BIO254:Recycle

INTRODUCTION

Synaptic vesicles are neurotransmitter-containing, membrane-bound organelles. They are initially generated in the cell bodies of neurons, and are subsequently transported along microtubules to axonal terminals. Depolarization of presynaptic nerve terminals leads to calcium influx into the cell. The calcium influx triggers the vesicles to fuse with the plasma membrane and release their neurotransmitter into the synaptic cleft. The rate at which vesicles can be transported from the soma to axon terminals is not sufficient for continuous neural activity. This lead to the hypothesis of SYNAPTIC VESICLE RECYCLING. Synaptic vesicle recycling is the process of transmitter release, followed by endocytosis of the vesicular membrane and refilling of the newly formed vesicles with transmitter in the nerve terminal. The idea of vesicle recycling was initially proposed by Bittner and Kennedy in 1970 (1). In 1973 Heuser and Reese were the first to demonstrate recycling of synaptic vesicles at nerve terminals (2). Using electron microscopy (EM) to visualize isolated frog neuromuscular junctions pre and post nerve stimulation, they showed that depletion of synaptic vesicles following nerve stimulation correlated with an increase in plasma membrane surface area. Prolonged stimulation resulted in appearance of endosomes (cisternae) within the cell, which disappeared after rest as vesicles reappeared in the nerve terminals. To determine the timing of movement through these stages of vesicular recycling, they filled the synaptic cleft with horseradish peroxidase (HRP) and again using EM, looked at HRP localization after nerve stimulation. They found HRP in endocytosed coated vesicles immediately after nerve stimulation, followed by localization to endosomes and finally, an hour after stimulation, in synaptic vesicles. Importantly, these experiments were conducted in the relatively large frog NMJ and thus did not exclude other possible mechanisms for vesicle recycling in the much smaller mammalian central synapse.

Figure taken from Heuser and Reese, 1973

MECHANISMS OF VESICLE RECYCLING

Two additional mechanisms of synaptic vesicle recycling have been proposed in addition to the classical clathrin mediated endocytosis model demonstrated by Heuser and Reese. Also based on ultrastructural evidence, Ceccarelli et al. proposed that vesicles fuse transiently with the terminal membrane and reform into functional vesicles (3). This idea later became known as the kiss-and-run hypothesis. The kiss-and-run hypothesis states that transmitter is released through the transient formation of a fusion pore between the vesicle and cell membrane. While each vesicle takes approximately 14 seconds to complete a cycle through the complete fusion model, the kiss-and-run mode occurs in less than one second.

The first direct evidence for the kiss-and-run hypothesis was provided independently by Aravanis, Pyle and Tsien and by Gandhi and Stevens in 2003 (4,5). Both groups used fluorescent markers to track individual vesicles in hippocampal neurons following an electrical stimulation.

Aravanis et al. used FM-dye labeling to study single vesicular events in hippocampal neurons. Cultures were bathed in FM1-43 and electrically stimulated, causing the dye to be endocytosed through vesicle recycling. FM1-43 was then removed from the extracellular solution, causing it to wash out from the plasma membrane but remain in the protected endocytosed vesicles. Cultures were then stimulated a second time, causing exocytosis and dye release from the labeled vesicles. Aravanis et al. found that upon fusion only a portion of the dye was released from an individual vesicle, and further, another quick electrical stimulation caused slightly more dye release. However if the latency between the two shocks was more than 23 seconds, further release was not seen. These results supported a mechanism slower than the kiss-and-run model (too fast for dye to leave vesicles), but faster than the complete fusion model seen in the frog NMJ.

Gandhi and Stevens used a pH sensitive GFP, pHluorin, fused to the intravesicular domain of synaptobrevin, to monitor vesicle recycling. This fusion protein, synaptopHluorin, fluoresces at high pHs, such as in the synaptic cleft. At low pHs, such as in synaptic vesicles, fluorescence is decreased. Monitoring the timing of fluorescence they concluded there are three modes of vesicle recycling. The first two are similar involving a vesicle that does not collapse and a fusion pore that remains open for less than a second (kiss-and-run) or 8-21 seconds (compensatory). They termed the third and longest (45 seconds) mechanism the stranded mode. In the stranded model, the vesicle membrane incorporates into plasma membrane, but cannot be endocytosed until another action potential causes an additional necessary increase in calcium.

Based on these studies, a kiss-and-run mechanism seemed to dominate vesicle recycling in the mammalian central nervous system. Recently, however, Granseth et al. demonstrated the classical clathrin-mediated mechanism predominates in the hippocampus (6). Using a similar technique as Gandhi and Stevens, Granseth et al. designed sypHy by fusing pHluorin to synaptophysin, another vesicle membrane protein. Using sypHy they found a time constant of 15 seconds for endocytosis. To determine if clathrin is necessary for vesicle recycling, they disrupted the classical pathway by two methods, RNAi knockdown of the clathrin heavy chain and overexpression of a dominant-negative form of the clathrin-adaptor protein, AP180. The resulting complete blockage of endocytosis supported their hypothesis that the clathrin mediated mechanism predominately functions in hippocampal cultures.

It remains unclear whether the clathrin coated endocytosis model or the kiss-and-run model of synaptic vesicle recycling predominates in the mammalian central nervous system. Currently the exocytosis-endocytosis pathway originally proposed in 1973 prevails and it will be interesting to see if the kiss-and-run model remains important in the intact central nevous system.

REFERENCES

1. Bittner,G.D. & Kennedy,D. Quantitative aspects of transmitter release. J. Cell Biol. 47, 585-592 (1970).

2. Heuser,J.E. & Reese,T.S. Evidence for recycling of synaptic vesicle membrane during transmitter release at the frog neuromuscular junction. J. Cell Biol. 57, 315-344 (1973).

3. Ceccarelli,B., Hurlbut,W.P. & Mauro,A. Turnover of transmitter and synaptic vesicles at the frog neuromuscular junction. J. Cell Biol. 57, 499-524 (1973).

4. Aravanis,A.M., Pyle,J.L. & Tsien,R.W. Single synaptic vesicles fusing transiently and successively without loss of identity. Nature 423, 643-647 (2003).

5. Gandhi,S.P. & Stevens,C.F. Three modes of synaptic vesicular recycling revealed by single-vesicle imaging. Nature 423, 607-613 (2003).

6. Granseth,B., Odermatt,B., Royle,S.J. & Lagnado,L. Clathrin-mediated endocytosis is the dominant mechanism of vesicle retrieval at hippocampal synapses. Neuron 51, 773-786 (2006).

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25 April 2024