Synapses can fire at very rapid rates. Synapses also need to fire reliably and they are often located far away from the soma where most protein synthesis occurs. Perhaps as one mechanism to enable the synapse to function under these conditions is that synaptic vesicles are recycled. Synaptic vesicles undergo repeated rounds of synthesis and fusion locally. This synaptic vesicle is highly regulated and a molecular understanding of many of the steps in the cycle is emerging from the combined efforts many years of molecular, cellular, genetic and biochemical studies.
The events of the synaptic vesicle cycle can be divided into steps:
Trafficking to the synapse
Synaptic vesicle components are initially trafficked to the synapse using members of the kinesin motor family. In C. elegans the major motor for synaptic vesicles is UNC-104. There is also evidence that other proteins such as UNC-16/Sunday Driver regulate the use of motors for transport of synaptic vesicles. Transport vesicles used to traffic synaptic vesicle components problably differ in composition from mature synaptic vesicles though these differences have not been well defined. Another question that has not been well resolved is how motors release cargo at synapses. Finally, though there is substancial evidence that active zone components are also trafficked to synaspes on vesicles, the nature of the motors that perform this transport remains unclear.
Transmitter Loading
Once at synaptic sites, vesicles are loaded with neurotransmitter. Loading of transmitter is an active processes requiring a neurotransmitter transporter and a vacuolar-type proton pump ATPase that provides a pH and electrochemical gradient. These transporters are selective for different classes of transmitters. Interestingly, the identity of many of these transporters was determined through the molecular characterization of C. elegans mutants. Characterization of unc-17 and unc-47, which encode the vesicular acetylcholine transporter and vesicular GABA transporter, defined the founding members of these two families of transporters. To what extent loading is regulated to modulate transmitter release is not known.
Docking
Next, loaded vesicles must dock near release sites. Docking is a step of the cycle that we know little about. Though many proteins on vesicles and at release sites have been identified, none of the identified protein interactions that occur between vesicle proteins and proteins at release sites can account for docking. Mutants in rab-3 and unc-18 alter vesicle docking or vesicle organization at release sites, but they do not completely disrupt docking. Perhaps surprisingly, the SNAREs, which are thought to mediate fusion, do not appear to be involved in the docking process.
Priming
When vesicles initially dock they are not fusion competent. Vesicles first need to be primed so that they are able to fuse rapidly in response to calcium influx. This priming step is thought to involve the formation of partially assembled SNARE complexes. The proteins UNC-13 and Rim participate in this event. UNC-13 is thought to stimulate the change of the t-SNARE syntaxin from a closed conformation to an open conformation, which stimulates the assembly of v-SNARE /t-SNARE complexes. Rim also appears to regulate priming, but is not essential for the step.
Fusion
Primed vesicles fuse very quickly in response to calcium elevations in the cytoplasm. This fusion event is thought to be mediated directly by the SNAREs and driven by the energy provided from SNARE assembly. The calcium-sensing trigger for this event is the calcium-binding synaptic vesicle protein synaptotagmin. The ability of SNAREs to mediate fusion in a calcium-dependent manner recently has been reconstituted in vitro. Consistent with SNAREs being essential for the fusion process, v-SNARE and t-SNARE mutants of C. elegans are lethal and even viable hypomorphic mutants exhibit almost no evoked release. Similarly, mutants in Drosphila and knockouts in mouse indicate that these SNARES play a critical role in synaptic exocytosis. However, our understanding of fusion is far from complete. One major issue in the field is defining the contribution of incompete fusion events (also refered to as kiss-and-run) where a fusion pore forms briefly to allow exit of transmitter without complete fusion of the vesicle and plasma membranes. There is substantial evidence that kiss and run occurs at least at some synapses. Studies examining the rates of endocytosis suggest that both very rapid and slow endocytosis occur at synapses. Analysis of SNAP-25 and synaptobrevin mouse knockout mutants revealed that although evoked release was completely disrupted, that some spontaneous fusion events continued. Similarly, C. elegans null mutants lacking the v-SNARE synaptobrevin and the t-SNARE SNAP-25 still are capable of some movements, in contrast to completely paralysis seen in t-SNARE syntaxin mutants. Thus, whether the neuronal SNAREs are absolutely required for all exocytotic events at the synapse remains unresolved.
Endocytosis
Synaptic vesicle proteins that have been incorporated into the plasma membrane after fusion are retrieved by endocytosis. A large cohort of proteins have been identified which participate in these events including endophilin, synaptojanin, synaptotagmin, dynamin, clathrin, AP180, as well as others. Rates of endocytosis vary widely in different preparations and also vary depending on the stimulus intensity. A variety of evidence suggests that different pathways may be utilized even at the same synapse. One of the major outstanding questions remains whether synaptic vesicle membranes are selectively endocytosed as synaptic vesicle entities or as precursors with other membrane proteins which must then traffic through an endosomal compartment to yield mature vesicles.
Neurotransmitter clearance
After transmitter is released and binds to receptor on the postsynaptic membrane, it must be cleared to permit subsequent signaling. Some transmitters like dopamine are transported back into the the neuron using plasma membrane transporters. Other transmitters including acetylcholine and some neuropeptides are broken down in the synaptic cleft. Acetyl cholineesterase does this job at the neuromuscular junction. In C. elegans, aldicarb, an inhibitor of the esterase, has been a powerful pharmacological tool for both isolating mutants that disrupt the transmission process and as an simple indirect method for quantifying the defects in the exocytosis of transmitter. Wild type animals placed on aldicarb are unable to control muscle contraction and eventually die due to hypercontraction of muscle. By contrast, mutants even with relatively modest defects in synaptic transmission have altered responses to aldicarb. Thus, by selecting for mutants resistant to the effects of aldicarb, Jim Rand’s lab, Erik Jorgensen’s lab, my lab as well as others, have been able to isolate a large number of mutants in the components of the synaptic transmission apparatus. These mutants have provided a wealth of genetic tools for the study of synaptic transmission in C. elegans.