Synaptophysin 1 and 2 and synaptogyrin 1 and 3 constitute a major family of synaptic vesicle membrane proteins expressed widely, possibly in all synaptic vesicles throughout the animal kingdom (Jahn et al., 1985; Stenius, 1995; Fernández-Chacón and Südhof, 1999; Takamori et al., 2006). Synaptogyrin 2, also known as cellugyrin, is non-neuronal (Janz and Südhof, 1998). The widespread expression of neuronal family members suggests a fundamental role in synaptic transmission, but what that might be is not known.

Family members bind to the vSNARE synaptobrevin 2/VAMP 2, which is a core component of the machinery that catalyzes membrane fusion during synaptic vesicle exocytosis (Söllner et al., 1993; Calakos and Scheller, 1994; Washbourne et al., 1995; Edelmann et al., 1995; Becher et al., 1999; Khvotchev and Südhof, 2004). And, overexpression of family members potently inhibited neurotransmitter release in a cell line (Sugita et al., 1999). Despite this, the hypothesis that the native function might involve negative regulation of exocytosis has not been pursued extensively, possibly because no clear evidence was found for increases in neurotransmitter release at synapses from synaptophysin 1 and synaptogyrin 1 single and double knockouts (McMahon et al., 1996; Janz et al., 1999; Abraham et al., 2006; Stevens et al., 2012). Instead, recent research has been focused on a variety of mechanisms that operate downstream of exocytosis, including: endocytosis of membrane; and/or recycling of proteins thought to be needed to catalyze subsequent rounds of exocytosis (Kwon and Chapman, 2011; Gordon et al., 2011; Rajappa et al., 2016). However, the previous studies, at least in mammals, involved exogenous expression or genetic deletion of synaptophysin 1 and synaptogyrin 1, whereas possible compensatory activity of synaptophysin 2 and synaptogyrin 3 has never been assessed (McInnes et al., 2018).

Here, we report that individual action potentials trigger exocytosis of a higher fraction of the readily releasable vesicles at a variety of synapse types from quadruple knockout mice (QKO) where all four neuronal family members have been deleted (see Materials and methods). No deficit was detected in other presynaptic parameters that control function such as the capacity of the readily releasable pool (RRP) for storing vesicles and the timing of vesicle recruitment to the RRP during light or heavy use. The results suggest strongly that family members play an inhibitory role at the level of exocytosis rather than the facilitatory downstream role that is currently envisioned. A follow-on analysis of double and triple knockouts showed that synaptophysin 1 and synaptogyrin 3 can compensate for missing family members, whereas synaptophysin 2 seemed to play a dominant negative role.

QKO mice appeared to develop normally when housed in individually ventilated cages, and produced litters of normal size. However, adults were prone to convulsions, sometimes causing death, especially after being startled. We were not able to maintain the colony in a second facility where ventilated cages were not available, suggesting that the ventilation system aided survival, possibly by producing continuous white noise that limited startling. A quantitative western blot analysis of homogenized tissue and purified synaptosomes from QKO brains revealed a selective decrease in VAMP 2 levels, but no major changes in a wide variety of other synaptic proteins (Figure 1); a decrease in VAMP 2 levels was detected previously in synaptophysin 1 single knockouts (McMahon et al., 1996).

Figure 1 with 1 supplement see all

Download asset Open asset

Elevated neurotransmitter release at calyx of Held synapses from QKO mice

In a first set of experiments to determine the primary functional deficit, we found that calyx of Held synapses from QKO mice were substantially stronger than WT when action potentials were evoked in the afferent axon at low frequency (Figure 2A–B). Excitatory postsynaptic currents (EPSCs) recorded in voltage clamped principal neurons of the medial nucleus of the trapezoid body (MNTB) had a greater quantal content (Figure 2C), indicating that the synapses were stronger because of exocytosis of transmitter from more presynaptic vesicles, in-line with the presynaptic locus of expression of synaptophysin family members. Spontaneous quantal release was elevated by a similar amount (Figure 2D–E), and the size of quantal events was elevated by a smaller amount (Figure 2F). No significant alterations were detected in the time courses of the EPSCs evoked with low-frequency stimulation or in spontaneous events (Figure 2—figure supplement 1).

Figure 2 with 1 supplement see all

Download asset Open asset

Elevated probability of release, with no alteration in RRP size

The number of vesicles that undergo exocytosis when action potentials are fired at low frequency is determined by two factors that seem to be controlled independently: the number of vesicles within a readily releasable pool, termed RRP content and possibly determined by the number of sites in the active zone area of the plasma membrane where vesicles dock; and the mean probability of release per vesicle (${\overline{p}}_v$) within the RRP (Figure 3A, left); the abbreviation $P_r$ has been used to denote the same concept in some other studies but is also sometimes used instead to denote the probability of release per synapse, which is the mathematical product of ${\overline{p}}_v$ and RRP content, and thus a different concept. To determine which of the parameters was altered, we stimulated at 300Hz for 300ms (90 action potentials; Figure 3B). The difference in synaptic strength disappeared quickly, by the 4th action potential (Figure 3B–C), and there was no difference in the total number of quanta released during the first 150ms of the trains (Figure 3D). The result suggests that the RRP content was not altered at QKO synapses because 150ms is enough to nearly completely exhaust the RRP (Mahfooz et al., 2016).

Figure 3 with 1 supplement see all

Download asset Open asset

Technically, the number of quanta released during trains that exhaust the RRP is not a perfect measure of RRP content because new vesicles are continually recruited and contents released during ongoing stimulation (Figure 3A, right). However, the amount of recruitment can be estimated by a variety of methods; two that have been proposed for this purpose are plotted in Figure 3D (i.e. curves marked ‘S’ and ‘M’). To our knowledge, the two span the full range of quantitative models that have been proposed; theory ‘S’ was proposed earlier, by Schneggenburger et al. (1999), whereas ‘M’ incorporates the conclusion of Mahfooz et al. (2016) that the RRP has a fixed capacity and vesicles are recruited to vacant spaces, such as empty release sites, as illustrated in Figure 3A (right panel). Although the various methods produced a variety of estimates for the amount of recruitment during the trains, all methods agreed that the amount was not different at QKO compared to at WT synapses (e.g. intercepts of curves ‘S’ and ‘M’ with ordinate-axis in Figure 3D). This result confirms that the initial RRP content was not altered at QKO synapses.

In contrast, ${\overline{p}}_v$ is calculated by dividing the number of quanta released after isolated action potentials by the RRP content, and was approximately double at QKO synapses (Figure 3E). These results show that removing synaptophysin family members increased the value of ${\overline{p}}_v$ without altering RRP content. Taken together, they suggest that endogenous synaptophysin family members inhibit neurotransmission downstream of vesicle recruitment to the RRP.

No alteration in the timing of vesicle priming

The conclusion is consistent with early experiments where exogenous expression of family members inhibited exocytosis (Sugita et al., 1999). However, one of the current hypotheses is that at least synaptophysin 1 plays a post-exocytosis role in clearing components of spent vesicles from the release machinery. The idea is that clearance determines the rate at which new vesicles can be recruited to the RRP during heavy use (Kwon and Chapman, 2011; Gordon et al., 2011; Rajappa et al., 2016). If so, removing family members would have produced a deficit in the rate at which vesicles are recruited to the RRP at later times during the trains of 300 Hz stimulation, which drove multiple rounds of exocytosis (see Legend of Figure 3D), and during subsequent rest intervals. But no such deficits were seen at QKO synapses.

That is, the methods used above to estimate the total amount of vesicle recruitment during trains additionally produce estimates of the ongoing rate of recruitment during heavy use (arrows in Figure 3A, right). All produced matching estimates for QKO and WT synapses (Figure 3F); see Figure 3—figure supplement 1 for control experiments verifying that the analyses were not confounded by postsynaptic mechanisms such as glutamate receptor desensitization.

The absence of a deficit in recruitment during extended stimulation could additionally be deduced without referencing any of the methods simply from the observation that steady state quantal output after the 150th ms of 300 Hz stimulation was similar at QKO synapses compared to WT (53.4 ± 4.7 quanta/action potential vs 59.2 ± 11.7). Nor did we find any alteration in the timing of RRP replenishment during rest intervals that followed 300 Hz stimulation (Figure 4). These results do not support the hypothesis that synaptophysin family members play a critical post-exocytosis role in facilitating ongoing vesicle priming and release, at least at calyx of Held synapses.

Figure 4

Download asset Open asset

Elevated ${\overline{p}}_v$ pertains to both high and low $p_v$ vesicles

The results so far suggest that synaptophysin family members ordinarily play an inhibitory role in neurotransmission downstream of vesicle recruitment to the RRP and upstream of neurotransmitter release, consistent with the possibility that the action is directly at the level of exocytosis. However, some current models include intervening priming steps between recruitment and exocytosis.

That is, the initial idea was that the RRP is a homogeneous pool (Elmqvist and Quastel, 1965; Vere-Jones, 1966), but there is now widespread agreement that some readily releasable vesicles are released more slowly than others during repetitive stimulation owing to a lower probability of release (Wu and Borst, 1999; Sakaba and Neher, 2001; Moulder and Mennerick, 2005). For clarity, we refer to the readily releasable vesicles with high and low probability of release as high and low $p_v$ vesicles, respectively, but note that it is possible that there are more than only two classes (Mahfooz et al., 2016; Taschenberger et al., 2016); elsewhere, low $p_v$ vesicles have been termed slow-releasing, or reluctantly releasable.

Both high and low $p_v$ vesicles seem to be immediately releasable, at least in the calyx of Held preparation examined here, and the timing of exocytosis is tightly synchronized to action potentials (Mahfooz et al., 2016). Our own models continue to treat recruitment to the RRP as the final vesicle trafficking step upstream of exocytosis (Mahfooz et al., 2016). the idea is that low and high $p_v$ vesicles are docked to distinct types of release sites (Figure 5A; Hu et al., 2013; Müller et al., 2015; Böhme et al., 2016). However, recent models of other research groups include additional mechanisms where individual vesicles already within the RRP reversibly transition between a variety of primed states distinguished by a range of release probabilities (Lee et al., 2013; Neher, 2017). If so, synaptophysin family members might either: limit the rate of transition of one of the forward steps, decreasing the fraction of vesicles in a high $p_v$ state; or directly inhibit exocytosis of low and high $p_v$ vesicles alike.

Figure 5 with 2 supplements see all

Download asset Open asset

To determine whether the action is at the level of exocytosis or upstream, we conducted frequency jump experiments where high $p_v$ vesicles are first eliminated from the RRP with submaximal stimulation of 50 or 100 Hz, leaving a standing population of low $p_v$ vesicles that can then be released by subsequent 300 Hz stimulation (Figure 5A). The experimental design was developed previously for isolating the kinetics of release of low $p_v$ vesicles at WT calyces of Held (Mahfooz et al., 2016). In the present case, the full experiment consisted of $6$ types of interleaved trials: frequency jumps where 1000 ms of 50 Hz stimulation was followed by 200 ms of 300 Hz stimulation and a matched control where the stimulating frequency was maintained at 50 Hz throughout (Figure 5B–C); two additional types of frequency jumps where 500 ms or 750 ms of 100 Hz stimulation was followed by 200 ms of 300 Hz stimulation, along with a matched control where the stimulating frequency was maintained at 100 Hz throughout (Figure 5—figure supplement 1A); and an additional control where stimulation was 300 Hz for 300 ms, with no prior stimulation for at least 1 min (subset of data in Figures 2–3).

Figure 5B–C and Figure 5—figure supplement 1A show that 300 Hz stimulation transiently increased the rate of release following 50 Hz or 100 Hz stimulation, extending to QKO calyces of Held the previous finding that 100 Hz stimulation is not sufficiently frequent to exhaust the RRP at WT synapses (Mahfooz et al., 2016). To determine the ${\overline{p}}_v$ value for the vesicles remaining in the RRP, we first isolated the increase in release by subtracting the time-averaged steady state response recorded during the matched trials when the stimulation frequency was not increased (gray data points in Figure 5C and Figure 5—figure supplement 1A; the amount of increase - termed the differential release - is plotted in Figure 5D and Figure 5—figure supplement 1B). The value for ${\overline{p}}_v$ was then calculated by dividing the differential release after the first action potential during 300 Hz stimulation by the RRP content at the start of 300 Hz stimulation. The RRP content at the start of 300 Hz stimulation was estimated by subtracting the amount of recruitment to the RRP during 300 Hz stimulation from the total.

As above for Figure 3D–E, the estimate for the amount of recruitment during 300 Hz stimulation depended partly on assumptions about mechanism that continue to be debated (lines marked ‘M’ vs ‘S’ in Figure 5D and Figure 5—figure supplement 1B). However, all methods produced ${\overline{p}}_v$ values for the vesicles remaining in the RRP that were higher for QKO synapses compared to WT (Figure 5E). The effect can be seen most clearly in the cumulative plots of the differential release measurements in Figure 5F, which are normalized so that the theoretical curves produced by the ‘M’ and ‘S’ theories intersect the y-axis at $\frac{1}{{\overline{p}}_v}$ (see Legend). Note that the effect is not as readily apparent in Figure 5C–D because the standing state fullness of the RRP was lower at QKO synapses (Figure 5—figure supplement 2) - which is an expected consequence of the higher ${\overline{p}}_v$ value (Mahfooz et al., 2016) - and because of the large bin size in Figure 5C.

The ${\overline{p}}_v$ values for both WT and QKO were approximately 3-fold lower during 50 or 100 Hz stimulation compared to the corresponding values when the RRP was full (compare Figure 5E to Figure 3E), confirming that high $p_v$ vesicles were eliminated. And indeed, almost all of the vesicles remaining within the RRP during both 50 and 100 Hz stimulation must have been in the low $p_v$ state because 100 Hz stimulation depleted the RRP to a greater extent (Figure 5—figure supplement 2), but did not further lower the value for ${\overline{p}}_v$ (Figure 5E).

These results do not distinguish between the classes of models with single and multiple sequential vesicle priming steps mentioned above, but do indicate that synaptophysin family members inhibit exocytosis of low and high $p_v$ vesicles alike. The results therefore strongly suggest that family members inhibit neurotransmission downstream of the final step in vesicle priming, which is consistent with the increase in spontaneous exocytosis seen in the absence of action potentials documented in Figure 2D–E. The analysis is based on the premise that low $p_v$ vesicles are immediately releasable, which seems likely because release of neurotransmitter continues to be tightly synchronized to action potentials during frequency jumps that are initiated after the high $p_v$ vesicles have been eliminated, and because synapses with more low $p_v$ vesicles express more paired pulse facilitation when the RRP is full (Figure 5B and Figure 5—figure supplement 1C; Mahfooz et al., 2016). Nevertheless, we did additionally considered the alternate scenario where the low $p_v$ vesicles are not immediately releasable (Miki et al., 2016; Gustafsson et al., 2019), but found it was not compatible with the results in Figure 5D and Figure 5—figure supplement 1B, as explained in the Legend of Figure 5—figure supplement 1.

Elevated ${\overline{p}}_v$ at Schaffer collateral synapses from QKO mice

We next conducted experiments analogous to Figures 3–4 on synapses between Schaffer collaterals and CA1 pyramidal neurons of the hippocampus. We stimulated with 20 Hz trains instead of 300 Hz because 20 Hz is frequent enough to nearly completely empty the RRP owing to ∼20-fold slower recruitment of new vesicles during ongoing stimulation compared to the calyx of Held (Wesseling et al., 2002; Mahfooz et al., 2016).

Estimating the quantal content of responses from individual Schaffer collaterals is not as straightforward as for the calyx of Held because the number of afferent axons activated during a typical experiment is an unknown function of the strength of the individual pulses of stimulation (Figure 6A). Nevertheless, similar to at the calyx of Held, postsynaptic responses depressed more rapidly at QKO synapses compared to WT (Figure 6B–C). A kinetic analysis indicated that ${\displaystyle {\overline{p}}_v}$ was approximately double (Figure 6D–E), whereas the timing of vesicle recruitment to the RRP during ongoing stimulation was not altered (Figure 6F). And, no difference was detected in the time course of RRP replenishment during subsequent rest intervals (Figure 6G). These results extend to hippocampal synapses the conclusion that the machinery that catalyzes synaptic vesicle exocytosis becomes more efficient after removing synaptophysin family proteins, whereas the timing of vesicle recruitment to the RRP is not altered. The comparison between calyces of Held and Schaffer collateral synapses is a good test for generality across synapse types because, in nature, Schaffer collateral synapses are typically used at frequencies that are ∼15-fold lower in addition to striking morphological and molecular differences and the ∼20-fold difference in the timing of vesicle trafficking already noted above (Ranck, 1973; Hermann et al., 2007; Borst and Soria van Hoeve, 2012).

Figure 6

Download asset Open asset

Higher throughput assay in primary cell culture

To confirm that synaptic vesicle exocytosis is increased at QKO hippocampal synapses when action potentials are fired at low frequency, and to assess the contribution of each of the four synaptophysin family members, we then developed an optical imaging assay in primary cell culture with higher throughput than the electrophysiological assays (see Figure 7A and diagram atop Figure 7B). We first loaded the recycling synaptic vesicles with FM4-64 dye during 60 s of 20 Hz electrical stimulation (Gaffield and Betz, 2006; see Figure 7—figure supplement 1 for example images). We then monitored destaining with time-lapse fluorescence imaging during low frequency (0.2 Hz) stimulation in the absence of dye, followed by near complete destaining with a second 20 Hz train.

Figure 7 with 1 supplement see all

Download asset Open asset

We reasoned that destaining would be directly proportional to ${\overline{p}}_v$ during low-frequency stimulation because the RRP would remain almost completely full. In contrast, we reasoned that destaining would not be influenced at all by ${\overline{p}}_v$ during 20 Hz stimulation which empties the RRP in less than the 4 s interval between acquisition of successive images, after which transmitter release is no-longer influenced by ${\overline{p}}_v$ and is instead rate-limited by vesicle recruitment to the RRP (Wesseling et al., 2002; see Figure 7A).

And indeed, QKO synapses destained almost 2-fold more than WT during the 0.2 Hz stimulation (Figure 7A–C), confirming that ${\overline{p}}_v$ is elevated. And, no differences were detected during subsequent 20 Hz stimulation (not shown), or during 20 Hz stimulation when the 0.2 Hz train was omitted (Figure 7D), confirming that the timing of vesicle recruitment to the RRP was not altered. Furthermore, no differences between QKO and WT were detected in the amount of staining during loading, further supporting the conclusion of no alterations in rate-limiting steps in vesicle trafficking in QKO synapses.

Synaptophysin family proteins are widely expressed in synaptic vesicle membranes, with more individuals per vesicle than the extensively studied synaptotagmins, although likely less than the vSNARE VAMP 2 and homologs (Takamori et al., 2006; Wilhelm et al., 2014). Despite the abundance, information about the role in presynaptic function has been elusive. Here, we show that the efficiency of the release machinery is elevated at a variety of synapse types from knockout mice where all four neuronal family members have been deleted, with no indication of any alteration in RRP content or the timing of vesicle recruitment to the RRP during light or heavy use. The new results strongly suggest that synaptophysin family members modulate function at the level of exocytosis. If so, the native action likely includes inhibition, especially when the results are taken together with an earlier study where exogenous synaptophysin 1 and synaptogyrin 1 potently inhibited exocytosis (Sugita et al., 1999; but see Alder et al., 1995).

The concern that the elevated ${\overline{p}}_v$ seen at QKO synapses might instead reflect complicating developmental or compensatory mechanisms that are not directly related to the native function of the missing proteins is countered by the following. First, the alteration is unusually specific; ${\overline{p}}_v$ was elevated without desynchronizing the relationship between action potentials and neurotransmitter release, or disrupting other vesicle trafficking mechanisms that control presynaptic function such as the capacity of the RRP for storing synaptic vesicles and the timing of vesicle recruitment to vacancies within the RRP. Second, the alteration was robust across a variety of synapse types embedded within neuronal networks that are subject to dissimilar developmental forces, including the networks formed by neurons grown in dissociated cell culture. Third, besides the decrease in VAMP 2, we did not detect substantial changes in levels of a variety of other proteins that have been implicated in exocytosis, including syntaxin 1, SNAP-25, RIM 1/2, Munc13-1, Rab 3a, and complexin 1/2. And fourth, synaptophysin 2 seemed to inhibit the function of other family members, which argues against the specific concern that removing the other family members increased the fusogenicity of synaptic vesicles mechanically, by simply exposing space on the surface vesicular membranes.

Notably, however, no elevation in ${\overline{p}}_v$ was detected in neuromuscular junctions after deleting the family from C. elegans or Drosophila - and exocytosis of neurotransmitter was increased rather than decreased after expressing exogenous synaptophysin 1 in Xenopus - and it seems unlikely that an entire family of proteins would have an unrelated function in the different species (Alder et al., 1995; Abraham et al., 2006; Stevens et al., 2012). One possibility is that family members function as bi-directional regulators of exocytosis where the directionality is modulated by second messengers, possibly via phosphorylation of the tyrosine residues along the C-terminal tail. In any case, we anticipate that the cause of the discrepancies between species will become clear when more is known about the mechanism.

Indeed, even basic information about the mechanism remains to be elucidated. One possibility is that synaptophysin family members might interact directly with catalysis as outlined in Rothman et al. (2017); see also Adams et al. (2015). However, when bound to synaptophysin 1, VAMP 2 was excluded from the core SNARE complex consisting of VAMP 2, syntaxin 1, and SNAP25 that catalyzes exocytosis (Edelmann et al., 1995), and the combination of syntaxin 1 and SNAP25 could disrupt the binding between synaptophysin 1 and VAMP 2 in an enriched vesicle preparation (Siddiqui et al., 2007). A second possibility is that family members might lessen SNARE complex formation simply by restricting the availability of VAMP 2. However, we are not aware of evidence that exocytosis can be modulated in this manner, and indeed, lowering levels of the SNAP-25 component paradoxically increased ${\overline{p}}_v$ in at least one study (Antonucci et al., 2013).

In any case, the relevance of the reduction in VAMP 2 levels in purified QKO synaptosomes to the increase in exocytosis seen at intact synapses in the functional assays is not known, and is counter-intuitive given that VAMP 2 is necessary for exocytosis. Intriguingly, the reduction is in-line with a previous study where exogenous VAMP 2 could be driven to synaptic vesicles by co-expressing synaptophysin 1 (Pennuto et al., 2003). The results do not suggest that synaptophysin family members are required for targeting VAMP 2 to synaptic vesicles, but are consistent with the possibility that VAMP 2 exists in two pools within vesicle membranes: one of which is stabilized by binding to synaptophysin family members; and the other by a different factor that remains to be identified. On the other hand, it is possible that the reduction in QKO synaptosomes resulted from depletion from plasma membrane rather than synaptic vesicles because significant amounts of VAMP 2 are consistently found in plasma membranes (Sankaranarayanan et al., 2000), and synaptosomes contain plasma membrane in addition to synaptic vesicles.

It is not clear how or if the small elevation in size of spontaneous responses seen both here and after deleting synaptogyrin from Drosophila is related to the elevated probability of release (Stevens et al., 2012). We cannot rule out a postsynaptic mechanism, but vesicles were larger in the Drosophila mutants, which might play a role. Possibly also relevant: Vesicles were also larger in synapses from knockouts of other presynaptic proteins involved in exocytosis, including VAMP 2, SNAP-25, and Munc13-1/2; the increase in radius was <10%, but even this small increase would translate to an increase in volume of 30%, which is more than the elevation in quantal size seen here (Imig et al., 2014).

Our results are in-line with previous reports of a role for synaptophysin family members in short-term synaptic plasticity (Janz et al., 1999; Kwon and Chapman, 2011; Rajappa et al., 2016). However, the decreased paired-pulse facilitation and increased paired-pulse depression seen in single, double and quadruple knockouts at a variety of synapse types do not necessarily indicate defects in mechanisms underlying short-term plasticity, but instead may result from the baseline elevation of ${\overline{p}}_v$. And indeed, paired-pulse facilitation could be unmasked at QKO synapses by lowering extracellular Ca²⁺, which lowers the baseline (Figure 8). In any case, the elevation in ${\overline{p}}_v$ at QKO synapses cannot be attributed solely to the superpriming phenomenon proposed in Taschenberger et al. (2016) because the frequency jump experiments documented in Figure 5 indicated that the elevated release probability pertained to both low and high $p_v$ vesicles, whereas superpriming is thought to pertain exclusively to vesicles with high $p_v$.

Figure 8

Download asset Open asset

The evidence against substantial deficits in the timing of vesicle recruitment to the RRP at QKO synapses seems to be strong. In particular, receptor desensitization mechanisms that could occlude differences between QKO and WT in the electrophysiological studies were ruled out with additional experiments in Figure 3—figure supplement 1 for calyx of Held and in Wesseling et al. (2002) for Schaffer collateral synapses. And, the evidence in Figure 7D relies on logic that avoids postsynaptic mechanisms altogether. However, our results do not conflict with the evidence for biochemical and cell biological alterations previously seen downstream of exocytosis in synaptophysin 1 knockouts (Kwon and Chapman, 2011; Gordon et al., 2011; Rajappa et al., 2016), although the results do indicate that any such downstream alterations would not affect the timing of vesicle recruitment to the RRP.

The selectivity of the increase in ${\overline{p}}_v$, with no change in the timing of vesicle recruitment or the size of the RRP, suggests that synaptophysin family members normally dampen synaptic connection strength when synapses are used at low frequencies. However, their effective role during the type of burst firing that occurs routinely in vivo would be more complex owing to slower depletion of the RRP as a direct consequence of reduced transmitter release. One way to characterize this sort of functional complexity is so called redistribution of synaptic efficacy where a decrease in synaptic strength at the beginning of a train of action potentials serves to enhance the strength later on (Markram and Tsodyks, 1996). Intriguingly, it seems that some activity-dependent form of redistribution of synaptic efficacy can be induced at a broad range of synapse types. For example, long-lasting bidirectional redistribution can be induced at cortical and hippocampal synapses by some of the same experimental protocols used to activate standard long-term potentiation and depression mechanisms at other synapse types (Markram and Tsodyks, 1996; Sjöström et al., 2003; Yasui et al., 2005; Monday et al., 2018). And, although the terminology was different, other reports have suggested that the mechanisms underlying post-tetanic potentiation and a third type of short-term plasticity termed augmentation have a similar re-distributive effect (Stevens and Wesseling, 1999; Garcia-Perez and Wesseling, 2008; Lee et al., 2010). Going forward it will be interesting to determine if synaptophysin family members or other selective regulators of ${\overline{p}}_v$ such as GIT 1/2 or Mover are involved in any of these phenomena (Körber et al., 2015; Montesinos et al., 2015).

Knockout and WT control mice were obtained from two independent crosses. For the experiments in Figures 1–5 and 7, QKO, matched WT controls, and the ten logically possible double and triple knockouts were bred out in three or four generations by crossing a germline CRE expressing line (Jackson labs catalog number 008454) with Jackson line 008415, which carries targeted knockouts of synaptophysin 1 and synaptogyrin 1 genes and floxed conditional mutations of synaptophysin 2 and synaptogyrin 3; the CRE transgene was eliminated during the process. QKO and WT mice for experiments in Figure 6 were obtained directly from Dr. Thomas Südhof.

All data analyzed for this study are included in the manuscript and supporting files. Source data files are available at Dryad (doi:10.5061/dryad.rn91r08).

1. 1. Raja MK
   2. Martinez-Turrillas R
   3. del Olmo-Cabrera S

   (2018) Dryad Digital Repository

   Data from: Elevated synaptic vesicle release probability in synaptophysin/gyrin family quadruple knockouts.

   https://doi.org/10.5061/dryad.rn91r08

1. 1. Abraham C
   2. Hutter H
   3. Palfreyman MT
   4. Spatkowski G
   5. Weimer RM
   6. Windoffer R
   7. Jorgensen EM
   8. Leube RE

   (2006) Synaptic tetraspan vesicle membrane proteins are conserved but not needed for synaptogenesis and neuronal function in Caenorhabditis elegans

   PNAS 103:8227–8232.

   https://doi.org/10.1073/pnas.0509400103

   • PubMed
   • Google Scholar
2. 1. Adams DJ
   2. Arthur CP
   3. Stowell MHB

   (2015) Architecture of the synaptophysin/Synaptobrevin complex: structural evidence for an entropic clustering function at the synapse

   Scientific Reports 5:1–9.

   https://doi.org/10.1038/srep13659

   • Google Scholar
3. 1. Alder J
   2. Kanki H
   3. Valtorta F
   4. Greengard P
   5. Poo MM

   (1995) Overexpression of synaptophysin enhances neurotransmitter secretion at Xenopus neuromuscular synapses

   The Journal of Neuroscience 15:511–519.

   https://doi.org/10.1523/JNEUROSCI.15-01-00511.1995

   • PubMed
   • Google Scholar
4. 1. Antonucci F
   2. Corradini I
   3. Morini R
   4. Fossati G
   5. Menna E
   6. Pozzi D
   7. Pacioni S
   8. Verderio C
   9. Bacci A
   10. Matteoli M

   (2013) Reduced SNAP-25 alters short-term plasticity at developing glutamatergic synapses

   EMBO reports 14:645–651.

   https://doi.org/10.1038/embor.2013.75

   • PubMed
   • Google Scholar
5. 1. Atluri PP
   2. Regehr WG

   (1998) Delayed release of neurotransmitter from cerebellar granule cells

   The Journal of Neuroscience 18:8214–8227.

   https://doi.org/10.1523/JNEUROSCI.18-20-08214.1998

   • PubMed
   • Google Scholar
6. 1. Becher A
   2. Drenckhahn A
   3. Pahner I
   4. Margittai M
   5. Jahn R
   6. Ahnert-Hilger G

   (1999) The synaptophysin-synaptobrevin complex: a hallmark of synaptic vesicle maturation

   The Journal of Neuroscience 19:1922–1931.

   https://doi.org/10.1523/JNEUROSCI.19-06-01922.1999

   • PubMed
   • Google Scholar
7. 1. Böhme MA
   2. Beis C
   3. Reddy-Alla S
   4. Reynolds E
   5. Mampell MM
   6. Grasskamp AT
   7. Lützkendorf J
   8. Bergeron DD
   9. Driller JH
   10. Babikir H
   11. Göttfert F
   12. Robinson IM
   13. O'Kane CJ
   14. Hell SW
   15. Wahl MC
   16. Stelzl U
   17. Loll B
   18. Walter AM
   19. Sigrist SJ

   (2016) Active zone scaffolds differentially accumulate Unc13 isoforms to tune Ca(2+) channel-vesicle coupling

   Nature Neuroscience 19:1311–1320.

   https://doi.org/10.1038/nn.4364

   • PubMed
   • Google Scholar
8. 1. Borst JG
   2. Soria van Hoeve J

   (2012) The calyx of Held synapse: from model synapse to auditory relay

   Annual Review of Physiology 74:199–224.

   https://doi.org/10.1146/annurev-physiol-020911-153236

   • PubMed
   • Google Scholar
9. 1. Calakos N
   2. Scheller RH

   (1994)

   Vesicle-associated membrane protein and synaptophysin are associated on the synaptic vesicle

   The Journal of Biological Chemistry 269:24534–24537.

   • PubMed
   • Google Scholar
10. 1. Chowdhury D
    2. Marco S
    3. Brooks IM
    4. Zandueta A
    5. Rao Y
    6. Haucke V
    7. Wesseling JF
    8. Tavalin SJ
    9. Pérez-Otaño I

    (2013) Tyrosine phosphorylation regulates the endocytosis and surface expression of GluN3A-containing NMDA receptors

    Journal of Neuroscience 33:4151–4164.

    https://doi.org/10.1523/JNEUROSCI.2721-12.2013

    • PubMed
    • Google Scholar
11. 1. Edelmann L
    2. Hanson PI
    3. Chapman ER
    4. Jahn R

    (1995) Synaptobrevin binding to synaptophysin: a potential mechanism for controlling the exocytotic fusion machine

    The EMBO Journal 14:224–231.

    https://doi.org/10.1002/j.1460-2075.1995.tb06995.x

    • PubMed
    • Google Scholar
12. 1. Elmqvist D
    2. Quastel DM

    (1965) A quantitative study of end-plate potentials in isolated human muscle

    The Journal of Physiology 178:505–529.

    https://doi.org/10.1113/jphysiol.1965.sp007639

    • PubMed
    • Google Scholar
13. 1. Evans GJ
    2. Cousin MA

    (2005) Tyrosine phosphorylation of synaptophysin in synaptic vesicle recycling

    Biochemical Society Transactions 33:1350–1353.

    https://doi.org/10.1042/BST0331350

    • PubMed
    • Google Scholar
14. 1. Fernández-Chacón R
    2. Südhof TC

    (1999) Genetics of synaptic vesicle function: toward the complete functional anatomy of an organelle

    Annual Review of Physiology 61:753–776.

    https://doi.org/10.1146/annurev.physiol.61.1.753

    • PubMed
    • Google Scholar
15. 1. Fiuza M
    2. González-González I
    3. Pérez-Otaño I

    (2013) GluN3A expression restricts spine maturation via inhibition of GIT1/Rac1 signaling

    PNAS 110:20807–20812.

    https://doi.org/10.1073/pnas.1312211110

    • PubMed
    • Google Scholar
16. 1. Gaffield MA
    2. Betz WJ

    (2006) Imaging synaptic vesicle exocytosis and endocytosis with FM dyes

    Nature Protocols 1:2916–2921.

    https://doi.org/10.1038/nprot.2006.476

    • PubMed
    • Google Scholar
17. 1. Garcia-Perez E
    2. Wesseling JF

    (2008) Augmentation controls the fast rebound from depression at excitatory hippocampal synapses

    Journal of Neurophysiology 99:1770–1786.

    https://doi.org/10.1152/jn.01348.2007

    • PubMed
    • Google Scholar
18. 1. Gordon SL
    2. Leube RE
    3. Cousin MA

    (2011) Synaptophysin is required for synaptobrevin retrieval during synaptic vesicle endocytosis

    Journal of Neuroscience 31:14032–14036.

    https://doi.org/10.1523/JNEUROSCI.3162-11.2011

    • PubMed
    • Google Scholar
19. 1. Gustafsson B
    2. Ma R
    3. Hanse E

    (2019) The small and dynamic Pre-primed pool at the release site; A useful concept to understand release probability and Short-Term synaptic plasticity?

    Frontiers in Synaptic Neuroscience 11:.

    https://doi.org/10.3389/fnsyn.2019.00007

    • PubMed
    • Google Scholar
20. 1. Hermann J
    2. Pecka M
    3. von Gersdorff H
    4. Grothe B
    5. Klug A

    (2007) Synaptic transmission at the calyx of Held under in vivo like activity levels

    Journal of Neurophysiology 98:807–820.

    https://doi.org/10.1152/jn.00355.2007

    • PubMed
    • Google Scholar
21. 1. Hu Z
    2. Tong XJ
    3. Kaplan JM

    (2013) UNC-13L, UNC-13S, and tomosyn form a protein code for fast and slow neurotransmitter release in Caenorhabditis elegans

    eLife 2:e00967.

    https://doi.org/10.7554/eLife.00967

    • PubMed
    • Google Scholar
22. 1. Imig C
    2. Min SW
    3. Krinner S
    4. Arancillo M
    5. Rosenmund C
    6. Südhof TC
    7. Rhee J
    8. Brose N
    9. Cooper BH

    (2014) The morphological and molecular nature of synaptic vesicle priming at presynaptic active zones

    Neuron 84:416–431.

    https://doi.org/10.1016/j.neuron.2014.10.009

    • PubMed
    • Google Scholar
23. 1. Jahn R
    2. Schiebler W
    3. Ouimet C
    4. Greengard P

    (1985) A 38,000-dalton membrane protein (p38) present in synaptic vesicles

    PNAS 82:4137–4141.

    https://doi.org/10.1073/pnas.82.12.4137

    • PubMed
    • Google Scholar
24. 1. Janz R
    2. Südhof TC
    3. Hammer RE
    4. Unni V
    5. Siegelbaum SA
    6. Bolshakov VY

    (1999) Essential roles in synaptic plasticity for synaptogyrin I and synaptophysin I

    Neuron 24:687–700.

    https://doi.org/10.1016/S0896-6273(00)81122-8

    • PubMed
    • Google Scholar
25. 1. Janz R
    2. Südhof TC

    (1998) Cellugyrin, a novel ubiquitous form of synaptogyrin that is phosphorylated by pp60c-src

    The Journal of Biological Chemistry 273:2851–2857.

    https://doi.org/10.1074/jbc.273.5.2851

    • PubMed
    • Google Scholar
26. 1. Khvotchev MV
    2. Südhof TC

    (2004) Stimulus-dependent dynamic homo- and heteromultimerization of synaptobrevin/VAMP and synaptophysin

    Biochemistry 43:15037–15043.

    https://doi.org/10.1021/bi048290+

    • PubMed
    • Google Scholar
27. 1. Kohansal-Nodehi M
    2. Chua JJ
    3. Urlaub H
    4. Jahn R
    5. Czernik D

    (2016) Analysis of protein phosphorylation in nerve terminal reveals extensive changes in active zone proteins upon exocytosis

    eLife 5:e14530.

    https://doi.org/10.7554/eLife.14530

    • PubMed
    • Google Scholar
28. 1. Körber C
    2. Horstmann H
    3. Venkataramani V
    4. Herrmannsdörfer F
    5. Kremer T
    6. Kaiser M
    7. Schwenger DB
    8. Ahmed S
    9. Dean C
    10. Dresbach T
    11. Kuner T

    (2015) Modulation of presynaptic release probability by the Vertebrate-Specific protein mover

    Neuron 87:521–533.

    https://doi.org/10.1016/j.neuron.2015.07.001

    • PubMed
    • Google Scholar
29. 1. Kwon SE
    2. Chapman ER

    (2011) Synaptophysin regulates the kinetics of synaptic vesicle endocytosis in central neurons

    Neuron 70:847–854.

    https://doi.org/10.1016/j.neuron.2011.04.001

    • PubMed
    • Google Scholar
30. 1. Lee JS
    2. Ho WK
    3. Lee SH

    (2010) Post-tetanic increase in the fast-releasing synaptic vesicle pool at the expense of the slowly releasing pool

    The Journal of General Physiology 136:259–272.

    https://doi.org/10.1085/jgp.201010437

    • PubMed
    • Google Scholar
31. 1. Lee JS
    2. Ho WK
    3. Neher E
    4. Lee SH

    (2013) Superpriming of synaptic vesicles after their recruitment to the readily releasable pool

    PNAS 110:15079–15084.

    https://doi.org/10.1073/pnas.1314427110

    • PubMed
    • Google Scholar
32. 1. Mahfooz K
    2. Singh M
    3. Renden R
    4. Wesseling JF

    (2016) A Well-Defined readily releasable pool with fixed capacity for storing vesicles at calyx of held

    PLOS Computational Biology 12:e1004855.

    https://doi.org/10.1371/journal.pcbi.1004855

    • PubMed
    • Google Scholar
33. 1. Markram H
    2. Tsodyks M

    (1996) Redistribution of synaptic efficacy between neocortical pyramidal neurons

    Nature 382:807–810.

    https://doi.org/10.1038/382807a0

    • PubMed
    • Google Scholar
34. 1. Matteoli M
    2. Takei K
    3. Cameron R
    4. Hurlbut P
    5. Johnston PA
    6. Südhof TC
    7. Jahn R
    8. De Camilli P

    (1991) Association of Rab3A with synaptic vesicles at late stages of the secretory pathway

    The Journal of Cell Biology 115:625–633.

    https://doi.org/10.1083/jcb.115.3.625

    • PubMed
    • Google Scholar
35. 1. McInnes J
    2. Wierda K
    3. Snellinx A
    4. Bounti L
    5. Wang YC
    6. Stancu IC
    7. Apóstolo N
    8. Gevaert K
    9. Dewachter I
    10. Spires-Jones TL
    11. De Strooper B
    12. De Wit J
    13. Zhou L
    14. Verstreken P

    (2018) Synaptogyrin-3 mediates presynaptic dysfunction induced by tau

    Neuron 97:823–835.

    https://doi.org/10.1016/j.neuron.2018.01.022

    • PubMed
    • Google Scholar
36. 1. McMahon HT
    2. Bolshakov VY
    3. Janz R
    4. Hammer RE
    5. Siegelbaum SA
    6. Südhof TC

    (1996) Synaptophysin, a major synaptic vesicle protein, is not essential for neurotransmitter release

    PNAS 93:4760–4764.

    https://doi.org/10.1073/pnas.93.10.4760

    • PubMed
    • Google Scholar
37. 1. Miki T
    2. Malagon G
    3. Pulido C
    4. Llano I
    5. Neher E
    6. Marty A

    (2016) Actin- and Myosin-Dependent vesicle loading of presynaptic docking sites prior to exocytosis

    Neuron 91:808–823.

    https://doi.org/10.1016/j.neuron.2016.07.033

    • PubMed
    • Google Scholar
38. 1. Monday HR
    2. Younts TJ
    3. Castillo PE

    (2018) Long-Term plasticity of neurotransmitter release: emerging mechanisms and contributions to brain function and disease

    Annual Review of Neuroscience 41:299–322.

    https://doi.org/10.1146/annurev-neuro-080317-062155

    • PubMed
    • Google Scholar
39. 1. Montesinos MS
    2. Dong W
    3. Goff K
    4. Das B
    5. Guerrero-Given D
    6. Schmalzigaug R
    7. Premont RT
    8. Satterfield R
    9. Kamasawa N
    10. Young SM

    (2015) Presynaptic deletion of GIT proteins results in increased synaptic strength at a mammalian central synapse

    Neuron 88:918–925.

    https://doi.org/10.1016/j.neuron.2015.10.042

    • PubMed
    • Google Scholar
40. 1. Moulder KL
    2. Mennerick S

    (2005) Reluctant vesicles contribute to the total readily releasable pool in glutamatergic hippocampal neurons

    Journal of Neuroscience 25:3842–3850.

    https://doi.org/10.1523/JNEUROSCI.5231-04.2005

    • PubMed
    • Google Scholar
41. 1. Müller M
    2. Genç Ö
    3. Davis GW

    (2015) RIM-binding protein links synaptic homeostasis to the stabilization and replenishment of high release probability vesicles

    Neuron 85:1056–1069.

    https://doi.org/10.1016/j.neuron.2015.01.024

    • PubMed
    • Google Scholar
42. 1. Neher E

    (2017) Some subtle lessons from the calyx of held synapse

    Biophysical Journal 112:215–223.

    https://doi.org/10.1016/j.bpj.2016.12.017

    • PubMed
    • Google Scholar
43. 1. Neher E
    2. Sakaba T

    (2001) Combining deconvolution and noise analysis for the estimation of transmitter release rates at the calyx of held

    The Journal of Neuroscience 21:444–461.

    https://doi.org/10.1523/JNEUROSCI.21-02-00444.2001

    • PubMed
    • Google Scholar
44. 1. Pennuto M
    2. Bonanomi D
    3. Benfenati F
    4. Valtorta F

    (2003) Synaptophysin I controls the targeting of VAMP2/synaptobrevin II to synaptic vesicles

    Molecular Biology of the Cell 14:4909–4919.

    https://doi.org/10.1091/mbc.e03-06-0380

    • PubMed
    • Google Scholar
45. 1. Rajappa R
    2. Gauthier-Kemper A
    3. Böning D
    4. Hüve J
    5. Klingauf J

    (2016) Synaptophysin 1 clears synaptobrevin 2 from the presynaptic active zone to prevent Short-Term depression

    Cell Reports 14:1369–1381.

    https://doi.org/10.1016/j.celrep.2016.01.031

    • PubMed
    • Google Scholar
46. 1. Ranck JB

    (1973) Studies on single neurons in dorsal hippocampal formation and septum in unrestrained rats. I. behavioral correlates and firing repertoires

    Experimental Neurology 41:461–531.

    https://doi.org/10.1016/0014-4886(73)90290-2

    • PubMed
    • Google Scholar
47. 1. Rothman JE
    2. Krishnakumar SS
    3. Grushin K
    4. Pincet F

    (2017) Hypothesis - buttressed rings assemble, clamp, and release SNAREpins for synaptic transmission

    FEBS Letters 591:3459–3480.

    https://doi.org/10.1002/1873-3468.12874

    • PubMed
    • Google Scholar
48. 1. Sakaba T

    (2006) Roles of the fast-releasing and the slowly releasing vesicles in synaptic transmission at the calyx of Held

    Journal of Neuroscience 26:5863–5871.

    https://doi.org/10.1523/JNEUROSCI.0182-06.2006

    • PubMed
    • Google Scholar
49. 1. Sakaba T
    2. Neher E

    (2001) Calmodulin mediates rapid recruitment of fast-releasing synaptic vesicles at a calyx-type synapse

    Neuron 32:1119–1131.

    https://doi.org/10.1016/S0896-6273(01)00543-8

    • PubMed
    • Google Scholar
50. 1. Sankaranarayanan S
    2. De Angelis D
    3. Rothman JE
    4. Ryan TA

    (2000) The use of pHluorins for optical measurements of presynaptic activity

    Biophysical Journal 79:2199–2208.

    https://doi.org/10.1016/S0006-3495(00)76468-X

    • PubMed
    • Google Scholar
51. 1. Schneggenburger R
    2. Meyer AC
    3. Neher E

    (1999) Released fraction and total size of a pool of immediately available transmitter quanta at a calyx synapse

    Neuron 23:399–409.

    https://doi.org/10.1016/S0896-6273(00)80789-8

    • PubMed
    • Google Scholar
52. 1. Siddiqui TJ
    2. Vites O
    3. Stein A
    4. Heintzmann R
    5. Jahn R
    6. Fasshauer D

    (2007) Determinants of synaptobrevin regulation in membranes

    Molecular Biology of the Cell 18:2037–2046.

    https://doi.org/10.1091/mbc.e07-01-0049

    • PubMed
    • Google Scholar
53. 1. Sjöström PJ
    2. Turrigiano GG
    3. Nelson SB

    (2003) Neocortical LTD via coincident activation of presynaptic NMDA and cannabinoid receptors

    Neuron 39:641–654.

    https://doi.org/10.1016/S0896-6273(03)00476-8

    • PubMed
    • Google Scholar
54. 1. Söllner T
    2. Whiteheart SW
    3. Brunner M
    4. Erdjument-Bromage H
    5. Geromanos S
    6. Tempst P
    7. Rothman JE

    (1993) SNAP receptors implicated in vesicle targeting and fusion

    Nature 362:318–324.

    https://doi.org/10.1038/362318a0

    • PubMed
    • Google Scholar
55. 1. Stenius K

    (1995) Structure of synaptogyrin (p29) defines novel synaptic vesicle protein

    The Journal of Cell Biology 131:1801–1809.

    https://doi.org/10.1083/jcb.131.6.1801

    • Google Scholar
56. 1. Stevens RJ
    2. Akbergenova Y
    3. Jorquera RA
    4. Littleton JT

    (2012) Abnormal synaptic vesicle biogenesis in Drosophila synaptogyrin mutants

    Journal of Neuroscience 32:18054–18067.

    https://doi.org/10.1523/JNEUROSCI.2668-12.2012

    • PubMed
    • Google Scholar
57. 1. Stevens CF
    2. Wesseling JF

    (1999) Augmentation is a potentiation of the exocytotic process

    Neuron 22:139–146.

    https://doi.org/10.1016/S0896-6273(00)80685-6

    • PubMed
    • Google Scholar
58. 1. Sugita S
    2. Janz R
    3. Südhof TC

    (1999) Synaptogyrins regulate Ca2+-dependent exocytosis in PC12 cells

    Journal of Biological Chemistry 274:18893–18901.

    https://doi.org/10.1074/jbc.274.27.18893

    • PubMed
    • Google Scholar
59. 1. Takamori S
    2. Riedel D
    3. Jahn R

    (2000) Immunoisolation of GABA-specific synaptic vesicles defines a functionally distinct subset of synaptic vesicles

    The Journal of Neuroscience 20:4904–4911.

    https://doi.org/10.1523/JNEUROSCI.20-13-04904.2000

    • PubMed
    • Google Scholar
60. 1. Takamori S
    2. Rhee JS
    3. Rosenmund C
    4. Jahn R

    (2001) Identification of differentiation-associated brain-specific phosphate transporter as a second vesicular glutamate transporter (VGLUT2)

    The Journal of Neuroscience 21:RC182.

    https://doi.org/10.1523/JNEUROSCI.21-22-j0002.2001

    • PubMed
    • Google Scholar
61. 1. Takamori S
    2. Holt M
    3. Stenius K
    4. Lemke EA
    5. Grønborg M
    6. Riedel D
    7. Urlaub H
    8. Schenck S
    9. Brügger B
    10. Ringler P
    11. Müller SA
    12. Rammner B
    13. Gräter F
    14. Hub JS
    15. De Groot BL
    16. Mieskes G
    17. Moriyama Y
    18. Klingauf J
    19. Grubmüller H
    20. Heuser J
    21. Wieland F
    22. Jahn R

    (2006) Molecular anatomy of a trafficking organelle

    Cell 127:831–846.

    https://doi.org/10.1016/j.cell.2006.10.030

    • PubMed
    • Google Scholar
62. 1. Taschenberger H
    2. Woehler A
    3. Neher E

    (2016) Superpriming of synaptic vesicles as a common basis for intersynapse variability and modulation of synaptic strength

    PNAS 113:E4548–E4557.

    https://doi.org/10.1073/pnas.1606383113

    • PubMed
    • Google Scholar
63. 1. Thévenaz P
    2. Ruttimann UE
    3. Unser M

    (1998) A pyramid approach to subpixel registration based on intensity

    IEEE Transactions on Image Processing 7:27–41.

    https://doi.org/10.1109/83.650848

    • PubMed
    • Google Scholar
64. 1. Vere-Jones D

    (1966) Simple stochastic models for the release of quanta of transmitter from a nerve terminal

    Australian Journal of Statistics 8:53–63.

    https://doi.org/10.1111/j.1467-842X.1966.tb00164.x

    • Google Scholar
65. 1. Washbourne P
    2. Schiavo G
    3. Montecucco C

    (1995) Vesicle-associated membrane protein-2 (synaptobrevin-2) forms a complex with synaptophysin

    Biochemical Journal 305:721–724.

    https://doi.org/10.1042/bj3050721

    • PubMed
    • Google Scholar
66. 1. Wesseling JF
    2. Lo DC
    3. Dc L

    (2002) Limit on the role of activity in controlling the release-ready supply of synaptic vesicles

    The Journal of Neuroscience 22:9708–9720.

    https://doi.org/10.1523/JNEUROSCI.22-22-09708.2002

    • PubMed
    • Google Scholar
67. 1. Wilhelm BG
    2. Mandad S
    3. Truckenbrodt S
    4. Kröhnert K
    5. Schäfer C
    6. Rammner B
    7. Koo SJ
    8. Claßen GA
    9. Krauss M
    10. Haucke V
    11. Urlaub H
    12. Rizzoli SO

    (2014) Composition of isolated synaptic boutons reveals the amounts of vesicle trafficking proteins

    Science 344:1023–1028.

    https://doi.org/10.1126/science.1252884

    • PubMed
    • Google Scholar
68. 1. Wu LG
    2. Borst JG

    (1999) The reduced release probability of releasable vesicles during recovery from short-term synaptic depression

    Neuron 23:821–832.

    https://doi.org/10.1016/s0896-6273(01)80039-8

    • PubMed
    • Google Scholar
69. 1. Yasui T
    2. Fujisawa S
    3. Tsukamoto M
    4. Matsuki N
    5. Ikegaya Y

    (2005) Dynamic synapses as archives of synaptic history: state-dependent redistribution of synaptic efficacy in the rat hippocampal CA1

    The Journal of Physiology 566:143–160.

    https://doi.org/10.1113/jphysiol.2005.086595

    • PubMed
    • Google Scholar

Author details

1. Mathan K Raja

   Department of Neuroscience, Universidad de Navarra, Pamplona, Spain

   Contribution

   Investigation, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-7727-6994

2. Julia Preobraschenski

   Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany

   Contribution

   Formal analysis, Investigation, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

3. Sergio Del Olmo-Cabrera

   Institute for Neurosciences CSIC-UMH, San Juan de Alicante, Spain

   Contribution

   Investigation, Writing—review and editing

   Competing interests

   No competing interests declared

4. Rebeca Martinez-Turrillas

   Department of Neuroscience, Universidad de Navarra, Pamplona, Spain

   Contribution

   Investigation, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

5. Reinhard Jahn

   Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany

   Contribution

   Formal analysis, Supervision, Investigation, Methodology, Writing—review and editing

   Competing interests

   Reviewing editor, eLife

   "This ORCID iD identifies the author of this article:" 0000-0003-1542-3498

6. Isabel Perez-Otano

   1. Department of Neuroscience, Universidad de Navarra, Pamplona, Spain
   2. Institute for Neurosciences CSIC-UMH, San Juan de Alicante, Spain

   Contribution

   Formal analysis, Investigation, Writing—original draft, Writing—review and editing

   Competing interests

   No competing interests declared

7. John F Wesseling

   1. Department of Neuroscience, Universidad de Navarra, Pamplona, Spain
   2. Institute for Neurosciences CSIC-UMH, San Juan de Alicante, Spain

   Contribution

   Conceptualization, Resources, Data curation, Software, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Methodology, Writing—original draft, Project administration, Writing—review and editing

   For correspondence

   johnfwesseling@gmail.com

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-7565-2594

• 3,413

  views

• 461

  downloads

• 43

  citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.