Background: Neuronal synapses accumulate clusters of vesicles at active zones. These clusters are comprised of several functional pools. The reserve pool of synaptic vesicles serves to replenish the ready releasable pool during strong activation of synapses and to sustain neurotransmitter release. Overexpression and/or mutations in genes encoding the proteins organizing synaptic vesicles in the reserve pool in synapses, such as synapsin and intersectin are implicated in several neurological disorders, such as Down syndrome, epilepsy, and autism. Recent studies demonstrated that interaction between these proteins is regulated by a phosphorylation-dependent autoinhibitory switch, which is present both in invertebrate and vertebrate synapses [1], [2]. How exactly these proteins interact at different stages of the synaptic vesicle cycle and during synaptic plasticity is still largely unknown.
Aims of the study: To identify possible evolution steps in vesicle clustering leading to the accumulation of vesicles into the reserve pool at the synaptic active zone.
Methods: Experiments were performed in river lampreys (Lampetra fluviatilis). These animals have synapses that are established by reticulospinal axons on dendrites of spinal neurons and characterized by large synaptic vesicle clusters, which accumulate on the surface of the axons. Isolated preparations of the lamprey spinal cord were prepared as earlier described [3]. Synapses were stimulated at 5 Hz for 30 min with an exracellular electrode and fixed directly following stimulation or left at rest from 30 min to 1h. Preparations were fixed in glutaraldehyde and embedded in resins as earlier described [3]. Serial ultrathin sections were cut from embedded specimens, stained and examined in Tecnai 12 electron microscope. For immunocytochemistry the specimens were fixed in 4% paraformaldehyde with 0,5% paraformaldehyde for 4h and then embedded into LRGold and labeled wiht antibodies as described previously [3].
Results: We found organized clusters of vesicles floating in the axoplasmic matrix in axons left at rest after stimulation. These clusters could be small or composed of hundreds of vesicles. The common feature of these clusters was in presence of electron-dense fibrils interspacing vesicles and passing through entire clusters. 3D analysis of these structures revealed that vesicle are linked to these electron dense fibrils by thin filaments. These floating clusters were not labeled with anti-actin antibodies, but displayed labeling with synapsin and intersectin antibodies as synaptic vesicles clusters at active zones as revealed by post-embedding immunogold techniques. In several instances such organization of vesicles was observed within reserve pools of vesicles at active zones strongly thus suggesting that the two synaptic vesicle clustering mechanisms are linked functionally.
Conclusions: Our studies describe a novel organization of synaptic vesicles in vertebrate axons, which occurs outside synaptic active zones. Our studies show that these clusters of vesicles derive from synapses and are formed through a reorganization of the intravesicular protein matrix clustering synaptic vesicles in the reserve pool. Our data suggest that this evolution of the intravesicular matrix of the reserve pool underlies a novel form of synaptic plasticity.

This study was supported by RSF grant, project 16-15-10273.