Different mechanisms of synapsin-induced vesicle clustering at inhibitory and excitatory synapses.

Synapsins cluster synaptic vesicles (SVs) to provide a reserve pool (RP) of SVs that maintains synaptic transmission during sustained activity. However, it is unclear how synapsins cluster SVs. Here we show that either liquid-liquid phase separation (LLPS) or tetramerization-dependent cross-linking can cluster SVs, depending on whether a synapse is excitatory or inhibitory. Cell-free reconstitution reveals that both mechanisms can cluster SVs, with tetramerization being more effective. At inhibitory synapses, perturbing synapsin-dependent LLPS impairs SV clustering and synchronization of gamma-aminobutyric acid (GABA) release, while preventing synapsin tetramerization does not. At glutamatergic synapses, the opposite is true: synapsin tetramerization enhances clustering of glutamatergic SVs and mobilization of these SVs from the RP, while synapsin LLPS does not. Comparison of inhibitory and excitatory transmission during prolonged synaptic activity reveals that synapsin LLPS serves as a brake to limit GABA release, while synapsin tetramerization enables rapid mobilization of SVs from the RP to sustain glutamate release.