SUPER-RESOLUTION IMAGING OF FUNCTIONAL SYNAPTIC VESICLE POOLS

Neurotransmitter release occurs through a tightly-regulated process known as exocytosis, which can either occur via the fusion of synaptic vesicles with the plasma membrane after depolarisation of the presynaptic terminal, or through the spontaneous fusion of vesicles. The specific identity of the vesicles underlying these two forms of release has been a subject of controversy for decades, with differing views as to whether these forms of release share a common vesicle pool or arise from two separate pools. Due to the limits imposed on conventional microscopy methods by the diffraction of light, the distribution of these pools within synapses has yet to be directly investigated. Super-resolution imaging methods such as stochastic optical reconstruction microscopy (STORM) circumvent the limits imposed by the diffraction barrier and thus offer the possibility to investigate the nanoarchitecture of the synapse. We have performed live labelling of synaptic vesicle pools in primary rat neuronal cultures using the genetically-encoded reporter biosyn in combination with simultaneous 2-colour 3D-dSTORM imaging to investigate whether the two forms of release are supported by different vesicle pools. Geometric analyses of the resulting point clouds have revealed that spontaneously released vesicles are more diffusely distributed than their evoked counterparts within boutons. Further, we find that on average spontaneously released vesicles are localised further away from key active zone proteins (e.g., bassoon) than those released by depolarisation. Thus, we provide direct evidence of distinct spatial organisation for these two pools, supporting the hypothesis that these two forms of release are sustained by independent pools.