We assess energy barrier changes by fitting release kinetics in response to hypertonic sucrose. Mimicking activation of the C2A domain of the Ca2+-sensor Synaptotagmin-1 (Syt1), by adding a positive charge (Syt1D232N) or increasing its hydrophobicity (Syt14W), lowers the energy barrier. Removing Syt1 or impairing its release inhibitory function (Syt19Pro) increases spontaneous release without affecting the fusion barrier. Both phorbol esters and tetanic stimulation potentiate synaptic strength, and lower the energy barrier equally well in the presence and absence of Syt1. We propose a model where tetanic stimulation activates Syt1-independent mechanisms that lower the energy barrier and act additively with Syt1-dependent mechanisms to produce PTP by exerting multiplicative effects on release rates.Previously, we showed that modulation of the energy barrier for synaptic vesicle fusion boosts release rates supralinearly . Here we show that mouse hippocampal synapses employ this principle to trigger Ca Changes in the energy barrier were probed in several mutant variants of Syt1 and during PTP, using hypertonic sucrose (HS) stimulation. We found that mimicking activation of Syt1’s Ca2+-sensor for synaptic vesicle release in many synapses , and change in the fusion energy barrier (ΔEa (RT)) (For detailed methodology see: 2+ bound) does not increase the energy barrier for synaptic vesicle fusion, despite its inhibitory effect on spontaneous release.Syt1 is well established to be the fast Casynapses . Howeversynapses . This insynapses , suggestsynapses . Alterna+-sensor , reducintuations , but liktuations  or dendrtuations . Howevertuations , as obsetuations . Voltagetuations , while ftuations . As desctuations  can be o2+-sensing C2 domains is fixed, selectively impairing inhibition of spontaneous release, without affecting evoked release  through repetitive stimulation also leads to a reduction. We assessed changes in the fusion barrier after induction of PTP in the presence and absence of Syt1. PTP is a form of STP which lasts tens of seconds to minutes , allowine by 39% , even the by 39% . This ree by 39% . PTP ince by 39% , and ince by 39% , indicate by 39% . These re by 39% , and sho2+. In Syt1 KO autapses, PTP also increased vesicle release probability  pathway .Several mechanisms have been proposed to explain the inhibitory effect of Syt1 on spontaneous release . These i2+ binding lowers the fusion barrier  reduction when Syt1 is fully activated after binding 5 Ca2+ ions, adding 10 charges in total (−1) reduction during AP-induced release in hippocampal neurons (2+-dependent SNARE binding (2+ levels. Modeling these scenarios (reduced electrostatic repulsion or increased Ca2+-dependent SNARE binding) with the energy barrier model, predicted in both cases an increase in Ca2+ sensitivity with little effect on cooperativity  was shown to reduce Ca2+ sensitivity in the same study  priming of vesicles, spatial distribution of vesicles or detailed Ca2+ dynamics, nor the effect of Syt1 or other sensors on these processes. Furthermore, it does not explicitly describe all successive steps in the fusion pathway  pups of both sexes, obtained by caesarean section of pregnant female mice. For this, previously described Synaptotagmin-1 knockout  or C57BLHippocampi from WT and Syt1 KO mice were isolated, collected in ice-cold Hank’s buffered salt solution  buffered with 1 mM HEPES (Invitrogen), and digested for 20 min with 0.25% trypsin (Invitrogen) at 37°C. After washing, neurons were dissociated using a fire-polished Pasteur pipette and resuspended in Neurobasal medium supplemented with 2% B-27, 1% HEPES, 0.25% GlutaMAX, and 0.1% Penicillin-Streptomycin . Neurons were counted in a Fuchs-Rosenthal chamber and plated at 1.5K per well in a 12-well plate. Neuronal cultures were maintained in Neurobasal medium supplemented with 2% B-27, 1% HEPES, 0.25% GlutaMAX, and 0.1% Penicillin-Streptomycin , at 37°C in a 5% CO2 humidified incubator.Autaptic hippocampal cultures were prepared as described previously . BrieflyFor rescue experiments, Syt1 KO neurons were infected at DIV4 with a synapsin-promoter-driven lentiviral vector expressing either Syt1 9Pro , Syt1 D232N, Syt1 4W , or wild type Syt1. The experimental groups were masked during the experiment. The code was broken after statistical analysis.m = −70 mV) were performed at room temperature with borosilicate glass pipettes (2–5 MΩ) filled with (in mM) 125 K+-gluconic acid, 10 NaCl, 4.6 MgCl2,4 K2-ATP, 15 creatine phosphate, 1 EGTA, and 10 units/mL phosphocreatine kinase (pH 7.30). External solution contained in mM: 10 HEPES, 10 glucose, 140 NaCl, 2.4 KCl, 4 MgCl2 . 4 mM CaCl2 was used externally in all experiments, unless otherwise specified. Inhibitory neurons were identified and excluded based on the decay of postsynaptic currents. Recordings were acquired with a MultiClamp 700B amplifier, Digidata 1440 A, and pCLAMP 10.3 software (Molecular Devices). Only cells with an access resistance <15 MΩ (80% compensated) and leak current of <300 pA were included. EPSCs were elicited by a 0.5 ms depolarization to 30 mV.Whole-cell voltage-clamp recordings  in bath and external solution was exchanged for 0 mM CaCl2. A decrease in spontaneous release during incubation was used as a positive control. For PDBu experiments, sucrose applications were performed as usual, after which neurons were incubated with 1 μM PDBu , and sucrose applications were repeated.For experiments including the cell permeable CaHippocampal neurons were fixed with 3.7% formaldehyde (Electron Microscopy Sciences) after two weeks in culture. After washing with PBS, cells were permeated with 0.5% Triton X-100 for 5 min and incubated in 2% normal goat serum/0.1% Triton X-100 for 30 min to block aspecific binding. Cells were incubated for 1 hr at room temperature with primary antibodies directed against MAP2 and vGlut1 to visualize dendrite morphology and synapses. The following antibodies were used: polyclonal chicken anti-MAP2 , polyclonal rabbit vGlut1 , polyclonal guinea pig vGlut1 , monoclonal mouse VAMP2 , polyclonal rabbit Synaptotagmin-1 , or polyclonal chicken GFP . After washing with PBS, cells were incubated for 1 hr at room temperature with second antibodies conjugated to Alexa dyes  and washed again. Coverslips were mounted with DABCO-Mowiol (Invitrogen) and imaged with a confocal LSM510 microscope (Carl Zeiss) using a 40 × oil immersion objective with 0.7 × zoom at 1024 × 1024 pixels. Neuronal morphology was analyzed using a published automated image analysis routine , and ImaAutaptic hippocampal neuron cultures of WT and Syt-1 KO mice (E18) grown on glass cover slips were fixed (DIV14) for 90 min at room temperature with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4). After fixation, cells were washed three times for 5 min with 0.1 M cacodylate buffer (pH 7.4), post-fixed for 1 hr at room temperature with 1% OsO4/1% KRu(CN)6. After dehydration through a series of increasing ethanol concentrations, cells were embedded in Epon and polymerized for 48 hr at 60°C. After polymerization of the Epon, the coverslip was removed by alternately dipping it in hot water and liquid nitrogen. Cells of interest were selected by observing the flat Epon-embedded cell monolayer under the light microscope and mounted on pre-polymerized Epon blocks for thin sectioning. Ultrathin sections (80 nm) were cut parallel to the cell monolayer, collected on single-slot, formvar-coated copper grids, and stained in uranyl acetate and lead citrate using a LEICA EM AC20 stainer. Synapses were randomly selected at low magnification using a JEOL 1010 electron microscope. For each condition, the number of docked synaptic vesicles, total synaptic vesicle number, postsynaptic density and active zone length were measured on digital images taken at 80,000-fold magnification using analySIS software (Soft Imaging System). The observer was blinded for the genotype. For all morphological analyses, we selected only clearly recognizable synapses with intact synaptic plasma membranes with a recognizable pre- and postsynaptic area and clearly defined synaptic vesicle membranes. Synaptic vesicles were defined as docked if there was no distance visible between the synaptic vesicle membrane and the active zone membrane. The active zone membrane was recognized as a specialized part of the presynaptic plasma membrane that contained a clear density opposed to the postsynaptic density and docked synaptic vesicles. Cells were cultured from six different WT and seven different Syt-1 KO mice. Approximately 25 synapses were analyzed per culture stemming from one animal.https://github.com/vhuson/viewEPSC (https://doi.org/10.7554/eLife.05531.031). In all figures, stimulation artefacts have been removed. For evoked release, total charge was calculated by integrating the current from the end of the stimulation until the start of the next pulse. HS-induced responses were fitted with a minimal vesicle state model as described previously  was set to match spontaneous release in Syt1 WT at 0mM extracellular Ca2+ and 20µM BAPTA-AM. All other parameters .Offline analysis was performed using custom‐written software routines in Matlab R2018b (Mathworks). Software routines for analysis of mEPSCs and electrically evoked release is available at viewEPSC , softwareviously . This meic model . An RRP  In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.eLife. Your article has been reviewed by Richard Aldrich as the Senior Editor, a Reviewing Editor, and three reviewers. The reviewers have opted to remain anonymous.Thank you for submitting your article "Post-tetanic potentiation lowers the energy barrier for synaptic vesicle fusion independently of Synaptotagmin-1" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission. In recognition of the fact that revisions may take longer than the two months we typically allow, until the research enterprise restarts in full, we will give authors as much time as they need to submit revised manuscripts.Cornelisse and colleagues have previously reported a method to study the final step of the neurotransmitter release process, the rates of vesicle fusion by fitting a kinetic model to hypertonic solution-induced release events determined experimentally. Assuming that the fusion energy barrier is imposed by various intermediate steps of the vesicle fusion reaction, the authors have proposed that the additive effects of overcoming this energy barrier, for example, by binding of Ca to the calcium sensor Synaptotagmin-1 (Syt1), could drive the supralinear relationship between Ca and release. Following from the previous work, Huson et al. now show that Syt1 lowers the fusion energy barrier independently of its fusion clamp function, and together with slower Ca sensor(s) for asynchronous release, Syt1 also serves to reduce the fusion energy barrier during short-term plasticity (STP).The manuscript has been reviewed by three experts, all of whom find the study to be technically well done and very interesting. However, several major concerns have been raised. We feel that all the issues can be addressed by further analysis and careful rewriting of the manuscript without the need for additional experiments. Notably the reviewers have unanimously agreed that the clarity of the paper will be significantly improved by presenting the data first, then interpreting the data within the framework of the model. Additionally, the assumptions and limitations of the model should be clearly explained in order to make the model more useful.The individual reviewer comments that warrant consideration for the revision are as follows:Reviewer 1:1) There is no evidence that the kinetic parameter k2_HS that is extracted from an empirical fit to HS-induced release profile has the same Arrhenius dependence as the rate constant for evoked (AP-induced) release k2_ev . Such evidence could be obtained by varying temperature and making Arrhenius plots for the two rate constants. It is also unclear why the frequency factor A should be fixed. It could be, e.g. calcium-dependent . However, in light of the extraordinary situation, these experiments are not necessary, nor likely feasible in the next few months. In any case, the authors should cite some temperature studies from the literature; these would at least indicate what energy barriers are at play, and how they compare to the energy reductions the authors estimate for the various conditions .2) HS-induced release is a convenient tool to measure the amount of readily-releasable vesicles (RRP), but we run into a conceptual problem (and confusion) in Syt1 KOs (and possibly other mutants). The pool that is releasable by ~500 mM sucrose correlates well with the size of the pool releasable by AP trains in wild-type neurons. But the correlation breaks down in Syt1 KOs: the HS-induced pool is nearly the same, but AP-induced releasable pool is much smaller. This is not surprising, because HS-induced release is calcium-independent, whereas AP-induced release requires calcium (and its sensor(s)). RRP (estimated by HS application) is often taken to mean the pool of "primed" vesicles, but can we have priming without Syt1? These issues arise when we push the interpretation of HS-induced release too far. As a result, the model creates more issues than it clarifies. The simplest way to deal with this is to first present the results (some of which are interesting on their own) in a model-independent manner, and then present a possible interpretation within the framework of the model (and explaining the limitations of the model). The alternative (framing everything around the model) would require much more work to put the model on more solid ground (and may fail).3) Evidence for a second sensor is very circumstantial. Syt7 is mentioned as a strong candidate. Does overexpression of Syt7 lead to more facilitation? If yes, this could lend support to the claim that PTP is due to two sensors that are activated simultaneously.If Syt7 or any other calcium sensor (in addition to Syt1) is the factor "g" that binds the vesicle in Figure 1C, why does it not interact with calcium, like Syt1?Similarly, for Syt1 D232N, how would the scheme be modified, and what calcium cooperativity would be predicted? How does the predicted cooperativity compare with the actual one (from the literature)?4) Subsection “Synaptotagmin-1 inhibits spontaneous release without changing the fusion energy barrier”. The claim that despite its inhibitory effect on spontaneous release, Syt1 that is not calcium-bound does not increase the energy barrier for synaptic vesicle fusion, seems to contradict the basic assumption of the model (release rate ~exp(E/RT)). The problem is that the fraction of the RRP depleted by 250 mM sucrose is taken as a proxy for the energy barrier height. The curves in Figure 2K,L, cross at 250 mM sucrose, so by this criterion, the energy barrier is not changed. This is an example of over-reliance on the model to interpret the data.The conclusion that "the spontaneous release rate can be modulated independently from the energy barrier" does not make sense. One of the basic assumptions of the model seems to be wrong (l+ being the spontaneous release rate).Can the authors please indicate on Figure 2H (and other figures) when HS is applied? It is not clear if there is an additional delay in the KO or the traces are simply shifted.Figure 2—figure supplement 1 top row is a repeat of Figure 2A,B,C.5) The subtitle "Spontaneous release rates are partially regulated by energy barrier independent factors" again does not make sense. Do the authors mean spontaneous release may be due to a different pathway with its own barrier?To avoid spontaneous Ca fluctuations leading to spontaneous fusion (a possible explanation), couldn't the authors repeat the experiments at 0 mM external calcium?6) Discussion section: "Syt1 does not inhibit spontaneous release by increasing the fusion energy barrier" it does, at 0 mM HS. Also see the comments above.Since Syt1's two roles on supressing spontaneous release and promoting evoked release can be dissociated with the 9Pro mutant, perhaps the authors should use the 9Pro mutant as their reference, not the WT. It would be interesting to see a replot of data vs 9Pro.7) Discussion section: There is some vague discussion about electrostatics, membrane bending and effects of curvature that might affect the energy barrier for fusion. Is the D232N mutant able to penetrate bilayers in response to calcium binding to C2? Or is it constitutively inserted into the membrane?8) Subsection “Fusion energy barrier model for STP”, "Fusion energy barrier model for STP""multiplication of membrane bound Syt1 and Syt7 corresponds to the facilitation produced by Syt7". Syt1*Syt7=facilitation?Reviewer 2:1) The authors investigated the relationship between evoked release and changes in the fusion energy barrier in Syt1KO cultures overexpressing the SytD232N mutant to find a convincing correlation between the increase in evoked release and the lowering of the energetic barrier. They had more difficulties to demonstrate a similar straightforward relationship with the Syt1 4W mutant, because the decrease in the energetic barrier is only detected for low HS concentrations (250mM), perhaps reflecting that the readout of the approach to measure the fusion energy barrier is more complex than initially envisioned. I think it would be helpful and informative to state those limitations which, however, do not invalidate the approach.2+ solution went too much in the limit in my opinion and it should require experimental demonstration or at least to be well documented in the literature. I wonder if such hypothetical Ca2+ fluctuations could be abolished using higher BAPTA concentrations. On the other hand, it could be that the changes in the fusion energy barrier in the context of spontaneous release are different (or not picked up) to the changes in the context of HS stimulation or different pool of vesicles are involved in each condition.2) They observed higher spontaneous release in Syt1KO and Syt1 9Pro expressing synapses which is difficult to conciliate with their measurements indicating that the fusion energy barrier is unchanged. The explanation of rapid local fluctuations resistant to BAPTA in 0mM Ca3) To interrogate the involvement of Syt1 in PTP makes sense in light of a previous publication by Verhage's group showing PKC-dependent phosphorylation of Syt1 in short term plasticity. The part of the study devoted to the analysis to the fusion energy barrier involvement in PTP is the most comprehensive, relevant and convincing part of the study in which conclusions are best supported by the results. It is very interesting and i opens new perspectives to investigate potential PTP mediators and its role in the modulation of the fusion energy barrier in future studies.Reviewer 3:1) Although the model is interesting, it is difficult to evaluate the values such as k2, max and δ E calculated from the model. It may be nice to show raw data such as time to peak, delay, integral of EPSCs and its dependence on sucrose concentration. What I mean here is that the overall results should be verified from experimental results, independent of modelling.2) It is a bit difficult to understand why the RRP size looks smaller in Syt1 KO and Syt14W mutants but not in D232N (at least it looks like this from representative traces). The authors need some arguments. This may affect calculation of release probability.3) In some experiments, the sucrose concentration was raised to 1M, but in some experiments, it was raised to 500 mM. Why?4) The authors describe that spontaneous mEPSC rates are higher in Syt1 KO, but others did not see the effect, though they used the same preparation. The authors did not provide an explanation and it is puzzling.5) Post-tetanic potentiation and the effects of phorbol esters are known to be regulated by PKC, Munc13/18, and are known to be independent of synaptotagmins. The authors need more explanation for novelty here.6) Phorbol esters are known to change the RRP size (Stevens and colleagues), but the authors have seen somewhat different (representative traces seem to indicate the increase in the RRP size). The authors need some explanation.7) Figure 2—figure supplement 1 top panel. The same figure as Figure 2? Reviewer 1:1) There is no evidence that the kinetic parameter k2_HS that is extracted from an empirical fit to HS-induced release profile has the same Arrhenius dependence as the rate constant for evoked (AP-induced) release k2_ev . Such evidence could be obtained by varying temperature and making Arrhenius plots for the two rate constants. It is also unclear why the frequency factor A should be fixed. It could be, e.g. calcium-dependent . However, in light of the extraordinary situation, these experiments are not necessary, nor likely feasible in the next few months.2+ channels, Ca2+ binding to the sensor, and calcium dynamics, which are all temperature sensitive. We and others have previously studied the effect of temperature on AP-induced release  and found for instance that peak release-rates were not affected by a ten-degrees increase in temperature, while charge transfer was even reduced at higher temperatures. This was best explained by more effective Ca2+ buffering and Ca2+ clearance at higher temperatures . To account for all these confounding temperature effects of processes not related to the fusion barrier would require several complicated experiments that we feel are beyond the scope of this paper. We thank the reviewer for acknowledging the extraordinary situation we are currently in, which prevents executing these experiments in due time.We agree that a direct comparison between HS-induced and AP-induced release rates at different temperatures would be interesting. However, the interpretation would be complicated since AP-induced release depends on more factors than the energy barrier alone, such as AP-shape, kinetics of the CaIn any case, the authors should cite some temperature studies from the literature; these would at least indicate what energy barriers are at play, and how they compare to the energy reductions the authors estimate for the various conditions .2+ binding (Discussion section), and how it can explain most of the increase in release probability during PTP or PDBU stimulation, in line with our model (Discussion section).We use RT as units for energy, which corresponds to 0.585 kcal/mol. We have now cited recent studies that have either used lipid bilayer fusion assays at different temperatures or course-grained simulations for an estimate of the fusion barrier, which was found to be around 30 RT (Discussion section). For comparison, we included values for the energy barrier reductions we found for the various conditions in the text, which all ranged between 0.2 to 0.6 RT. We discuss how these relatively small changes compare to the energy barrier effects that were predicted for the activation of Syt1 by Ca2) HS-induced release is a convenient tool to measure the amount of readily-releasable vesicles (RRP), but we run into a conceptual problem (and confusion) in Syt1 KOs (and possibly other mutants). The pool that is releasable by ~500 mM sucrose correlates well with the size of the pool releasable by AP trains in wild-type neurons. But the correlation breaks down in Syt1 KOs: the HS-induced pool is nearly the same, but AP-induced releasable pool is much smaller. This is not surprising, because HS-induced release is calcium-independent, whereas AP-induced release requires calcium (and its sensor(s)). RRP (estimated by HS application) is often taken to mean the pool of "primed" vesicles, but can we have priming without Syt1? These issues arise when we push the interpretation of HS-induced release too far. As a result, the model creates more issues than it clarifies. The simplest way to deal with this is to first present the results (some of which are interesting on their own) in a model-independent manner, and then present a possible interpretation within the framework of the model (and explaining the limitations of the model). The alternative (framing everything around the model) would require much more work to put the model on more solid ground (and may fail).We agree that different definitions of priming exist, which may lead to different interpretations of the data. We stated more explicitly in the text (Results section) which definition we used. To circumvent unnecessary confusion, we adopted the suggestion of the reviewer to rewrite the paper and present the data first before presenting the model and interpreting the data in this framework. In addition, we explain the limitations of the model more extensively (Discussion section). We feel this has improved the clarity of the paper and balanced the discussion about the model.3) Evidence for a second sensor is very circumstantial. Syt7 is mentioned as a strong candidate. Does overexpression of Syt7 lead to more facilitation? If yes, this could lend support to the claim that PTP is due to two sensors that are activated simultaneously.If Syt7 or any other calcium sensor (in addition to Syt1) is the factor "g" that binds the vesicle in Figure 1C, why does it not interact with calcium, like Syt1?We briefly mention Syt7 as a sensor for paired-pulse facilitation (PPF) in the context of a more general energy barrier framework for STP. We are not aware of overexpressing studies but cite several studies that show that ablation of Syt7 did not affect normal transmission but eliminated PPF (Discussion section). However, we did not intent to suggest that Syt7 is a sensor for PTP, which occurs at a slower time scale than PPF and most likely involves a different pathway. To emphasize that the data and model we present here is for PTP we explicitly say so in the text (Results section) and in the discussion (Discussion section). Since we argue that the DAG pathway is most likely involved in the production of PTP we have adapted Figure 7C  accordingly, showing the activation of the second sensor by DAG.Similarly, for Syt1 D232N, how would the scheme be modified, and what calcium cooperativity would be predicted? How does the predicted cooperativity compare with the actual one (from the literature)?2+ sensitivity, and included a new supplemental figure  with a modified scheme. Based on an electrostatic energy barrier model proposed by the Sorensen lab  we modeled the reduction of the energy barrier, as a consequence of adding a charge to the C2A domain, as half of the reduction associated with the binding of one Ca2+ ion, which adds two charges. In addition, we modeled the two-fold increase in Ca2+-dependent SNARE binding found by Pang et al.  as a two-fold increase in factor f. These changes resulted in an increase in Ca2+ sensitivity, with higher release rates at all Ca2+ concentrations, with little effect on the apparent cooperativity (see new text in the Results section). We discuss how these predictions compare to previous findings in the literature  (Discussion section). We conclude that both mechanisms can explain the increase in Ca2+ sensitivity without big changes in cooperativity, but that increased Ca2+-dependent SNARE binding is more likely, given the other results in this study, and previous findings in literature (Discussion section).We have explored two mechanisms in the model by which the D232N mutant can increase Ca4) Subsection “Synaptotagmin-1 inhibits spontaneous release without changing the fusion energy barrier”. The claim that despite its inhibitory effect on spontaneous release, Syt1 that is not calcium-bound does not increase the energy barrier for synaptic vesicle fusion, seems to contradict the basic assumption of the model (release rate ~exp(E/RT)). The problem is that the fraction of the RRP depleted by 250 mM sucrose is taken as a proxy for the energy barrier height. The curves in Figure 2K, L, cross at 250 mM sucrose, so by this criterion, the energy barrier is not changed. This is an example of over-reliance on the model to interpret the data.The conclusion that "the spontaneous release rate can be modulated independently from the energy barrier" does not make sense. One of the basic assumptions of the model seems to be wrong (l+ being the spontaneous release rate).We agree that our conclusion that "the spontaneous release rate can be modulated independently from the energy barrier" is strictly speaking not correct. All fusion events occur by overcoming the fusion barrier, and increasing spontaneous release requires always that the energy barrier is reduced, at least locally and temporarily in individual synapses. What we want to argue here is that the inhibition of spontaneous release by Syt1 is not caused by an increase in the energy barrier in the presence of Syt1, as assessed with HS. We use more precise wording to describe this observation in the result section. Based on this finding we conclude indeed that the assumption in the allosteric model  that l+ represents the spontaneous release rate constant does not hold for hippocampal autapses, and that other mechanisms also contribute the mEPSC frequency (Discussion section). In our previous paper  we came to the same conclusions based on a different data set. In our answer to point 5 below and in the Discussion section we further elaborate on possible mechanisms that contribute to spontaneous release.Can the authors please indicate on Figure 2H (and other figures) when HS is applied? It is not clear if there is an additional delay in the KO or the traces are simply shifted.To avoid unnecessary empty space in the figures, the traces are plotted on top of each other and indeed shifted. There is no additional delay in the KO. We have quantified the delay with respect to the onset, as well as other kinetic properties of the traces , for all HS responses and added these data in supplemental figures to the original figures. In all plotted traces HS is applied at the start of the displayed trace for 7s (until the onset of the decay back to base-line in the traces). We tried to indicate the application interval in the graphs but felt that this did not added to the clarity of the figures. Instead we now explicitly mention in the figure captions when HS was applied.Figure 2—figure supplement 1 top row is a repeat of Figure 2A,B,C.These panels were left in Figure 2—figure supplement 1 by mistake and have been removed.5) The subtitle "Spontaneous release rates are partially regulated by energy barrier independent factors" again does not make sense. Do the authors mean spontaneous release may be due to a different pathway with its own barrier?To avoid spontaneous Ca fluctuations leading to spontaneous fusion (a possible explanation), couldn't the authors repeat the experiments at 0 mM external calcium?We agree that this statement is factually not correct (as discussed above), and changed to subsection “Changes in spontaneous release do not directly correspond to changes in the energy barrier as assessed with hypertonic sucrose”. As described in our previous paper , and now more extensively explained in the Discussion section, we suggest that stochastic calcium fluctuations could contribute to spontaneous release by briefly activating Ca sensors locally in individual synapses. In this scenario, changes in the frequency of Ca fluctuations or in the sensitivity of the release machinery to these fluctuations, e.g. by unclamping a high-affinity Ca sensor, would also change the spontaneous fusion rate, but in an additive manner, not in a multiplicative manner as l+ would do. These additive changes in release rate would be negligible compared to release rates induced by sucrose and not translate to changes in the fusion barrier.Our sucrose experiments with the 9Pro mutant were performed in 0 mM external calcium and in the presence of 20µM BAPTA-AM, to avoid spontaneous Ca fluctuations, as also suggested by the reviewer. We discuss these results and possible interpretations now in more detail in the Discussion section. Under these conditions spontaneous release was reduced in WT glutamatergic autapses, but to a lesser extent than reported for GABAergic neurons it seemed , although we did not do a direct comparison. Also, spontaneous release remained significantly higher in the 9Pro mutant compared to WT, under these conditions. This might suggest that BAPTA loading was insufficient to block all spontaneous events. We also include an alternative explanation, as suggested by this reviewer and reviewer 2, that spontaneous release occurs through a separate pathway. This opens the possibility that effects on the fusion barrier of genetic manipulations that increase spontaneous release could be missed with our sucrose assay when only a small subset of vesicles is affected, or when these vesicles are somehow less sensitive to our HS assay (Discussion section).6) Discussion section: "Syt1 does not inhibit spontaneous release by increasing the fusion energy barrier" it does, at 0 mM HS. Also see the comments above.This relates to point 4 and 5 discussed above. In agreement with our answers to these points we have adapted the title to "Syt1 does not inhibit spontaneous release by increasing the fusion energy barrier as assessed with hypertonic sucrose".Since Syt1's two roles on supressing spontaneous release and promoting evoked release can be dissociated with the 9Pro mutant, perhaps the authors should use the 9Pro mutant as their reference, not the WT. It would be interesting to see a replot of data vs 9Pro.We agree that the 9Pro mutant is of important value to study the inhibitory role of Syt1 separately from its activating role. This is why we included it in our study to investigate the relation between changes in spontaneous release and changes in the fusion barrier. For research questions concerning the activation role we think the unmutated Syt1 WT is a better control. Moreover, we always make sure for all our experiments that cell cultures we use for experimental and control groups are from the same preparation (KO-rescue experiments) or littermate pups from the same week (KO vs WT experiments). Therefore, we cannot replot our data using the 9Pro data from different experiments as a control, and many new experiments would be required, which is not feasible given the current COVID situation.7) Discussion section: There is some vague discussion about electrostatics, membrane bending and effects of curvature that might affect the energy barrier for fusion. Is the D232N mutant able to penetrate bilayers in response to calcium binding to C2? Or is it constitutively inserted into the membrane?Biochemical properties of the Syt1 D232N mutant were studied in . The ability to penetrate bilayers, either in response to Ca or constitutively, was not directly tested. However, no difference was found in phospholipid binding at different Ca-concentrations between Syt1 WT and D232N. In hindsight, this is not in line with one of our suggestions in the discussion that increased membrane interactions of this mutant would reduce the fusion barrier by expelling interlamellar water, which was therefore left out. This also clarifies the discussion.8) Subsection “Fusion energy barrier model for STP”, "Fusion energy barrier model for STP""multiplication of membrane bound Syt1 and Syt7 corresponds to the facilitation produced by Syt7". Syt1*Syt7=facilitation?This refers to the observation in  that the amount membrane bound Syt1 multiplied with the amount of membrane bound Syt7, when taken as a proxy for sensor activation, can account for the facilitation observed in the same synapses. This is in line with the energy barrier model for STP, which predicts that in a dual sensor model additive effects of different sensors on the fusion barrier lead to multiplicative effects on the fusion rate. We have clarified this in the Discussion section.Reviewer 2:1) The authors investigated the relationship between evoked release and changes in the fusion energy barrier in Syt1KO cultures overexpressing the SytD232N mutant to find a convincing correlation between the increase in evoked release and the lowering of the energetic barrier. They had more difficulties to demonstrate a similar straightforward relationship with the Syt1 4W mutant, because the decrease in the energetic barrier is only detected for low HS concentrations (250mM), perhaps reflecting that the readout of the approach to measure the fusion energy barrier is more complex than initially envisioned. I think it would be helpful and informative to state those limitations which, however, do not invalidate the approach.This is a valid point. We have addressed the limitations of our HS method now more explicitly in the discussion. We restructured our discussion on the possible interaction between the mechanisms by which HS and Syt1 reduce the fusion energy barrier, as an explanation for why changes in the energy barrier are not detected beyond 250mM for the Syt1 mutants . We also address more specifically why the enhanced hydrophobicity of Syt1 4W could have opposite effects at higher sucrose concentrations (Discussion section). Furthermore, also in response to point 5 of reviewer 1 and point 2 of this reviewer, we discuss how our method is insensitive to possible differential effects on evoked and spontaneous release if these are governed by different pathways/vesicle pools (Discussion section).2+ solution went too much in the limit in my opinion and it should require experimental demonstration or at least to be well documented in the literature. I wonder if such hypothetical Ca2+ fluctuations could be abolished using higher BAPTA concentrations. On the other hand, it could be that the changes in the fusion energy barrier in the context of spontaneous release are different (or not picked up) to the changes in the context of HS stimulation or different pool of vesicles are involved in each condition.2) They observed higher spontaneous release in Syt1KO and Syt1 9Pro expressing synapses which is difficult to conciliate with their measurements indicating that the fusion energy barrier is unchanged. The explanation of rapid local fluctuations resistant to BAPTA in 0mM CaThis is a fair point, similar to point 5 of reviewer 1. Since other studies were successful in reducing spontaneous release by 95% using 10uM BAPTA-AM and 0mM Ca, we considered our explanation of local Ca fluctuations being BAPTA resistant less likely and took this out of the discussion. We now consider the possibility that our BAPTA loading was not sufficient to block all spontaneous events (Discussion section). We also included an alternative explanation, as suggested by this reviewer and reviewer 1, that spontaneous release occurs through a separate pathway. This opens the possibility that effects on the fusion barrier of genetic manipulations that increase spontaneous release could be missed with our sucrose assay when only a small subset of vesicles is affected, or when these vesicles are somehow less sensitive to our HS assay (Discussion section).3) To interrogate the involvement of Syt1 in PTP makes sense in light of a previous publication by Verhage's group showing PKC-dependent phosphorylation of Syt1 in short term plasticity. The part of the study devoted to the analysis to the fusion energy barrier involvement in PTP is the most comprehensive, relevant and convincing part of the study in which conclusions are best supported by the results. It is very interesting and i opens new perspectives to investigate potential PTP mediators and its role in the modulation of the fusion energy barrier in future studies.We thank the reviewer for these kind remarks and agree that our findings could shed new light on mechanisms that drive STP.Reviewer 3:1) Although the model is interesting, it is difficult to evaluate the values such as k2, max and δ E calculated from the model. It may be nice to show raw data such as time to peak, delay, integral of EPSCs and its dependence on sucrose concentration. What I mean here is that the overall results should be verified from experimental results, independent of modelling.We think we might have created some confusion due to the original layout of the paper, where we first presented the dual sensor model and then the data. The dual sensor model we present in this paper is not the model we used to fit HS responses. Instead we used a simpler release model, without sensors, in our fitting method that we previously developed . To avoid this confusion, and also in response to point 2 of reviewer 1, we changed the order of the manuscript, and first present the data before presenting the dual sensor model. In our previous paper , to which this study is follow up, we have put a lot of effort to optimize and validate our fitting method, which we feel has two advantages over other analysis methods. Firstly, it quantifies parameters such as RRP size, priming rate constant, fusion rate constant and energy barrier changes, which give direct information about (changes in) the release process. Secondly, it allows for more accurate quantification of release parameters: RRP is determined more precisely, due to a more realistic correction for vesicle replenishment, and arbitrary choices about the onset of the HS response, introducing additional variation in parameters such as “time-to-peak” and “delay” are circumvented. We have emphasized these advantages in the Materials and methods section. For these reasons, we choose our fit method over other analysis methods, and felt that a presentation of more HS parameters would complicate the text further. Nevertheless, for completeness and comparison with other HS studies we included seven new supplementary figures, where we provide statistics of the HS integral , 10-90% rise-time, delay of HS onset (delay of the onset of the HS induced current with respect the start of the HS stimulus), time-to-peak (time of the peak current with respect to the start of the HS stimulation), and time-to-peak minus delay.2) It is a bit difficult to understand why the RRP size looks smaller in Syt1 KO and Syt14W mutants but not in D232N (at least it looks like this from representative traces). The authors need some arguments. This may affect calculation of release probability.We did not find significant changes in RRP size for any of these mutants . The HS responses for 500mM and higher might seem smaller in the Syt1 KO and Syt1 4W, given the lower peaks in the typical examples, but these are the consequence of slower release rates at these concentrations . Slower release kinetics produce lower, but wider peaks, leaving the “area under the curve”, used for fitting of the RRP , the same. Interestingly, release kinetics in the D232N mutant is slightly higher at 500mM, producing higher and sharper peaks. We tried to find the most representative traces for our typical examples, but it has proven difficult to always exactly match all features in the same example. This is due to the variation in the data, as indicated by the individual data points in the boxplots, and the fact that we always select our example traces for all HS concentrations from the same cell.3) In some experiments, the sucrose concentration was raised to 1M, but in some experiments, it was raised to 500 mM. Why?The two experiments the reviewer is referring to were carried out at the beginning of this study and covered a broader range of sucrose concentrations to include data points beyond 500mM, since we did not know exactly what to expect. During the course of the study we found that stimulations of 750mM and 1M were more challenging than 500mM and less, while the assay was most sensitive at concentrations around 250mM. Therefore, to be efficient, we carried out most of our experiments with 250mM and 500mM sucrose during the rest of the study.4) The authors describe that spontaneous mEPSC rates are higher in Syt1 KO, but others did not see the effect, though they used the same preparation. The authors did not provide an explanation and it is puzzling.Our finding that spontaneous release is increased autapses in the absence of Syt1 was recently confirmed by an independent study form our institute , and a study from the Sorensen lab , and is in line with previous studies in network cultures. It remains enigmatic why the latter group and the Chapman lab previously found a discrepancy between the Syt1 KO phenotype in networks and autapses. It is difficult for us to speculate about a specific explanation without information about the experimental details in these studies. In the discussion we have included a reference to the new autapse studies and provide two general explanations for the difference in Syt1KO autapse phenotypes (Discussion section).5) Post-tetanic potentiation and the effects of phorbol esters are known to be regulated by PKC, Munc13/18, and are known to be independent of synaptotagmins. The authors need more explanation for novelty here.Although, the involvement of PKC, Munc13, and Munc18 in PTP and phorbol ester potentiation has been studied, it was not known whether Syt1 is required for the reduction in the energy barrier after PTP and phorbol ester stimulation. Our experiments are the first to show that modulation of the fusion barrier by PTP/phorbol esters can occur in the absence of Syt1, which is an important assumption of our energy barrier model for PTP. We now briefly explain the rationale of these experiments in the text (Results section).6) Phorbol esters are known to change the RRP size (Stevens and colleagues), but the authors have seen somewhat different (representative traces seem to indicate the increase in the RRP size). The authors need some explanation.Stevens and Sullivan, 1998 indeed reported increased RRP size after PDBu application, measured with 500mM sucrose. However, several later studies did not replicate this finding and reported unchanged pool sizes after phorbol ester application . We mention this now in the Discussion section. Responses to 500mM sucrose in the typical examples might appear larger after PDBU application due to faster release kinetics of release. This produces sharper and higher peaks, while the total charge transfer is not changed. Furthermore, results for individual cells showed some variation in their response to PDBU as depicted by the individual data points in Figure 6E. Since we always select our example traces for all conditions  from the same cell, some examples might deviate slightly from the average values.7) Figure 2—figure supplement1 top panel. The same figure as Figure 2?These panels were left in Figure 2—figure supplement 1 by mistake and have been removed.


This article reports the results from a feasibility study of an intervention (‘E-PLAYS’) aimed at supporting children who experience difficulties with social communication. E-PLAYS is based around a dyadic computer game, which aims to develop collaborative and communication skills. A pilot study found that when E-PLAYS was delivered by researchers, improvements on communication test scores and on collaborative behaviours were observed. The aim of this study was to ascertain the feasibility of running a full-scale trial to test the effectiveness of E-PLAYS in a National Health Service (NHS) setting with delivery by speech and language therapists and teaching assistants.The study was a two-arm feasibility cluster-randomised controlled trial of the E-PLAYS intervention with a treatment as usual control arm. Data relating to recruitment and retention, treatment fidelity, acceptability to participants, suitability of outcomes and feasibility of collecting health economic measures and of determining cost-effectiveness were collected.Speech and language therapists selected suitable children (ages 4–7 years old) from their caseload. E-PLAYS intervention  was then delivered by teaching assistants overseen by speech and language therapists. The control group received usual care. Assessments included blinded language measures and observations, non-blinded teacher-reported measures of peer relations and classroom behaviour and non-blinded parent-reported use of health and education resources and quality of life.Planned recruitment was for 70 children, in the event, 50 children were recruited which was sufficient for feasibility purposes. E-PLAYS was very highly rated by children, teaching assistants and speech and language therapists and treatment fidelity did not pose any issues. We were able to collect health economic data which suggests that E-PLAYS would be a low-cost intervention.Based on recruitment, retention and adherence rates and our outcome measures, a full-scale randomised controlled trial estimated appears feasible and warranted to assess the effectiveness of E-PLAYS for use by the NHS and schools.ISRCTN 14818949 (retrospectively registered). What uncertainties existed regarding the feasibility? The study aimed to establish the feasibility of delivering the E-PLAYS intervention in schools. An earlier pilot study by the authors had demonstrated that, when delivered by researchers, an earlier version of E-PLAYS produced significant gains in pragmatic language test scores and communication skills compared to a control group. The areas of uncertainty concerned delivery of E-PLAYS by speech and language therapists and teaching assistants and the impact that this may have on intervention fidelity and acceptability, also the ease of recruitment for a large-scale study.What are the key feasibility findings? Acceptability was very high and intervention fidelity was good. However, there were some challenges in relation to recruitment and retention. Recruitment was via speech and language therapists who examined their caseloads for children fitting the inclusion and exclusion criteria and then approached schools. One unintended consequence of this recruitment strategy was that the demographic profiles of the parents of the children recruited were largely White British, graduates and in fulltime employment. This was not reflective of the North East London area in which we were recruiting which was ethnically highly-diverse and where several boroughs experience high levels of poverty. However, this was consistent with previous research which has found that well-educated parents with higher socio-economic status are better able to access services such as speech and language therapy.What are the key implications of the feasibility findings for the main study? Based on the outcomes of our study, a full-scale trial appears feasible and warranted to assess the effectiveness of E-PLAYS for use by the NHS and schools. The low-cost, computer-based nature of E-PLAYS makes it highly suitable for national distribution. E-PLAYS was designed ultimately to be widely shared and we have used existing, easily updated technology which is available within the NHS and in primary schools. A vast number of commercially-available games are targeted at parents of children with autism spectrum disorders and other children with social communication impairments. Reviewers of the computer game literature have urgently called for large-scale studies to ensure these are based on a sound evidence-base. A possible recruitment alternative would be to approach schools directly without using speech and language services as an intermediary. It is likely that this would reach a wider demographic in terms of socio-economic status and ethnicity.Children with social communication impairments struggle to communicate effectively in social contexts; our E-PLAYS intervention aims to provide them with support. Difficulties with social communication can manifest themselves as an inability to maintain a topic of conversation or take turns appropriately, misunderstanding of non-literal language such as jokes, irony or sarcasm, failure to make inferences and repair communication breakdowns. Responding appropriately, interpreting others’ communications whilst at the same time understanding society’s norms and expectations constitutes pragmatic language skill , 2.linguistic component  of social communication.The terms ‘social communication’ and ‘pragmatic language’ are often used interchangeably. Recent systematic reviews of interventions for ‘social communication impairments’ or ‘pragmatic language impairments’ , 4 have Children with social communication impairments are highly heterogeneous. The Diagnostic and Statistical Manual of Mental Disorders  and thIt is important to remember, however, that many children will experience difficulties with social communication without ever receiving diagnoses and there are indications that this is particularly the case for children of low socio-economic status , 12, 13.Pragmatic language difficulties can have profound and long-lasting effects on emotional functioning and peer relations , 15. ChiSchools aim to facilitate children’s learning and train team-building skills for adulthood through collaborative activities –22. CollStudies which have observed peer collaborations involving children with social communication impairments –29 have creation and development of these activities. Technology can fill this gap and be used to regulate turn-taking, participation and enforce the rules  very helpful (SLT 12)Simply written. Easy with diagrams (TA4)When it was unclear, I looked at the pictures and it helped me (TA7)Step by step explanations also very easy to follow (TA10)Similarly, teaching assistants commented positively on the manual and especially on the illustrations:He (name of child receiving intervention) loved it… he really did love it. (TA4)The game is brilliant (TA8)It does make it fun that it is a game and it does help them and make them want todo it (TA9)There were few problems with running the game itself, which had been designed to be easy to load and use. During focus groups, teaching assistants were overwhelmingly enthusiastic about the game.I think they are more relaxed with peers than an adult (TA2)…he really listened well; he listened better to (partner name) than he does in class. (TA8)Teaching assistants were especially positive about the involvement of typically-developing partner children and stressed that both the partners and the focal children enjoyed the game.Children appeared to like all aspects of the game, particularly finding the ‘hidden treasures’. Six of the nine children interviewed rated the game as ‘good’, ‘really good’ or ‘brilliant’. Teaching assistants explained that one of the children who rated the game as ‘not very good’ was upset at having to finish (because of school timetabling) the game sooner than they wanted and the two others were ‘having a bad day’ but did in general at other times enjoy E-PLAYS.A further indication of the acceptability and popularity of E-PLAYS is that after completion of data collection, control group schools were offered the use of E-PLAYS. All control group schools took up this offer; furthermore, many of the intervention group schools asked to continue using E-PLAYS, with the result that we extended the licence to all participating schools until December 2019. In total, nine of the 14 participating schools (2 of 6 in the intervention and 7 of 8 in the control group) were still using E-PLAYS 12 months after study completion.During observations of the teaching assistants, we found that all were able to use the software and set up the game and all were able to keep the children on task. Less positively, only three of the observed teaching assistants followed manual recommendations to pause to give the children time to respond to questions and remind children of the collaborative nature of the task. Almost all teaching assistants tended to intervene with the children more than recommended instead of allowing the children to explore the game.The unanticipated delays in recruitment had a detrimental, knock-on effect on intervention delivery. E-PLAYS is a 12-session intervention with one (recommended) session per week. Due to the recruitment delays, many schools were left with only the end of the summer term in which to deliver 12 sessions of E-PLAYS and this was often less than 12 weeks. Schools were therefore asked to deliver as many sessions as was reasonably practicable. The mean number of sessions received by the children in the intervention group was 6.2 (SD 4.3). Five children randomised to the intervention group did not receive any E-PLAYS sessions. This was due to one school being unable to commence E-PLAYS for one child and another school (four children) withdrawing immediately following the baseline visit due to flooding at the school. Excluding the children from the school which withdrew, then the mean number of sessions received was 7.4 (SD 3.6). However, once schools were able to begin delivering E-PLAYS, the average frequency with which they delivered the E-PLAYS sessions was within the recommended range. The mean frequency of session completion was 1.1 sessions per week (range 0.3–2.6). Recommended duration for the sessions where the focal child played with the teaching assistant was 30 min ; actual mean duration was 25.50 (SD 11.6) min. Recommended duration for child and classmate peer partner sessions was 15 min ; actual mean duration was 28.72 (SD 12.47) min.Children in the intervention group generally received the early sessions with declining numbers receiving the later sessions; all 19 children received session 1 but only six children received session 12. Peer sessions required as much adult input (in terms of supervision) as did the teaching assistant only sessions so this was not a factor impacting delivery. To illustrate, the number of children receiving session 4 (teaching assistant only) = 17, session 5 (peer session) = 16, session 6 (peer session) = 12 and session 7 (teaching assistant only) = 11 and session 12 (peer session) = 6.From the point of view of participant burden and ease of administration, these outcome measures were generally found to be suitable. Importantly, the TPS, CELF-5 and CCT were blinded independent measures and we were able to maintain blinding. On two occasions school staff did inadvertently unblind research assistants despite being reminded. On these occasions, a different (blinded) research assistant was substituted to administer the outcome measure.Table Scores in general are typical of children with language disorders. Of the 49 children with TPS scores at baseline, 45 (91.8%) had scores below the 25th percentile of the standardisation sample used in the development of the TPS and 26 (53.1%) had scores below the 10th percentile. Similarly, 48 (96%) of the children in the sample had baseline scores on the CELF-5 Recalling Sentences subscale either at or below the mean score obtained by the standardisation sample, with 34 (68%) having scores more than one standard deviation below the standardisation sample mean score. This was also reflected in the teacher-completed instruments. Of the 41 children with a valid baseline score for the CCC-2 General Communication Composite, all of them had scores below the 20th percentile of the standardisation sample, with 37 (90.2%) of these having scores below the 10th percentile. Of the 42 children with a valid SDQ Prosocial scale score at baseline, 35 (83.3%) had scores below the mean score for British children aged 5–10 years  and 23 (54.8%) had scores more than one standard deviation below this mean.We successfully achieved inter-rater reliability between the research assistants (weighted kappa = 0.69). According to guidelines proposed by Landis and Koch (1977) , kappa vTable By comparison to the EQ-5D-Y (proxy-1), the PedsQL provides more in-depth information concerning emotional, social and school functioning, all dimensions that are likely to be affected by social communication impairments.Little NHS, Social Services and educational services use was reported across both trial arms either through the NHS or privately. Similarly, low levels of use were reported for home-based assistance, voluntary services or education and childcare services. The most widely used resource was special educational needs coordinator (SENCO) though the mean number of contacts was still very low: 2 (SD 3.5) in the comparator group and less than 1 (SD 0.5) in the E-PLAYS intervention group.From an NHS perspective, low input is required from health care professionals to deliver E-PLAYS. We anticipate, therefore, the cost to the NHS to be small meaning if the intervention was effective it would likely represent good value for money. Schools are not required to purchase or provide any additional resources to facilitate the delivery of E-PLAYS. There was some time required to set up the intervention initially in schools and attend the training. Teaching assistants spent around 10 min preparing for each session. Schools participating in the feasibility study were not required to pay for the E-PLAYS software; however, in a full-scale trial, it is possible that schools would incur an annual licencing fee of approximately £50 per school to cover software maintenance costs. As with the NHS, costs for schools would be low.1 30.0 to 80.6) and 47.8 (95% CI1 23.5 to 93.3), respectively. These suggest a reasonable estimate of the standard deviation of 7.0 . Conditional on the fixed effects, the intra-cluster correlation for SLT was essentially 0. In order to be conservative, we assume an intra-cluster correlation of 0.05 in the following calculation which, together with a (conservative) estimate of the mean cluster size of 5, gives an estimated design effect due to clustering of 1.20. The observed correlations between the baseline TPS score and the TPS scores at week 20 and week 40 were strong at 0.84 (95% CI2 0.63 to 0.89), respectively. We can account for a more conservative correlation of 0.6 in our sample size calculation by multiplying the sample size required for an unadjusted analysis by(1 − 0.62). Finally, we assume a conservative rate of attrition of 25%. These parameters mean that 356 children would be required for 90% power in a two-sided test of size 5%. Assuming an average of 5 children per SLT, approximately 71 NHS SLTs would need to be recruited and randomised.The variance of the available TPS scores at baseline, week 20 and week 40 was 51.3   found thShould E-PLAYS ultimately become nationally available and distributed, it is important that SLTs and teaching assistants can use it autonomously. For maximum fidelity, E-PLAYS was web-based and manualised with the content for each session specified step-by-step. The manual was generally well-liked and sessions were delivered without major problems thereby showing promise for good fidelity in a national roll-out. Delays tended to occur around the commencement of the intervention, chiefly due to staff shortages. However, once schools began with E-PLAYS, they were keen to continue.We were able to gain precise assessment of treatment delivery through automatic recording by the E-PLAYS software. This recording was supplemented with live observations of the teaching assistants delivering E-PLAYS. These two methods confirmed that delivery was acceptable in terms of number and length of sessions.Pragmatic language skills are notoriously difficult to measure; the problem being that they manifest themselves only during dynamic social interaction, thus rendering testing with standardised questionnaires largely unachievable . FurtherA key outcome of language and communication skill-learning is measuring subsequent use of the skill in contexts other than the one in which it was learned; skills can fail to generalise to novel contexts. Wieckowski and White’s (2017)  systematThe suitability of our measures was indicated by the good response and retention rates. Also, completion of the measures was excellent with very few missing items; almost all were analysable see Fig. . CompariAt present, neither of the instruments tested (ED-5D-Y proxy-1 and PedsQL) can be used to calculate QALYs that are required for a utility analysis, nor is there an alternative that can be used for this age range. However, EuroQol are currently looking to establish a value set for children and this may be available in the near future. On this basis, we would plan to include both the EQ-5D-Y proxy-1 and PedsQL in a full trial to ensure the best representation of health-related quality of life with the view of calculating QALYs if possible. Completion rates for EQ-5D proxy-1 and PedsQl were very similar to each other but PedsQL provides more information on social and emotional functioning.As the feasibility study was conducted with a small number of participants in a geographically small area, the low uptake of resources may reflect low levels of availability within this area. This is at odds with previous research; health economic evaluations have been called for in this field as it has been shown that healthcare costs are 36% higher for children with language disorders aged 4–5 years . In a fuThe low-cost, computer-based nature of E-PLAYS makes it highly suitable for national distribution. E-PLAYS was designed ultimately to be widely shared and we have used existing, easily updated technology which is available within the NHS and in primary schools. A vast number of commercially-available games are targeted at parents of children with autism spectrum disorders and other children with social communication impairments . RevieweParticular strengths of this study are that we have included independent, blinded measures and also a measure of generalisation of the skills taught. Through the use of technology, we were also able measure delivery of the intervention sessions precisely; other studies have been obliged to depend on less reliable methods such as teacher reports and diary methods.Fidelity was further assessed via direct live observation of teaching assistants delivering some of the E-PLAYS sessions. Teaching assistants knew that they were being observed and this may have impacted on their behaviour when delivering these particular sessions. Possible alternatives could involve video-recording every session and then selecting random sessions for fidelity assessment. This would have the advantage that teaching assistants would not know which sessions were to be observed and could not alter their behaviour accordingly. However, this would have considerable resource implications for a large-scale study requiring either that a research assistant attend every session to record it or that teaching assistants were asked to make all the recordings themselves. A further drawback would be that teaching assistants and children would only use E-PLAYS with constant observation, thereby possibly not reflecting natural usage of the intervention which could impact on outcomes. A limitation of our study concerns the demographic makeup of our sample. Demographics were taken at the 40-week data collection point thereby probably resulting in a lower response rate than had we collected this information at baseline. We acknowledge that this is a shortcoming of our study and that the value of this data is correspondingly reduced. Responses came primarily from white British participants with an above-average level of education; this did not reflect the highly diverse areas of London in which the study took place. However, our finding is consistent with those of Safer-Lichtenstein et al.’s systematic review (2019)  who repoBased on the outcomes of our study, a full-scale trial appears feasible and warranted to assess the effectiveness of E-PLAYS for use by the NHS and schools. Participants in this study  enjoyed using E-PLAYS; overall acceptability was high. Few concerns were reported regarding participation in a future trial. However, strategies to improve recruitment rate and retention and to recruit a more diverse sample would need to be adopted. Despite some limitations, this feasibility study prepares the ground for one of the first interventions for children with social communication impairments to harness the potential of computer technology. A full-scale trial would result in making a valuable resource available as well as advancing our knowledge of how to design technological interventions tailored for this group of children.Ethical approval was obtained on 4th December 2017 . The study opened to recruitment on 1st January 2018 and completed recruitment on 30th March 2018.


Leishmania parasites. However, the temporal dynamics of a positive LST under different epidemiologic scenarios and whether it relates to the protection against the recurrence of an overt disease are not fully documented.Leishmanin Skin Test (LST) is considered as a useful indicator of past infection by Leishmania (L.) major infection and disease and changes in LST reactivity. The two foci were both endemic for Cutaneous Leishmaniasis (CL) due to L. major, but contrasted in their history for this disease (ie: an old focus versus a recent focus).We report here on a population based prospective study conducted on 2686 individuals living in two foci located in Central Tunisia, to assess over a one-year epidemiologic season, the incidence of We found that most infections occurred in the new focus  with an incidence rate of CL lesions 2.4 times higher than in the old focus. Likewise, the rates of LST reactivity reversion and loss, in the new focus, were 99/1000[38–116] person-years and 14/1000[8–21] person-years, respectively. Loss of LST reactivity was not noticed in the old focus. Interestingly, the incidence rates of symptomatic infection did not differ significantly according to the LST status at enrolment (negative versus positive) between the combined foci and the new one.L. major cryptic infection. However, the instability of the LST positivity in new foci should be considered as an important confounder of the outcome of this infection when developing a research protocol for vaccine trial.Our findings confirm LST as a good tool for assessing  L major transmission. Our results demonstrate that people living in an old CL focus with intense and sustained exposure to L major, develop long-lasting immunity against the parasite resulting in protection against overt disease; This feature markedly differs from that observed in more recent CL focus where overt CL occurs regardless of LST status. Our study highlights that although LST is a useful epidemiological tool for monitoring leishmania transmission rate over time, its use as a surrogate marker of protection against recurrence of CL or as an inclusion criterion in vaccine trials, calls caution. Moreover, the intensity and past history of Leishmania transmission in the study site should be anticipated as important confounders of the outcome of infection and should be carefully addressed in the design of anti-Leishmania vaccine trials.This population-based cohort study is the first one conducted in the Old World which compares the incidence of Zoonotic Cutaneous Leishmaniasis (ZCL) to the temporal dynamics of LST in two foci contrasting in their past history for  Leishmania (L.) major with rodents as animal reservoir and occurs in Central and Southern Tunisia  / (PTR),rate of LST reversion:Number of persons with positive LST1 and negative LST2 / PTR,rate of LST reactivity loss:Number of persons with LST reversion in which there was at least a 5 mm decrease in the mean reaction size of LST2 compared to LST1/ PTR,incidence of Leishmaniasis cases (symptomatic infection):Number of persons without any active skin lesion at enrolment that later develop an active lesion diagnosed as leishmaniasis/ PTR.Person-time at risk (PTR) was calculated differently for infection and disease rates, because persons were regularly under surveillance for disease but assessed only two times for infection: at enrolment before the ZCL transmission season, and at the end of follow up, one year later just before the next transmission season. As for disease, a subject is eligible to contribute person-time to the study as long as that person is disease-free i.e. still remains at risk of developing the first lesion of interest. As for infection, the time contributed by each subject at risk was assumed to be one year.Rates of LST conversion, reversion, and loss of LST reactivity were measured only on the pool of persons who were tested twice (i.e. for LST1 and LST2). To contribute to person-time at risk, participants were required to be LST1 negative for LST conversion, and LST1 positive for reversion and for loss of LST reactivity.The LST reactivity at enrolment was considered to refine the estimation of the incidence of leishmaniasis cases according to LST result at baseline.Rates with 95% Confidence Interval (CI) were calculated for both “focus category” and “alternative focus category” and stratified by age groups and gender. The Relative Risk (RR) of developing a CL lesion was also calculated for each risk factor .The sampling design was taken into account in the analysis. Data were weighted to reduce the potential for bias due to nonresponse. Weights were calculated by means of the French INSEE software CALMAR 2 . Raking 2 test or fisher's exact test for variables expressed in frequencies. A 5% significance level was adopted (p≤0.05).Between-group comparisons were carried out using T-tests, analysis of variance (ANOVA), or Mann-Witney test for continuous variables, and χData were analyzed using STATA/IC 11.0 .A house to house survey recruited 2686 individuals with 2186 and 500 individuals living in the new and old focus respectively. LST1 was done and read in 1688 and 405 individuals in either focus, respectively. Most missing individuals were absent during household visits. Leishmania parasites but with salient differences between the two foci: LST1 positivity was 2.3 times greater in the old focus (99%) than in the new focus (43%); (p<10−3).The weighted LST1 results showed that 56% of the population under study have been exposed to Between the first and the second LST, 363 subjects were lost from follow-up: one case died, 115 declined receiving LST2, and 247 others were not present in the dwelling during LST2 visits (17.3%). Hence, only 1395 (82.6%) individuals from the new focus and 335 (82.7%) from the old one were re-tested before the start of the next transmission season . The LSTAmong the 1730 LST participants tested before and after one-year epidemiologic season, 218 were LST1 negative at baseline but converted to LST2 positive and 98% among them lived in the new focus.−3), but did not significantly differ according to gender.The incidence rate of infection (defined as LST conversion rate) was 295/1000 person-years at risk overall, and 290/1000 person-years in the new focus where most individuals LST negative at baseline were living . In the Of the 1114 individuals who were tested LST positive at enrolment, 61 reverted their LST to negative after one epidemiologic season with a mean decrease in the reaction size of 6.84 mm [6.26–7.34]. All of these 61 subjects were living in the new focus.Overall, the rate of LST reversion was 56/1000 person-years at risk. It was also higher in persons over 25 years old (p = 0.036) and in females compared to males (p = 0.029).We observed a loss of LST reactivity in 47 out of 1730 (2.7%) people who had LST1 and LST2, none of them had any comorbidity or used any immunosuppressive drug. As already noted, such reversion was not recorded in the old focus.−3). No statistically significant difference was found in rates of LST reactivity loss between males and females.The rate of LST reactivity loss was 8/1000 person-years in both foci combined. The higher rates were found in the age groups under 25-year old . If one considers the 1730 subjects who had both LST1 and LST2 measures, 50 developed patent CL lesion. The ten other CL cases occurred in the 363 participants (2.8%) who were tested for LST1 and had a clinical follow up, but were not tested for LST2. Only one of these ten incident cases occurred in the old focus.Leishmania amastigotes in all incident cases (100%). Culture in SLC medium was done in 43 cases out of 60. It led to the growth of promastigotes in 27 samples (62.8%), was negative in 11 (25.6%), and was contaminated in five (11.6%).Microscopic examination of smeared exudates from cutaneous sores, detected −3). About 50% of cases occurred in the range 5–15 years. Of the 60 cases of active CL, 25 (42%) were males and 35 (58%) females (p = NS).The incidence rate of leishmaniasis during the study period in the two foci combined was 29/1000 person-years at risk . The risImportantly, the incidence rates of symptomatic infection did not differ significantly according to the results of LST at enrolment (either negative or positive) and this was also true when combining data from both foci, as well as when considering only data from the new focus .L major (an old focus and a more recent one) to better understand over a one year follow up, the natural history and dynamics of infection and the predictive value of LST for overt disease occurrence. We show that the incidence of infection, as assessed by LST conversion rate, was high in the study area, but individuals were at higher risk of developing symptomatic leishmaniasis in the new (young) focus only. Likewise, the rate of LST reversion and reactivity loss was significantly higher in the new focus. Loss of LST reactivity was not noticed in the old focus. Most importantly, the occurrence of overt CL in both foci was irrelevant to the pre-existent LST status.This cohort study was conducted in two types of foci endemic for ZCL due to Leishmania infection (defined as the rate of LST conversion) was 295/1000 person-years, of whom 97% exhibited no symptoms as the incidence rate of overt CL was 29/1000 person-years. This figure highlights the fact that in the two study areas, the very large majority of individuals bitten by infected sandflies, will remain asymptomatic. Strictly speaking, we cannot exclude that our survey may have missed some cutaneous sores appearing beyond the end point of the study, either because the inoculation of the parasite by sandfly bite occurred lately during the transmission season, or because of a very long incubation period of the disease or because reactivation of latent parasites as occasionally reported [This study is the first one conducted in North Africa to prospectively estimate the incidence of CL infection at the populational level using a representative sample of exposed individuals. When the present study was implemented in 2009–2010, one focus was categorized as recent/young (i.e. LCZ cases have appeared in this area for less than 5 years), compared to the old focus where LCZ cases were reported since several decades earlier. Even if the status of that focus is no longer young today, the concentric spatial extension of ZCL in Central Tunisia since 1980 [reported . Indeed,reported , 14. Howreported , 16.Leishmania strains [Leishmania strains, the type of leishmanin used and the characteristics of the study population.Similar results were reported in a prospective study conducted in Colombia which investigated the dynamics of CL infection and clinical manifestations using LST conversion . Study a strains , 12. It  strains . Unfortu strains . One shoLeishmania parasites [Some studies have suggested that one single administration of LST, is capable by itself, to induce an immune response in the recipient or stimulate a pre-existing immune response to arasites , 19. A rWhen incidence rates were analysed according to age, there was a higher rate of LST conversion in older people over age 25, while the rate of LST reactivity loss and the risk of symptomatic infection were higher among younger ages. These interesting findings are consistent with many prior reports which indicate that older persons with a longer average duration of stay in endemic areas might have had longer exposure to infective sandfly bites which continuously boost their immune response, compared to younger people , 20, 21.ZCL control is difficult due to the complexity of transmission cycle and the costliness of effective strategies compared to the limited resources available in developing countries. Hence, vaccine development would be the most cost effective control strategy for CL. So far, attempts to develop such vaccine have failed for several reasons , 23. TheLeishmania was continuing for more than 25 years, whereas in the new focus, a significant LST reversion (to negative) was observed following one single epidemiologic season. Thus, persons living in an old focus who got infected long ago and were continuously exposed to the parasite, are more likely to display a long lasting positive LST reactivity [Our longitudinal study allowed us to evaluate the dynamics of LST reactivity under two different past history of transmission. The individual LST positivity appeared fairly stable over time in old endemic areas where transmission of activity  and LST activity  and as cLeishmania DNA nor revivable Leishmania parasite could be recovered from the cutaneous biopsy of the scar of healed cutaneous leishmaniasis due to L major [Leishmania in ZCL due to L major is a possible option, at least in some patients. In these cases particularly, the longevity of LST reactivity will rely on the continuous exposure of the patients to Leishmania antigens through repeated biting by infected sand flies as experienced in an old established focus with intense transmission. Possibly, a short history of exposure to infected sandfly bites as in a recent focus may lead to a more or less rapid decay and even a loss of LST reactivity. The lack of stability of the positive LST reaction in new (young) foci may be confusing while selecting candidates for vaccine trials.A rapid decay of a weak memory T cells response over the one year follow up long term memory responses are dependent on the persistence of antigenic stimulation by residual parasites. In the mouse model of leishmaniasis, once parasites are eliminated, mice lost their immunity indicating that persistent parasites are important in maintaining a long term cell-mediated immune response . Hence,  L major . Such rePhlebotomus papatasi sandfly bites, express higher IgG antibodies to sandfly saliva antigens and IFN-γ levels compared to those living in a new focus [As there is evidence that exposure to sandfly saliva may play a protective role –32, the ew focus .Several studies have looked at the relation between lesions characteristics  and Leishmania antigens. When comparing the LST reactivity with in vitro lymphoproliferative responses to Leishmania antigens we have observed occasional patients with negative LST (<5mm) with otherwise good lymphoproliferative responses [Several hypotheses may account for this unusual phenotype. One possibility is that these patients will develop a positive reaction later than 3 months after lesion appearance. Another possibility is that the cut off value of 5 mm that segregates between the negative and positive LST reactions may not be valid to all individuals. Some CL patients may develop a weak LST reaction of less than 5 mm possibly and this phenotype does not preclude development of a potent T cell immunity to esponses . SimilarL. major invaded the population and reached epidemic dimensions in 1993, more than 20 years after the discovery of this disease in Central Tunisia [The present study has yielded different findings than those obtained through previous cohort study performed by our group in Dhiba and Remada in Southern-East of Tunisia . These t Tunisia . This pr Tunisia . The disLeishmania antigens possibly triggered by a previous infection but it does not support that it is a marker of protection against disease recurrence. Hence LST positivity should not be considered as a surrogate marker of protective immunity [We found that a negative LST at time of enrolment was not predictive of a significant increased risk of developing overt CL. The risk of developing a new active lesion was almost similar in LST negative individuals supposed to be susceptible to infection and in LST positive ones supposed to be resistant with the former group displaying a higher increase in mean LST reaction size. Our data support that LST positivity might indicate a sensitisation to immunity , 40, 41.Leishmania (Viannia) braziliensis and a negative LST, had a 3.4 fold higher risk of relapse after successful treatment, compared to CL patients with a positive LST [In a study reported from Iran , 273 voltive LST . It was tive LST . The impLeishmania parasites is a result of a complex interplay between the host immune system and the inoculated parasites [Leishmania in humans, are still poorly understood. Our study stresses the importance of investigating the mechanisms of immunity that efficiently protect people against symptomatic infection, by electively targeting human populations living in old endemic foci where they are continuously exposed to parasites (re) inoculated by infective sandfly bites. This population may have developed on the long term stable mechanisms of resistance against the disease that are not yet fully developed in people living in more recent foci as evidenced by the temporal fluctuations in LST reactivity in such setting. This information will be crucial for the optimal design of anti-Leishmania vaccines trials.In humans, the outcome of infection by arasites , 43–46. S1 Checklist(DOC)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.