How does Ca2+ trigger the release of neurotransmitters?

From gene expression to cell differentiation; from neurotransmitter release to synaptic plasticity – Ca2+ is a ubiquitous ion which plays a vital role in a variety of processes throughout the nervous system, and indeed throughout the majority of biological systems. Here we will focus on just one of the many roles of Ca2+ in the nervous system – the triggering of neurotransmitter/neuropeptide release from vesicles in the pre-synaptic terminal into the synaptic cleft. The role of Ca2+ in neurotransmitter release has been at the forefront of neuroscience research for decades, leading to the accumulation of a substantial amount of scientific literature on the subject.

Voltage-clamp experiments on the giant synapse of the squid found a direct correlation between the amount of neurotransmitter released and the amount of Ca2+ that enters the pre-synaptic terminal, and that neurotransmitter release was inhibited when pre-synaptic Ca2+ channels were blocked (Llinás et al., 1972). Furthermore, subsequent experiments showed that, even in the absence of an action potential, microinjection of Ca2+ into the pre-synaptic terminal is sufficient to trigger neurotransmitter release, and microinjection of Ca2+ chelators prevents neurotransmitter release upon the arrival of an action potential (Hall, 1992). Thus, research began to focus increasingly on the mechanisms by which Ca2+ is able to trigger the release of neurotransmitters.

The first step to triggering release of neurotransmitters is the packaging of small synaptic vesicles (~40-60nm in diameter) with neurotransmitter molecules, which is carried out via active transport (Purves et al., 2001). The vesicles then move towards the active zone – the site of neurotransmitter release – and are docked close to the plasma membrane and primed so that they are ready to respond to Ca2+. An action potential in the pre-synaptic neuron then causes an influx of Ca2+ at the pre-synaptic terminal – due to the opening of voltage-gated Ca2+ channels – and it is this Ca2+ influx which triggers a series of reactions leading ultimately to the fusion of the synaptic vesicles with the plasma membrane and the release of their contents (neurotransmitter molecules) across the membrane and into the synaptic cleft. The neurotransmitter molecules will then diffuse across the synaptic cleft and bind to receptor proteins on the plasma membrane of the post-synaptic neuron, leading ultimately to the generation of an action potential in that cell. The membrane proteins forming the Clathrin-coated vesicle are then endocytosed and may be recycled locally to form new synaptic vesicles – known as the kiss-and-run pathway. Alternatively, the vesicles may remain at the active zone and be reacidified and refilled with neurotransmitter, whilst remaining within the readily releasable pool – known as the kiss-and-stay pathway.

At rest, there is a higher concentration of Ca2+ ions outside the neuron. As an action potential passes down the axon and arrives at the pre-synaptic nerve terminal, voltage-gated Ca2+ channels open, allowing Ca2+ to enter through the channels down its concentration gradient. As Ca2+ ions diffuse into the terminal, they begin to accumulate around the mouth of open Ca2+ channels at a radius spanning tens of nanometres (Simon & Llinás, 1985). The rate at which they reach and bind to Ca2+–binding proteins – the next step in the neurotransmitter release signalling cascade – is of course variable, and depends on the spatial arrangement of Ca2+ channels relative to the Ca2+–binding proteins. The release of fast-acting neurotransmitters such as glutamate or acetylcholine (ACh) was originally thought to be mediated via Ca2+ ‘nanodomains’ – a domain of concentrated Ca2+ ions spanning less than 100 nanometres which interact directly with Ca2+–binding proteins in close proximity – as opposed to Ca2+ ‘microdomains’ – a cluster of Ca2+ channels constituting a domain of Ca2+ ions extending over >100 nanometres and thus interacting with Ca2+–binding proteins within an area spanning around 1μm2. The functional relevance of this is that nanodomain coupling provides greater efficacy and more crucially, significantly greater speeds of synaptic transmission than microdomain coupling (Eggermann et al., 2012). This dogma was proposed in light of evidence that BAPTA (a fast-acting Ca2+ chelator) inhibited neurotransmitter release from the presynaptic terminal with far greater potency than EGTA (a slower-acting Ca2+ chelator), indicating that the Ca2+–binding protein triggering neurotransmitter release binds Ca2+ rapidly and is located very close to Ca2+ channels (Adler et al., 1991). Likewise, it was assumed that the release of slower-acting neurotransmitters is mediated via Ca2+ microdomains.

However, later research on the mammalian Calyx of Held synapse contradicted this, providing strong evidence that microdomain coupling mediates the very rapid release of neurotransmitter at this synapse (Borst & Sakmann, 1996). Comparisons of the number of effective vesicles released in response to postsynaptic currents with the presynaptic Ca2+ current during an action potential indicated that, for each vesicle released, over 60 Ca2+ channels were opened. Furthermore, concentrations of EGTA as low as 1mM significantly inhibited neurotransmitter release – making it almost as potent as BAPTA at this synapse. Therefore, the results concluded that Ca2+ must be diffusing across a greater distance, and that the activation of multiple channels is necessary for rapid neurotransmitter release at this synapse – i.e. the release is mediated via microdomain coupling. Later research corroborated that while vesicles appear to be randomly distributed at the active zone, Ca2+ channels are clustered, with vesicles averaging around 100 nanometres from the Ca2+ domains (Meinrenken et al., 2002). Furthermore, the release probability of a population of vesicles varies during an action potential (Sakaba & Neher, 2001), likely due to the varying concentrations of Ca2+ reaching vesicles across a microdomain. This may in part account for the time-variances observed in neurotransmitter release across different pre-synaptic terminals.

Thus, very rapid exocytosis can be initiated by concentrations of Ca2+ as low as 5-10μM, or as high as 100-200μM (Augustine, 2001). There are clearly marked variations in the concentrations of Ca2+ required by different pre-synaptic terminals to trigger neurotransmitter release. There are two main factors which determine these variations – the first of which is the spatial organisation of Ca2+ channels in the pre-synaptic active zones, as discussed above. The second is the rate at which Ca2+ binds to various Ca2+–binding proteins which modulate neurotransmitter release. Indeed, Meinrenken’s model proposed that the only step in the neurotransmitter release cascade whose speed is dependent on Ca2+ concentration is the binding of Ca2+ to the “calcium sensor” – Synaptotagmin (Meinrenken et al., 2002).

Before discussing Synaptotagmin, perhaps the most crucial Ca2+–binding protein for vesicle fusion / exocytosis of neurotransmitter, it is important to consider other proteins involved earlier in the neurotransmitter release cascade which depend on the binding of Ca2+ to function. During the release process, there are two significant Ca2+ influxes – one which leads to vesicle docking/priming at the active zone, and a second which activates fusion proteins via Ca2+–dependent processes at the active zone, leading to exocytosis of neurotransmitter into the synaptic cleft.

Following the first influx of Ca2+, Synapsin – a protein which binds both to the cytoskeleton and to vesicles, thus preventing vesicles from moving to the active zone before a Ca2+ signal arrives – is phosphorylated by Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) (Ceccaldi et al., 1995), due to a conformational change in CaM kinase II in which an auto-inhibitory sequence is directly relieved by Ca2+/calmodulin. The phosphorylation of Synapsin releases vesicles from the cytoskeleton, allowing them begin their journey towards the active zone. Rab3A/Rab3B (GTP-binding proteins) are thought to then guide vesicles towards the active zone and aid in preparation for docking – a process in which vesicles and pre-synaptic membrane phospholipids arrange into a fusion-ready state (Leenders et al., 2001).

Following the second influx of Ca2+, proteins in the membrane of the vesicle, including Synaptobrevin and Synaptophysins, are thought to be involved in the formation of a ‘fusion pore’ – essentially a cytoplasmic bridge connecting the lumen inside the vesicle with the extracellular synaptic cleft, through which exocytosis will occur – by interacting with proteins in the pre-synaptic terminal plasma membrane such as Syntaxin, SNAP-25 and the putative Physophilins (Woodman, 1997; Thomas & Betz, 1990). Crucially, in the absence of the second Ca2+ influx, no fusion pore is formed.

Synaptotagmin – another protein found on the vesicle membrane – owes its reputation as the ‘calcium sensor’ for neurotransmitter release to the presence of its Ca2+ binding domains which, when bound to Ca2+, allows the protein to bind to phospholipids in the plasma membrane. While not participating directly in the fusion of the vesicle membrane with the plasma membrane, Synaptotagmin may help to overcome the electrostatic repulsion between the two membranes (Domanska et al., 2010), as well as trigger conformational changes in other proteins necessary for fusion. Research found that mutations of Synaptotagmin’s C-terminus Ca2+–binding domain C2A caused a reduction in neurotransmitter release which was directly correlated with the Ca2+–dependent binding of Synaptotagmin to phospholipids of the plasma membrane (Fernández-Chacón et al., 2001), indicating the importance of this step in the release of neurotransmitter. Additionally, mutations of the C2B Ca2+–binding domain in vivo (Drosophila), inhibits neurotransmitter release by disrupting the Ca2+–dependent self-oligomerisation of Synaptotagmins (Fukuda et al., 2000), which is crucial to Synaptotagmins function in triggering vesicle fusion by initiating the assembly of ‘SNARE’ (soluble NSF attachment protein receptor) complexes (Littleton, Bai, et al., 2001) – the next step in the neurotransmitter release cascade. The molecular model in which Synaptotagmin triggers neurotransmitter release in a Ca2+–dependent manner through interactions with the plasma membrane phospholipid bilayer and the initiation of SNARE complex formation is supported by evidence that the speed at which Synaptotagmin carries out these reactions in response to Ca2+ is rapid enough for the kinetic constraints of synaptic vesicle fusion (Davis et al., 1999).

Synaptotagmin is thought to initiate SNARE complex formation by binding to Syntaxin (a.k.a. t-SNARE) (de Wit et al., 2009) – a protein found in the pre-synaptic plasma membrane. Thus, this interaction is also crucial in the targeting of the vesicle membrane to the plasma membrane. Mutations preventing the binding of Syntaxin lead to reduced neurotransmitter release (Wu et al., 1999). The interaction is highly Ca2+–dependent, since it is the binding of Ca2+ to Synaptotagmin which increases its affinity for Syntaxin approximately hundredfold (Chapman et al., 1995). The formation of the SNARE complex also involves N-ethylmaleimide sensitive fusion proteins (NSF) and cytoplasmic SNAPs (soluble NSF attachment proteins) (Morgan & Burgoyne, 2009).

The three main components of the SNARE complex are Syntaxin (t-SNARE), Synaptobrevin (a.k.a. v-SNARE, or vesicle associated membrane proteins (VAMP1/2)) and SNAP-25 (Synaptosomal-associated protein 25). Syntaxin is found on the plasma membrane, along with SNAP-25, while Synaptobrevin is found on the vesicle membrane. Once in proximity, these three proteins each contribute α-helices which progressively wrap around each other to form a ‘coiled-coil’ quaternary structure – SNAP-25 contributes two α-helices, while Syntaxin and Synaptobrevin each contribute one (Chapman et al., 1994). The biomechanical wrapping of these α-helices pulls the two membranes closer together, since Syntaxin is associated with the plasma membrane and Synaptobrevin with the vesicle membrane. This, along with Synaptotagmin’s interactions with the plasma membrane (and undoubtedly numerous other reactions), bring the two membranes close enough so that proteins on the vesicle membrane – including Synaptophysins – may interact with proteins on the plasma membrane – perhaps including Physophilins – and form a fusion pore, through which neurotransmitter can diffuse upon sufficient dilation. The SNARE complex is then disassembled by NSF, an ATPase (Littleton, Barnard, et al., 2001). However, since none of the known SNARE-mediated reactions are directly influenced by Ca2+ (Augustine, 2001), none of this can occur unless Synaptotagmin is first bound to Ca2+. The SNARE reactions essentially rely on this binding – hence Synaptotagmin’s reputation as the ‘calcium sensor’ for neurotransmitter release. Furthermore, the binding of Synaptotagmin to SNAP-25 is essential for Ca2+–dependent neurotransmitter exocytosis (Zhang et al., 2002). A further, interesting role of SNAP-25 is its ability to inhibit the Ca2+ sensitivity of Synaptotagmin in GABAergic neurons (Verderio et al., 2004), functioning to regulate intracellular Ca2+ dynamics and possibly the amount of neurotransmitter released in response to Ca2+.

Finally, a further role of Synaptotagmin is to displace the protein ‘complexin’ from the SNARE complex. Complexin effectively blocks neurotransmitter release by incorporating an α-helix domain into the SNARE complex α-helices, preventing the biomechanical ‘zippering’ and the formation of the ‘coiled-coil’ structure, effectively inhibiting vesicle fusion (Giraudo et al., 2009). Ca2+–bound Synaptotagmin binds to the SNARE complex, causing the inhibitory ‘clamp’ effect of complexin to be relieved (Schaub et al., 2006), allowing proper SNARE complex formation and successful vesicle fusion. Thus, the ‘clamping’ effect of complexins serves to prevent spontaneous vesicle fusion in the absence of Ca2+, allowing greater control of neurotransmitter release. Furthermore, mutations of complexin in Caenorhabditis elegans cause not only a two-fold increase in vesicle fusion in the absence of Ca2+ but also an almost complete loss of effective fusion in response to a Ca2+ influx; thus it was proposed that complexin may be involved in the stabilisation of docked vesicles at the plasma membrane (Hobson et al., 2011). Similar findings were found in experiments with Drosophila complexin-/- mutants, along with indications that complexin may have a further role in regulating the size of both immediate/readily releasable vesicle pools (Jorquera et al., 2012). Additional proteins such as Munc18 bind to SNARE complexes and are crucial for vesicle fusion – with Munc13 removing an inhibitory clamp of Munc18 in the SNARE complex in a similar Ca2+–dependent manner as above (Rizo & Südhof, 2012).

Note that both Synaptotagmin’s interaction with the SNARE complex to relieve the ‘clamping’ effect of complexin and Synaptotagmin’s interaction with the phospholipids of the plasma membrane are Ca2+–dependent. However, different isoforms of Synaptotagmin require varying concentrations of Ca2+ to bind to phospholipids/Syntaxin (Li et al., 1995), suggesting that the use of different Synaptotagmin isoforms in synapses found across different neuronal circuits may play a role in specificity / precise modulation of neurotransmitter release.

Thus, the release of fast acting, small neurotransmitters (e.g. ACh, glutamate, GABA) from small synaptic vesicles (SSVs) is highly dependent on local changes in Ca2+ concentration at Ca2+ channel microdomains, with exocytosis triggered by a cascade of reactions essentially beginning with the phosphorylation of Synapsin by Ca2+–activated CaM kinase II, and the binding of Ca2+ to Synaptotagmin. However, such local changes in Ca2+ concentration are insufficient to cause the release of larger signalling molecules such as neuropeptides, which are carried in large dense-core vesicles (LDCVs) (~90-250nm in diameter) as opposed to SSVs (~35-50nm).

While the exocytosis of neuropeptides from LDCVs has long been known to be Ca2+ dependent (Iversen et al., 1978), the exact mechanisms by which LDCVs fuse with the pre-synaptic plasma membrane and release neuropeptides are unknown. Exocytosis from LDCVs requires greater, sustained Ca2+ currents – first to mobilise the vesicles towards the active zone, then to trigger fusion and exocytosis (Südhof, 2008). The first step is necessary since LDCVs, unlike SSVs, are not found localised around active zones; instead, they are scattered around the pre-synaptic nerve terminal (Salio et al., 2006). However, the precise mechanisms by which LDCVs move to the active zone remain unknown, although some of the mechanisms may be shared with SSVs. Exocytosis of peptides from Chromaffin granules – organelles similar to LDCVs and found in neuroendocrine cells of the adrenal gland (Borges et al., 2010) – has been extensively studied and is known to involve SNARE and Munc18 proteins, similar to SSV exocytosis. A known mechanistic difference is the Synaptotagmin isoforms involved in triggering release. Chromaffin granule exocytosis requires both Synaptotagmin-I – also involved in SSV exocytosis – and Synaptotagmin-VII – not involved in SSV exocytosis (Südhof, 2008). However, it is uncertain whether the Ca2+ sensor for all LDCVs is Synaptotagmin. Furthermore, in addition to Ca2+ influx via voltage-gated Ca2+ channel microdomains, some neuropeptides (e.g. oxytocin) can also be released from dendrites, triggered by the mobilisation of Ca2+ from intracellular stores (Ludwig et al., 2002).

One protein essential for the fusion of LDCVs with the pre-synaptic plasma membrane is ‘Ca2+-dependent activator protein for secretion’ (CAPS). CAPS contains a domain which binds to phospholipids of the plasma membrane, and another domain which is thought to associate with LDCVs – and functions in parallel with SNARE proteins (Grishanin et al., 2002). Since LDCVs are not localised to Ca2+ channel microdomains, and Ca2+ is rapidly buffered as it travels into and throughout the cell, yet the Ca2+ concentration required for LDCV exocytosis is significantly lower than is required for SSV exocytosis (Verhage et al., 1991), the proteins mediating neuropeptide release must have a significantly higher affinity for Ca2+ than those mediating neurotransmitter release from SSVs. Thus, it is thought that neuropeptides are released in response to a large, general increase in Ca2+ concentration throughout the cytoplasm. Such general changes in Ca2+ concentration require high-frequency/burst-patterned firing, in contrast to the single action potentials which can trigger SSV exocytosis. It is thought that the requirement for numerous, successive depolarisations leading to larger accumulations of intracellular Ca2+ is due to the longer latencies observed in the release of neuropeptides (30-2000ms) compared to neurotransmitters (0.3-1ms) (Bergquist & Ludwig, 2009). In any case, the mechanisms underlying the release of neuropeptides from LDCVs remains a key focus of modern neuroscience.

Thus, Ca2+ appears to be the key driver for triggering vesicle mobilisation, docking/priming, fusion and ultimately neurotransmitter release into the synaptic cleft, highlighting its importance in the functioning of neurons throughout the nervous system. While some of the key mechanistic players in the release of neurotransmitters have been identified – Ca2+ channel microdomains, Synapsin, Rab3A/B, Synaptotagmin (the ‘calcium sensor’), SNARE proteins including Synaptobrevin, Syntaxin and SNAP-25, Synaptophysin, Munc18/13 and complexin – the mechanisms underlying the release of neuropeptides are not yet fully understood. Nonetheless, the Ca2+–dependent release of neurotransmitters and neuropeptides from the pre-synaptic terminal remains a rapidly-changing, exciting area of study – and likely will be for years to come.


References: 

Adler, E. M., Augustine, G. J., Duffy, S. N. and Charlton, M. P. (1991) ‘Alien intracellular calcium chelators attenuate neurotransmitter release at the squid giant synapse’, The Journal of Neuroscience, 11(6), pp. 1496–1507.

Augustine, G. J. (2001) ‘How does calcium trigger neurotransmitter release?’, Current Opinion in Neurobiology, 11(3), pp. 320–326.

Bergquist, F. and Ludwig, M. (2009) ‘Neuropeptide Release’, 1st ed. Encyclopedia of Neuroscience, Academic Press.

Borges, R., Pereda, D., Beltrán, B., Prunel, M., Rodríguez, M. and Machado, J. D. (2010) ‘Intravesicular Factors Controlling Exocytosis in Chromaffin Cells’, Cellular and Molecular Neurobiology, 30(8), pp. 1359–1364.

Borst, J. G. G. and Sakmann, B. (1996) ‘Calcium influx and transmitter release in a fast CNS synapse’, Nature, 383, pp. 431–434.

Ceccaldi, P.-E., Grohovaz, F., Benfenati, F., Chieregatti, E. and Greengard, P. (1995) ‘Dephosphorylated Synapsin I Anchors Synaptic Vesicles to Actin Cytoskeleton: An Analysis by Videomicroscopy’, The Journal of Cell Biology, 128(5), pp. 905–912.

Chapman, E. R., An, S., Barton, N. and Jahn, R. (1994) ‘SNAP-25, a t-SNARE which binds to both syntaxin and synaptobrevin via domains that may form coiled coils.’, The Journal of Biological Chemistry, 269, pp. 27427–27432.

Chapman, E. R., Hanson, P. I., An, S. and Jahn, R. (1995) ‘Ca2+ Regulates the Interaction between Synaptotagmin and Syntaxin 1’, The Journal of Biological Chemistry, 270, pp. 23667–23671.

Davis, A. F., Bai, J., Fasshauer, D., Wolowick, M. J., Lewis, J. L. and Chapman, E. R. (1999) ‘Kinetics of Synaptotagmin Responses to Ca2+ and Assembly with the Core SNARE Complex onto Membranes’, Neuron, 24(2), pp. 363–376.

Domanska, M. K., Kiessling, V. and Tamm, L. K. (2010) ‘Docking and Fast Fusion of Synaptobrevin Vesicles Depends on the Lipid Compositions of the Vesicle and the Acceptor SNARE Complex-Containing Target Membrane’, Biophysical Journal, 99(9), pp. 2936–2946.

Eggermann, E., Bucurenciu, I., Goswami, S. P. and Jonas, P. (2012) ‘Nanodomain coupling between Ca2+ channels and sensors of exocytosis at fast mammalian synapses’, Nature Reviews Neuroscience, 13, pp. 7–21.

Fernández-Chacón, R., Königstorfer, A., Gerber, S. H., García, J., Matos, M. F., Stevens, C. F., Brose, N., Rizo, J., Rosenmund, C. and Südhof, T. C. (2001) ‘Synaptotagmin I functions as a calcium regulator of release probability’, Nature, 410, pp. 41–49.

Fukuda, M., Kabayama, H. and Mikoshiba, K. (2000) ‘Drosophila AD3 mutation of synaptotagmin impairs calcium-dependent self-oligomerization activity’, FEBS Letters, 482(3), pp. 269–272.

Giraudo, C. G., Garcia-Diaz, A., Eng, W. S., Chen, Y., Hendrickson, W. A., Melia, T. J. and Rothman, J. E. (2009) ‘Alternative Zippering as an On-Off Switch for SNARE-Mediated Fusion’, Science, 323(5913), pp. 512–516.

Grishanin, R. N., Klenchin, V. A., Loyet, K. M., Kowalchyk, J. A., Ann, K. and Martin, T. F. J. (2002) ‘Membrane Association Domains in Ca2+-dependent Activator Protein for Secretion Mediate Plasma Membrane and Dense-core Vesicle Binding Required for Ca2+-dependent Exocytosis’, The Journal of Biological Chemistry, 277, pp. 22025–22034.

Hall, Z. W. (1992) An Introduction to Molecular Neurobiology, Sunderland, Massachusetts, Sinauer Associates, [online] Available from: http://www.cell.com/cell/comments/0092-8674(93)90154-I (Accessed 29 December 2015).

Hobson, R. J., Liu, Q., Watanabe, S. and Jorgensen, E. M. (2011) ‘Complexin Maintains Vesicles in the Primed State in C. elegans’, Current Biology, 21(2), pp. 106–113.

Iversen, L. L., Iversen, S. D., Bloom, F., Douglas, C., Brown, M. and Vale, W. (1978) ‘Calcium-dependent release of somatostatin and neurotensin from rat brain in vitro’, Nature, 273, pp. 161–163.

Jorquera, R. A., Huntwork-Rodriguez, S., Akbergenova, Y., Cho, R. W. and Littleton, J. T. (2012) ‘Complexin Controls Spontaneous and Evoked Neurotransmitter Release by Regulating the Timing and Properties of Synaptotagmin Activity’, The Journal of Neuroscience, 32(50), pp. 18234–18245.

Leenders, A. G. M., Lopes da Silva, F. H., Ghijsen, W. E. J. M. and Verhage, M. (2001) ‘Rab3A Is Involved in Transport of Synaptic Vesicles to the Active Zone in Mouse Brain Nerve Terminals’, Molecular Biology of the Cell, 12, pp. 3095–3102.

Li, C., Ullrich, B., Zhang, J. Z., Anderson, R. G. W., Brose, N. and Südhof, T. C. (1995) ‘Ca2+-dependent and -independent activities of neural and non-neural synaptotagmins’, Nature, 375, pp. 594–599.

Littleton, J. T., Bai, J., Vyas, B., Desai, R., Baltus, A. E., Garment, M. B., Carlson, S. D., Ganetzky, B. and Chapman, E. R. (2001) ‘Synaptotagmin Mutants Reveal Essential Functions for the C2B Domain in Ca2+-Triggered Fusion and Recycling of Synaptic Vesicles In Vivo’, The Journal of Neuroscience, 21(5), pp. 1421–1433.

Littleton, J. T., Barnard, R. J. O., Titus, S. A., Slind, J., Chapman, E. R. and Ganetzky, B. (2001) ‘SNARE-complex disassembly by NSF follows synaptic-vesicle fusion’, Proceedings of the National Academy of Sciences, 98(21), pp. 12233–12238.

Llinás, R., Blinks, J. R. and Nicholson, C. (1972) ‘Calcium Transient in Presynaptic Terminal of Auid Giant Synapse: Detection with Aequorin’, Science, 176(4039), pp. 1127–1129.

Ludwig, M., Sabatier, N., Bull, P. M., Landgraf, R., Dayanithi, G. and Leng, G. (2002) ‘Intracellular calcium stores regulate activity-dependent neuropeptide release from dendrites’, Nature, 418, pp. 85–89.

Meinrenken, C. J., Borst, J. G. G. and Sakmann, B. (2002) ‘Calcium Secretion Coupling at Calyx of Held Governed by Nonuniform Channel–Vesicle Topography’, The Journal of Neuroscience, 22(5), pp. 1648–1667.

Morgan, A. and Burgoyne, R. D. (2009) ‘NSF and SNAPs’, 1st ed. Encyclopedia of Neuroscience, Academic Press, [online] Available from: http://www.sciencedirect.com/science/article/pii/B9780080450469013711 (Accessed 26 December 2015).

Purves, D., Augustine, G. J., Fitzpatrick, D., Katz, L. C., LaMantia, A.-S., McNamara, J. O. and Williams, S. M. (eds.) (2001) Neuroscience, 2nd ed. Sunderland, MA, Sinauer Associates.

Rizo, J. and Südhof, T. C. (2012) ‘The Membrane Fusion Enigma: SNAREs, Sec1/Munc18 Proteins, and Their Accomplices—Guilty as Charged?’, Annual Reviews, 28, pp. 279–308.

Sakaba, T. and Neher, E. (2001) ‘Quantitative Relationship between Transmitter Release and Calcium Current at the Calyx of Held Synapse’, The Journal of Neuroscience, 21(2), pp. 462–476.

Salio, C., Lossi, L., Ferrini, F. and Merighi, A. (2006) ‘Neuropeptides as synaptic transmitters’, Cell and Tissue Research, 326(2), pp. 583–598.

Schaub, J. R., Lu, X., Doneske, B., Shin, Y.-K. and McNew, J. A. (2006) ‘Hemifusion arrest by complexin is relieved by Ca2+-synaptotagmin I’, Nature Structural & Molecular Biology, 13, pp. 748–750.

Simon, S. M. and Llinás, R. R. (1985) ‘Compartmentalization of the submembrane calcium activity during calcium influx and its significance in transmitter release’, Biophysical Journal, 48(3), pp. 485–498.

Südhof, T. C. (2008) Pharmacology of Neurotransmitter Release, Starke, K. (ed.), Handbook of Experimental Pharmacology, Springer.

Thomas, L. and Betz, H. (1990) ‘Synaptophysin binds to physophilin, a putative synaptic plasma membrane protein’, Journal of Cell Biology, 111, pp. 2041–2052.

Verderio, C., Pozzi, D., Pravettoni, E., Inverardi, F., Schenk, U., Coco, S., Proux-Gillardeaux, V., Galli, T., Rossetto, O., Frassoni, C. and Matteoli, M. (2004) ‘SNAP-25 Modulation of Calcium Dynamics Underlies Differences in GABAergic and Glutamatergic Responsiveness to Depolarization’, Neuron, 41(4), pp. 599–610.

Verhage, M., McMahon, H. T., Ghijsen, W. E. J. M., Boomsma, F., Scholten, G., Wiegant, V. M. and Nicholls, D. G. (1991) ‘Differential release of amino acids, neuropeptides, and catecholamines from isolated nerve terminals’, Neuron, 6(4), pp. 517–524.

de Wit, H., Walter, A. M., Milosevic, I., Gulyás-Kovács, A., Riedel, D., Sørensen, J. B. and Verhage, M. (2009) ‘Synaptotagmin-1 Docks Secretory Vesicles to Syntaxin-1/SNAP-25 Acceptor Complexes’, Cell, 138(5), pp. 935–946.

Woodman, P. G. (1997) ‘The roles of NSF, SNAPs and SNAREs during membrane fusion’, BBA Molecular Cell Research, 1357(2), pp. 155–172.

Wu, M. N., Fergestad, T., Lloyd, T. E., He, Y., Broadie, K. and Bellen, H. J. (1999) ‘Syntaxin 1A Interacts with Multiple Exocytic Proteins to Regulate Neurotransmitter Release In Vivo’, Neuron, 23(3), pp. 593–605.

Zhang, X., Kim-Miller, M. J., Fukuda, M., Kowalchyk, J. A. and Martin, T. F. J. (2002) ‘Ca2+-Dependent Synaptotagmin Binding to SNAP-25 Is Essential for Ca2+-Triggered Exocytosis’, Neuron, 34(4), pp. 599–611.