[show abstract][hide abstract] ABSTRACT: Using pHluorin-tagged synaptic vesicle proteins we have examined the partitioning of these probes into recycling and nonrecycling pools at hippocampal nerve terminals in cell culture. Our studies show that for three of the major synaptic vesicle components, vGlut-1, VAMP-2, and Synaptotagmin I, approximately 50-60% of the tagged protein appears in a recycling pool that responds readily to sustained action potential stimulation by mobilizing and fusing with the plasma membrane, while the remainder is targeted to a nonrecycling, acidic compartment. The fraction of recycling and nonrecycling (or resting) pools varied significantly across boutons within an individual axon, from 100% resting (silent) to 100% recycling. Single-bouton bleaching studies show that recycling and resting pools are dynamic and exchange between synaptic boutons. The quantitative parameters that can be extracted with the approaches outlined here should help elucidate the potential functional role of the resting vesicle pool.
[show abstract][hide abstract] ABSTRACT: Presynaptic nerve terminals rely heavily on membrane traffic to maintain efficient neurotransmission between cells. It is often assumed that, as neurons can fire action potentials at high frequency, the cell biological machinery for vesicle cycling must be highly specialized. Here, we examine the demands that are placed on the recycling machinery in three model systems used to characterize vertebrate vesicle recycling--small hippocampal synapses, calyx-type brainstem synapses, and ribbon-type sensory synapses--and the molecular pathways thought to underlie certain aspects of the vesicle cycle.
Trends in Cell Biology 09/2006; 16(8):413-20. · 11.72 Impact Factor
[show abstract][hide abstract] ABSTRACT: During recycling of synaptic vesicles (SVs), the retrieval machinery faces the challenge of recapturing SV proteins in a timely and precise manner. The significant dilution factor that would result from equilibration of vesicle proteins with the much larger cell surface would make recapture by diffusional encounter with the endocytic retrieval machinery unlikely. If SV proteins exchanged with counterparts residing at steady state on the cell surface, the dilution problem would be largely avoided. In this scenario, during electrical activity, endocytosis would be driven by the concentration of a pre-existing pool of SVs residing on the axonal or synaptic surface rather than the heavily diluted postfusion vesicular pool. Using both live cell imaging of endogenous synaptotagmin Ia (sytIa) as well as pHluorin-tagged sytIa and VAMP-2, we show here that synaptic vesicle proteins interchange with a large pool on the cell axonal surface whose concentration is approximately 10-fold lower than that in SVs.
[show abstract][hide abstract] ABSTRACT: During sustained action potential (AP) firing at nerve terminals, the rates of endocytosis compared to exocytosis determine how quickly the available synaptic vesicle pool is depleted, in turn influencing presynaptic efficacy. Mechanisms, including rapid kiss-and-run endocytosis as well as local, preferential recycling of docked vesicles, have been proposed as a means to allow endocytosis and recycling to keep up with stimulation. We show here that, for CNS nerve terminals at physiological temperatures, endocytosis is sufficiently fast to avoid vesicle pool depletion during continuous AP firing at 10 Hz. This endocytosis-exocytosis balance persists for turnover of the entire releasable pool of vesicles and allows for efficient escape of FM 4-64, indicating that it is a non-kiss-and-run endocytic event. Thus, under physiological conditions, the sustained speed of vesicle membrane retrieval for the entire releasable pool appears to be sufficiently fast to compensate for exocytosis, avoiding significant vesicle pool depletion during robust synaptic activity.