Vesicle dynamics: how synaptic proteins regulate different modes of neurotransmission

Authors

Errata

This article is corrected by:

  1. Errata: Corrigendum Volume 128, Issue 2, 340, Article first published online: 10 December 2013

Address correspondence and reprint requests to Jesica Raingo, Multidisciplinary Institute of Cell Biology (IMBICE), CIC-PBA CONICET, Argentina. E-mails: jraingo@imbice.org.ar; jraingo@gmail.com

Abstract

Central synapses operate neurotransmission in several modes: synchronous/fast neurotransmission (neurotransmitters release is tightly coupled to action potentials and fast), asynchronous neurotransmission (neurotransmitter release is slower and longer lasting), and spontaneous neurotransmission (where small amounts of neurotransmitter are released without being evoked by an action potential). A substantial body of evidence from the past two decades suggests that seemingly identical synaptic vesicles possess distinct propensities to fuse, thus selectively serving different modes of neurotransmission. In efforts to better understand the mechanism(s) underlying the different modes of synaptic transmission, many research groups found that synaptic vesicles used in different modes of neurotransmission differ by a number of synaptic proteins. Synchronous transmission with higher temporal fidelity to stimulation seems to require synaptotagmin 1 and complexin for its Ca2+ sensitivity, RIM proteins for closer location of synaptic vesicles (SV) to the voltage operated calcium channels (VGCC), and dynamin for SV retrieval. Asynchronous release does not seem to require functional synaptotagmin 1 as a calcium sensor or complexins, but the activity of dynamin is indispensible for its maintenance. On the other hand, the control of spontaneous neurotransmission remains less clear as deleting a number of essential synaptic proteins does not abolish this type of synaptic vesicle fusion. VGCC distance from the SV seems to have little control on spontaneous transmission, while there is an involvement of functional synaptic proteins including synaptotagmins and complexin. Recently, presynaptic deficits have been proposed to contribute to a number of pathological conditions including cognitive and mental disorders. In this review, we evaluate recent advances in understanding the regulatory mechanisms of synaptic vesicle dynamics and in understanding how different molecular substrates maintain selective modes of neurotransmission. We also highlight the implications of these studies in understanding pathological conditions.

Abbreviations used
RIM1/2

Rab3-interacting molecule type 1 and 2

RP

reserve pool or recycling pool

RRP

readily releasable pool

SNARE

soluble NSF attachment protein receptor

SV

synaptic vesicles

VGCC

voltage operated calcium channels

Central synapses contain a relatively small number of synaptic vesicles (SV) therefore SV need to be efficiently recycled to maintain reliable synaptic transmission. SV undergo three trafficking steps during neurotransmission: vesicle fusion or exocytosis, vesicle retrieval or endocytosis, and local recycling for reuse (Sudhof 1995, 2000; Kavalali 2006). The first event that occurs when action potentials arrive to the presynaptic terminals is a Ca2+ influx mediated by voltage-gated Ca2+ channels (VGCC). SVs sense the rise in Ca2+ and fuse with the plasma membrane, thereby releasing their chemical contents into a narrow cleft and relaying the information to the post-synaptic neurons. However, the initial step of neurotransmission at central synapses is fairly selective as only 10–20% of action potentials trigger neurotransmitter release (Goda and Sudhof 1997). Upon completion of fusion events, vesicles are rapidly retrieved back to the synaptic terminal and undergo maintenance routines, including neurotransmitter refilling, to accommodate multiple rounds of neurotransmission. Given that central synapses possess limited SV and only a small fraction of them actually serves as the workhorses of neurotransmission (Harata et al. 2011) (Zucker and Regehr), efficient SV endocytosis and recycling are important tasks to accomplish.

In central synapses, different modes of neurotransmission exist depending on timing as well as Ca2+ sensitivity. Besides the tightly synchronous release events that underlie fast synaptic transmission, there are rather less-regulated events such as spontaneous release of SVs, which can be observed at rest independent of action potentials (Katz and Miledi 1967), and delayed asynchronous release that occurs tenths to hundreds of milliseconds after re-polarization (Del Castillo and Katz 1954) (Fig. 1). In past years, many research groups have studied the mechanisms and physiological roles underlying these different modes of neurotransmission. This review will cover the various regulatory strategies that central synapses employ to balance between the excessive use and the sufficient supply of SV depending on modes of synaptic transmission and highlight their significance in pathological conditions.

Figure 1.

The figure illustrates the different modes of neurotransmission with cartoons representing the synaptic vesicle dynamics and real recordings to show the responses and time frame of each mode. The synaptic terminal cartoon is filled with synaptic vesicles (SVs) that are more prone to fuse spontaneously (green), or under stimulation during synchronous (orange) or asynchronous (yellow) neurotransmitter release. (a) This panel shows a cartoon representing the SVs recycling at rest and a mini inhibitory post-synaptic current (mIPSC) recording in hippocampal primary culture. (b) This panel shows a cartoon representing SVs recycling under electrical stimulation and an inhibitory post-synaptic current (IPSC) recording from hippocampal primary culture under field electrode stimulation at 10 Hz, (i) points to the 1st IPSC, (ii) points to the 45th IPSC recorded, and (iii) corresponds to the late neurotransmitter release after the stimulation stops. The (i) and (ii) IPSC traces are enlarged in the inset to display the differences in the noise level and timing. During stimulation, ongoing spontaneous transmission events are ignored for the simplicity.

The dynamic synaptic vesicle pools

The ultra-structural analysis of small central synapses suggests that SV appear morphologically uniform and ubiquitous in the presynaptic terminal (Mundigl and De Camilli 1999). Small central synaptic terminals contain hundreds of SV; however, most of them are known to be ‘dormant’ because they do not seem to be involved in synaptic transmission (Sudhof 2004; Harata et al. 2011; Rizzoli and Betz 2005). Only a small fraction (5–10%) of SV, collectively called the readily releasable pool (RRP), is set for fusion (Rizzoli and Betz 2005). Morphologically, SV belonging to the RRP are located in close proximity to the release site. The distance to the active zone determines the likelihood of release at small central synapses (Marra et al. 2012), although the close location of SV to active zone does not necessarily guarantee their fusion potency in larger synapses such as neuromuscular junction (Rizzoli and Betz 2005). Functionally, these vesicles readily respond to Ca2+-independent hypertonic stimulation (Rosenmund and Stevens 1996) as well as in synchrony to rapid Ca2+-dependent stimulation or increases in intra-synaptic Ca2+ (Murthy and Stevens 1999; Schneggenburger et al. 1999; Wu and Borst 1999). Another fraction of SV, often referred as the reserve pool or recycling pool (RP), serve as a reservoir for the RRP (Rizzoli and Betz 2005). This pool of vesicles is proposed to be responsible for replenishing the RRP on demand in a calcium-dependent manner because once the RRP is depleted, slower release kinetics are observed, presumably mediated by SV mobilized from the RP (Stevens and Wesseling 1998; Wang and Kaczmarek 1998). This classification in two pools of SV is a rather simplified model that only works for the fast SV release that is highly synchronized with action potentials. Other types of SV pools have been defined, such as a reluctant pool and the spontaneously recycling pool. Reluctant SV are documented when some SV, which do not respond to Ca2+-dependent action potentials, are drawn out by mechanistic secretagogues such as hypertonicity (Moulder and Mennerick 2005).

Functional diversity in SV pools has been observed in different types of synapses as well as modes of neurotransmission. For example, a pool of SV has been proposed to be designated to selectively carry out transmission at rest (spontaneous transmission) but not in response to action potentials in hippocampal neurons (Sara et al. 2005; Fredj and Burrone 2009; Chung et al. 2010). Similar observations were made in cortical inhibitory synapses (Mathew et al. 2005; Hablitz et al. 2011), suggesting that this could be a more general case. On the other hand two different pools of vesicles are responsible for synchronous (fast) and asynchronous (slow) SV release at the calyx of held at a wide range of Ca2+ concentrations, implying that there are distinctive properties in the release machinery that determine the timing of SV release (Sakaba 2006; Wolfel et al. 1999). We now have evidence that the SV membrane proteins participating in release determine the identity of a particular SV type and determine its mode of release (Raingo et al. 2012; Ramirez et al. 2012). A large collection of experiments that have genetically manipulated essential synaptic proteins support that the idea that there is great diversity in SV dynamics, which is achieved by possible heterogeneity in molecular composition (Takamori et al. 2000b, 2006), therefore in release probability (Wolfel et al. 2007), and/or alternatively, their position relative to VGCC (Wadel et al. 2011). Here, we summarize tentative molecular players that likely actively regulate SV dynamics and that also define functional differences between different types of SV.

Features of different neurotransmission modes

Fast evoked neurotransmitter release is tightly coupled to the action potential invasion in time (Sabatini and Regehr 1996). Synchronous neurotransmitter release is a key process to preserve timing and high fidelity of neuronal communication. However, neurotransmitter quanta may also be released with a delay in response to Ca2+ entry (Barrett and Stevens 1972), named as asynchronous release. This loosely coupled fusion is driven by residual Ca2+ at the microdomain of presynaptic terminal, presumably because of the temporary saturation in Ca2+ buffering capacity upon relatively large and rapid Ca2+ influx (Lu and Trussell 2000; Hefft and Jonas 2005; Maximov and Sudhof 2005; Sun et al. 2007). Although asynchronous release is generally present after a train of action potentials as a delayed return of the post-synaptic current level to the baseline, it occurs also after a single action potential (Iremonger and Bains 2007). Thus, to compare the degree of asynchronicity of neurotransmitter release among different synapses or conditions one can measure (i) the cumulative integration of total charge transferred in post-synaptic currents recorded during a train of stimulation, (ii) the increase in the variance of consecutive post-synaptic current episodes evoked during a high frequency train of stimulation and also, (iii) the integrative current that remains after the cessation of stimulation (Raingo et al. 2012) (see Fig. 1). Interestingly, some synapses seem to solely rely on this mode of delayed transmission. For example, cholecystokinin-containing GABAergic interneurons in the hippocampus have depend on a 3 : 1 ratio of asynchronous to synchronous release to obtain a long-lasting inhibitory input into principal neurons (Hefft and Jonas 2005).

Asynchronous neurotransmitter release has been proposed to be important for information discrimination. The selective disruption in synchronous neurotransmitter release seen in the vesicular calcium sensor synaptotagmin 1 (syt I) knock down mice model resulted in alterations in the frequency of hippocampal theta oscillation (Buzsaki 2002). In this model, viral delivery of short hairpin RNA sequences were used to knock down syt I to the hippocampus Since theta activity is implicated in hippocampus-dependent learning and memory processes, animals were tested in traditional fear conditioning tasks for memory retrieval. These animals displayed no impairment in fear memory acquisition; however, when syt I was knocked down in the prefrontal cortex instead of the hippocampus, fear memory retrieval was significantly impaired (Xu et al. 2012). This study suggests that there is a specific contribution of each mode of transmission for higher brain functions, such as cognition, as well as that might be differential contribution of the same types of transmission in different areas of the brain. It is thus valid to postulate that asynchronous release serves as a low pass filter in some brain areas such as hippocampus but not in others, such as entorhinal and prefrontal cortex, where more time-defined patterns of information coding seem to be required (Buzsaki 2012).

Synchronous neurotransmitter release depends on several synaptic proteins such as syt I and complexin (Yang et al. 2010; Martin et al. 2011; Xu et al. 2012). Animals with mutations in syt I die soon after birth and exhibit a severe reduction in synchronous release while leaving the asynchronous component relatively intact, suggesting the independent function of syt I as the selective calcium sensor for synchronous fusion events (Geppert et al. 1994; Maximov and Sudhof 2005). Specific interactions in a given synaptic protein could also play independent roles in different modes of neurotransmission. The mutations in the calcium binding C2B domain of syt I have been shown to disrupt synchronous release while calcium binding to the C2A domain attenuates asynchronous release (Yoshihara et al. 2010). Observations coming from the analysis of another synaptic protein, complexin, provide further support that synchronous transmission is maintained by a selective set of synaptic proteins. Disrupted complexin decreased synchronous release while augmenting spontaneous as well as asynchronous release, indicating the selective involvement of complexin in promoting synchronous release. In addition, the observation that knock down of complexin not only protected but also resulted in enhanced spontaneous transmission suggests a possible competition between two types of transmission (Yang et al. 2010; Martin et al. 2011).

In addition, spontaneous neurotransmission has recently attracted significant attention. Spontaneous transmission (also known as ‘miniature release’ or ‘minis’) is defined as neurotransmission occurring independently of action potentials, which generally refers to very low rate of SV fusion. A consensus is emerging that spontaneous transmission may serve several important roles in neuronal communication. It has been shown that, in brain slices, spontaneous GABA release could provide continuous background inhibition (Otis et al. 2004). In the cochlear nucleus, spontaneous GABAergic transmission may set the inhibitory tone of post-synaptic neurons (Lu and Trussell 2011). Spontaneous transmission has been also suggested to regulate receptor clustering (Saitoe et al. 2001) and to even trigger action potentials when membrane resistance is high (Carter and Regehr 2002; Sharma and Vijayaraghavan 2003). Furthermore, there is convincing evidence that spontaneous release could suppress the local dendritic protein translation machinery, thereby monitoring receptor composition at synapses (Sutton et al. 2004, 2006, 2007). More surprisingly, a subset of ionotropic NMDA-type glutamate receptors seems to be selectively activated by this particular mode of release, implying that spontaneous transmission may impact various aspects of neuronal communication (Atasoy et al. 2008).

The initial studies employing FM dye to selectively label the recycling of SV during trains of stimulation or at rest suggested their preferential reuse for one mode of transmission or the other. However, the observations made in a set of experiments by sequential FM dye loading during evoked and subsequent spontaneous vesicle recycling have been challenged by observations using simultaneous monitoring of two colors of FM dyes (Groemer and Klingauf 2007). Later, the discrepancy in the two studies was ascribed to the possible structural differences in two FM dyes used in these studies (Chung et al. 2010). The long-tailed FM1-43 seems to label an additional population of SV when used at rest, while the shorter FM2-10 dye labels the genuine pool of SV that are recycled spontaneously. Optical experiments using FM dyes also revealed different time scales of SV mobilization at rest and during stimulation in inhibitory synapses, generalizing the initial proposal of separate pools of SV for these two modes of neurotransmission (Mathew et al. 2008; Hablitz et al. 2009).

Completely independent approaches using biosyn, a biotinylated Vesicle associated membrane protein type 2 (VAMP2) probe, labeling with streptadivin (Mathew et al. 2008; Fredj and Burrone 2009) also supports the idea of a pool of SV assigned for spontaneous transmission [but see (Hua et al. 2011)]. More recently, another study using the biosyn labeling approach has expanded this idea and showed that separate SV pools maintain evoked and spontaneous transmission throughout neuronal development independent of post-synaptic contacts, suggesting presynaptic autonomous characteristic of segregated SV pools (Andreae et al. 2012).

Collectively, the studies discussed here propose that the identity and function of particular SV are determined by their preferred fusion mode possible determination. Further investigation is anticipated to reconcile some inconsistent observations.

Hallmark of neurotransmission modes: differential regulation by Ca2+

The entry of Ca2+ through VGCC is a key determinant for fast SV release at central synapses. In response to action potential arrival and VGCC activation, the Ca2+ concentration increases across the synaptic terminal and reaches the docking sites or active zones. The dynamics of this diffusion process may alter the synchronicity of vesicle fusion.

One of the most commonly used methods to monitor asynchronous release is detecting the occurrence of delayed post-synaptic currents after high frequency field electrical stimulation. Thus, initial studies have associated asynchronous release with vesicle fusion caused by high Ca2+ concentrations built by high frequency action potentials and/or low Ca2+ buffering at the presynaptic terminals. Therefore, asynchronous release was expected to be more probable at presynaptic terminals with a low probability of release that can maintain a high vesicle reservoir even after high activity periods. Alternatively, asynchronous release could arise from a pool of vesicles that differ in their calcium sensitivity. Under certain conditions Ca2+ influx could selectively activate synchronous or asynchronous release. At least three mechanisms have been proposed:

  1. The temporal resolution between Ca2+ influx induced by action potentials and SV fusion coupled to Ca2+ influx differs in synchronous and asynchronous release. The temporal precision largely depends on the distance between SV and VGCC, which matters for the timing of SV fusion. Fast synchronous neurotransmission is mediated by SV coupled to VGCC, while recruitment of new vesicles to the proximity of VGCC seems to contribute to the delayed asynchronous release. Thus, the distance between docked SV and VGCC could determine the timing of release, rather than differences in SV maturation or calcium sensitivity of SV (Wadel et al. 2007; Sakaba 2008). In this regard, the Rab3-interacting molecule types 1 and 2 (RIM1/2) are cytoskeletal matrix proteins enriched at presynaptic terminals. RIM1/2 contain PSD-95/Discs-large/ZO-1 (PDZ) domains that interact with the C-termini of presynaptic VGCC α-subunits (CaV2.1 and CaV2.2), targeting them to the active zone. Thus, RIM-induced high VGCC density speeds up release and promotes a tighter coupling between vesicles and VGCC (Han et al. 2011). Knocking down all RIMs subtypes ablates neurotransmitter release because of SV priming disruption and reduced VGCC density at the terminals. Sudhof and colleagues have shown that the PDZ domain of RIM is enough to target VGCC to the active zones and to recover synchronicity and normal calcium dependency of release in RIMs-deficient synapses (Kaeser et al. 2011).
  2. Calcium-dependent modulation of VGCC activity could contribute to asynchronous release at the synaptic terminals. Few and collaborators found that presynaptic VGCC CaV2.1 and CaV2.2 can maintain delayed Ca2+ currents during re-polarization, giving rise to slow tail currents (500 ms of duration) that could contribute to asynchronous release. This asynchronous current measured after depolarizing pulses increased over consecutive trials. Its sensitivity to the slow Ca2+ chelator EGTA and its decay time after high frequency stimulation match the properties of delayed asynchronous neurotransmitter release (Few et al. 2012), suggesting that accumulation of Ca2+ can activate further VGCC to maintain slower, asynchronous release.
  3. C2 domain proteins work as calcium sensors for neurotransmitter release. Several mechanisms have been proposed for this, but general consensus exists about the proteins that clamp release at rest and accelerate fusion at high Ca2+ concentrations (Walter et al. 2011). Syt I deficient neurons have no reduction in asynchronous neurotransmission, indicating that a different calcium sensor must exist for slow kinetic calcium-dependent SV release. Recently, a specific calcium sensor for asynchronous release has been proposed: Doc2, suggesting a distinct regulation in slow and fast SV release (Yao et al. 2011).

In the case of spontaneous neurotransmission, the frequency of SV release events increases at higher Ca2+ concentrations at presynaptic terminals. The participation of VGCC in controlling spontaneous neurotransmission, however, seems to be dependent on synaptic terminals. In neocortical neurons, spontaneous GABA release requires the opening of several types of VGCC; this is in contrast to spontaneous glutamate release that is not initiated by VGCC Ca2+ influx (Williams et al. 2012). Yet, the calcium sensors responsible for spontaneous release are less understood. There is a clear dependence of syt I but there are other models implicating a soluble currently unknown calcium sensor (Walter et al. 2011). Interestingly, spontaneous release seems to require Doc2 proteins but in a calcium independent manner, pointing to a dual model of spontaneous release control, one calcium dependent and one calcium independent (Pang et al. 2011).

In summary, calcium sensitivity of the different modes of neurotransmission is heterogeneous. The differences arise from differential interaction between VGCC and docked SV, as well as usage of different Ca2+ sensors, which could impact the timing of SV release.

Distinct recycling routes depending on the usage of SVs

Molecular evidence dissecting different types of transmission comes from one of the key molecular players implicated in SV recycling: Dynamin. Dynamin has been proposed to play an essential role in SV recycling because of its specialized role in scission events during re-formation of SV. Yet, when a dynamin 1 null mutant mice line was generated in 2007 (Ferguson et al. 2007), the mild phenotype of dynamin 1-lacking synapses came as a surprise: These mice exhibited typical development and had little deficit in excitatory synaptic transmission in cultured cortical synapses (Ferguson et al. 2007). Dynamin 1 deficiency caused rather mild yet significant deficits merely in SV endocytosis upon high frequency stimulation. Similar observations were made in dynamin 3 lacking synapses, another major isoform of dynamin in the brain (Raimondi et al. 2011). Later, double knock out animals were generated, which lacked both brain-specific dynamins (dynamin 1 and 3) (Raimondi et al. 2011; Lou et al. 2012). These synapses exhibited defective synchronous release although some levels of release remained (Raimondi et al. 2011). The dynamin-independent recycling pathway has also been proposed to be present in the calyx of Held synapses as multiple rounds of stimulation could induce spontaneous recovery of SV endocytosis in the presence of non-hydrolyzable GTP or GDP analogs such as GTPγS or GDPβS as well as of a dynamin inhibitor (Xu et al. 2012).

Notably, few alterations were observed in spontaneous transmission in these dynamin-lacking synapses (Ferguson et al. 2007; Xu et al. 2012; Raimondi et al. 2011). This aspect was further investigated by acutely inhibiting the GTPase activity of dynamin in hippocampal cultures. The acute inhibition of dynamin activity using a small molecular inhibitor, dynasore, attenuates both synchronous and asynchronous release while leaving spontaneous transmission almost intact (Chung et al. 2010). This observation suggests that common recycling pathways are utilized to maintain synchronous and asynchronous release (Chung et al. 2010). A recent study in neuromuscular junction synapses using another acute dynamin inhibitor, Dyngo 4A has reported contradicting observations that dynamin inhibition by either Dyngo-4A or dynasore has little effect on nerve evoked transmission (Harper et al. 2011). This observation suggests further complexity in SV usage between central and peripheral nervous system. Therefore, in central synapses, even though the lack of complexins enhanced both asynchronous and spontaneous release, the requirement of dynamin may differentiate these pools of vesicles giving rise to asynchronous versus spontaneous vesicle fusion events. Moreover, the conflicting observations from experiments with FM dyes mentioned could be reconciled by employing acute inhibition paradigm of dynamin activity. The additional FM dye uptake at rest by FM1-43 seems to be dynamin dependent. Given that spontaneous transmission remained undisturbed in the presence of dynasore, this observation suggests that the extra FM1-43 labeling at rest described earlier possibly includes an additional retractable component, and it lends further support to the notion that different pools of SVs can be distinguished depending on their propensity to fuse.

Molecular identity of synaptic vesicles as determinant of neurotransmission modes

In past years, a growing body of evidence suggests that specific proteins present at the SV are enriched at distinct vesicle pools and may determine the ‘character’ of SV to favor a certain mode of neurotransmission over others (Table 1). The list of these proteins includes several SNARE (soluble NSF attachment protein receptor) proteins. It is well established that a protein complex containing four SNARE motifs including vesicular and plasma membrane SNAREs is required for SV release. A stable SNARE complex requires three glutamate residues located within the SNARE motif contributed by Q-SNARE proteins and one arginine residue contributed by an R-SNARE protein. The canonical vesicular SNARE protein (vSNARE), VAMP2, is an R-SNARE with a short N terminal region (brevin family). There are also several SNARE proteins with longer N terminal domains (longins) present at the SV (Takamori et al. 2006). Hua et al. have shown that a longin vSNARE, tetanus toxin-insensitive vesicle associated membrane protein (VAMP7), labels a different pool of vesicles from the one labeled by VAMP2. Although the authors failed to observe any significant trafficking of VAMP7 in its native form, they found that a shorter mutant of VAMP7 without the long N terminus domain can increase spontaneous release without affecting electrical evoked release, demonstrating that manipulations of the SV protein collection can modify their routes (Hua et al. 2011). This study suggests that the long N terminus can act as a brake to vSNARE protein mobilization within the SV cycle. Using another approach, we studied if non-canonical vSNARE proteins can drive SV release in VAMP2-deficient neurons. Our work showed that at least two different SNARE proteins present at SV, Vps10p-tail-interactor1a (Vti1a), and VAMP4 (Takamori et al. 2006) can selectively restore spontaneous (Ramirez et al. 2012) and asynchronous release (Raingo et al. 2012) in VAMP2-deficient synapses, respectively. We also found that over-expression of these SV proteins could alter the extent of spontaneous and asynchronous release. In the case of spontaneous release, Ramirez and colleagues have shown that vesicles containing Vti1a, which controls endosomal trafficking, preferentially undergo spontaneous release, while they are not mobilized by action potential stimulation. On the other hand, expression of VAMP4, another SNARE protein present at endosomal pathways, modulates the degree of synchronicity of SV release. VAMP4 expression favors asynchronous release, without altering fast synchronous release. This study also found that VAMP4-containing SNARE complexes do not interact with syt I indicating that there must be a different calcium sensor responsible for asynchronous fusions. These data point to an alternative pathway to maintain asynchronous release in a syt I-independent manner, supporting that other calcium sensor protein must play a central role in determining the specificity of protein–protein interactions between synaptic players. Taken together, these results indicate that the conformation of the SNARE complex helps determine the dominate mode of release at a given presynaptic terminal. This provides further complexity in the SV dynamics by proposing that different vesicle pools may drive fast synchronous and delayed asynchronous release (Otsu et al. 2004). One open question is what drives localization of non-canonical SNARE proteins, such as VAMP7, VAMP4, and VtiI at synapses. The answer may reside in the protein structures. In the case of VAMP7, which is mainly targeted to the SV reserved pool, it is demonstrated to interact with the clathrin adaptor AP3, suggesting distinct SV trafficking pathway in maintenance of different SV pools.

Table 1. Tentative molecular markers differentially govern each mode of neurotransmission
ConditionModes of transmissionCa2+ sensorv-SNARE isoformOther SV proteinsRecycling
At restSpontaneous transmissionSoluble Ca2+ sensor?Vti1a  
During stimulationSynchronous transmissionSynaptotagmin-1VAMP-2RIM complexinDynamin dependent
Asynchronous transmissionSynaptotagmin-1 DOC2VAMP-4 Dynamin dependent

Pathological implications

Here, we summarized recent advances in understanding the dynamic regulation of SV usage during different types of neurotransmission. SV dynamics seem more complicated than they initially appeared. Spontaneous and asynchronous releases have long been considered as variations of fast synchronous release. On the basis of recent evidence, we outlined a new scenario where these different modes of neurotransmission operate via separate fusion machineries and trafficking mechanisms. Even though it is largely unknown how central synapses mechanically maintain different pools of SV at the molecular level, we now know that neurons can selectively modulate the degree of contribution of the types of release to inter-neuronal information transfer by employing separate pools of SV for each mode of transmission. In addition, it is obvious that synaptic deficits could be detrimental for brain function, particularly if there is a selective disruption of one mode of neurotransmission. As anticipated, analyses of synaptic deficits have given us clues into the circuitry of pathological brains, including those with neurodegenerative diseases as well as mental disorders. For example, VAMP7-lacking animals have been shown to exhibit increased anxiety, indicating that VAMP7 mediated neurotransmission has an important role in higher brain functions (Danglot et al. 2012). Neurons in a part of the brain called the lateral habenula, a subset of neurons which projects to the dopamine reward system, exhibit potentiated spontaneous transmission and enhanced release probability during evoked transmission in rodent models of depression (Li et al. 2011). This study suggests that abnormal synaptic vesicle recycling could contribute to the patho-physiology of depressive disorders. It is important to note that selective disruption in a given pool of SV assigned for a specific mode of neurotransmission could contribute to the development of mental disorders including anxiety disorders, depression and addiction. It was recently shown that selective blockade of spontaneous excitatory transmission in the hippocampus produced an anti-depressant like effect in animal models of depression, indicating that there is a specific contribution of spontaneous transmission in the expression of depressive behaviors (Autry et al. 2011). Drugs of abuse have also been proposed to not only reshape synaptic circuitry so that the brain is more sensitive to the drug of abuse but also may mobilize more SVs – thereby mediating enhanced synaptic efficacy in the addicted brain (Venton et al. 2006).

Taken together, a better understanding of the regulation of SV dynamics would elucidate the complexity of neurotransmission under physiological conditions as well as uncover steps in the SVs dynamics that can be affected under pathological conditions.

Acknowledgement

We thank Dr. Ege Kavalali and Dr. Summer Allen for critically reading this manuscript. This study was supported by a grant of the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (C.C. 2011-A010-0043 and 2012-A010-0030) and by grants of the National Agency of Science and Technology promotion, FONCyT, Argentina (J.R. PICT2010-1589 and PICT2011-1816). The authors declare no conflict of interest.

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