calcium-binding double C2 domain (Doc2)-like protein family to selectively reduce spontaneous neurotransmission in cultured mouse and rat neurons. Activity-evoked responses appear normal while both excitatory and inhibitory spontaneous events exhibit reduced frequency. Excitatory miniature postsynaptic currents (mEPSCs), but not miniature inhibitory postsynaptic currents (mIPSCs), increase in amplitude after quadruple knockdown. This increase in synaptic efficacy correlates with reduced phosphorylation levels of eukaryotic elongation factor 2 and also requires the presence of elongation factor 2 kinase. Together, these data suggest that spontaneous neurotransmission independently contributes to the regulation of synaptic efficacy, and action potential-evoked and spontaneous neurotransmission can be segregated at least partially on a molecular level.