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 unknown how synapsins cluster SVs. Here we show that either liquid-liquid phase separation (LLPS) or tetramerization-dependent cross-linking can cluster SVs, depending upon whether a synapse is excitatory or inhibitory. Cell-free reconstitution revealed that both mechanisms can cluster SVs, with tetramerization bring more effective. At inhibitory synapses, perturbing synapsin-dependent LLPS impairs SV clustering and synchronization of GABA release, while perturbing 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 revealed 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.