Synaptic vesicle recycling in Vivo

The basic mechanism of neurotransmitter release at synapses is relatively well understood: upon arrival of an action potential, calcium influx into the nerve terminal triggers fusion of synaptic vesicles with the plasma membrane, resulting in the release of neurotransmitter from the vesicle interior into the synaptic cleft. The neurotransmitter diffuses to the postsynaptic cell, where it binds its respective receptors and evokes a change in membrane potential. At the presynaptic side, the vesicle membrane is retrieved and refilled with neurotransmitter, in what is termed vesicle recycling. Surprisingly, synapses can contain up to half a million vesicles, most of which can be forced to undergo recycling under high frequency stimulation in many preparations in vitro. However, whether and how they are involved in neurotransmission in vivo is unknown. The aim of this project was therefore to determine the number of vesicles used by a living animal during a defined time period. Vesicle use in vivo was monitored by three different approaches: first, the fluorescent dye FM 1-43 was injected into a living animal, which was then allowed to behave freely for a defined amount of time, during which the dye was taken up by recycling vesicles. At the end of the observation period, the organ of interest was dissected and photo-oxidized, a procedure that allows for the identification and quantification of labelled vesicles by electron microscopy. Using this technique, I found that only about 1-5% of all vesicles had undergone recycling over a few hours, in animal models ranging from nematodes and insects over fish, amphibians and birds to mammals. This limited vesicle use was confirmed by two independent experimental approaches, imaging of pHluorin Drosophila larvae combined with injection of the proton pump inhibitor bafilomycin and electron microscopy of the endocytic Drosophila mutant shibire. To determine by what molecular mechanism the majority of the vesicles might be prevented from participating in recycling, a Drosophila knockout strain of the vesicle-associated protein synapsin was investigated. Synapsin was found to restrict the mobility of the non-recycling vesicle population. In addition, synapsin deletion resulted in a substantial increase in the percentage of vesicles recycling in vivo, indicating that synapsin is one of the molecular players involved in distinguishing actively recycling and inactive vesicles. Finally, the functional role of the non-recycling vesicle population was investigated. It was shown that these vesicles might function as a molecular buffer, retaining soluble proteins involved in vesicle recycling in the synapse. Using immunostaining and stimulated emission depletion (STED) microscopy, the vesicle cluster (consisting largely of nonrecycling vesicles) was found to concentrate many accessory molecules, and this interaction was also confirmed by immunoblotting. In line with the buffer model, vesicle cluster disruption by black widow spider venom (BWSV) treatment resulted in protein loss into the axon. Further experiments using the calcium ionophore ionomycin indicated that molecular buffering is probably controlled by calcium, as was also confirmed biochemically. This would provide an explanation for how the vesicle cluster can provide these soluble molecules to a fused actively recycling vesicle upon demand. In summary, this project revealed that the majority of synaptic vesicles do not function in neurotransmitter release in vivo. Instead, they support synaptic transmission indirectly by ensuring the availability of accessory molecules for the recycling of the few active vesicles.