Functionally heterogeneous synaptic vesicle ... - Wiley Online Library

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J Physiol 594.4 (2016) pp 825–835

The Journal of Physiology

Neuroscience

TO P I C A L R E V I E W

Functionally heterogeneous synaptic vesicle pools support diverse synaptic signalling ´ Simon Chamberland and Katalin Toth Quebec Mental Health Institute, Department of Psychiatry and Neuroscience, Université Laval, Quebec City, Quebec, Canada, G1J 2G3

Molecular heterogeneity

Distinct modes of vesicle recycling

Functional heterogeneity

Abstract Synaptic communication between neurons is a highly dynamic process involving specialized structures. At the level of the presynaptic terminal, neurotransmission is ensured by fusion of vesicles to the membrane, which releases neurotransmitter in the synaptic cleft. Depending on the level of activity experienced by the terminal, the spatiotemporal properties of calcium invasion will dictate the timing and the number of vesicles that need to be released. Diverse presynaptic firing patterns are translated to neurotransmitter release with a distinct temporal feature. Complex patterns of neurotransmitter release can be achieved when different vesicles respond to distinct calcium dynamics in the presynaptic terminal. Specific vesicles from different pools are recruited during various modes of release as the particular molecular composition of their membrane proteins define their functional properties. Such diversity endows the presynaptic terminal with the ability to respond to distinct physiological signals

Simon Chamberland is currently completing a PhD in Neuroscience at Université Laval. He ´ has been working on synaptic transmission in Katalin Toth’s lab since 2012, combining electrophysiological approaches with two-photon calcium imaging to study the details of glutamate release from mossy fibre terminals. He is interested in the mechanisms supporting the speed and precision ´ earned her PhD in of neurotransmitter release and neuronal communication. Katalin Toth 1995 from the Eötvös Loránd University in Budapest, Hungary, under the supervision of Tamás F. Freund, after completing an undergraduate degree in Biology. She then spent the next 2 years as postdoctoral fellow in the laboratory of Richard Miles at the Pasteur Institute in Paris, France, and studied the properties of synaptic interactions between connected pairs of neurons. She moved to the National Institutes of Health in the USA where she worked with Chris J. McBain on the plastic properties of hippocampal networks. She established her laboratory in 2000 at Laval University where she is currently a Professor in the Department of Psychiatry and Neuroscience. Her research is focused on presynaptic release mechanisms and information processing at hippocampal mossy fibres.

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DOI: 10.1113/JP270194

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via the mobilization of specific subpopulation of vesicles. There are several mechanisms by which a diverse vesicle population could be generated in single presynaptic terminals, including distinct recycling pathways that utilize various adaptor proteins. Several additional factors could potentially contribute to the development of a heterogeneous vesicle pool such as specialized release sites, spatial segregation within the terminal and specialized delivery pathways. Among these factors molecular heterogeneity plays a central role in defining the functional properties of different subpopulations of vesicles. (Received 8 February 2015; accepted after revision 23 November 2015; first published online 28 November 2015) ´ Corresponding author K. Toth: Quebec Mental Health Institute, Department of Psychiatry and Neuroscience, Faculty of Medicine, Université Laval, 2601 chemin de la Canardière, Quebec City, QC, Canada, G1J 2G3. Email: [email protected] Abstract figure legend Single presynaptic terminals transmit information via functionally distinct modes of neurotransmitter release. Functional heterogeneity in the presynaptic terminal is largely supported by the existence of distinct pools of vesicles expressing unique sets of vesicle membrane proteins. These distinct pools can be generated via different modes of vesicle recycling pathways.

Introduction

Highly specialized functions of various subcellular elements endow neurons with a wide range of physiological information-processing capabilities. Dendrites detect incoming information and axons transmit the processed information. A single axon has several hundreds of synaptic terminals each containing large numbers of synaptic vesicles. Morphologically these vesicles are quite similar: their shape, size and spatial distribution is rather uniform. However, in spite of the uniform morphological features of synaptic vesicles, their functional heterogeneity was suggested by several recent studies (Sakaba & Neher, 2001a; Hua et al. 2011). This functional heterogeneity can be envisioned via the integration of different sets of proteins into the vesicular membrane (Takamori et al. 2006). This complexity within the presynaptic terminal could play a key role in various forms of synaptic communication. There are several factors that can define distinct vesicle pools. Vesicles are released during different presynaptic activities, they contribute to various forms of release, they have different sets of membrane proteins, and they can be generated via diverse endocytotic recycling pathways. How do these factors contribute to the functionally diverse forms of neurotransmitter release exhibited by presynaptic terminals and what is the relationship between these categories? Functional heterogeneity among vesicles based on their availability for release

Traditionally vesicles are sorted into categories based on their availability for release and the degree of difficulty to trigger their fusion with the membrane (Fig. 1A). According to these criteria, three pools of synaptic vesicles were identified as the readily releasable pool (RRP), the recycling pool and the reserve or resting

pool (Sudhof, 2000; Zucker & Regehr, 2002; Rizzoli & Betz, 2005; Alabi & Tsien, 2012). While the number and distribution of synaptic vesicles in the three pools differs substantially between organisms and synapses, several common characteristics are observed and will be emphasized below. Vesicles belonging to the RRP are the first to be recruited during neuronal activity. Most of these vesicles are docked at the active zone or located in its vicinity, where activation of release machinery can trigger their release with short latency (Schikorski & Stevens, 2001; Dittman & Ryan, 2009). Although this pool accounts for only a small fraction of the total number of vesicles available (1–2%) in several synapses, including CA1 hippocampal excitatory synapses (Schikorski & Stevens, 1997) and the frog neuromuscular junction (Rizzoli & Betz, 2005), their availability for release and their fast recycling allow them to support the majority of physiological neurotransmission. Vesicles located in the recycling pool are not exocytosed right away during weak neuronal activity. Vesicles in this pool are largely scattered within the synaptic terminal but display high mobility. Synaptic vesicles in the recycling pool encompass a larger fraction of the total number of vesicles than the RRP and represent approximately 10–20% of the total vesicles available in several synapses, including hippocampal presynaptic terminals (Harata et al. 2001). Together, these characteristics suggest that the recycling pool acts as an effective backup mechanism to secure synaptic activity on longer time scales. Although it largely dominates the other pools in terms of numbers (80–90% of total synaptic vesicles), mobilization of the resting pool vesicles to the membrane requires strong activity, demanding tens of seconds of high-frequency electrical stimulation (Richards et al. 2000; Denker et al. 2009). The conditions required for its complete depletion remain mysterious at certain synapses, including the calyx of Held, as approximately 50% of the pool could never be released C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society

The functional heterogeneity of synaptic vesicle pools

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(de Lange et al. 2003; Fernandez-Alfonso & Ryan, 2008; Wyatt & Balice-Gordon, 2008). Why should a larger portion of vesicles inside synaptic terminals participate minimally in neurotransmission? Intriguingly, experiments performed for longer periods of time in vivo or using physiological firing frequencies suggest that all vesicles from the reserve pool could be released. Experiments performed at the frog neuromuscular junction for periods up to 8 h using low-frequency stimulation (2 Hz) were able to induce fusion of the full vesicle pool (Ceccarelli et al. 1972; Betz

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& Henkel, 1994) and challenge the view that the resting pool contributes little to physiological neurotransmission (Denker et al. 2011). As such, the frontiers between the recycling pool and the resting pool may be thinner than previously thought. Vesicles could move through vesicle pools in a state-dependent manner. This evidence supports the notion that vesicles evolve as they move through different pools following endocytosis (Denker & Rizzoli, 2010). The level of activity exhibited by the presynaptic terminal will also influence the form of endocytosis released vesicles will go through. Vesicles can recycle via

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Figure 1. Heterogeneity among vesicles Synaptic vesicles in the presynaptic terminal can be sorted into distinct pools based on how strongly the terminal needs to be stimulated for their release (A), via which endocytotic pathway they recycle (B), the form of neurotransmitter release they contribute to (C), and the particular sets of membrane protein vesicles expressed (D). RRP, readily releasable pool. C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society

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three distinct mechanisms: during the kiss-and-run type of endocytosis vesicles do not fuse completely with the plasma membrane (Fesce et al. 1994); clathrin-dependent recycling involves full vesicle fusion and endocytosis via clathrin-coated vesicles; and bulk-endocytosis occurs when vesicles recycle via endosome-like intermediate organelles (for review see: Rizzoli, 2014; Jähne et al. 2015; Kononenko & Haucke, 2015). Kiss-and-run and clathrin-dependent endocytosis occur at lower activity levels; however, bulk endocytosis is usually activated after prolonged, extensive activity. In this way, the composition of the vesicle pool based on the route of recycling is a result of the previous history of the synapse (Fig. 1B). Spatial localization of vesicles in the terminal

Why are certain pools easier to release than others? It is tempting to speculate that physical distance from the active zone determines how likely it is to release vesicles from specific pools. Vesicles belonging to the RRP need to be docked at the active zone to be released with millisecond precision. In accordance with this function, a portion of vesicles belonging to the RRP are found close to the active zone (Schikorski & Stevens, 2001). Considering the increased level of activity required for mobilization of the recycling and resting pool, the most obvious hypothesis would be that the recycling and resting pools are located gradually further away from the active zone (Fig. 2A). While this is the case at the drosophila neuromuscular junction (NMJ) (Kuromi & Kidokoro, 1998), no evidence supports the precise spatial segregation of the recycling and the resting pools in CNS synapses (Rizzoli & Betz, 2005). Indeed, calcium uncaging experiments showed that the position of vesicles within the terminal could not explain the release properties of distinct pools (Schneggenburger et al. 1999; Bollmann et al. 2000; Schneggenburger & Neher, 2000; Denker & Rizzoli, 2010). Functional heterogeneity among vesicles based on their contribution to particular release type

Three modes of vesicle release govern chemical synaptic transmission: spontaneous, synchronous and asynchronous release (Fig. 1C). Here again, the activity pattern of the presynaptic terminal plays a key role in determining through which release type neurotransmission occurs. Indeed, activity-dependent release of neurotransmitters is tightly regulated by the spatiotemporal dynamics of calcium elevation in the presynaptic terminal. Spontaneous release, fast synchronous release and asynchronous release are sequentially observed with increasing intraterminal calcium concentration. The three different modes of vesicle release can be associated with specific neuronal network states

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and serve specialized physiological roles for intercellular communication. For instance, spontaneously occurring release of vesicles was demonstrated to be a key element regulating homeostatic plasticity (Sutton et al. 2006). Synchronous neurotransmitter release is well-known to pace neuronal network activity on a very precise time scale. A striking example is the generation of hippocampal oscillations by interconnected interneuron networks. Indeed, synchronous release of GABA at interneuron-to-interneuron synapses is essential in generating hippocampal gamma oscillations (Bartos et al. 2002). On the other hand, many studies support the notion that the longer-lasting and delayed asynchronous release is ideal to smooth the synaptic transmission over a longer period of time. For example, asynchronous glutamate release contributes to up states in striatal medium spiny neurons (Plenz & Kitai, 1998). Consistent with a role of elevating the general level of activity in neuronal networks, asynchronous glutamate release was found to sustain network reverberations in cultured hippocampal neurons (Lau & Bi, 2005). Given that different modes of release support specific physiological functions, vesicles liberated during a given mode of release could originate from designated pools; this provides an interesting model combining structure and physiological function. It would be straightforward to propose that the RRP mediates spontaneous and fast synchronous release while the recycling and resting pools are progressively recruited during asynchronous release. As whether various forms of release utilize the same vesicle pool or not is currently extensively debated, both possibilities will be explored. First, vesicles contributing to different types of release could originate from the same pools (Prange & Murphy, 1999; Groemer & Klingauf, 2007). Several lines of evidence suggest that spontaneously and synchronously released vesicles share a common origin (Groemer & Klingauf, 2007). Initial studies by Prange & Murphy (1999) positively correlated the probability of spontaneous synaptic activity with the synchronous release probability, indicating that the two modes of release shared a common vesicle pool. By imaging labelled vesicles in hippocampal neurons, Groemer & Klingauf (2007) showed that vesicles released spontaneously and following stimulation emerged from the same pool. Furthermore, studies indicate that fast synchronous and asynchronous release recruit vesicles from the same pool (Hagler & Goda, 2001; Otsu et al. 2004). Using electrophysiological approaches in cultured hippocampal neurons, Hagler & Goda (2001) found that asynchronous release could deplete the vesicle pool mediating fast synchronous release. In accordance with this view, Otsu et al. (2004) showed that both synchronous and asynchronous release compete for the same pool of vesicles. Therefore, both release modes could access the same pool of vesicles in discrete time windows. C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society


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On the other hand, reports show that distinct pools contribute to different types of physiological activity (Sara et al. 2005; Fredj & Burrone, 2009). Indeed, a large body of evidence demonstrates that spontaneous and synchronous releases are mediated by specific pools (Sara et al. 2005; Fredj & Burrone, 2009). Using tracking of fluorescently labelled vesicles and immunohistochemistry, Sara et al. (2005) showed that vesicles were recycled at rest and that, following fusion, spontaneously released vesicles were directed to a small pool of vesicles. Furthermore, this specific pool would then be more likely to provide vesicles that would be released during the subsequent event of spontaneous release. In accordance with these results, fast synchronous and asynchronous release do not compete for the recruitment of vesicles at glutamatergic synapses in the nucleus accumbens (Hjelmstad, 2006). Furthermore, exocytosed vesicles during evoked synchronous or asynchronous release are segregated from spontaneously released vesicles (Chung

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et al. 2010). According to these latter studies, vesicles released during specific types of activity would originate from distinct pools. The studies that lead to these opposing views on vesicle heterogeneity are using similar technical approaches. The reason behind this discrepancy is not immediately apparent: however, an interesting idea proposing that developmental factors could be behind these disparate results has recently emerged (Truckenbrodt & Rizzoli, 2014). While at the moment this debate is not resolved, recent findings identifying distinct regulatory processes associated with spontaneous release such as postsynaptic signalling and vesicle fusion are increasingly difficult to reconcile with the idea of a homogeneous vesicle population (Sutton et al. 2006; Sutton & Schuman, 2006; Leitz & Kavalali, 2014). On the postsynaptic side, can different modes of release requiring different pools of vesicles recruit specific signalling cascades? Postsynaptic cells are modulated differently by spontaneously released synaptic

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Figure 2. Potential mechanisms of functional heterogeneity within the presynaptic terminal Heterogeneity on the presynaptic side could be achieved if vesicles situated at various distances from the active zone responded to different levels of calcium (A), if certain active zones or synapses are specialized to support distinct forms of release (B), if pools are composed of vesicles exhibiting particular sets of membrane proteins endowing them with unique functional properties (C), or if filaments within the terminal ensure the delivery of distinct vesicles to the active zone (D). C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society

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signals compared with action potential-triggered events; spontaneous release can influence homeostatic plasticity via the regulation of dendritic protein synthesis through the deactivation of eukaryotic elongation factor 2 (eEf2) kinase (Sutton et al. 2006; Sutton & Schuman, 2006). Endocytosis of spontaneously released vesicles shows kinetic properties that are different from those after action potential invasion of the terminal, while their fusion is distinctly regulated (Leitz & Kavalali, 2014). Heterogeneity among vesicles based on their molecular composition

Which feature of synaptic vesicles determines under which conditions they are released? Differences in morphological features such as diameter or shape could be envisioned as a significant factor in determining when a vesicle is released. However, ultrastructural studies failed to identify a key morphological parameter that would define different vesicle pools (Rizzoli & Betz, 2005). Similar to neuronal membranes (Stoeckenius, 1962), synaptic vesicles are composed of proteins embedded in a lipid bilayer and exhibit a 60% protein to lipid ratio (Takamori et al. 2006). Although neither the protein/lipid ratio nor the basic structure composition can distinguish between functionally different synaptic vesicles (Takamori et al. 2000), the complex protein structures assembled on the surface of vesicles coupled with a vast repertoire of proteins could support synaptic vesicle specialization (Poudel & Bai, 2014). Indeed, a potential source of vesicle diversity lies within the combination of membrane proteins that is incorporated into vesicle membranes. It was revealed that over 400 different proteins can be found on synaptic vesicles (Takamori et al. 2006; Gronborg et al. 2010). This wide array of possible proteins can provide the molecular cues to distinguish vesicles and associate them to their respective pools of belonging. Incorporation of specific proteins in addition to the required basic elements (Takamori et al. 2006; Dittman & Ryan, 2009) at the vesicle surface could be envisaged to explain how vesicles within the same presynaptic terminal possess different functional properties (Fig. 1D). Synapsin is a molecular marker for vesicles in the reserve pool and these vesicles did not seem to participate in physiological neurotransmission (Denker et al. 2011). As such, fewer than 5% of the total vesicles were recycled over hours of neuronal activity (Denker et al. 2011). However, the number of vesicles being recycled was increased to 30% for the same amount of activity in drosophilas that did not express synapsin. Therefore, synapsin appears to be specifically incorporated in vesicles belonging to the reserve pool. Additionally, it is considered to control the entry and the exit of synaptic vesicles from the reserve pool (Verstegen et al. 2014).

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As additional support for vesicle sorting based on their molecular composition, zinc was found to be only present in a subpopulation of vesicles in hippocampal mossy fibre presynaptic terminals. Evidence also shows that the presence of zinc-containing vesicles was crucial in maintaining normal levels of spontaneous glutamatergic activity (Lavoie et al. 2011), but that these vesicles were preferentially released during high-frequency stimulation. Thus zinc-containing vesicles contribute to specific modes of neurotransmission. This is consistent with the concept that these vesicles belong to a specific pool of vesicles. How is this molecular heterogeneity of molecule incorporation in synaptic vesicles created? In addition to vesicles, zinc staining was also found in synaptic endosomes at mossy fibre to CA3 pyramidal cell synapses, suggesting that zinc-containing vesicles were derived from bulk endocytosis (Lavoie et al. 2011). Therefore, these zinc-containing synaptic vesicles could be generated through a specific recycling pathway. In return, this suggests that the recycling pathway could be responsible for tagging vesicles with the appropriate proteins or molecules to direct them to specific pools. In accordance with this idea, vesicles derived from distinct recycling pathways populate specific pools in terminals of cerebellar granule neurons (Cheung et al. 2010). Potential mechanisms of distinct modes of neurotransmitter release

How is a certain mode of release selected over another in presynaptic terminals? Synapses can be specialized to a specific mode of release or support multiple modes of release. First, the demonstration that spontaneous and evoked release can co-exist in the same synaptic terminal implies that highly specialized molecular machinery can discriminate between different modes of release (Ramirez & Kavalali, 2011). One such example is the calcium affinity of sensors and their position relative to the calcium source, which dictates the recruitment of a specific pool of vesicles during a given type of activity (Wen et al. 2010) (Fig. 2B). On the other hand, recent findings suggest that specialized active zones in the same synaptic terminal independently support spontaneous and evoked neurotransmitter release at the fly NMJ (Melom et al. 2013). Additionally, an interesting avenue for the separation of function of release modes could be the specialization of synapses in mediating a specific mode of release. Indeed, recent evidence indicates that evoked and spontaneous neurotransmitter release are mediated at different sets of synapses expressing specific postsynaptic receptors in drosophila NMJ (Peled et al. 2014). In addition, neurotransmitter release from these highly specialized terminals activates different sets of postsynaptic receptors, consistent with data showing that spontaneous release and fast synchronous release activate different sets of postsynaptic C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society

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receptors in cultured neurons (Atasoy et al. 2008). Taken together, these results support the fact that certain synapses are specialized to mediate either spontaneous or evoked release. While these two modes of release can originate from the same synaptic terminal, distinct active zones can also mediate spontaneous and evoked release to activate specific postsynaptic receptors. Synchronous and asynchronous release are differentially affected by slow and fast calcium chelators (EGTA and BAPTA) (Adler et al. 1991; Cummings et al. 1996; Atluri & Regehr, 1998; Lu & Trussell, 2000). This differential effect is generally interpreted as asynchronous release resulting from the slow expansion of the localized calcium entry near the active zones (Borst & Sakmann, 1996; Meinrenken et al. 2002). However, according to data obtained from zebrafish NMJ, the two types of release could also be triggered by calcium originating from different sources. While synchronous release was triggered by calcium entry via P/Q-type channels localized in synaptic boutons, asynchronous release was boosted by calcium sources from off-synaptic locations (Wen et al. 2013). While distinct release sites and various calcium sources could explain some aspects of various forms of neurotransmitter release occurring in response to different stimuli, none of these scenarios explain why and how these vesicles respond to different calcium dynamics. In addition, it is also unclear at the moment whether these contributors play a key role in small CNS synapses. Spatial segregation of distinct modes of release would need to occur in the same small terminal or various boutons would need to specialize to certain types of release; currently neither of these scenarios are supported by experimental evidence. Molecular determinants of functional heterogeneity

The molecular machinery involved in spontaneous, synchronous and asynchronous release also appears to be distinct (Fig. 2C), as shown by the selective expression of proteins on vesicles preferentially recruited during specific modes of release. For example, Vps10p-tail-interactor-1a is selectively found on a population of vesicles from the resting pool that supports spontaneous neurotransmitter release (Ramirez et al. 2012). While VAMP2-expressing vesicles were released during synchronous release (Schoch et al. 2001), VAMP4 was selectively observed on synaptic vesicles associated with asynchronous release (Raingo et al. 2012). Molecular heterogeneity of vesicles also makes it possible to distinguish between vesicles originating from the same pool. Both synchronous and asynchronous release can be mediated by vesicles from the RRP (Sun et al. 2007), but the calcium sensors involved were different. In this report, the authors demonstrated that deletion of synaptotagmin 2 abolished synchronous release, but C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society

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asynchronous release was kept intact. An additional example of the selectivity of sensors for a given type of release is found at GABAergic synapses. Deletion of the calcium sensor synaptotagmin 1 blocked synchronous release of glutamate and GABA, while asynchronous release was unaffected (Maximov & Sudhof, 2005). Confirming these findings regarding specific sensors being associated with particular release types, at the zebrafish NMJ, knockdown of synaptotagmin 7 decreased the asynchronous release while synaptotagmin 2 was key in mediating fast synchronous release (Wen et al. 2010). A similar conclusion was reached using cultured hippocampal neurons. In this study, synaptotagmin 7 ablation in synaptotagmin 1-deficient synapses suppressed asynchronous release (Bacaj et al. 2013). In contrast to its function in asynchronous release, synaptotagmin 7 was rather suggested to regulate Ca2+ -dependent synaptic vesicle replenishment in mammalian synaptic terminals (Liu et al. 2014). Similarly to synaptotagmin 7, the role of Doc2, a calcium sensor, in synaptic functions is intensely debated. Recent findings show that Doc2 is required for spontaneous activity (Groffen et al. 2010; Pang et al. 2011; Walter et al. 2011). Indeed, Doc2 deletion results in significant decrease in the frequency of spontaneous EPSCs, while its deletion has no effect on asynchronous release. In sharp contrast with the aforementioned findings, Doc2 has been identified as a calcium sensor that is involved in asynchronous release in hippocampal neurons (Yao et al. 2011). Based on the kinetics of Doc2 interaction with calcium, the authors linked its activity to asynchronous release. Furthermore, the level of asynchronous release was intimately linked with the expression of Doc2. These studies adhering specific calcium sensors to distinct modes of release support the idea that inclusion of distinct sets of sensors to the vesicle membrane could link different presynaptic calcium dynamics with distinct release modes. Specific sensors and their position relative to the source of calcium could dictate through which mode of release the vesicle will fuse to the presynaptic terminal, even when vesicles belong to the same pool. Theoretical data suggest that heterogeneity among vesicles is necessary for neurotransmission over a wide range of physiological activities (Sakaba & Neher, 2001b). In order to maintain an appropriate level of neurotransmitter release, modelling studies predict that at least two distinct pools of vesicles need to exist in presynaptic terminals. At the calyx of Held, two pools of vesicles can be recruited in an activity-dependent manner (Sakaba & Neher, 2001a). First, fast-releasing vesicles were emptied quickly following activity but were recycled more slowly and their recovery was strongly associated with the level of Ca2+ in the terminal (Sakaba & Neher, 2001a). On the other hand, slow-releasing vesicles were recruited later during activity, but were

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endocytosed faster from the membrane following activity (Sakaba & Neher, 2001a). Consistent with their timing of fusion with the membrane, fast-releasing vesicles are involved in synchronous release while slowly releasing vesicles mediate asynchronous release (Sakaba, 2006). Molecular markers expressed on vesicles could dictate such precise recruitment of vesicle pools during neurotransmission. In support of this idea is the fact that both pools respond differently to pharmacological treatments (Sakaba & Neher, 2001a). Additionally, expression of various V-SNARE proteins could predict the fate of vesicles (Scheuber et al. 2006; Fig. 2A). For example, VAMP7 is highly expressed in resting pool vesicles, while VGLUT1 is found in vesicles from the recycling pool. Not only do these vesicles demonstrate molecular heterogeneity, but VAMP7-positive vesicles are also more likely to be released during spontaneous activity than VGLUT1-containing vesicles (Hua et al. 2011). Therefore, the release of specific vesicles during different modes of neurotransmission appears guided by the differential expression of vesicular proteins. The heterogeneous populations of synaptic vesicles are preferentially released in response to different intensities of synaptic activity. How subsets of vesicles contribute to neurotransmission and their role in neuronal coding is currently being actively investigated. Different calcium sensors could promote the fusion of vesicles in function of the activity sensed in the presynaptic terminal (Fig. 2C). How are different pools of vesicles generated following membrane fusion and vesicle recycling?

Recent experiments tracking synaptic vesicles in real-time indicate that vesicles will be reassigned to their pool of

Figure 3. Distinct physiological signals trigger the release of different subpopulations of vesicles Molecular heterogeneity (green and orange) of synaptic vesicles could endow a single presynaptic terminal with the ability to respond to different (A and B) physiological stimuli with the release of distinct sets of vesicles.

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origin following neurotransmitter release and endocytosis (Park et al. 2012). Since vesicles could be sorted into pools without spatial consideration, certain mechanisms should exist to allow for the allocation of vesicles to the right pool. Although vesicles are recycled and sorted in their respective pool of origin, our current understanding of how this process occurs is rather limited. Expression of specific molecular markers could be ideally suited to sort vesicles in their respective pools (Lavoie et al. 2011). In agreement with this view, data demonstrate that an amino acid sequence present in the VGAT transporter is required for proper shipping of the vesicle to its pool of residence (Santos et al. 2013). Further evidence for molecule-guided vesicle sorting is the effect of the presence of adaptor protein 3 (AP-3) on vesicles, which routes vesicles to the correct pool (Voglmaier et al. 2006; Voglmaier & Edwards, 2007). The functionally distinct role of these vesicles was demonstrated in a subsequent study. Vesicles generated via AP-3-dependent recycling contribute to asynchronous release demonstrating how various recycling pathways can contribute to the generation of a functionally diverse vesicle pool in single presynaptic terminals (Evstratova et al. 2014). Alternatively, one could envision special delivery routes for vesicles contributing to various forms of release working similarly to conveyor belts. Filaments connecting vesicles within the presynaptic terminal were visualized using high-pressure freezing (Siksou et al. 2007) (Fig. 2D). While it is intriguing to imagine that these filaments could support various forms of release, experimental evidence regarding the mechanism of action by which the cytomatrix regulates transmitter release is currently not available. Conclusion

Multiple studies support the existence of various functionally distinct vesicle pools in presynaptic terminals. This heterogeneity has been linked to spatial segregation in large NMJ terminals; however, no definite spatial position can be attributed to these pools within small CNS synapses. Heterogeneity of vesicles cannot be explained by morphological characteristics, but rather by specific expression of vesicular proteins. Molecular heterogeneity among vesicles can explain how small CNS synapses utilizing limited vesicle pools can exhibit different forms of neurotransmitter release in response to distinct physiological stimuli (Fig. 3). When vesicles are specifically tagged with membrane proteins they can respond to a particular physiological stimulus. With the utilization of a heterogeneous vesicle population even a small presynaptic terminal can show a wide range of responses, depending on the level of activity, by releasing specific populations of vesicles. This vast heterogeneity of possible responses widens the range over which the synapse can transmit physiological information. Various forms C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society

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of neurotransmitter release and vesicle endocytosis are tightly linked to the level of activity experienced by the presynaptic terminal. Therefore, the composition of a heterogeneous vesicle pool at any given moment is the direct result of the previous history of the neuron; this could potentially allow the presynaptic terminal to adapt to changes in neuronal activity levels by altering the ratio between different vesicle pools.

Future directions

In recent years we have seen a steady increase in the number of studies that identified various processes and molecules that are specific for certain subgroups of vesicles. Identification of the molecular signatures of functionally distinct vesicle groups will be necessary steps towards answering several questions regarding the functional importance of vesicle heterogeneity. First, the proper understanding of downstream effects of various modes of neurotransmitter release relies on the demonstration of selective stimulation of distinct signalling pathways. Second, identification of the unique sets of membrane proteins will also be crucial for determining the mechanisms by which distinct pools are regulated by calcium dynamics. And third, molecular ‘fingerprints’ of various vesicle groups will be vital for obtaining a complete picture of the biogenesis of various vesicle groups. It will also be important to understand how rigid these groups are, how previous activity can alter the composition of vesicle pools, and via which mechanism this activity-dependent response can be achieved. Vesicle heterogeneity is only one, albeit crucial, element of the complex output patterns single terminals can generate; other factors such as the composition of ion channels and signalling cascades in a given terminal endow different types of presynaptic terminals with functional properties that are uniquely suited to their function.

References Adler EM, Augustine GJ, Duffy SN & Charlton MP (1991). Alien intracellular calcium chelators attenuate neurotransmitter release at the squid giant synapse. J Neurosci 11, 1496–1507. Alabi AA & Tsien RW (2012). Synaptic vesicle pools and dynamics. Cold Spring Harb Perspect Biol 4, a013680. Atasoy D, Ertunc M, Moulder KL, Blackwell J, Chung C, Su J & Kavalali ET (2008). Spontaneous and evoked glutamate release activates two populations of NMDA receptors with limited overlap. J Neurosci 28, 10151–10166. Atluri PP & Regehr WG (1998). Delayed release of neurotransmitter from cerebellar granule cells. J Neurosci 18, 8214–8227.


833

Bacaj T, Wu D, Yang X, Morishita W, Zhou P, Xu W, Malenka RC & Sudhof TC (2013). Synaptotagmin-1 and synaptotagmin-7 trigger synchronous and asynchronous phases of neurotransmitter release. Neuron 80, 947–959. Bartos M, Vida I, Frotscher M, Meyer A, Monyer H, Geiger JR & Jonas P (2002). Fast synaptic inhibition promotes synchronized gamma oscillations in hippocampal interneuron networks. Proc Natl Acad Sci USA 99, 13222–13227. Betz WJ & Henkel AW (1994). Okadaic acid disrupts clusters of synaptic vesicles in frog motor nerve terminals. J Cell Biol 124, 843–854. Bollmann JH, Sakmann B & Borst JG (2000). Calcium sensitivity of glutamate release in a calyx-type terminal. Science 289, 953–957. Borst JG & Sakmann B (1996). Calcium influx and transmitter release in a fast CNS synapse. Nature 383, 431–434. Ceccarelli B, Hurlbut WP & Mauro A (1972). Depletion of vesicles from frog neuromuscular junctions by prolonged tetanic stimulation. J Cell Biol 54, 30–38. Cheung G, Jupp OJ & Cousin MA (2010). Activity-dependent bulk endocytosis and clathrin-dependent endocytosis replenish specific synaptic vesicle pools in central nerve terminals. J Neurosci 30, 8151–8161. Chung C, Barylko B, Leitz J, Liu X & Kavalali ET (2010). Acute dynamin inhibition dissects synaptic vesicle recycling pathways that drive spontaneous and evoked neurotransmission. J Neurosci 30, 1363–1376. Cummings DD, Wilcox KS & Dichter MA (1996). Calcium-dependent paired-pulse facilitation of miniature EPSC frequency accompanies depression of EPSCs at hippocampal synapses in culture. J Neurosci 16, 5312–5323. de Lange RP, de Roos AD & Borst JG (2003). Two modes of vesicle recycling in the rat calyx of Held. J Neurosci 23, 10164–10173. Denker A, Bethani I, Krohnert K, Korber C, Horstmann H, Wilhelm BG, Barysch SV, Kuner T, Neher E & Rizzoli SO (2011). A small pool of vesicles maintains synaptic activity in vivo. Proc Natl Acad Sci USA 108, 17177–17182. Denker A, Krohnert K & Rizzoli SO (2009). Revisiting synaptic vesicle pool localization in the Drosophila neuromuscular junction. J Physiol 587, 2919–2926. Denker A & Rizzoli SO (2010). Synaptic vesicle pools: an update. Front Synaptic Neurosci 2, 135. Dittman J & Ryan TA (2009). Molecular circuitry of endocytosis at nerve terminals. Annu Rev Cell Dev Biol 25, 133–160. Evstratova A, Chamberland S, Faundez V & Toth K (2014). Vesicles derived via AP-3-dependent recycling contribute to asynchronous release and influence information transfer. Nat Commun 5, 5530. Fernandez-Alfonso T & Ryan TA (2008). A heterogeneous ‘resting’ pool of synaptic vesicles that is dynamically interchanged across boutons in mammalian CNS synapses. Brain Cell Biol 36, 87–100. Fesce R, Grohovaz F, Valtorta F & Meldolesi J (1994). Neurotransmitter release: fusion or ’kiss-and-run’? Trends Cell Biol 4, 1–4. Fredj NB & Burrone J (2009). A resting pool of vesicles is responsible for spontaneous vesicle fusion at the synapse. Nat Neurosci 12, 751–758.

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Groemer TW & Klingauf J (2007). Synaptic vesicles recycling spontaneously and during activity belong to the same vesicle pool. Nat Neurosci 10, 145–147. Groffen AJ, Martens S, Diez Arazola R, Cornelisse LN, Lozovaya N, de Jong AP, Goriounova NA, Habets RL, Takai Y, Borst JG, Brose N, McMahon HT & Verhage M (2010). Doc2b is a high-affinity Ca2+ sensor for spontaneous neurotransmitter release. Science 327, 1614–1618. Gronborg M, Pavlos NJ, Brunk I, Chua JJ, Munster-Wandowski A, Riedel D, Ahnert-Hilger G, Urlaub H & Jahn R (2010). Quantitative comparison of glutamatergic and GABAergic synaptic vesicles unveils selectivity for few proteins including MAL2, a novel synaptic vesicle protein. J Neurosci 30, 2–12. Hagler DJ Jr & Goda Y (2001). Properties of synchronous and asynchronous release during pulse train depression in cultured hippocampal neurons. J Neurophysiol 85, 2324–2334. Harata N, Ryan TA, Smith SJ, Buchanan J & Tsien RW (2001). Visualizing recycling synaptic vesicles in hippocampal neurons by FM 1–43 photoconversion. Proc Natl Acad Sci USA 98, 12748–12753. Hjelmstad GO (2006). Interactions between asynchronous release and short-term plasticity in the nucleus accumbens slice. J Neurophysiol 95, 2020–2023. Hua Z, Leal-Ortiz S, Foss SM, Waites CL, Garner CC, Voglmaier SM & Edwards RH (2011). v-SNARE composition distinguishes synaptic vesicle pools. Neuron 71, 474–487. Jähne S, Rizzoli SO & Helm MS (2015). The structure and function of presynaptic endosomes. Exp Cell Res 335, 172–179. Kononenko NL & Haucke V (2015). Molecular mechanisms of presynaptic membrane retrieval and synaptic vesicle reformation. Neuron 85, 484–496. Kuromi H & Kidokoro Y (1998). Two distinct pools of synaptic vesicles in single presynaptic boutons in a temperature-sensitive Drosophila mutant, shibire. Neuron 20, 917–925. Lau PM & Bi GQ (2005). Synaptic mechanisms of persistent reverberatory activity in neuronal networks. Proc Natl Acad Sci USA 102, 10333–10338. Lavoie N, Jeyaraju DV, Peralta MR 3rd, Seress L, Pellegrini L & Toth K (2011). Vesicular zinc regulates the Ca2+ sensitivity of a subpopulation of presynaptic vesicles at hippocampal mossy fiber terminals. J Neurosci 31, 18251–18265. Leitz J & Kavalali ET (2014). Fast retrieval and autonomous regulation of single spontaneously recycling synaptic vesicles. Elife 3, e03658. Liu H, Bai H, Hui E, Yang L, Evans CS, Wang Z, Kwon SE & Chapman ER (2014). Synaptotagmin 7 functions as a Ca2+ -sensor for synaptic vesicle replenishment. Elife 3, e01524. Lu T & Trussell LO (2000). Inhibitory transmission mediated by asynchronous transmitter release. Neuron 26, 683–694. Maximov A & Sudhof TC (2005). Autonomous function of synaptotagmin 1 in triggering synchronous release independent of asynchronous release. Neuron 48, 547–554. Meinrenken CJ, Borst JG & Sakmann B (2002). Calcium secretion coupling at calyx of Held governed by nonuniform channel-vesicle topography. J Neurosci 22, 1648–1667.

J Physiol 594.4

Melom JE, Akbergenova Y, Gavornik JP & Littleton JT (2013). Spontaneous and evoked release are independently regulated at individual active zones. J Neurosci 33, 17253–17263. Otsu Y, Shahrezaei V, Li B, Raymond LA, Delaney KR & Murphy TH (2004). Competition between phasic and asynchronous release for recovered synaptic vesicles at developing hippocampal autaptic synapses. J Neurosci 24, 420–433. Pang ZP, Bacaj T, Yang X, Zhou P, Xu W & Sudhof TC (2011). Doc2 supports spontaneous synaptic transmission by a Ca2+ -independent mechanism. Neuron 70, 244–251. Park H, Li Y & Tsien RW (2012). Influence of synaptic vesicle position on release probability and exocytotic fusion mode. Science 335, 1362–1366. Peled ES, Newman ZL & Isacoff EY (2014). Evoked and spontaneous transmission favored by distinct sets of synapses. Curr Biol 24, 484–493. Plenz D & Kitai ST (1998). Up and down states in striatal medium spiny neurons simultaneously recorded with spontaneous activity in fast-spiking interneurons studied in cortex-striatum-substantia nigra organotypic cultures. J Neurosci 18, 266–283. Poudel KR & Bai J (2014). Synaptic vesicle morphology: a case of protein sorting? Curr Opin Cell Biol 26, 28–33. Prange O & Murphy TH (1999). Correlation of miniature synaptic activity and evoked release probability in cultures of cortical neurons. J Neurosci 19, 6427–6438. Raingo J, Khvotchev M, Liu P, Darios F, Li YC, Ramirez DM, Adachi M, Lemieux P, Toth K, Davletov B & Kavalali ET (2012). VAMP4 directs synaptic vesicles to a pool that selectively maintains asynchronous neurotransmission. Nat Neurosci 15, 738–745. Ramirez DM & Kavalali ET (2011). Differential regulation of spontaneous and evoked neurotransmitter release at central synapses. Curr Opin Neurobiol 21, 275–282. Ramirez DM, Khvotchev M, Trauterman B & Kavalali ET (2012). Vti1a identifies a vesicle pool that preferentially recycles at rest and maintains spontaneous neurotransmission. Neuron 73, 121–134. Richards DA, Guatimosim C & Betz WJ (2000). Two endocytic recycling routes selectively fill two vesicle pools in frog motor nerve terminals. Neuron 27, 551–559. Rizzoli SO (2014). Synaptic vesicle recycling: steps and principles. EMBO J 33, 788–822. Rizzoli SO & Betz WJ (2005). Synaptic vesicle pools. Nat Rev Neurosci 6, 57–69. Sakaba T (2006). Roles of the fast-releasing and the slowly releasing vesicles in synaptic transmission at the calyx of Held. J Neurosci 26, 5863–5871. Sakaba T & Neher E (2001a). Calmodulin mediates rapid recruitment of fast-releasing synaptic vesicles at a calyx-type synapse. Neuron 32, 1119–1131. Sakaba T & Neher E (2001b). Quantitative relationship between transmitter release and calcium current at the calyx of Held synapse. J Neurosci 21, 462–476. Santos MS, Park CK, Foss SM, Li H & Voglmaier SM (2013). Sorting of the vesicular GABA transporter to functional vesicle pools by an atypical dileucine-like motif. J Neurosci 33, 10634–10646.


J Physiol 594.4


Sara Y, Virmani T, Deak F, Liu X & Kavalali ET (2005). An isolated pool of vesicles recycles at rest and drives spontaneous neurotransmission. Neuron 45, 563–573. Scheuber A, Rudge R, Danglot L, Raposo G, Binz T, Poncer JC & Galli T (2006). Loss of AP-3 function affects spontaneous and evoked release at hippocampal mossy fiber synapses. Proc Natl Acad Sci USA 103, 16562–16567. Schikorski T & Stevens CF (1997). Quantitative ultrastructural analysis of hippocampal excitatory synapses. J Neurosci 17, 5858–5867. Schikorski T & Stevens CF (2001). Morphological correlates of functionally defined synaptic vesicle populations. Nat Neurosci 4, 391–395. Schneggenburger R, Meyer AC & Neher E (1999). Released fraction and total size of a pool of immediately available transmitter quanta at a calyx synapse. Neuron 23, 399–409. Schneggenburger R & Neher E (2000). Intracellular calcium dependence of transmitter release rates at a fast central synapse. Nature 406, 889–893. Schoch S, Deák F, Königstorfer A, Mozhayeva M, Sara Y, Südhof TC & Kavalali ET (2001). SNARE function analyzed in synaptobrevin/VAMP knockout mice. Science 294, 1117–1122. Siksou L, Rostaing P, Lechaire JP, Boudier T, Ohtsuka T, Fejtova A, Kao HT, Greengard P, Gundelfinger ED, Triller A & Marty S (2007). Three-dimensional architecture of presynaptic terminal cytomatrix. J Neurosci 27, 6868–6877. Stoeckenius W (1962). Structure of the plasma membrane. An electron-microscope study. Circulation 26, 1066–1069. Sudhof TC (2000). The synaptic vesicle cycle revisited. Neuron 28, 317–320. Sun J, Pang ZP, Qin D, Fahim AT, Adachi R & Sudhof TC (2007). A dual-Ca2+ -sensor model for neurotransmitter release in a central synapse. Nature 450, 676–682. Sutton MA, Ito HT, Cressy P, Kempf C, Woo JC & Schuman EM (2006). Miniature neurotransmission stabilizes synaptic function via tonic suppression of local dendritic protein synthesis. Cell 125, 785–799. Sutton MA & Schuman EM (2006). Dendritic protein synthesis, synaptic plasticity, and memory. Cell 127, 49–58. Takamori S, Holt M, Stenius K, Lemke EA, Gronborg M, Riedel D, Urlaub H, Schenck S, Brugger B, Ringler P, Muller SA, Rammner B, Grater F, Hub JS, De Groot BL, Mieskes G, Moriyama Y, Klingauf J, Grubmuller H, Heuser J, Wieland F & Jahn R (2006). Molecular anatomy of a trafficking organelle. Cell 127, 831–846. Takamori S, Riedel D & Jahn R (2000). Immunoisolation of GABA-specific synaptic vesicles defines a functionally distinct subset of synaptic vesicles. J Neurosci 20, 4904–4911. Truckenbrodt S & Rizzoli SO (2014). Spontaneous vesicle recycling in the synaptic bouton. Front Cell Neurosci 8, 409. Verstegen AM, Tagliatti E, Lignani G, Marte A, Stolero T, Atias M, Corradi A, Valtorta F, Gitler D, Onofri F, Fassio A &


835

Benfenati F (2014). Phosphorylation of synapsin I by cyclin-dependent kinase-5 sets the ratio between the resting and recycling pools of synaptic vesicles at hippocampal synapses. J Neurosci 34, 7266–7280. Voglmaier SM & Edwards RH (2007). Do different endocytic pathways make different synaptic vesicles? Curr Opin Neurobiol 17, 374–380. Voglmaier SM, Kam K, Yang H, Fortin DL, Hua Z, Nicoll RA & Edwards RH (2006). Distinct endocytic pathways control the rate and extent of synaptic vesicle protein recycling. Neuron 51, 71–84. Walter AM, Groffen AJ, Sorensen JB & Verhage M (2011). Multiple Ca2+ sensors in secretion: teammates, competitors or autocrats? Trends Neurosci 34, 487–497. Wen H, Hubbard JM, Rakela B, Linhoff MW, Mandel G & Brehm P (2013). Synchronous and asynchronous modes of synaptic transmission utilize different calcium sources. Elife 2, e01206. Wen H, Linhoff MW, McGinley MJ, Li GL, Corson GM, Mandel G & Brehm P (2010). Distinct roles for two synaptotagmin isoforms in synchronous and asynchronous transmitter release at zebrafish neuromuscular junction. Proc Natl Acad Sci USA 107, 13906–13911. Wyatt RM & Balice-Gordon RJ (2008). Heterogeneity in synaptic vesicle release at neuromuscular synapses of mice expressing synaptopHluorin. J Neurosci 28, 325–335. Yao J, Gaffaney JD, Kwon SE & Chapman ER (2011). Doc2 is a Ca2+ sensor required for asynchronous neurotransmitter release. Cell 147, 666–677. Zucker RS & Regehr WG (2002). Short-term synaptic plasticity. Annu Rev Physiol 64, 355–405.

Additional information Competing interests None declared.

Funding This work was supported by a Canadian Institutes of Health Research (CIHR) Operating grant (MOP-81142) to K.T. S.C. was supported by Natural Sciences and Engineering Research Council of Canada (NSERC) and Centre Thématique de Recherche en Neuroscience (CTRN) fellowships.

Acknowledgements The figures were created by SciLight (www.scilight.eu).