Vesicle Priming in a SNAP

Abstract

In this issue of Neuron, Burgalossi et al. (2010) investigate synaptic vesicle priming using presynaptic Ca²⁺ uncaging at a small, glutamatergic, central synapse. Combining this technique with mouse genetics, the authors demonstrate that vesicle priming during ongoing neural activity can be limited by the recycling of recently used SNARE complexes.

The redistribution of charges over the plasma membrane of nerve terminals upon arrival of an action potential leads to the formation of a brief (~ 1ms), local elevation of the intracellular calcium concentration ([Ca²⁺]_i) in the close vicinity of open Ca²⁺ channels, and finally in synchronous fusion of synaptic vesicles (Schneggenburger and Neher, 2005). Importantly, only a fraction of vesicles located at or near the plasma membrane are competent to undergo action potential induced fusion at any point in time. These are referred to as ‘primed’ vesicles and the total number of primed vesicles is referred to as the release competent-, or primed vesicle pool. At many synapses, the size of the primed vesicle pool becomes limiting during ongoing activity because repetitive synaptic stimulation causes depletion of this pool, thereby causing a decrease in synaptic strength referred to as short-term depression (Neher and Sakaba, 2008).

What are the factors that determine the number of primed, release-competent vesicles at rest and during ongoing activity? Two proteins, Munc-13 and CAPS, have been shown to be crucial for vesicle priming. These proteins are important for the initiation of SNARE complex formation and help to stabilize synaptic vesicles in a fusion competent state (Südhof, 2004; Jockusch et al., 2007). The priming process is also regulated by [Ca²⁺]_i (Neher and Sakaba, 2008). Recent studies have provided evidence that priming may include a calcium sensor/effector complex formed by calmodulin and munc-13 (Junge et al., 2004). While these data define several molecular determinants of a primed vesicle, they do not illustrate why only a small fraction of synaptic vesicles are primed at any point in time, or why this pool becomes depleted during ongoing neural activity. One possibility is that many fusion competent vesicles exist at any given time, but only those in close proximity to calcium channels are released because of the low affinity of the Ca²⁺ sensor for synchronous vesicle fusion (Neher and Sakaba, 2008). Might other molecular determinants of primed vesicles become limiting during ongoing neural activity? In this issue of Neuron, Burgalossi and colleagues provide evidence that the recycling of SNARE complex proteins could be involved.

Once a release-competent vesicle has fused, its SNARE complexes are thought to be dissociated and recycled by the action of a complex consisting of the ATPase NSF and its SNAP co-factors (Jahn et al., 2003). One tends to think of SNARE proteins as being in vast excess and, therefore, they would not become limiting during the vesicle cycle. However, analysis of a temperature sensitive mutation in the Drosophila homologue of NSF, the comatose mutation, revealed pronounced synaptic depression during repetitive stimulation (Siddiqi and Benzer, 1976). These and other data suggest that SNARE recycling might become limiting during the vesicle cycle. However, little is known about when during the vesicle cycle NSF might function. There is even evidence that NSF could have an acute function prior to vesicle fusion (Kuner et al., 2008). If one could quantify the rate of vesicle priming and combine this with molecular manipulation of NSF, or its co-factors α-/β-SNAP, then it would be possible to test the intriguing possibility that SNARE recycling is an important and limiting factor in determining the number of fusion competent, primed vesicles during ongoing activity. This is what Burgalossi et al. (2010) achieve in their work, published in the current issue of Neuron.

In order to isolate and measure the rate of vesicle priming, Burgalossi and colleagues use presynaptic flash photolysis of caged Ca²⁺ (Ca²⁺ uncaging) in a manner first pioneered at neurosecretory cells and the neuromuscular junction, and successfully applied at central synapses like the calyx of Held (Schneggenburger and Neher, 2005). In order to investigate vesicle priming, it is advantageous to release all primed vesicles irrespective of their localization relative to calcium channels. Ca²⁺ uncaging enables the production of a spatially homogenous Ca²⁺ elevation so that the Ca²⁺ sensors of all vesicles ‘see’ the same [Ca²⁺]_i elevation. The consequence is that all of the primed, fusion competent vesicles are released.

For technical reasons, Ca²⁺ uncaging had never before been applied to study synaptic transmission at a small glutamatergic CNS terminal. Therefore, Burgalossi and colleagues first validate the technique and their measurements at hippocampal autapses. Flash photolysis of caged Ca²⁺ was used to generate step-like, prolonged presynaptic [Ca²⁺]_i elevations, which induced an EPSC with two kinetic components – a fast and a slow component. The authors provide evidence that the fast release component reflects the release of the entire fusion competent, primed vesicle pool when [Ca²⁺]_i was elevated to > 20 μM. First, the fast phase is abolished by prior perfusion of the synapse with high-sucrose saline. It is well established that a high-sucrose challenge induces an osmotic shock that releases ‘readily-releasable’ vesicles at or near the presynaptic membrane (Rosenmund and Stevens, 1996). Second, the authors use Ca²⁺ uncaging in priming-deficient CAPS1/2 double-knockout (DKO) autapses (Jockusch et al., 2007). It is thought that CAPS1/2 are necessary to stabilize primed, fusion-competent vesicles but that vesicle priming can persist in the absence of CAPS1/2 due to the activity of the calmodulin/Munc-13 complex. Surprisingly, when Burgalossi et al. apply Ca²⁺ uncaging in CAPS1/2 DKO autapses, they find that CAPS-deficient neurons have only a slow phase of release; the fast component is completely missing. This indicates that the fast phase of the flash-evoked response corresponds to the release of the sucrose-sensitive primed pool, whereas the slow phase should correspond to refilling of this pool. Therefore, Ca²⁺ uncaging can be used to report the size of the fusion competent primed vesicle pool (fast component), the repopulation of the primed vesicle pool (rise/amplitude of the slow component), and the exhaustion of this pool (decay of the slow component) at hippocampal autapses.

Having explored Ca²⁺ uncaging at autapses, and extended our understanding of CAPS1/2 function, Burgalossi et al. then turn to the question of SNARE recycling and vesicle priming. The authors studied priming in a hypomorphic α-/β-SNAP mouse mutant in which α-/β-SNAP levels are reduced by ~70% compared to controls. Interestingly, the reduction of α-/β-SNAP levels, which lead to an accumulation of assembled SNARE complexes in brain homogenates of mutant mice, selectively reduced the amplitude of the slow release component while leaving the fast component unaffected. This indicates that the repopulation of the fusion competent, primed vesicle pool is impaired by a reduction in recycled SNARE complexes (Figure 1). These and other experiments performed by the authors provide evidence that impaired SNARE complex recycling limits the rate of vesicle priming during persistent neural activity thereby leading to increased short-term depression. However, the importance of SNAP-mediated SNARE complex disassembly in synaptic transmission seems to be primarily confined to ongoing neural activity, because baseline synaptic transmission, and thus priming at rest, was largely unchanged.

Figure 1.

(A) Ca²⁺ uncaging was used to produce prolonged, spatially homogenous presynaptic [Ca²⁺]_i elevations (top), which produced EPSCs (bottom) recorded from wild-type (black), and α-/β-SNAP mutant autapses (gray). The inset shows a magnification of the early phase of the EPSC.

(B) Top: In wild-type, the step-like [Ca²⁺]_i rise leads to the release of all primed vesicles (green), and evokes an EPSP that is rapidly rising (A). Vesicles that are not primed (red) cannot be released. Middle: After fusion, SNARE complexes (t-SNAREs shown in black) are disassembled by the action of NSF and SNAPs (not shown). Bottom: In the continued presence of elevated [Ca²⁺]_i, newly primed vesicles (green) undergo fusion with slower kinetics as seen by the slow component of the EPSC (A).

(C) In α-/β-SNAP mutants, the number of newly primed vesicles is limited because of a shortage in free SNARE complexes. This is reflected by a decrease in the amplitude of the slow release component (gray trace in A) and illustrated by the red vesicle that cannot be released because of a cis-SNARE complex (bottom).

Finally, the authors make one further foray into analysis of calcium-dependent vesicle fusion. In addition to the authors’ analyses of CAPS1/2 and α-/β-SNAP mutant mice, they also used Ca²⁺ uncaging to study release at Synaptotagmin-1 deficient terminals and argue against the existence of a uniquely primed ‘asynchronous’ vesicle pool (Maximov and Südhof, 2005).

Conclusions and Outlook

This paper makes important technical and experimental advances regarding how synaptic vesicle release is controlled under baseline conditions and during continuous synaptic activity. For example, although two distinct kinetic release components in response to prolonged presynaptic depolarizations, or Ca²⁺ uncaging, have been studied at other synapses, the underlying molecular mechanisms are not well understood (Neher and Sakaba, 2008). Based on the absence of a fast release component in priming-deficient CAPS1/2 DKO mutants, Burgalossi and colleagues define the fast phase of the flash-evoked response at hippocampal autapses as the release of the primed vesicle pool, and the slow phase as the continuous release of newly primed vesicles. The combination of this biophysical assay with further genetic manipulation should continue to be a powerful method to gain new insights into the molecular mechanisms underlying priming and release.

Many questions also arise. For example, Burgalossi et al. provide evidence for a post-fusion role of SNARE complex disassembly in vesicle fusion during ongoing neural activity. However, they also observe a slight decrease in the number of release-competent vesicles and a slight increase in transmitter release probability in SNAP mutants. This might open up the possibility of a more rapid effect of NSF-mediated SNARE complex recycling that could occur prior to fusion (Kuner et al., 2008). Further work is needed to discern the function of NSF/SNAP in synaptic vesicle fusion. The analyses using α-/β-SNAP mutant mice also raise interesting questions. Does the availability of recycled SNARE complexes define the number of available release sites for vesicle priming at wild type synapses? The authors provide some evidence that this could be the case during continuous synaptic activity. How are SNARE complexes and release sites re-established during re-priming? Could newly primed vesicles be fused with fewer SNARE complexes, as recently suggested (Mohrmann et al., 2010)? Does the requirement for SNARE complex recycling via NSF suggest that the fusion of vesicles, at least following Ca²⁺ uncaging, is mediated through full fusion? Finally, there is the question of whether priming is modulated in vivo and, if so, how. Intraterminal Ca²⁺ levels modulate priming (Neher and Sakaba, 2008), but are there other signals that change the priming state of a synapse? For instance, it is known that PKC activation of munc-13 promotes priming. Are there in vivo conditions during which the priming state of synapses is modulated by extrinsic factors? As we gain a greater understanding of the events that constrain and modulate the vesicle cycle, we may eventually come to understand how characteristic properties of short-term synaptic plasticity are established at synaptic terminals throughout the central and peripheral nervous systems