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.

Comments on revisions:

The authors have adequately addressed my earlier comments and questions.

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 and 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 non-permanent 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 probability 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, doesn't these suggests 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.

Fig. 4B 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 a increased paired pulse depression in presence of OAG, that 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.

Comments on revisions:

I am satisfied with the reply of the authors and I do not have any further points of concern.

Reviewer #3 (Public review):

The results are consistent with the main claim that facilitation is caused by overfilling a readily releasable pool, but alternative interpretations continue to seem more likely, especially when the current results are taken together with previous studies. Key doubts could be resolved with a single straightforward experiment (see below).

The central issue is the interpretation of paired pulse depression that occurs when the interval between action potentials is 25 ms, but not when 50. To summarize: a similar phenomenon was observed at Schaffer collateral synapses (Dobrunz and Stevens, 1997), but was interpreted as evidence for a decrease in pv. Ca2+-channel inactivation was proposed as the mechanism, but this was not proven. The key point for evaluating the current study is that Dobrunz and Stevens specifically ruled out the kind of decrease in pocc that is the keystone premise of the current study because the depression occurred independently of whether or not the first action potential elicited exocytosis. Of course, the mechanism might be different at layer 2/3 cortical synapses. But, it seems reasonable to hope that the older hypothesis would be ruled out for the cortical synapses before concluding that the new hypothesis must be correct.

The old and new hypotheses could be distinguished from each other cleanly with a straightforward experiment. Most/maybe all central synapses strengthen a great amount when extracellular Ca2+ is increased from 1.3 to 2 mM, even when intracellular Ca2+ is buffered with EGTA. According to the authors' model, this is only possible when pv is low, and so could not occur at synapses between layer 2/3 neurons. Because of this, confirmation that increasing extracellular Ca2+ does not change synaptic strength would support the hypothesis that baseline pv is high, as the authors claim, and the support would be impressive because large changes have been seen at every other type of synapse where this has been studied (to my knowledge at least). In contrast, the Ca2+ imaging experiment that has been added to the new version of the manuscript does not address the central issue because a wide range of mechanisms could, in principle, decrease release without involving prior exocytosis or altering bulk Ca2+ signals, including: a small decrease in nano-domain Ca2+, which wouldn't be detected because nano-domains contribute a minuscule amount to the bulk signal during Ca2+-imaging; or even very fast activity-dependent undocking of synaptic vesicles, which was reported in the same Kusick et al, 2020 study that is central to the LS/TS terminology adopted by the authors.

Additional points:

(1) A new section in the Discussion (lines 458-475) suggests that previous techniques employed to show that augmentation and facilitation are caused by increases in pv did not have the resolution to distinguish between pv and pocc, but this is misleading. The confusion might be because the terminology has changed, but this is all the more reason to clarify this section. The previous evidence for increases in pv - and against increases in pocc - is as follows: The residual Ca2+ that drives augmentation decreases the latency between the onset of hypertonic solution and onset of the postsynaptic response by about 150 ms, which is large compared to the rise time of the response. The decrease indicates that the residual Ca2+ drives a decrease in the energy barrier that must be overcome before readily releasable vesicles can undergo exocytosis, which is precisely the type of mechanism that would enhance pv. In contrast, an increase in pocc could change the rise time, but not the latency. There is a small change in the rise time, but this could be caused by changes in either pv or pocc, and one of the studies (Garcia-Perez and Wesseling, 2008) showed that augmentation occluded facilitation, even at times when pocc was reduced by a factor of 3, which would seem to argue against parallel increases in both pv and pocc.

(2) Similar evidence from hypertonic stimulation indicates that Phorbol esters increase pv, but I am not aware of evidence ruling out a parallel increase in pocc.

Comments on revisions:

There are at least two straightforward ways to address the main concern.

The first would be experiments analogous to those in Dobrunz and Stevens that show that - unlike at Schaffer collateral synapses - paired pulse depression at L2/3 synapses requires neurotransmitter release. I proposed this in the first round, but realized since that a simpler and more powerful strategy would be to test directly that pv is/is-not near 1.0 in 1.2 mM Ca2+ simply by increasing to 2 mM Ca2+ (and showing that synaptic strength does-not/does change). This would be powerful because the increase in Ca2+ greatly increases synaptic strength at Schaffer collaterals by about 2.5-fold. Concerns about a confounding elevation in the basal intracellular Ca2+ concentration could be easily neutralized by pre-treating with EGTA-AM, which the authors have already done for other experiments.

Author response:

The following is the authors’ response to the original reviews

eLife Assessment

This valuable work explores how synaptic activity encodes information during memory tasks. All reviewers agree that the quality of the work is high. Although experimental data do support the possibility that phospholipase diacylglycerol signaling and synaptotagmin 7 (Syt7) dynamically regulate the vesicle pool required for presynaptic release, concerns remain that the central finding of paired pulse depression at very short intervals was more likely caused by Ca²⁺ channel inactivation than pool depletion. Overall, this is a solid study with valuable findings, but the results warrant consideration of alternative interpretations.

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 the most critical concerns was a possible involvement of Ca²⁺ channel inactivation in the strong paired pulse depression (PPD). Meanwhile, we have measured total (free plus buffered) calcium increments induced by each of first four APs in 40 Hz trains at axonal boutons of prelimbic layer 2/3 pyramidal cells. We found that first four Ca²⁺ increments were not different from one another, 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 (p_v) 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 p_v in the present study is the 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.

We below described our point-by-point replies to the Reviewers’ comments.

Public Reviews:

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:

(1) 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 release probability as an alternative.

Quantal content (m) depends on n * p_v, where n = RRP size and p_v =vesicular release probability. The value for p_v 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 RRP_hyper) may encompass not only AP-evoked fast-releasing vesicles (TS vesicle) but also reluctant vesicles (LS vesicles). Because we measured p_v based on AP-evoked EPSCs such as strong paired pulse depression (PPD) and associated failure rates, p_v 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 (p_occ) 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 p_occ, among which N is a fixed parameter but p_occ 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), p_occ is not a fixed parameter. Therefore, release probability should be re-defined as p_occ * p_v. Given that N is fixed, 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 p_occ rather than p_v, because p_v 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 p_v of reluctant vesicles.

We imagine that the Reviewer meant vesicular release or fusion probability (p_v) by ‘release probability’. If so, p_v (of TS vesicles) cannot be a major player in STF, because the baseline p_v 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 p_v is close to unity (Fig. S8).

Conventional models for facilitation assume a post-AP residual Ca²⁺-dependent step increase in p_v of RRP (Dittman et al., 2000) or reluctant vesicles (Turecek et al., 2016). Given that p_v of TS vesicles is close to one, an increase in p_v of TS vesicles cannot account for facilitation. The possibility for activity-dependent increase in fusion probability of LS vesicles (denoted as p_v,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 p_v,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 p_{v v,LS} that reside in a distinct pool. Because the increase in p_v,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 p_v,LS that occurs in parallel with STF. Strong PPD, indicative of high p_v, 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 p_v,LS. One may argue that STF may be mediated by a drastic step increase of p_v,LS from zero to one, but it is not distinguishable from conversion of LS to TS vesicles.

To address the reviewer’s concern, we incorporated these perspectives into Discussion and further clarified the reasoning behind our conclusions.

References

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.

Please note that papers cited in the manuscript are not repeated here.

(2) 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 p_v and p_occ, 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 (= p_occ x p_v). Our novel finding is that the increase in release probability should be attributed to an increase in p_occ, not to that in p_v.

(3) 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 p_v is already close to one, and thus it cannot be further increased by OAG. Instead, the baseline p_occ seems to be increased by OAG.

Reference

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.

(4) 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 p_v, particularly in light of observations in Drosophila synapses (Guan et al., 2020; Fujii et al., 2021), in which Syt7 mutants exhibited elevated initial p_v. However, it is important to note that these findings markedly differ from those in mammalian systems, where the role of Syt7 in regulating initial p_v has been extensively studied. In rodents, consistent evidence indicates that Syt7 does not significantly affect initial p_v, 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 p_v already near its maximal level, approaching a value of 1. Consequently, it is unlikely that the loss of Syt7 could further elevate the initial p_v. Instead, such effects are more plausibly explained by alternative mechanisms, such as alterations in vesicle replenishment dynamics, rather than a direct influence on p_v.

References

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:

(1) 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 added 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 p_v 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, p_occ in the present study is the 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 p_v close to 1 (Fig. S8 and associated text). Moreover, strong PPD, indicating release of vesicles with high p_v, 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 p_v 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 p_occ (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 p_v 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 p_v or for a gradual increase in p_v of reluctant vesicles during short-term facilitation.

Following statement was added to Discussion in the revised manuscript

“Previous studies suggested that an increase in p_v 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 ‘RRP_hyper’). 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 p_v 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, p_v in the present study indicates the fusion probability of TS vesicles. From the same reasons, p_occ 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 p_occ (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 RRP_hyper.”

(2) 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.

Figure 2—figure supplement 2 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 incorporated these results into the revised version of our manuscript to provide evidence against the Ca²⁺ channel inactivation.

(3) 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 p_fusion of reluctant vesicles. It should be noted, however, that Maschi and Klyachko (2020) showed a distribution of release probability (p_r) within a single active zone rather than a heterogeneity in p_fusion of individual docked vesicles. Therefore both p_occ and p_v of TS vesicles would contribute to the p_r 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 p_v 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 p_v vesicles and for a gradual increase in fusion probability of reluctant vesicles.

To address the reviewers’ concern, we incorporated the perspectives into Discussion and further clarified the reasoning behind our conclusions.

(4) 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 (p_r). Given that both p_v and p_occ are regulated by [Ca²⁺]_i, low external [Ca²⁺] may affect not only p_v but also p_occ, both of which would contribute to low p_r. Under such conditions, it would be plausible that the baseline p_r becomes much lower than 0.1 due to low p_v and p_occ (for instance, p_v decreases from 1 to 0.5, and p_occ from 0.3 to 0.1, then p_r = 0.05), and then p_r (= p_v x p_occ) 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 p_v is close to one, p_r depends p_occ, 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.

Reference

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.

Please note that papers cited in the manuscript are not repeated here.

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 p_occ 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 the manuscript, 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).

Reference

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 p_occ 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.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

Comments:

(1) While the authors claim that Syt7-mediated facilitation is connected to the behavioral deficits they observed, this link is still somewhat speculative. This manuscript could benefit from further discussions of other alternative mechanisms to consider.

We added following statement to Discussion of the revised manuscript:

“The acquisition of trace fear memory was impaired by inhibition of persistent activity in mPFC during trace period (Gilmartin et al., 2013). The similar deficit observed in Syt7 KD animals is consistent with the hypothesis that STF provides bi-stable ensemble activity in a recurrent network (Mongillo et al., 2012). Nevertheless, alternative mechanisms may be responsible for the behavioral deficit. Not only recurrent network but also long-range loop between the mPFC and the mediodorsal (MD) thalamus play a critical role in maintaining persistent activity within the mPFC especially for a delay period longer than 10 s (Bolkan et al., 2017). Prefrontal L2/3 is heavily innervated by MD thalamus, and L2/3-PCs subsequently relay signals to L5 cortico-thalamic (CT) neurons (Collins et al., 2018). Given that L2/3 is an essential component of the PFC-thalamic loop, loss of STF at recurrent synapses between L2/3 PCs may lead to insufficient L2/3 inputs to L5 CT neurons and failure in the reverberant PFC-MD thalamic feedback loop. Therefore, not only L2/3 recurrent network but also its output to downstream network should be considered as a possible network mechanism underlying behavioral deficit caused by Syt7 KD L2/3.”

(2) The authors mention that Syt7 contributes to persistent activity during working memory tasks but focus on using only a trace fear conditioning task. However, it would be interesting to see if their results are generalizable to other working memory tasks (i.e. a delayed alternation task).

We thank to Reviewer for the insightful suggestion. Trace fear conditioning (tFC) shares behavioral properties with working memory (WM) tasks in that tFC is vulnerable to attentional distraction and to the load of WM task. In general WM tasks including delayed alternation tasks such as a T-maze task need persistent activity of ensemble neurons representing target-specific information among multiple choices. Different from such WM tasks, tFC is not appropriate to examine target-specific ensemble activity. Because it is not trivial to examine in vivo recordings in KD animals during delayed alternation tasks, it will be appropriate to study the effect of Syt7 KD in a separate study.

(3) The figure legend in Figure 6A and 6B mentions dotted lines and broken lines in the figure. However, this is confusing, and it is unclear as to what these lines are referring to in the figure.

To avoid the confusion in the figure legend for Figure 6A and 6B, we corrected “dotted line” to " vertical broken line", and “broken lines” to “dashed parabolas”.

(4) The manuscript can benefit from close reading and editing to catch typos and improve general readability (i.e. line 173: the word "are" is repeated twice).

We corrected typographical errors throughout the manuscript and carefully read the manuscript to improve readability. A revised version reflecting these corrections has been prepared and will be resubmitted for your consideration.

Reviewer #3 (Recommendations for the authors):

The points in this section are all minor.

(1) Line 44: Define release probability (p_r) more clearly. Authors use it to mean p_v*p_occ, but others routinely use it to mean p_v*p_occ*N.

We understand that the Reviewer meant “others routinely use it to mean p_v”. At this statement, we meant conventional definition of release probability, which is release probability among vesicles of RRP. We think that it is not appropriate to re-define release probability as p_v * p_occ in this first paragraph of Introduction. Therefore we clarified this issue in Discussion as we mentioned in our reply to the 1st weakness issue raised by Reviewer #3.

(2) Line 82: For clarity, define better what recurrent excitatory synapses are. It seems that synapses between L2/3 PCs and local targets may all be recurrent?

Each of L2/3 and L5 of the prefrontal cortical layers harbors intralaminar recurrent excitatory synapses between pyramidal cells, called a recurrent network. Previous theoretical studies have proposed that a single layer recurrent network model can have bi-stable E/I balanced states (up- and down-states) if recurrent excitatory synapses display short-term facilitation (STF), and thus is able to temporally hold an information once external input shifts the network to the up-state. In this theory, synapses to local targets across layers are not considered and specific roles of L2/3 and L5 in working memory tasks are still elusive. For clarity, we added a statement at the beginning of the paragraph (line 82): “Each of layer 2/3 (L2/3) and layer 5 (L5) of neocortex displays intralaminar excitatory synapses between pyramidal cells comprising a recurrent network (Holmgren et al., 2003; Thomson and Lamy, 2007)”

(3) Cite earlier studies of short-term synaptic plasticity at synapses between L2/3 pyramidal neurons and local targets in mPFC. If there are none, take more explicit credit for being first.

As we mentioned in Introduction, previous studies on short-term plasticity (STP) at neocortical excitatory recurrent synapses have focused on synapses between L5 pyramidal cells (PCs) (Hemple et al. 2000; Wang et al. 2006; Morishima et al., 2011; Yoon et al., 2020). The local connectivity between L2/3 PCs in the somatosensory cortex has been elucidated by Homgren et al. (2003) and Ko et al. (2011). Although these study showed STP of EPSPs, it was at a fixed frequency or stimulus pattern at high external [Ca²⁺] (2 mM). There is a study on the frequency-dependence of STP of EPSP between L2/3-PCs (Feldmyer et al., 2006). Different from our study, Feldmyer et al., (2006) observed monotonous STD at all frequencies less than 50 Hz, but this study was done in the somatosensory cortex and at high external [Ca²⁺] (2 mM). To our knowledge, no previous study have investigated STP at recurrent excitatory synapses of L2/3 pyramidal cells of the mPFC especially at physiological external [Ca²⁺]. The present study, therefore, represents the first extensive investigation of STP at recurrent excitatory synapses in L2/3 of the mPFC under physiologically relevant external [Ca²⁺].

References

Feldmeyer D, Lubke J, Silver RA, Sakmann B (2002) Synaptic connections between layer 4 spiny neurone-layer 2/3 pyramidal cell pairs in juvenile rat barrel cortex: physiology and anatomy of interlaminar signalling within a cortical column. J Physiol 538:803-822.

Holmgren C, Harkany T, Svennenfors B, Zilberter Y (2003) Pyramidal cell communication within local networks in layer 2/3 of rat neocortex. J Physiol 551:139-153.

Ko H, Hofer SB, Pichler B, Buchanan KA, Sjöström PJ, Mrsic-Flogel TD (2011) Functional specificity of local synaptic connections in neocortical networks. Nature 473:87-91.

Morishima M, Morita K, Kubota Y, Kawaguchi Y (2011) Highly differentiated projection-specific cortical subnetworks. Journal of Neuroscience 31:10380-10391.

Wang Y, Markram H, Goodman PH, Berger TK, Ma J, Goldman-Rakic PS (2006) Heterogeneity in the pyramidal network of the medial prefrontal cortex. Nat Neurosci 9:534-542.

(4) I couldn't figure out the significance of Figure S3. Perhaps this could be explained better.

Optical minimal stimulation methods have not been previously documented in detail. This figure illustrates what parameters we should carefully examine in order to attain optical minimal stimulation, which hopefully stimulates a single afferent fiber. A single fiber stimulation by optical minimal stimulation is supported by the similarity of our estimate for the number of release sites (N) as the previous morphological estimate (Holler et al., 2021). For minimal stimulation, we used a collimated DMD-coupled LED was employed to restrict 470 nm illumination to a small and well-defined region within layer 2/3 of the prelimbic mPFC, and carefully adjusted the illumination radius such that one step smaller (by 1 μm) illumination results in failure to evoke EPSCs. Our typical illumination area ranged between 3–4 μm, as shown in Figure S3A. Under this minimal illumination area, we confirmed unimodal distributions for the EPSC parameters (amplitude, rise time, decay time and time to peak; Figure 3B-E). Otherwise, we excluded the recordings from analysis. We hope this explanation provides a clearer understanding of the figure's significance.

(5) Note that CTZ seems to alter p_r at some synapses.

We acknowledge that CTZ can increase release probability by blocking presynaptic K⁺ currents. Indeed, Ishikawa and Takahashi (2001) reported that CTZ slowed the repolarizing phase of presynaptic action potentials and the frequency of miniature EPSCs in the calyx synapses. Consistently, we observed a slight increase in the baseline EPSC amplitude, from 33.3 pA to 41.9 pA (p=0.045) following the application of 50 µM CTZ. However, given that vesicular release probability (p_v) is already close to 1 at the synapse of our interest, we believe that the observed effect is more likely attributed to an increase in release sites occupancy (p_occ), which would be reflected as an increase in miniature EPSC frequency in Ishikawa and Takahashi (2001). Given that PPR depends on p_v rather than p_occ, this increase in p_occ would not critically change our conclusion that AMPA receptor desensitization is not responsible for the strong PPD.

Reference

Ishikawa, T., & Takahashi, T. (2001). Mechanisms underlying presynaptic facilitatory effect of cyclothiazide at the calyx of Held of juvenile rats. The Journal of Physiology, 533(2), 423-431.

(6) Figure 8B. The result in Figure 8C seems important, but I couldn't figure out why behaviour was not altered during the acquisition phase summarized in Figure 8B. Perhaps this could be explained more clearly for non-experts.

Little difference in freezing behavior during acquisition has been also observed when prelimbic persistent firing was optogenetically inhibited (Gilmartin, 2013). Not only CS (tone) but also other sensory inputs (visual and olfactory etc.) and the spatial context could be a cue predicting US (shock). Moreover, during the acquisition phase, the presence of the electric shock inherently induces a freezing response as a natural defensive behavior, which may obscure specific behavioral changes related to the associative learning process. Therefore, the freezing behavior during acquisition cannot be regarded as a sign for specific association of CS and US. Instead, on the next day, we specifically evaluated the CS-US association of the conditioned animals by measuring freezing behavior in response to CS in a distinct context. We explicitly documented little difference between WT and KD animals during the acquisition phase in the relevant paragraph (line 397).