Physiological roles of endocytosis and presynaptic scaffold in vesicle replenishment at fast-signaling and slow-plastic synapses

Reviewer #1 (Public Review):

Summary:

The study examines the role of release site clearance in synaptic transmission during repetitive activity under physiological conditions in two types of central synapses, calyx of Held and hippocampal CA1 synapses. After acute block of endocytosis by pharmacology, deeper synaptic depression or less facilitation was observed in two types of synapses. Acute block of CDC42 and actin polymerization, which possibly inhibits the activity of Intersectin, affected synaptic depression at the calyx synapse, but not at CA1 synapses. The data suggest an unexpected, fast role of the site clearance in counteracting synaptic depression.

Strengths:

The study uses acute block of the molecular targets with pharmacology together with precise electrophysiology. The experimental results are clear cut and convincing. The study also examines the physiological roles of the site clearance using action potential-evoked transmission at physiological Ca and physiological temperature at mature animals. This condition has not been examined.

Weaknesses:

Pharmacology may have some off-target effects, though acute manipulation should be appreciated and the authors have tried several reagents to verify the overall conclusions.

Reviewer #2 (Public Review):

Summary:

In this manuscript, Mahapatra and Takahashi report on the physiological consequences of pharmacologically blocking either clathrin and dynamin function during compensatory endocytosis or of the cortical actin scaffold both in the calyx of Held synapse and hippocampal boutons in acute slice preparations

Strengths:

Although many aspects of these pharmacological interventions have been studied in detail during the past decades, this is a nice comprehensive and comparative study, which reveals some interesting differences between a fast synapse (Calyx of Held) tuned to reliably transmit at several 100 Hz and a more slow hippocampal CA1 synapse. In particular the authors find that acute disturbance of the synaptic actin network leads to a marked frequency-dependent enhancement of synaptic depression in the Calyx, but not in the hippocampal synapse This striking difference between both preparations is the most interesting and novel finding.

Weaknesses:

Unfortunately, however, these findings concerning the different consequences of actin depolymerization are not sufficiently discussed in comparison to the literature. My only criticism concerns the interpretation of the ML 141 and Lat B data. With respect to the Calyx data, I am missing a detailed discussion of the effects observed here in light of the different RRP subpools SRP and FRP. This is very important since Lee at al. (2012, PNAS 109 (13) E765-E774) showed earlier that disruption of actin inhibits the rapid transition of SRP SVs to the FRP at the AZ. The whole literature on this important concept is missing. Likewise, the role of actin for the replacement pool at a cerebellar synapse (Miki et al., 2016) is only mentioned in half a sentence. There is quite some evidence that actin is important both at the AZ (SRP to FRP transition, activation of replacement pool) and at the peri-active zone for compensatory endocytosis and release site clearance. Both possible underlying mechanisms (SRP to FRP transition or release site clearance) should be better dissected.

Reviewer #3 (Public Review):

The manuscript by Mahapatra and Takahashi addresses the role of presynaptic release site clearance during sustained synaptic activity. The authors characterize the effects of pharmacologically interfering with SV endocytosis (pre-incubation with Dynasore or Pitstop-2) on synaptic short-term plasticity (STP) at two different CNS synapses (calyx of Held synapses and hippocampal SC to CA1 synapses) using patch-clamp recordings in acute slices under experimental conditions designed to closely mimic a physiological situation (37{degree sign}C and 1.3 mM external [Ca2+]). Endocytosis blocker-induced changes in STP and in the recovery from short-term depression (STD) are compared to those seen after pharmacologically inhibiting actin filament assembly (pre-incubation with Latrunculin-B or the selective Cdc42 GTPase inhibitor ML-141). Presynaptic capacitance (Cm) recordings in calyx terminals were used to establish the effects of the pharmacological maneuvers on SV endocytosis.
Latrunculin-B and ML-141 neither affect SV endocytosis (assayed by Cm recordings) nor EPSC recovery following conditioning trains, but strongly enhances STD at calyx synapses. No changes in STP were observed at Latrunculin-B- or ML-141-treated SC to CA1 synapses.

Dynasore and Pitstop-2 slow down endocytosis, limit the total amount of exocytosis in response to long stimuli, enhance STD in response to 100 Hz stimulation, but profoundly accelerate EPSC recovery following conditioning 100 Hz trains at calyx synapses. At SC to CA1 synapses, Dynasore and Pitstop-2 reduce the extend of facilitation and lower relative steady-state EPSCs suggesting a change in the facilitation-depression balance in favor of the latter.

The authors use state-of-the art techniques and their data, which is clearly presented, leads to authors to conclude that endocytosis is universally important for clearance of release sites while the importance of scaffold protein-mediated site clearance is limited to 'fast synapses'.

Unfortunately, and perhaps not completely unexpected in view of the pharmacological tools chosen, there are several observations which remain difficult to understand:

(1) Blocking site clearance affects release sites that have previously been used, i.e. sites at which SV fusion has occurred and which therefore need to be cleared. Calyces use at most 20% of all release sites during a single AP, likely fewer at 1.3 mM external [Ca2+]. Even if all those 20% of release sites become completely unavailable due to a block of release site clearance, the 2nd EPSC in a train should not be reduced by >20% because ~80% of the sites cannot be affected. However, ~50% EPSC reduction was observed (Fig. 2B1, lower right panel) raising the possibility that Dynasore does more than specifically interfering with SVs endocytosis (and possibly Pitstop as well). Non-specific effects are also suggested by the observed two-fold increase in initial EPSC size in SC to CA1 synapses after Dynasore pre-incubation.

(2) More severe depression was observed at calyx synapses after blocking endocytosis which the authors attribute to a presynaptic mechanism affecting pool replenishment. When probing EPSC recovery after conditioning 100 Hz trains, a speed up was observed mediated by an "unknown mechanism" which is "masked in 2 mM [Ca2+]". These two observations, deeper synaptic depression during 100 Hz but faster recovery from depression following 100 Hz, are difficult to align and no attempt was made to find an explanation.

(3) To reconcile previous data reporting a block of Ca2+-dependent recovery (CDR) by Dynasore or Latrunculin (measured at 2 mM external [Ca2+]) with the data presented here (using 1.3 mM external [Ca2+]) reporting no effect or a speed up of recovery from depression, the authors postulate that "CDR may operate only when excessive Ca2+ enters during massive presynaptic activation" (page 10 line 244). While that is possible, such explanation ignores plenty of calyx studies demonstrating fiber stimulation-induced CDR and elucidating molecular pathways mediating fiber stimulation-induced CDR, and it also completely dismisses the strong change in recovery time course after 10 Hz conditioning (single exponential) as compared to 100 Hz conditioning (double exponential with a pronounced fast component).

Strong presynaptic stimuli such as those illustrated in Figs. 1B,C induce massive exocytosis. The illustrated Cm increase of 2 to 2.5 pF represents fusion of 25,000 to 30,000 SVs (assuming a single SV capacitance of 80 aF) corresponding to a 12 to 15% increase in whole terminal membrane surface (assuming a mean terminal capacitance of ~16 pF). Capacitance measurements can only be considered reliable in the absence of marked changes in series and membrane conductance. Documentation of the corresponding conductance traces is therefore advisable for such massive Cm jumps and merely mentioning that the first 450 ms after stimulation were skipped during analysis or referring to previous publications showing conductance traces is insufficient.
All bar graphs in Figures 1 through 6 and Figures S3 through S6 compare three or even four (Fig. 5C) conditions, i.e. one control and at least two treatment data sets. It appears as if repeated t-tests were used to run multiple two-group comparisons (i.e. using the same control data twice for two different comparisons). Either a proper multiple comparison test should be used or a Bonferroni correction or similar multiple-comparison correction needs to be applied.

Finally, the terminology of contrasting "fast-signaling" (calyx synapses) and "slow-plastic" (SC synapses) synapses seems to imply that calyx synapses lack plasticity, as does the wording "conventional bouton-type synapses involved in synaptic plasticity" (page 11, line 251). I assume, the authors primarily refer to the maximum frequencies these two synapse types typically transmit (fast-signaling vs slow-signaling)?

Author Response

The following is the authors’ response to the original reviews.

eLife assessment:

This important study combines a comparative approach in different synapses with experiments that show how synaptic vesicle endocytosis in nerve terminals regulates short-term plasticity. The data presented support the conclusions and make a convincing case for fast endocytosis as necessary for rapid vesicle recruitment to active zones. Some aspects of the description of the data and analysis are however incomplete and would benefit from a more rigorous approach. With more discussion of methods and analysis, this paper would be of great interest to neurobiologists and biophysicists working on synaptic vesicle recycling and short-term plasticity mechanisms.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

The study examines the role of release site clearance in synaptic transmission during repetitive activity under physiological conditions in two types of central synapses, calyx of Held and hippocampal CA1 synapses. After the acute block of endocytosis by pharmacology, deeper synaptic depression or less facilitation was observed in two types of synapses. Acute block of CDC42 and actin polymerization, which possibly inhibits the activity of Intersectin, affected synaptic depression at the calyx synapse, but not at CA1 synapses. The data suggest an unexpected, fast role of the site clearance in counteracting synaptic depression.

Strengths:

The study uses an acute block of the molecular targets with pharmacology together with precise electrophysiology. The experimental results are clear-cut and convincing. The study also examines the physiological roles of the site clearance using action potential-evoked transmission at physiological Ca and physiological temperature at mature animals. This condition has not been examined.

Weaknesses:

Pharmacology may have some off-target effects, though acute manipulation should be appreciated. Although this is a hard question and difficult to address experimentally, reagents may affect synaptic vesicle mobilization to the release sites directly in addition to blocking endocytosis.

To acutely block vesicle endocytosis, we utilized two different pharmacological tools, Dynasore and Pitstop-2, after testing their blocking spectra and potencies at the calyx presynaptic terminals and collected data of their common effects on target functions. Since the recovery from STD was faster at the calyx synapses in the presence of both endocytic blockers in physiological 1.3 mM [Ca2+] (Figure 2B), but not in 2.0 mM [Ca2+] (Figure S4), they might facilitate vesicle mobilization in physiological condition.

Reviewer #2 (Public Review):

Summary:

In this manuscript, Mahapatra and Takahashi report on the physiological consequences of pharmacologically blocking either clathrin and dynamin function during compensatory endocytosis or of the cortical actin scaffold both in the calyx of Held synapse and hippocampal boutons in acute slice preparations

Strengths:

Although many aspects of these pharmacological interventions have been studied in detail during the past decades, this is a nice comprehensive and comparative study, which reveals some interesting differences between a fast synapse (Calyx of Held) tuned to reliably transmit at several 100 Hz and a more slow hippocampal CA1 synapse. In particular, the authors find that acute disturbance of the synaptic actin network leads to a marked frequency-dependent enhancement of synaptic depression in the Calyx, but not in the hippocampal synapse. This striking difference between both preparations is the most interesting and novel finding.

Weaknesses:

Unfortunately, however, these findings concerning the different consequences of actin depolymerization are not sufficiently discussed in comparison to the literature. My only criticism concerns the interpretation of the ML 141 and Lat B data. With respect to the Calyx data, I am missing a detailed discussion of the effects observed here in light of the different RRP subpools SRP and FRP. This is very important since Lee et al. (2012, PNAS 109 (13) E765-E774) showed earlier that disruption of actin inhibits the rapid transition of SRP SVs to the FRP at the AZ. The whole literature on this important concept is missing. Likewise, the role of actin for the replacement pool at a cerebellar synapse (Miki et al., 2016) is only mentioned in half a sentence. There is quite some evidence that actin is important both at the AZ (SRP to FRP transition, activation of replacement pool) and at the peri-active zone for compensatory endocytosis and release site clearance. Both possible underlying mechanisms (SRP to FRP transition or release site clearance) should be better dissected.

The concept of FRP and SRP are derived from voltage-clamp step-depolarization experiments at calyces of Held in pre-hearing rodents at RT, which cannot be directly dissected in data of action-potential evoked EPSCs at post-hearing calyces at physiological conditions. However, we dissected as much by referring to related literatures in new paragraphs in Result section (p9-10), particularly on the different effects of Latrunculin application and experimental conditions by adding a new supplementary Figure (now S5). Regarding F-actin role in vesicle replenishment at cerebellar synapses, we added sentences in Discussion section (p14, last paragraph).

Reviewer #3 (Public Review):

General comments:

(1) While Dynasore and Pitstop-2 may impede release site clearance due to an arrest of membrane retrieval, neither Latrunculin-B nor ML-141 specifically acts on AZ scaffold proteins. Interference with actin polymerization may have a number of consequences many of which may be unrelated to release site clearance. Therefore, neither Latrunculin-B nor ML-141 can be considered suitable tools for specifically identifying the role of AZ scaffold proteins (i.e. ELKS family proteins, Piccolo, Bassoon, α-liprin, Unc13, RIM, RBP, etc) in release site clearance which was defined as one of the principal aims of this study.

In this study, we focused our analysis on the downstream activity of scaffold protein intersectin by comparing the common inhibitory effects of CDC42 and actin polymerization, by use of ML141 and Latrunculin B, respectively, on vesicle endocytosis and synaptic depression/ facilitation without addressing diverse individual drug effects. To avoid confusion we removed “AZ” from scaffold protein.

(2) Initial EPSC amplitudes more than doubled in the presence of Dynasor at hippocampal SC->CA1 synapses (Figure S2). This unexpected result raises doubts about the specificity of Dynasor as a tool to selectively block SV endocytosis.

It is possible that Dynasore might have unknown or off-target effects. However, the main conclusion is backed up by Pitstop-2.

(3) In this study, the application of Dynasore and Pitstop-2 strongly decreases 100 Hz steady-state release at calyx synapses while - quite unexpectedly - strongly accelerates recovery from depression. A previous study found that genetic ablation of dynamin-1 actually enhanced 300 Hz steady-state release while only little affecting recovery from depression (Mahapatra et al., 2016). A similar scenario holds for the Latrunculin-B effects: In this study, Latrunculin-B strongly increased steady-state depression while in Babu et al. (2020), Latrunculin-B did not affect steady-state depression. In Mahapatra et al. (2016), Latrunculin-B marginally enhanced steady-state depression. The authors need to make a serious attempt to explain all these seemingly contradicting results.

The latrunculin effect on STD can vary according to the condition of application and external [Ca2+], which we show in a new supplemental Figure S5. The latrunculin effect on the recovery from STD also varies with temperature, [Ca2+], and animal age, which affect Ca2+-dependent fast recovery component from depression. We added paragraphs for this issue in Results section (p9-10).

(4) The experimental conditions need to be better specified. It is not clear which recordings were obtained in 1.3 mM and which (if any?) in 2 mM external Ca. It is also unclear whether 'pooled data' are presented (obtained from control recordings and from separate recordings after pre-incubation with the respective drugs), or whether the data actually represent 'before'/'after' comparisons obtained from the same synapses after washing in the respective drugs. The exact protocol of drug application (duration of application/pre-incubation?, measurements after wash-out or in the continuous presence of the drugs?) needs to be clearly described in the methods and needs to be briefly mentioned in Results and/or Figure legends.

We added methodological explanations and reworded sentences in the text to be clear for pharmacological data derived from non-sequential separate experiments.

(5) The authors compare results obtained in calyx with those obtained in SC->CA1 synapses which they considered examples for 'fast' and 'slow' synapses, respectively. There is little information given to help readers understand why these two synapse types were chosen, what the attributes 'fast' and 'slow' refer to, and how that may matter for the questions studied here. I assume the authors refer to the maximum frequency these two synapse types are able to transmit rather than to EPSC kinetics?

Yes, the “fast and slow” naming features maximum operating frequency these synapses can transmit. We reworded “fast and slow” to “fast-signaling and slow-plastic” and added explanation in the text.

(6) Strong presynaptic stimuli such as those illustrated in Figures 1B and C induce massive exocytosis. The illustrated Cm increase of 2 to 2.5 pF represents a fusion of 25,000 to 30,000 SVs (assuming a single SV capacitance of 80 aF) corresponding to a 12 to 15% increase in whole terminal membrane surface (assuming a mean terminal capacitance of ~16 pF). Capacitance measurements can only be considered reliable in the absence of marked changes in series and membrane conductance. Since the data shown in Figs. 1 and 3 are central to the argumentation, illustration of the corresponding conductance traces is mandatory. Merely mentioning that the first 450 ms after stimulation were skipped during analysis is insufficient.

Conductance trace is shown with a trace of capacitance change induced by a square pulse in our previous paper (Yamashita et al, 2005 Science).

(7) It is essential for this study to preclude a contamination of the results with postsynaptic effects (AMPAR saturation and desensitization). AMPAR saturation limits the amplitudes of initial responses in EPSC trains and hastens the recovery from depression due to a 'ceiling effect'. AMPAR desensitization occludes paired-pulse facilitation and reduces steady-state responses during EPSC trains while accelerating the initial recovery from depression. The use of, for example, 1 mM kynurenic acid in the bath is a well-established strategy to attenuate postsynaptic effects at calyx synapses. All calyx EPSC recordings should have been performed under such conditions. Otherwise, recovery time courses and STP parameters are likely contaminated by postsynaptic effects. Since the effects of AMPAR saturation on EPSC_1 and desensitization on EPSC_ss may partially cancel each other, an unchanged relative STD in the presence of kynurenic acid is not necessarily a reliable indicator for the absence of postsynaptic effects. The use of kynurenic acid in the bath would have had the beneficial side effect of massively improving voltage-clamp conditions. For the typical values given in this MS (10 nA EPSC, 3 MOhm Rs) the expected voltage escape is ~30 mV corresponding to a change in driving force of 30 mV/80 mV=38%, i.e. initial EPSCs in trains are likely underestimated by 38%. Such large voltage escape usually results in unclamped INa(V) which was suppressed in this study by routinely including 2 mM QX-314 in the pipette solution. That approach does, however, not reduce the voltage escape.

Glutamate released during AP-evoked EPSCs does not saturate or desensitize postsynaptic receptors at post-hearing calyces of Held (Ishikawa et al, 2002; Yamashita et al, 2003) although it does in pre-hearing calyces (Yamashita et al, 2009). In fact, as shown in Figure S3, our results are essentially the same with or without kynurenate.

(8) In the Results section (pages 7 and 8), the authors analyze the time course into STD during 100 Hz trains in the absence and presence of drugs. In the presence of drugs, an additional fast component is observed which is absent from control recordings. Based on this observation, the authors conclude that '... the mechanisms operate predominantly at the beginning of synaptic depression'. However, the consequences of blocking or slowing site clearing are expected to be strongly release-dependent. Assuming a probability of <20% that a fusion event occurs at a given release site, >80% of the sites cannot be affected at the arrival of the second AP even by a total arrest of site clearance simply because no fusion has yet occurred. That number decreases during a train according to (1-0.2)^n, where n is the number of the AP, such that after 10 APs, ~90% of the sites have been used and may potentially be unavailable for new rounds of release after slowing site clearance. Perhaps, the faster time course into STD in the presence of the drugs isn't related to site clearance?

Enhanced depression at the beginning of stimulation indicates the block of rapid SV replenishment mechanism, which includes endocytosis-dependent site-clearance and scaffold-dependent vesicle translocation to release sites.

(9) In the Discussion (page 10), the authors present a calculation that is supposed to explain the reduced size of the second calyx EPSC in a 100 Hz train in the presence of Dynasore or Pitstop-2. Does this calculation assume that all endocytosed SVs are immediately available for release within 10 ms? Please elaborate.

We do not assume rapid endocytosed vesicle reuse within 10 ms as it requires much longer time for glutamate refilling (7s at PT; Hori & Takahashi, 2012). Instead, already filled reserved vesicles can rapidly replenish release sites if sites are clean and scaffold works properly. Results shown in Figure S6 also indicate that block of vesicle transmitter refilling has no immediate effect on synaptic responses.

(10) It is not clear, why the bafilomycin/folimycin data is presented in Fig. S5. The data is also not mentioned in the Discussion. Either explain the purpose of these experiments or remove the data.

These v-ATPase blockers, which block vesicular transmitter refilling, are reported to enhance EPSC depression at hippocampal synapses at RT and 2 mM [Ca2+] presumably because of lack of filled vesicles undergoing rapid vesicle recycling (eg Kiss & Run). We thought it important to determine whether these data have physiological relevance since such a mechanism might also regulate synaptic strength during repetitive transmission. However, our results did not support its physiological relevance. Since these results are not within our main questions, the negative results are shown it in supplementary Figure 6 and explained in the last paragraph of Result section (p11), but were not discussed further in Discussion section.

(11) The scheme in Figure 7 is not very helpful.

We updated the scheme to summarize our conclusion that vesicle replenishment through endocytosis-dependent site-clearance and scaffold-dependent mechanism independently co-operate to strengthen synaptic efficacy during repetitive transmission at calyx fast-signaling synapses. However, endocytic site clearance is solely required to support facilitation at slow-plastic hippocampal SC-CA1 synapses.

Recommendations for the authors:

First, my deep apologies for the long delay in reviewing your paper. All reviewers are now in agreement that the paper has valuable new information, but some methods are not described well and some results appear to be incompatible with previous results in the literature. The discussion of previous literature is also incomplete and not well-balanced. With more discussion of methods and literature strengthened this paper would be of great interest to neurobiologists and biophysicists working on synaptic vesicle recycling and short-term plasticity mechanisms. We ask that you address the comments and revise your paper before we can fully recommend the paper as being an important contribution with compelling evidence and a strong data set that supports the conclusions.

We explained methods more explicitly. Apparent incompatibility with previous results is now explained and discussed with new supplementary data.

Major:

(1) In this study, the application of Dynasore and Pitstop-2 strongly decreased 100 Hz steady-state release at calyx synapses while - quite unexpectedly - it strongly accelerated recovery from depression. A previous study found that genetic ablation of dynamin-1 actually enhanced 300 Hz steady-state release while only little affecting recovery from depression (Mahapatra et al., 2016). A similar scenario holds for the Latrunculin-B effects: In this study, Latrunculin-B strongly increased steady-state depression while in Babu et al. (2020), Latrunculin-B did not affect steady-state depression. In Mahapatra et al. (2016), Latrunculin-B marginally enhanced steady-state depression. The authors need to make a serious attempt to explain all these seemingly contradicting results.

Lack of change in the recovery from depression in dynamin-1 knockout mice by Mahapatra et al (2016) is consistent with results in Figure S4 in 2 mM [Ca2+], whereas accelerated recovery by Dynasore (Figure 2B2) is observed in 1.3 mM [Ca2+] suggesting that it is masked in 2 mM [Ca2+] but revealed in physiological [Ca2+] (p7, top paragraph). In both cases, however, recovery from STD is not prolonged unlike Hosoi et al (2009).

The latrunculin issues are discussed in Results section with newly added Supplementary Figure S5 (p9-10).

(2) The experimental conditions need to be better specified. It is not clear which recordings were obtained in 1.3 mM and which (if any?) in 2 mM external Ca. It is also unclear whether 'pooled data' are presented (obtained from control recordings and from separate recordings after pre-incubation with the respective drugs), or whether the data actually represent 'before'/'after' comparisons obtained from the same synapses after washing in the respective drugs. The exact protocol of drug application (duration of application/pre-incubation?, measurements after wash-out or in the continuous presence of the drugs?) needs to be clearly described in the methods and needs to be briefly mentioned in Results and/or Figure legends.

We made these points clearer in Method section and Result section.

(3) Please cite and discuss briefly previous papers that have shown fast endocytosis in the calyx of Held with membrane capacitance measurements like Renden and von Gersdorff, J Neurophysiology, 98:3349, 2007 and Taschenberger et al., Neuron, 2002. These papers first showed exocytosis and endocytosis kinetics in more mature (hearing) mice calyx of Held and at higher physiological temperatures.

One of these literatures relevant to the present study is quoted in p4.

(4) The findings concerning the different consequences of actin depolymerization are not sufficiently discussed in comparison to the literature. My only criticism concerns the interpretation of the ML 141 and Lat B data. With respect to the Calyx data, I am missing a detailed discussion of the effects observed here in light of the different RRP subpools SRP and FRP. This is very important since Lee et al. (2012, PNAS 109 (13) E765-E774) showed earlier that disruption of actin inhibits the rapid transition of SRP SVs to the FRP at the AZ. The whole literature on this important concept is missing. Likewise, the role of actin for the replacement pool at a cerebellar synapse (Miki et al., 2016) is only mentioned in half a sentence. There is quite some evidence that actin is important both at the AZ (SRP to FRP transition, activation of replacement pool) and at the peri-active zone for compensatory endocytosis and release site clearance. Both possible underlying mechanisms (SRP to FRP transition or release site clearance) should be better dissected.

We added discussions on the issue of latrunculin in Result section by quoting previous literatures (p9-10). Since there is no direct evidence (by vesicle imaging) for the presence of FRP and SRP, these definitions derived from voltage clamp step-depolarization studies are difficult to incorporate into the dissection of synaptic depression in physiological conditions.

Reviewer #1 (Recommendations For The Authors):

I have no major comments, but the following issues may be addressed.

(1) The term "fast and slow" synapses may be relative and a bit confusing. I do not think hippocampal synapses are slow synapses.

We have replaced “fast and slow” by “fast-signaling and slow-plastic” to represent their functions and added explanation in the text.

(2) Off-target effects of pharmacological effects may be discussed. In this respect, bafilomycin experiments can be used to argue against the slow effects of vesicle cycling such as endocytosis, and vesicle mobilization. However, the effects on rapid vesicle mobilization cannot be excluded entirely. Because I cannot exclude the absence of off-target effects either (can be addressed by looking at single vesicle imaging at nano-scale, which is hard to do or looking at EM level quantitatively?), I feel this is a matter of discussion.

It is possible that Dynasore might have unknown or off-target effects. However, the main conclusion is backed up by Pitstop-2.

(3) Fig2 A2, B2 and Fig 4 A2 and B2. It is easier to plot the recovery only normalized to the initial value. Subtracting steady-state is somewhat confusing because the recovery looks faster after deeper depression, but this may be just apparent.

We have given values for both types of plots in Table 2, which indicates no essential difference in the recovery parameters.

Reviewer #2 (Recommendations For The Authors):

Line 51: Rajappa et al. (2016) investigated clearance deficits in synaptophysin KO mice (not synaptobrevin).

Corrected.

Line 54: intersectin is introduced as AZ scaffold protein, although in most of the literature, it is referred to as an endocytic scaffold protein (also in the cited one, e.g. Sakaba et al. 2013). At least, this should be discussed.

Since blockers of intersectin downstream protein activity has no effect on vesicle endocytosis (Figure 3 and Sakaba et al, 2013), we called it (presynaptic) scaffold protein instead of endocytic scaffold protein.

Reviewer #3 (Recommendations For The Authors):

Minor comments

Page 1, Title: I don't think the presented data address the role of the presynaptic scaffold in SV replenishment. In addition, 'SV replenishment' and 'site clearance' should not be used synonymously as it seems to be implied here.

In this study our focus was on the downstream activity of scaffold protein intersectin and since block of its downstream effector proteins CDC42 and actin activities do not obstruct the endocytic activity (Fig 3, and Sakaba et al., 2013), instead of naming it as “endocytic scaffold protein”, we adopted “presynaptic scaffold protein”.

We have corrected it in the text.

Page 2, Abstract: Clarify 'physiologically optimized condition' here and elsewhere in the manuscript.

Abstract: in physiologically optimized condition → in physiological temperature and Ca2+.

Page 3, line 62: I don't think 'the site-clearance hypothesis is widely accepted'. There are very few models that implement such a mechanism. Examples would be Pan & Zucker (2009) Neuron and Lin, Taschenberger & Neher 2022 (PNAS) which could be cited.

62: the site-clearance hypothesis is “widely accepted”→ “well supported”

Page 3 line 77: Please clarify 'fast synapses

77: fast synapses→fast-signaling synapses, added clarification in the text.

Page 4, line 100: Please clarify 'in the maximal rate'.

100: in the maxima rate→reached during 1-Hz stimulation.

Page 6, line 136: Please clarify 'to reduce the gap'.

136: To reduce the gap between these different results→To explore the reason for these different results

Page 7, line 157: I don't consider ML141 and Latrunculin-B 'scaffold protein inhibitors'.

157: scaffold protein inhibitors had no effect on→ reworded as “none of these inhibitors affected fast or slow endocytosis”.

Page 7, line 162: P-value missing.

162: p < 0.001 added.

Page 8, line 184: "Since both endocytic blockers and scaffold inhibitors enhanced synaptic depression with a similar time course" consider rephrasing. Sounds like you refer to the time course by which these drugs exert their effect after being applied.

184: Since both endocytic blockers and scaffold inhibitors enhance synaptic depression with a similar time course→Since the enhancement of synaptic depression by endocytic blockers or scaffold inhibitor occurred mostly at the early phase of synaptic depression.

Same on page 11, line 250: "At the calyx of Held, scaffold protein inhibitors significantly enhanced synaptic depression with a time course closely matching to that enhanced by endocytic blocker" Please consider rephrasing.

At the calyx of Held, scaffold protein inhibitors significantly enhanced synaptic depression with a time course closely matching to that enhanced by endocytic blocker →the early phase of synaptic depression like endocytic blockers

Page 13, line 318: Please clearly state which experiments were performed at 1.3 mM and which at 2 mM external Ca if two different concentrations were used during recordings.

320: Added text “Unless otherwise noted, EPSCs were recorded in 1.3 mM [Ca2+] aCSF at 37oC” in the methods.

Page 15: line 346: Reference in the wrong format.

346; (25) → (Yamashita et al, 2005)

Page 15: line 351: Do you mean to say every 10 s and every 20 s? Please clarify.

No, averaged at 10 ms and 20 ms, respectively as written.

Page 16, line 369: 1 mM kyn was present in only very few experiments shown in the supplemental figures. Please clarify.

368: In some experiments, to test in the presence of 1 mM kyn, if there is any difference in enhanced STD following endocytic block. However, as shown in Figure S3, our results are essentially the same with or without kynurenate, suggesting glutamate released during AP-evoked EPSCs does not saturate or desensitize postsynaptic receptors at post-hearing calyces of Held (Ishikawa et al, 2002; Yamashita et al, 2003) unlike in pre-hearing calyces (Yamashita et al, 2009).

Page 16, line 387: You cannot simply use multiple t-tests to compare a single control to multiple test conditions which seems to be the scenario here. Please correct or clarify.

Experimental protocols are clarified in Methods as “Experiments were designed as population study using different cells from separate brain slices under control and drug treatment, rather than on a same cell before and after the drug exposure.”

Table S1: 'Endo decay rate'. It's either the 'Endo rate' or the 'Deacy rate of delta Cm'. Please correct.

Corrected as Endocytosis rate (Endo rate).