Progressive overfilling of readily releasable pool underlies short-term facilitation at recurrent excitatory synapses in layer 2/3 of the rat prefrontal cortex

Reviewer #1 (Public review):

Shin et al. conduct extensive electrophysiological and behavioral experiments to study the mechanisms of short-term synaptic plasticity at excitatory synapses in layer 2/3 of the rat medial prefrontal cortex. The authors interestingly find that short-term facilitation is driven by progressive overfilling of the readily releasable pool, and that this process is mediated by phospholipase C/diacylglycerol signaling and synaptotagmin-7 (Syt7). Specifically, knockdown of Syt7 not only abolishes the refilling rate of vesicles with high fusion probability, but it also impairs the acquisition of trace fear memory.

Overall, the authors offer novel insight to the field of synaptic plasticity through well-designed experiments that incorporate a range of techniques.

Reviewer #2 (Public review):

Summary:

Shin et al aim to identify in a very extensive piece of work a mechanism that contributes to dynamic regulation of synaptic output in the rat cortex at the second time scale. This mechanism is related to a new powerful model is well versed to test if the pool of SV ready for fusion is dynamically scaled to adjust supply demand aspects. The methods applied are state-of-the-art and both address quantitative aspects with high signal to noise. In addition, the authors examine both excitatory output onto glutamatergic and GABAergic neurons, which provides important information on how general the observed signals are in neural networks, The results are compellingly clear and show that pool regulation may be predominantly responsible. Their results suggests that a regulation of release probability, the alternative contender for regulation, is unlikely to be involved in the observed short term plasticity behavior (but see below). Besides providing a clear analysis pof the underlying physiology, they test two molecular contenders for the observed mechanism by showing that loss of Synaptotagmin7 function and the role of the Ca dependent phospholipase activity seems critical for the short term plasticity behavior. The authors go on to test the in vivo role of the mechanism by modulating Syt7 function and examining working memory tasks as well as overall changes in network activity using immediate early gene activity. Finally, they model their data, providing strong support for their interpretation of TS pool occupancy regulation.

Strengths:

This is a very thorough study, addressing the research question from many different angles and the experimental execution is superb. The impact of the work is high, as it applies recent models of short term plasticity behavior to in vivo circuits further providing insights how synapses provide dynamic control to enable working memory related behavior through nonpermanent changes in synaptic output.

Weaknesses:

While this work is carefully examined and the results are presented and discussed in a detailed manner, the reviewer is still not fully convinced that regulation of release provability is not a putative contributor to the observed behavior. No additional work is needed but in the moment I am not convinced that changes in release probability are not in play. One solution may be to extend the discussion of changes in rules probability as an alternative.

Fig 3 I am confused about the interpretation of the Mean Variance analysis outcome. Since the data points follow the curve during induction of short term plasticity, aren't these suggesting that release probability and not the pool size increases? Related, to measure the absolute release probability and failure rate using the optogenetic stimulation technique is not trivial as the experimental paradigm bias the experiment to a given output strength, and therefore a change in release probability cannot be excluded.

Fig4B interprets the phorbol ester stimulation to be the result of pool overfilling, however, phorbol ester stimulation has also been shown to increase release probability without changing the size of the readily releasable pool. The high frequency of stimulation may occlude an increased paired pulse depression in presence of OAG, which others have interpreted in mammalian synapses as an increase in release probability.

The literature on Syt7 function is still quite controversial. An observation in the literature that loss of Syt7 function in the fly synapse leads to an increase of release probability. Thus the observed changes in short term plasticity characteristics in the Syt7 KD experiments may contain a release probability component. Can the authors really exclude this possibility? Figure 5 shows for the Syt7 KD group a very prominent depression of the EPSC/IPSC with the second stimulus, particularly for the short interpulse intervals, usually a strong sign of increased release probability, as lack of pool refilling can unlikely explain the strong drop in synaptic output.

Reviewer #3 (Public review):

Summary:

The report by Shin, Lee, Kim, and Lee entitled "Progressive overfilling of readily releasable pool underlies short-term facilitation at recurrent excitatory synapses in layer 2/3 of the rat prefrontal cortex" describes electrophysiological experiments of short-term synaptic plasticity during repetitive presynaptic stimulation at synapses between layer 2/3 pyramidal neurons and nearby target neurons. Manipulations include pharmacological inhibition of PLC and actin polymerization, activation of DAG receptors, and shRNA knockdown of Syt7. The results are interpreted as support for the hypothesis that synaptic vesicle release sites are vacant most of the time at resting synapses (i.e., p_occ is low) and that facilitation (and augmentation) components of short-term enhancement are caused by an increase in occupancy, presumably because of acceleration of the transition from not-occupied to occupied. The report additionally describes behavioural experiments where trace fear conditioning is degraded by knocking down syt7 in the same synapses.

Strengths:

The strength of the study is in the new information about short-term plasticity at local synapses in layer 2/3, and the major disruption of a memory task after eliminating short-term enhancement at only 15% of excitatory synapses in a single layer of a small brain region. The local synapses in layer 2/3 were previously difficult to study, but the authors have overcome a number of challenges by combining channel rhodopsins with in vitro electroporation, which is an impressive technical advance.

Weaknesses:

The question of whether or not short-term enhancement causes an increase in p_occ (i.e., "readily releasable pool overfilling") is important because it cuts to the heart of the ongoing debate about how to model short term synaptic plasticity in general. However, my opinion is that, in their current form, the results do not constitute strong support for an increase in p_occ, even though this is presented as the main conclusion. Instead, there are at least two alternative explanations for the results that both seem more likely. Neither alternative is acknowledged in the present version of the report.

The evidence presented to support overfilling is essentially two-fold. The first is strong paired pulse depression of synaptic strength when the interval between action potentials is 20 or 25 ms, but not when the interval is 50 ms. Subsequent stimuli at frequencies between 5 and 40 Hz then drive enhancement. The second is the observation that a slow component of recovery from depression after trains of action potentials is unveiled after eliminating enhancement by knocking down syt7. Of the two, the second is predicted by essentially all models where enhancement mechanisms operate independently of release site depletion - i.e., transient increases in p_occ, p_v, or even N - so isn't the sort of support that would distinguish the hypothesis from alternatives (Garcia-Perez and Wesseling, 2008, https://doi.org/10.1152/jn.01348.2007).

Regarding the paired pulse depression: The authors ascribe this to depletion of a homogeneous population of release sites, all with similar p_v. However, the details fit better with the alternative hypothesis that the depression is instead caused by quickly reversing inactivation of Ca2+ channels near release sites, as proposed by Dobrunz and Stevens to explain a similar phenomenon at a different type of synapse (1997, PNAS,
https://doi.org/10.1073/pnas.94.26.14843). The details that fit better with Ca2+ channel inactivation include the combination of the sigmoid time course of the recovery from depression (plotted backwards in Fig1G,I) and observations that EGTA (Fig2B) increases the paired-pulse depression seen after 25 ms intervals. That is, the authors ascribe the sigmoid recovery to a delay in the activation of the facilitation mechanism, but the increased paired pulse depression after loading EGTA indicates, instead, that the facilitation mechanism has already caused p_r to double within the first 25 ms (relative to the value if the facilitation mechanism was not active). Meanwhile, Ca2+ channel inactivation would be expected to cause a sigmoidal recovery of synaptic strength because of the sigmoidal relationship between Ca2+-influx and exocytosis (Dodge and Rahamimoff, 1967, https://doi.org/10.1113/jphysiol.1967.sp008367).

The Ca2+-channel inactivation hypothesis could probably be ruled in or out with experiments analogous to the 1997 Dobrunz study, except after lowering extracellular Ca2+ to the point where synaptic transmission failures are frequent. However, a possible complication might be a large increase in facilitation in low Ca2+ (Fig2B of Stevens and Wesseling, 1999, https://doi.org/10.1016/s0896-6273(00)80685-6).

On the other hand, even if the paired pulse depression is caused by depletion of release sites rather than Ca2+-channel inactivation, there does not seem to be any support for the critical assumption that all of the release sites have similar p_v. And indeed, there seems to be substantial emerging evidence from other studies for multiple types of release sites with 5 to 20-fold differences in p_v at a wide variety of synapse types (Maschi and Klyachko, eLife, 2020, https://doi.org/10.7554/elife.55210; Rodriguez Gotor et al, eLife, 2024, https://doi.org/10.7554/elife.88212 and refs. therein). If so, the paired pulse depression could be caused by depletion of release sites with high p_v, whereas the facilitation could occur at sites with much lower p_v that are still occupied. It might be possible to address this by eliminating assumptions about the distribution of p_v across release sites from the variance-mean analysis, but this seems difficult; simply showing how a few selected distributions wouldn't work - such as in standard multiple probability fluctuation analyses - wouldn't add much.

In any case, the large increase - often 10-fold or more - in enhancement seen after lowering Ca2+ below 0.25 mM at a broad range of synapses and neuro-muscular junctions noted above is a potent reason to be cautious about the LS/TS model. There is morphological evidence that the transitions from a loose to tight docking state (LS to TS) occur, and even that the timing is accelerated by activity. However, 10-fold enhancement would imply that at least 90 % of vesicles start off in the LS state, and this has not been reported. In addition, my understanding is that the reverse transition (TS to LS) is thought to occur within 10s of ms of the action potential, which is 10-fold too fast to account for the reversal of facilitation seen at the same synapses (Kusick et al, 2020, https://doi.org/10.1038/s41593-020-00716-1).

Individual points:

(1) An additional problem with the overfilling hypothesis is that syt7 knockdown increases the estimate of p_occ extracted from the variance-mean analysis, which would imply a faster transition from unoccupied to occupied, and would consequently predict faster recovery from depression. However, recovery from depression seen in experiments was slower, not faster. Meanwhile, the apparent decrease in the estimate of N extracted from the mean-variance analysis is not anticipated by the authors' model, but fits well with alternatives where p_v varies extensively among release sites because release sites with low p_v would essentially be silent in the absence of facilitation.

(2) Figure S4A: I like the TTX part of this control, but the 4-AP part needs a positive control to be meaningful (e.g., absence of TTX).

(3) Line 251: At least some of the previous studies that concluded these drugs affect vesicle dynamics used logic that was based on some of the same assumptions that are problematic for the present study, so the reasoning is a bit circular.

(4) Line 329 and Line 461: A similar problem with circularity for interpreting earlier syt7 studies.

Author Response:

We greatly appreciate invaluable and constructive comments from Editors and Reviewers. We also thank for their time and patience. We are pleased for our manuscript to have been assessed valuable and solid.

One of most critical concerns was a possible involvement of Ca2+ channel inactivation in the strong paired pulse depression (PPD). Meanwhile, we have already measured total (free plus buffered) calcium increments induced by each of first four APs in a 40 Hz train at axonal boutons of prelimbic layer 2/3 pyramidal cells. We found that first four Ca2+ increments were not different each other, arguing against possible contribution of Ca2+ channel inactivation to PPD. Please see our reply to the 2nd issue in the Weakness section of Reviewer #3.

The second critical issue was on the definition of ‘vesicular probability’. Previously, vesicular probability (pv) has been used with reference to the releasable vesicle pool which includes not only tightly docked vesicles but also reluctant vesicles. On the other hand, the meaning of pv in the present study was release probability of tightly docked vesicles. We clarified this point in our replies to the 1st issues in the Weakness sections of Reviewer #2 and Reviewer #3.

To other Reviews’ comments, we below described our point-by-point replies.

Reviewer #2 (Public review):

Summary:

Shin et al aim to identify in a very extensive piece of work a mechanism that contributes to dynamic regulation of synaptic output in the rat cortex at the second time scale. This mechanism is related to a new powerful model is well versed to test if the pool of SV ready for fusion is dynamically scaled to adjust supply demand aspects. The methods applied are state-of-the-art and both address quantitative aspects with high signal to noise. In addition, the authors examine both excitatory output onto glutamatergic and GABAergic neurons, which provides important information on how general the observed signals are in neural networks, The results are compellingly clear and show that pool regulation may be predominantly responsible. Their results suggests that a regulation of release probability, the alternative contender for regulation, is unlikely to be involved in the observed short term plasticity behavior (but see below). Besides providing a clear analysis pof the underlying physiology, they test two molecular contenders for the observed mechanism by showing that loss of Synaptotagmin7 function and the role of the Ca dependent phospholipase activity seems critical for the short term plasticity behavior. The authors go on to test the in vivo role of the mechanism by modulating Syt7 function and examining working memory tasks as well as overall changes in network activity using immediate early gene activity. Finally, they model their data, providing strong support for their interpretation of TS pool occupancy regulation.

Strengths:

This is a very thorough study, addressing the research question from many different angles and the experimental execution is superb. The impact of the work is high, as it applies recent models of short term plasticity behavior to in vivo circuits further providing insights how synapses provide dynamic control to enable working memory related behavior through nonpermanent changes in synaptic output.

Weaknesses:

While this work is carefully examined and the results are presented and discussed in a detailed manner, the reviewer is still not fully convinced that regulation of release provability is not a putative contributor to the observed behavior. No additional work is needed but in the moment I am not convinced that changes in release probability are not in play. One solution may be to extend the discussion of changes in rules probability as an alternative.

Quantal content (m) depends on n * pv, where n = RRP size and pv =vesicular release probability. The value for pv critically depends on the definition of RRP size. Recent studies revealed that docked vesicles have differential priming states: loosely or tightly docked state (LS or TS, respectively). Because the RRP size estimated by hypertonic solution or long presynaptic depolarization is larger than that by back extrapolation of a cumulative EPSC plot (Moulder & Mennerick, 2005; Sakaba, 2006) in glutamatergic synapses, the former RRP (denoted as RRPhyper) may encompass not only AP-evoked fast-releasing vesicles (TS vesicle) but also reluctant vesicles (LS vesicles). Because we measured pv based on AP-evoked EPSCs such as strong paired pulse depression (PPD) and associated failure rates, pv in the present study denotes vesicular fusion probability of TS vesicles not that of LS plus TS vesicles.

Recent studies suggest that release sites are not fully occupied by TS vesicles in the baseline (Miki et al., 2016; Pulido and Marty, 2018; Malagon et al., 2020; Lin et al., 2022). Instead the occupancy (pocc) by TS vesicles is subject to dynamic regulation by reversible rate constants (denoted by k1 and b1, respectively). The number of TS vesicles (n) can be factored into the number of release sites (N) and pocc, among which N is a fixed parameter but pocc depends on k1/(k1+b1) under the framework of the simple refilling model (see Methods). Because these refilling rate constants are regulated by Ca2+ (Hosoi, et al., 2008), pocc is not a fixed parameter. Therefore, release probability should be re-defined as pocc x pv. In this regard, the increase in release probability is a major player in STF. Our study asserts that STF by 2.3 times can be attributed to an increase in pocc rather than pv, because pv is close to unity (Fig. S8). Moreover, strong PPD was observed not only in the baseline but also at the early and in the middle of a train (Fig. 2 and 7) and during the recovery phase (Fig. 3), arguing against a gradual increase in pv of reluctant vesicles.

If the Reviewer meant vesicular release or fusion probability (pv) by ‘release provability’, pv (of TS vesicles) is not a major player in STF, because the baseline pv is already higher than 0.8 even if it is most parsimoniously estimated (Fig. 2). Moreover, considering very high refilling rate (23/s), the high double failure rate cannot be explained without assuming that pv is close to unity (Fig. S8).

Conventional models for facilitation assume a post-AP residual Ca2+-dependent step increase in pv of RRP (Dittman et al., 2000) or reluctant vesicles (Turecek et al., 2016). Given that pv of TS vesicles is close to one, an increase in pv of TS vesicles cannot account for facilitation. The possibility for activity-dependent increase in fusion probability of LS vesicles (denoted as pv,LS) should be considered in two ways depending on whether LS and TS vesicles reside in distinct pools or in the same pool. Notably, strong PPD at short ISI implies that pv,LS is near zero at the resting state. Whereas LS vesicles do not contribute to baseline transmission, short-term facilitation (STF) may be mediated by cumulative increase in pv, LS that reside in a distinct pool. Because the increase in pv,LS during facilitation recruits new release sites (increase in N), the variance of EPSCs should become larger as stimulation frequency increases, resulting in upward deviation from a parabola in the V-M plane, as shown in recent studies (Valera et al., 2012; Kobbersmed et al., 2020). This prediction is not compatible with our results of V-M analysis (Fig. 3), showing that EPSCs during STF fell on the same parabola regardless of stimulation frequencies. Therefore, it is unlikely that an increase in fusion probability of reluctant vesicles residing in a distinct release pool mediates STF in the present study.

For the latter case, in which LS and TS vesicles occupy in the same release sites, it is hard to distinguish a step increase in fusion probability of LS vesicles from a conversion of LS vesicles to TS. Nevertheless, our results do not support the possibility for gradual increase in pv,LS that occurs in parallel with STF. Strong PPD, indicative of high pv, was consistently found not only in the baseline (Fig. 2 and Fig. S6) but also during post-tetanic augmentation phase (Fig. 3D) and even during the early development of facilitation (Fig. 2D-E and Fig. 7), arguing against gradual increase in pv,LS. One may argue that STF may be mediated by a drastic step increase of pv,LS from zero to one, but it is not distinguishable from conversion of LS to TS vesicles.

To address the reviewer’s concern, we will incorporate these perspectives into the discussion and further clarify the reasoning behind our conclusions.

Moulder KL, Mennerick S (2005) Reluctant vesicles contribute to the total readily releasable pool in glutamatergic hippocampal neurons. J Neurosci 25:3842–3850.

Sakaba, T (2006) Roles of the fast-releasing and the slowly releasing vesicles in synaptic transmission at the calyx of Held. J Neurosci 26(22): 5863-5871.

Fig 3 I am confused about the interpretation of the Mean Variance analysis outcome. Since the data points follow the curve during induction of short term plasticity, aren't these suggesting that release probability and not the pool size increases? Related, to measure the absolute release probability and failure rate using the optogenetic stimulation technique is not trivial as the experimental paradigm bias the experiment to a given output strength, and therefore a change in release probability cannot be excluded.

Under the recent definition of release probability, it can be factored into pv and pocc, which are fusion probability of TS vesicles and the occupancy of release sites by TS vesicles, respectively. With this regard, our interpretation of the Variance-Mean results is consistent with conventional one: different data points along a parabola represent a change in release probability (= pocc x pv). Our novel finding is that the increase in release probability should be attributed to an increase in pocc, not to that in pv.

Fig4B interprets the phorbol ester stimulation to be the result of pool overfilling, however, phorbol ester stimulation has also been shown to increase release probability without changing the size of the readily releasable pool. The high frequency of stimulation may occlude an increased paired pulse depression in presence of OAG, which others have interpreted in mammalian synapses as an increase in release probability.

To our experience in the calyx of Held synapses, OAG, a DAG analogue, increased the fast releasing vesicle pool (FRP) size (Lee JS et al., 2013), consistent with our interpretation (pool overfilling). Once the release sites are overfilled in the presence of OAG, it is expected that the maximal STF (ratio of facilitated to baseline EPSCs) becomes lower as long as the number of release sites (N) are limited. As aforementioned, the baseline pv is already close to one, and thus it cannot be further increased by OAG. Instead, the baseline pocc seems to be increased by OAG.

Lee JS, et al., Superpriming of synaptic vesicles after their recruitment to the readily releasable pool. Proc Natl Acad Sci U S A, 2013. 110(37): 15079-84.

The literature on Syt7 function is still quite controversial. An observation in the literature that loss of Syt7 function in the fly synapse leads to an increase of release probability. Thus the observed changes in short term plasticity characteristics in the Syt7 KD experiments may contain a release probability component. Can the authors really exclude this possibility? Figure 5 shows for the Syt7 KD group a very prominent depression of the EPSC/IPSC with the second stimulus, particularly for the short interpulse intervals, usually a strong sign of increased release probability, as lack of pool refilling can unlikely explain the strong drop in synaptic output.

The reviewer raises an interesting point regarding the potential link between Syt7 KD and increased initial pv, particularly in light of observations in Drosophila synapses (Guan et al., 2020; Fujii et al., 2021), in which Syt7 mutants exhibited elevated initial pv. However, it is important to note that these findings markedly differ from those in mammalian systems, where the role of Syt7 in regulating initial pv has been extensively studied. In rodents, consistent evidence indicates that Syt7 does not significantly affect initial pv, as demonstrated in several studies (Jackman et al., 2016; Chen et al., 2017; Turecek and Regehr, 2018). Furthermore, in our study of excitatory synapses in the mPFC layer 2/3, we observed an initial pv already near its maximal level, approaching a value of 1. Consequently, it is unlikely that the loss of Syt7 could further elevate the initial pv. Instead, such effects are more plausibly explained by alternative mechanisms, such as alterations in vesicle replenishment dynamics, rather than a direct influence on pv.

Chen, C., et al., Triple Function of Synaptotagmin 7 Ensures Efficiency of High-Frequency Transmission at Central GABAergic Synapses. Cell Rep, 2017. 21(8): 2082-2089.

Fujii, T., et al., Synaptotagmin 7 switches short-term synaptic plasticity from depression to facilitation by suppressing synaptic transmission. Scientific reports, 2021. 11(1): 4059.

Guan, Z., et al., Drosophila Synaptotagmin 7 negatively regulates synaptic vesicle release and replenishment in a dosage-dependent manner. Elife, 2020. 9: e55443.

Jackman, S.L., et al., The calcium sensor synaptotagmin 7 is required for synaptic facilitation. Nature, 2016. 529(7584): 88-91.

Turecek, J. and W.G. Regehr, Synaptotagmin 7 mediates both facilitation and asynchronous release at granule cell synapses. Journal of Neuroscience, 2018. 38(13): 3240-3251.

Reviewer #3 (Public review):

Summary:

The report by Shin, Lee, Kim, and Lee entitled "Progressive overfilling of readily releasable pool underlies short-term facilitation at recurrent excitatory synapses in layer 2/3 of the rat prefrontal cortex" describes electrophysiological experiments of short-term synaptic plasticity during repetitive presynaptic stimulation at synapses between layer 2/3 pyramidal neurons and nearby target neurons. Manipulations include pharmacological inhibition of PLC and actin polymerization, activation of DAG receptors, and shRNA knockdown of Syt7. The results are interpreted as support for the hypothesis that synaptic vesicle release sites are vacant most of the time at resting synapses (i.e., p_occ is low) and that facilitation (and augmentation) components of short-term enhancement are caused by an increase in occupancy, presumably because of acceleration of the transition from not-occupied to occupied. The report additionally describes behavioural experiments where trace fear conditioning is degraded by knocking down syt7 in the same synapses.

Strengths:

The strength of the study is in the new information about short-term plasticity at local synapses in layer 2/3, and the major disruption of a memory task after eliminating short-term enhancement at only 15% of excitatory synapses in a single layer of a small brain region. The local synapses in layer 2/3 were previously difficult to study, but the authors have overcome a number of challenges by combining channel rhodopsins with in vitro electroporation, which is an impressive technical advance.

Weaknesses:

The question of whether or not short-term enhancement causes an increase in p_occ (i.e., "readily releasable pool overfilling") is important because it cuts to the heart of the ongoing debate about how to model short term synaptic plasticity in general. However, my opinion is that, in their current form, the results do not constitute strong support for an increase in p_occ, even though this is presented as the main conclusion. Instead, there are at least two alternative explanations for the results that both seem more likely. Neither alternative is acknowledged in the present version of the report.

The evidence presented to support overfilling is essentially two-fold. The first is strong paired pulse depression of synaptic strength when the interval between action potentials is 20 or 25 ms, but not when the interval is 50 ms. Subsequent stimuli at frequencies between 5 and 40 Hz then drive enhancement. The second is the observation that a slow component of recovery from depression after trains of action potentials is unveiled after eliminating enhancement by knocking down syt7. Of the two, the second is predicted by essentially all models where enhancement mechanisms operate independently of release site depletion - i.e., transient increases in p_occ, p_v, or even N - so isn't the sort of support that would distinguish the hypothesis from alternatives (Garcia-Perez and Wesseling, 2008, https://doi.org/10.1152/jn.01348.2007).

The apparent discrepancy in interpretation of post-tetanic augmentation between the present and previous papers [Sevens Wesseling (1999), Garcia-Perez and Wesseling (2008)] is an important issue that should be clarified. We noted that different meanings of ‘vesicular release probability’ in these papers are responsible for the discrepancy. We will add an explanation to Discussion on the difference in the meaning of ‘vesicular release probability’ between the present study and previous studies [Sevens Wesseling (1999), Garcia-Perez and Wesseling (2008)]. In summary, the pv in the present study was used for vesicular release probability of TS vesicles, while previous studies used it as vesicular release probability of vesicles in the RRP, which include LS and TS vesicles. Accordingly, pocc in the present study is occupancy of release sites by TS vesicles.

Not only double failure rate but also other failure rates upon paired pulse stimulation were best fitted at pv close to 1 (Fig. S8 and associated text). Moreover, strong PPD, indicating release of vesicles with high pv, was observed not only at the beginning of a train but also in the middle of a 5 Hz train (Fig. 2D), during the augmentation phase after a 40 Hz train (Fig 3D), and in the recovery phase after three pulse bursts (Fig. 7). Given that pv is close to 1 throughout the EPSC trains and that N does not increase during a train (Fig. 3), synaptic facilitation can be attained only by the increase in pocc (occupancy of release sites by TS vesicles). In addition, it should be noted that Fig. 7 demonstrates strong PPD during the recovery phase after depletion of TS vesicles by three pulse bursts, indicating that recovered vesicles after depletion display high pv too. Knock-down of Syt7 slowed the recovery of TS vesicles after depletion of TS vesicles, highlighting that Syt7 accelerates the recovery of TS vesicles following their depletion.

As addressed in our reply to the first issue raised by Reviewer #2 and the third issue raised by Reviewer #3, our results do not support possibilities for recruitment of new release sites (increase in N) having low pv or for a gradual increase in pv of reluctant vesicles during short-term facilitation.

Previous studies suggested that an increase in pv is responsible for post-tetanic augmentation (Stevens and Wesseling, 1999; Garcia-Perez and Wesseling, 2008) by observing invariance of the RRP size after tetanic stimulation. In these studies, the RRP size was estimated by hypertonic sucrose solution or as the sum of EPSCs evoked 20 Hz/60 pulses train (denoted as ‘RRPhyper’). Because reluctant vesicles (called LS vesicles) can be quickly converted to TS vesicles (16/s) and are released during a train (Lee et al., 2012), it is likely that the RRP size measured by these methods encompasses both LS and TS vesicles. In contrast, we assert high pv based on the observation of strong PPD and failure rates upon paired stimulations at ISI of 20 ms (Fig. 2 and Fig. S8). Given that single AP-induced vesicular release occurs from TS vesicles but not from LS vesicles, pv in the present study indicates the fusion probability of TS vesicles. From the same reasons, pocc denotes the occupancy of release sites by TS vesicles. Note that our study does not provide direct clue whether release sites are occupied by LS vesicles that are not tapped by a single AP, although an increase in the LS vesicle number may accelerate the recovery of TS vesicles. As suggested in Neher (2024), even if the number of LS plus TS vesicles are kept constant, an increase in pocc (occupancy by TS vesicles) would be interpreted as an increase in ‘vesicular release probability’ as in the previous studies (Stevens and Wesseling (1999); Garcia-Perez and Wesseling (2008)) as long as it was measured based on RRPhyper.

Regarding the paired pulse depression: The authors ascribe this to depletion of a homogeneous population of release sites, all with similar p_v. However, the details fit better with the alternative hypothesis that the depression is instead caused by quickly reversing inactivation of Ca2+ channels near release sites, as proposed by Dobrunz and Stevens to explain a similar phenomenon at a different type of synapse (1997, PNAS,
https://doi.org/10.1073/pnas.94.26.14843). The details that fit better with Ca2+ channel inactivation include the combination of the sigmoid time course of the recovery from depression (plotted backwards in Fig1G,I) and observations that EGTA (Fig2B) increases the paired-pulse depression seen after 25 ms intervals. That is, the authors ascribe the sigmoid recovery to a delay in the activation of the facilitation mechanism, but the increased paired pulse depression after loading EGTA indicates, instead, that the facilitation mechanism has already caused p_r to double within the first 25 ms (relative to the value if the facilitation mechanism was not active). Meanwhile, Ca2+ channel inactivation would be expected to cause a sigmoidal recovery of synaptic strength because of the sigmoidal relationship between Ca2+-influx and exocytosis (Dodge and Rahamimoff, 1967, https://doi.org/10.1113/jphysiol.1967.sp008367).

The Ca2+-channel inactivation hypothesis could probably be ruled in or out with experiments analogous to the 1997 Dobrunz study, except after lowering extracellular Ca2+ to the point where synaptic transmission failures are frequent. However, a possible complication might be a large increase in facilitation in low Ca2+ (Fig2B of Stevens and Wesseling, 1999, https://doi.org/10.1016/s0896-6273(00)80685-6).

We appreciate the reviewer's thoughtful comment regarding the potential role of Ca2+ channel inactivation in the observed paired-pulse depression (PPD). As noted by the Reviewer, the Dobrunz and Stevens (1997) suggested that the high double failure rate at short ISIs in synapses exhibiting PPD can be attributed to Ca2+ channel inactivation. This interpretation seems to be based on a premise that the number of RRP vesicles are not varied trial-by-trial. The number of TS vesicles, however, can be dynamically regulated depending on the parameters k1 and b1, as shown in Fig. S8, implying that the high double failure rate at short ISIs cannot be solely attributed to Ca2+ channel inactivation. Nevertheless, we acknowledge the possibility that Ca2+ channel inactivation may contribute to PPD, and therefore, we have further investigated this possibility. Specifically, we measured action potential (AP)-evoked Ca2+ transients at individual axonal boutons of layer 2/3 pyramidal cells in the mPFC using two-dye ratiometry techniques. Our analysis revealed no evidence for Ca2+ channel inactivation during a 40 Hz train of APs. This finding indicates that voltage-gated Ca2+ channel inactivation is unlikely to contribute to the pronounced PPD.

Author response image 1 below shows how we measured the total Ca2+ increments at axonal boutons. First we estimated endogenous Ca2+-binding ratio from analyses of single AP-induced Ca2+ transients at different concentrations of Ca2+ indicator dye (panels A to E). And then, using the Ca2+ buffer properties, we converted free [Ca2+] amplitudes to total calcium increments for the first four AP-evoked Ca2+ transients in a 40 Hz train (panels G-I). We will incorporate these results into the revised version of reviewed preprint to provide evidence against the Ca2+ channel inactivation.

Author response image 1.

On the other hand, even if the paired pulse depression is caused by depletion of release sites rather than Ca2+-channel inactivation, there does not seem to be any support for the critical assumption that all of the release sites have similar p_v. And indeed, there seems to be substantial emerging evidence from other studies for multiple types of release sites with 5 to 20-fold differences in p_v at a wide variety of synapse types (Maschi and Klyachko, eLife, 2020, https://doi.org/10.7554/elife.55210; Rodriguez Gotor et al, eLife, 2024, https://doi.org/10.7554/elife.88212 and refs. therein). If so, the paired pulse depression could be caused by depletion of release sites with high p_v, whereas the facilitation could occur at sites with much lower p_v that are still occupied. It might be possible to address this by eliminating assumptions about the distribution of p_v across release sites from the variance-mean analysis, but this seems difficult; simply showing how a few selected distributions wouldn't work - such as in standard multiple probability fluctuation analyses - wouldn't add much.

We appreciate the reviewer’s insightful comments regarding the potential increase in pfusion of reluctant vesicles. It should be noted, however, that Maschi and Klyachko (2020) showed a distribution of release probability (pr) within a single active zone rather than a heterogeneity in pfusion of individual docked vesicles. Therefore both pocc and pv of TS vesicles would contribute to the pr distribution shown in Maschi and Klyachko (2020).

The Reviewer’s concern aligns closely with the first issue raised by Reviewer #2, to which we addressed in detail. Briefly, new release site may not be recruited during facilitation or post-tetanic augmentation, because variance of EPSCs during and after a train fell on the same parabola (Fig. 3). Secondly, strong PPD was observed not only in the baseline but also during early and late phases of facilitation, indicating that vesicles with very high pv contribute to EPSC throughout train stimulations (Fig. 2, 3, and 7). These findings argue against the possibilities for recruitment of new release sites harboring low pv vesicles and for a gradual increase in fusion probability of reluctant vesicles.

To address the reviewers’ concern, we will incorporate the perspectives into Discussion and further clarify the reasoning behind our conclusions.

In any case, the large increase - often 10-fold or more - in enhancement seen after lowering Ca2+ below 0.25 mM at a broad range of synapses and neuro-muscular junctions noted above is a potent reason to be cautious about the LS/TS model. There is morphological evidence that the transitions from a loose to tight docking state (LS to TS) occur, and even that the timing is accelerated by activity. However, 10-fold enhancement would imply that at least 90 % of vesicles start off in the LS state, and this has not been reported. In addition, my understanding is that the reverse transition (TS to LS) is thought to occur within 10s of ms of the action potential, which is 10-fold too fast to account for the reversal of facilitation seen at the same synapses (Kusick et al, 2020, https://doi.org/10.1038/s41593-020-00716-1).

As the reviewer suggested, low external Ca2+ concentration can lower release probability (pr). Given that both pv and pocc are regulated by [Ca2+]i, low external [Ca2+] may affect not only pv but also pocc, both of which would contribute to low pr. Under such conditions, it would be plausible that the baseline pr becomes much lower than 0.1 due to low pv and pocc (for instance, pv decreases from 1 to 0.5, and pocc from 0.3 to 0.1, then pr = 0.05), and then pr (= pv x pocc) has a room for an increase by a factor of ten (0.5, for example) by short-term facilitation as cytosolic [Ca2+] accumulates during a train.

If pv is close to one, pr depends pocc, and thus facilitation depends on the number of TS vesicles just before arrival of each AP of a train. Thus, post-train recovery from facilitation would depend on restoration of equilibrium between TS and LS vesicles to the baseline. Even if transition between LS and TS vesicles is very fast (tens of ms), the equilibrium involved in de novo priming (reversible transitions between recycling vesicle pool and partially docked LS vesicles) seems to be much slower (13 s in Fig. 5A of Wu and Borst 1999). Thus, we can consider a two-step priming model (recycling pool -> LS -> TS), which is comprised of a slow 1st step (-> LS) and a fast 2nd step (-> TS). Under the framework of the two-step model, the slow 1st step (de novo priming step) is the rate limiting step regulating the development and recovery kinetics of facilitation. Given that on and off rate for Ca2+ binding to Syt7 is slow, it is plausible that Syt7 may contribute to short-term facilitation (STF) by Ca2+-dependent acceleration of the 1st step (as shown in Fig. 9). During train stimulation, the number of LS vesicles would slowly accumulate in a Syt7 and Ca2+-dependent manner, and this increase in LS vesicles would shift LS/TS equilibrium towards TS, resulting in STF. After tetanic stimulation, the recovery kinetics from facilitation would be limited by slow recovery of LS vesicles.

Wu, L.-G. and Borst J.G.G. (1999) The reduced release probability of releasable vesicles during recovery from short-term synaptic depression. Neuron, 23(4): 821-832.

Individual points:

(1) An additional problem with the overfilling hypothesis is that syt7 knockdown increases the estimate of p_occ extracted from the variance-mean analysis, which would imply a faster transition from unoccupied to occupied, and would consequently predict faster recovery from depression. However, recovery from depression seen in experiments was slower, not faster. Meanwhile, the apparent decrease in the estimate of N extracted from the mean-variance analysis is not anticipated by the authors' model, but fits well with alternatives where p_v varies extensively among release sites because release sites with low p_v would essentially be silent in the absence of facilitation.

Slower recovery from depression observed in the Syt7 knockdown (KD) synapses (Fig. 7) may results from a deficiency in activity-dependent acceleration of TS vesicle recovery. Although basal occupancy was higher in the Syt7 KD synapses, this does not indicate a faster activity-dependent recovery.

Higher baseline occupancy does not always imply faster recovery of PPR too. Actually PPR recovery was slower in Syt7 KD synapses than WT one (18.5 vs. 23/s). Under the framework of the simple refilling model (Fig. S8Aa), the baseline occupancy and PPR recovery rate are calculated as k1 / (k1 + b1) and (k1 + b1), respectively. The baseline occupancy depends on k1/b1, while the PPR recovery on absolute values of k1 and b1. Based on pocc and PPR recovery time constant of WT and KD synapses, we expect higher k1/b1 but lower values for (k1 +b1) in Syt7 KD synapses compared to WT ones.

Lower release sites (N) in Syt7-KD synapses was not anticipated. As you suggested, such low N might be ascribed to little recruitment of release sites during a train in KD synapses. But our results do not support this model. If silent release sites are recruited during a train, the variance should upwardly deviate from the parabola predicted under a fixed N (Valera et al., 2012; Kobbersmed et al. 2020). Our result was not the case (Fig. 3). In the first version of Ms, we have argued against this possibility in line 203-208.

As discussed in both the Results and Discussion sections, the baseline EPSC was unchanged by KD (Fig. S3) because of complementary changes in the number of docking sites and their baseline occupancy (Fig. 6). These findings suggest that Syt7 may be involved in maintaining additional vacant docking sites, which could be overfilled during facilitation. It remains to be determined whether the decrease in docking sites in Syt7 KD synapses is related to its specific localization of Syt7 at the plasma membrane of active zones, as proposed in previous studies (Sugita et al., 2001; Vevea et al., 2021).

(2) Figure S4A: I like the TTX part of this control, but the 4-AP part needs a positive control to be meaningful (e.g., absence of TTX).

The reason why we used 4-AP in the presence of TTX was to increase the length constant of axon fibers and to facilitate the conduction of local depolarization in the illumination area to axon terminals. The lack of EPSC in the presence of 4-AP and TTX indicates that illumination area is distant from axon terminals enough for optic stimulation-induced local depolarization not to evoke synaptic transmission. This methodology has been employed in previous studies including the work of Little and Carter (2013).

Little JP and Carter AG (2013) Synaptic mechanisms underlying strong reciprocal connectivity between the medial prefrontal cortex and basolateral amygdala. J Neurosci, 33(39): 15333-15342.

(3) Line 251: At least some of the previous studies that concluded these drugs affect vesicle dynamics used logic that was based on some of the same assumptions that are problematic for the present study, so the reasoning is a bit circular.

(4) Line 329 and Line 461: A similar problem with circularity for interpreting earlier syt7 studies.

(Reply to #3 and #4) We selected the target molecules as candidates based on their well-characterized roles in vesicle dynamics, and aimed to investigate what aspects of STP are affected by these molecules in our experimental context. For example, we could find that the baseline pocc and short-term facilitation (STF) are enhanced by the baseline DAG level and train stimulation-induced PLC activation, respectively. Notably, the effect of dynasore informed us that slow site clearing is responsible for the late depression of 40 Hz train EPSC. The knock-down experiments also provided us with information on the critical role of Syt7 in replenishment of TS vesicles. These approaches do not deviate from standard scientific reasoning but rather builds upon prior knowledge to formulate and test hypotheses.

Importantly, our conclusions do not rely solely on the assumption that altering the target molecule impacts synaptic transmission. Instead, our conclusions are derived from a comprehensive analysis of diverse outcomes obtained through both pharmacological and genetic manipulations. These interpretations align closely with prior literature, further validating our conclusions.

Therefore, the use of established studies to guide candidate selection and the consistency of our findings with existing knowledge do not represent a logical circularity but rather a reinforcement of the proposed mechanism through converging lines of evidence.