These analogously transmitted excitatory synaptic potentials augment presynaptic Ca++ influx upon arrival of an immediately following AP through a mechanism that involves a voltage-dependent priming of the Ca++ channels, leading to an increase in GABA release, without any modification in the presynaptic AP waveform or residual Ca++. Our work highlights the role of the axon in synaptic integration.Axons are equipped with the digital signaling capacity by which they generate and faithfully propagate action potentials (APs), and also with the analogue signaling capacity by which subthreshold activity in dendrites and soma is transmitted down the axon. Despite intense work, the extent and physiological role for subthreshold synaptic activity reaching the presynaptic boutons has remained elusive because of the technical limitation to record from them. To address this issue, we made simultaneous patch-clamp recordings from the presynaptic varicosities of cerebellar GABAergic interneurons together with their parent soma or postsynaptic target cells in young rat slices and/or primary cultures. Our  Synaptic integration stands at the core of neuronal signaling. During synaptic integration, neuronal information provided by the presynaptic neurons is processed, leading to a new encoding of signaling that takes into account both the activity of the presynaptic neurons and the intrinsic properties of the integrating neuron. In the classical view of synaptic integration, the tasks of various compartments of the neurons are sharply defined: the somatodendritic compartment gathers information from presynaptic neurons; the axon initial segment sets the threshold for action potential (AP) firing; and the axon transmits the new AP to presynaptic terminals. In recent years, however, several studies have uncovered substantial deviations from this simple picture , and today it is clear that individual neurons do not necessarily behave as the ‘platonic’ or ‘canonical’ neuron described by Coombs, Eccles, and Fatt in the middle 1950s .This conceptual evolution was in part due to the description of a significant electrical coupling between somatic and axonal compartments in the subthreshold voltage range . Although analogue signaling has been described in a variety of different preparations in mammals  the analogue coupling of somatodendritic synaptic activity to axon is substantial. By performing paired recordings from the presynaptic bouton and its postsynaptic target, we further show that this spontaneous synaptic activity coupled with an AP can affect transmitter release through a mechanism that involves the activation of voltage-dependent Ca++ channels (Cav), with no change in the presynaptic AP waveform or basal Ca++. Considered collectively with previous work, our findings highlight the importance of subthreshold coupling between neuronal compartments and further suggest that the process of synaptic integration is far more complex than classically envisaged, and that this is due to rich, largely unexplored signaling capabilities of the neuronal axon.Cerebellar MLIs allow to quantitatively study the extent and functional impact of analogue signaling because their presynaptic bouton can be directly recorded with the patch-clamp technique, as shown by , the orthodromically transmitted presynaptic APs induced by a somatic depolarization were observed in cell-attached and whole-cell voltage and current-clamp (CC) configurations: in cell-attached (B); an unclamped (or escaping) spike current in voltage-clamp , and a short train of APs recorded in CC .In order to assess directly the degree of coupling between the somatodendritic and the axonal compartments we performed paired whole-cell recordings from the soma and the intact presynaptic varicosities of cerebellar MLIs in an acute slice at near-physiological temperature (34°C). To do so we first patched the somatic compartment with an intracellular solution (IS) containing the fluorescent dye Alexa 594; after waiting for a few minutes for the dye diffusion we went on to patch a presynaptic varicosity with a pipette containing the same IS without Alexa dye. In the example shown in Figure 1B, CTaking advantage of the cell-attached recording configuration presented above, which offers the possibility to characterize the presynaptic AP in unperturbed conditions , we atten = 29 events, 50-s recording time, with an average coupling ratio  of 0.6 ± 0.07), have a longer risetime  and always appear later than the somatically recorded EPSPs . The fact that the final phase of decay, τ2, of the somatic and the presynaptic bouton events is the same, is also in accordance with the prediction by cable models: the decay cannot be slower than the membrane time constant of the cell  in paired somato-presynaptic bouton recordings. ed EPSPs . All of the cell .In the second type of experiment, we performed spot laser photolysis of MNI-glutamate . Laser pλ, of this distance-dependent relationship is 181 ± 14 µm . Considering that the total length of the MLI axon at PN14 is around 300 µm  depolarizing pulses can affect release. We thus wondered whether the analogically traveling spontaneous synaptic activity lasting only tens of ms see  could af++. By controlling the gating of presynaptic voltage-gated Ca++ channels (Cav), the duration of the presynaptic AP has a strong impact on the amount of released neurotransmitter (Vm) at the presynaptic varicosity would be 14 mV higher upon the 20 mV somatic depolarization, which is expected to decrease the driving force for sodium ions by about 10%. Altogether, these experiments indicate that the change in synaptic efficacy elicited by short, EPSP-like subthreshold depolarization prior to an AP does not involve any change in the presynaptic AP width.Previous studies in MLIs using longer , independently of whether the recording was performed from presynaptic varicosities located in the primary axon or axonal collaterals. The results presented above  and 4 in++pre) is induced that triggers a PSC  was triggered by a square depolarization (to 30 mV) preceded by 20 ms depolarizations to different voltages (−90 to −40 mV). ++pre (and the subsequent PSC) is highly dependent on the subthreshold voltage before the suprathreshold pulse: the more depolarized is the presynaptic Vm, the larger are the ICa++pre and subsequent release. A complete statistical analysis of the responses induced by the different pre-pulses is shown in ++pre and PSC) obtained with the different pre-pulses were compared between each other. Again, there is no evidence of any activation of the ICa++pre during the subthreshold depolarization. When a 3-ms interval is inserted between the subthreshold and suprathreshold pulses, the preceding subthreshold voltage levels do not have any influence on the ICa++pre and transmission . When the same protocol was applied to the granule cell terminal, there was no change in either of the two amplitudes  upon depolarizing pulses. When the presynaptic bouton is voltage-clamped to 0 mV for various durations, an inward ICa++pre develops with an activation time constant of around 3.0 ms and little inactivation (n = 15). On the other hand, as shown in ++pre starts to develop at Vm that are more depolarized than −40 mV, so it is unlikely that small depolarizations like the ones induced by EPSPs ) was simply expressed as a sigmoid function using two parameters, VCav50  and α (the factor reflecting steepness of the voltage-dependent activation), like Hodgkin–Huxley equations . Fitting of the Ca++ current characterized by the I–V curve presented in Ca++ value as +60 mV, yielded the two values for the free parameters: VCav50 = −17 mV and α = 0.2.What is then the mechanism by which the analogue–digital coupling of EPSPs with an AP increases Cav possesses four repeats of structural assembly, each of them having six transmembrane segments including the voltage-sensing S4 domains and the pore-forming regions of the channel. The (non-conductive) closed state of the channel can assume four different conformations, ‘a’ to ‘d’, represented in Ps(act) (but see Ps(act). Thus, the probability of voltage-dependent steady activation of each voltage sensor, Ps(steady act), can be represented as the 0.25th power of PCav (steady op). From the voltage-dependent probability change of PCav (steady op)  (the magenta trace), ‘closed’ , and ‘primed’ states ranging from (b) to (d) corresponding to closed Cav with partially active states in some of the four voltage sensors. Evidently, these ‘primed’ states of Cav clearly increase even at substantially hyperpolarized potentials.The α1 subunit of Ca but see , and thav), which is dependent on the on (kon) and off (koff) rate constants  and 0.65 (/ms), respectively. Consistently with previous studies showing that MLI boutons have axonal GABAA receptors causing autoreceptor currents upon presynaptic activation  condition where the probability of the voltage sensors to be in the activated position is at its minimum. The disappearance of the effect by a 3- to 5-ms interval after the subthreshold depolarization . Indeed, the presynaptic AP waveform is a critical determinant of synaptic strength because it sets the opening probability and duration of Cav and hence of release probability. This has been shown in a variety of preparations like the giant brainstem auditory synapse, the Calyx of Held . In a recent work,  showed trequency  and somarequency , which irequency , is in c++ is slow . In this sense, it is interesting to note that the kinetics of the presynaptic ICa boutons  and ~2 m boutons . This sltion see , does noated see , the ICaulse see  might reefore AP .A autoreceptors are part of the same activity-dependent mechanisms that link the production and release of GABA and the establishment of functional GABAergic contacts in the same neurons . After decapitation, the cerebellum was quickly removed in an ice-cold extracellular solution (ES), the cerebellum taken out and sagittal cerebellar slices (202 µm width) cut with a Leica vibroslicer (VT1200S). The slices were kept in a recovering chamber at 34°C until use. The composition of the ES was, in mM: NaCl 115, KCl 2.5, NaH2PO4 1.3, NaHCO3 26, glucose 25, Na-pyruvate 5, CaCl2 2, MgCl2 1, pH 7.4 when bubbled with carbogen (95% O2 and 5% CO2). For animals aged 12–21 days the same aforementioned ES was used for the recordings. For animals aged above 21 days old a KGluconate-based ES was used for the dissection  was also added to the somatic recording pipette. With this IS the somatic pipettes had resistances of around 6 MΩ and the axonal ones 25 MΩ. To record the Ca++ currents  (2 mM) and TTX (tetrodotoxin) (200 nM) were added to the ES in order to block K+ and Na+ voltage-dependent conductances.Both the soma and axon of MLIs were recorded with the patch-clamp technique  either icurrents  and 6 a n = 16, calculated from the recordings shown in ++pre onset kinetics (the time constant of the rising phase). This analysis, presented in ++pre kinetics.The average series resistance (Rs) in presynaptic bouton recordings was 82 ± 31 MΩ  or Narishige (PP-83) vertical pullers. For experiments in the slice, we first recorded from the soma with the Alexa 594 containing IS. After a 5- to 10-min waiting time the fluorescence illumination was turned on in order to identify the axon and a suitable, superficial varicosity. In order to patch the axon a second pipette containing the IS without Alexa was used. The axon was patched by looking at the image created by the camera with both the bright field and fluorescent lights on . PictureRecordings in slices were done in an upright Olympus BX51W equipped with a ×60, 1.0 numerical aperture objective (NA). Experiments were done at near-physiological temperature (≈34°C) with a Peltier system (Luigs & Neumann). Electrophysiological recordings from primary cultures were done in an inverted Olympus IX71 microscope equipped with a ×40 objective (NA 0.95). For experiments in slices, epifluorescence excitation was by light-emitting diode (LED) controlled by an OptoLED system (Cairn Research) at 572-/35 nm excitation and at 630/60 nm emission. Filters and dichroic were from Chroma Corporation . For experiments in cultures epifluorescence was by an LED light  at 450-/40 nm excitation and 510-/50 nm emission; fluorescent images were taken with Andor cameras (EMCCD Andor Ixon or a sCMOS Zyla 4.2).Salts were either from Sigma-Aldrich or Nacalai Tesque (Japan).l-glutamate; Tocris Biotechne, UK) with a 405-nm laser  following well-established procedures  and −53 mV (2nd EPSP) and the duration of the depolarization (time between the onset of the EPSP and the onset of the AP) is 40 ms. In the experiments where the axonal AP waveform was used , and the selection of each event was visually confirmed before subsequent analysis. The EPSPs' CR represents the ratio of the peak axonal and peak somatic depolarizations.EPSPs were detected with a threshold detection algorithm implemented by Dr. Taro Ishikawa as IgorPro extensions  at a given membrane potential Vm can be expressed by the following equation,The normalized steady CaPCav (steady op) is the steady probability of Ca++ channel opening at a certain Vm, and ECa++ is the equilibrium potential for Ca++.where PCav (steady op) was simply expressed using two parameters, VCav50 and α, the membrane potential for the half-maximal Cav opening and the factor reflecting the voltage sensitivity of channel activation, respectively:v channels contain four repeats of structural assembly, each of which has a voltage-sensing S4 domain. In order to study the voltage-dependent state change of each voltage sensor, we simply assumed that the four sensors are independently activated by voltage with an identical probability, Ps(act), and that the channel conducts only when the four voltage sensors are simultaneously active. Thus, the probability of the four sensors being in their active position simultaneously can be represented as the 4th power of Ps(act). From Ps(steady act), can be represented as the 0.25th power of PCav(stady op), as follows:Cav),The voltage-dependent state change between active and non-active states of an individual sensor takes place as defined by the velocity  and the voltage-dependent probability of the sensor state, that is, v = kon·Ps(steady act) (if Ps(steady act) > Ps(act)), or v = koff·(1 − Ps(steady act)) (if Ps(steady act) < Ps(act)).where a’ to ‘d’ (v (P(a), P(b), P(c), and P(d)) were calculated at each time point based on the Ps(act) as follows:According to the abovementioned assumptions, the (non-conductive) closed state of a single channel can assume four different conformations, ‘’ to ‘d’ , bottom:t) of 0.01–0.02 ms (as the sampling rate used in the experiments) using the Python programming language. The dt value was set so as to minimize the calculation error. The Python scripts used and the calculated data are available as Supplementary files online.The above equations were numerically solved by the Euler method with a time step , α = 0.2,kon = 0.55 (/ms), and koff = 0.65 (/ms), respectively, which were used for the rest of the simulations.Based on the assumption that the ECa–V curve  and the on pulse  yielded Data are presented as mean ± SD unless otherwise stated. Statistical significance was tested with the Wilcoxon signed-rank test for paired and the Wilcoxon–Mann–Whitney test for non-paired data. The difference between groups was considered significant when p < 0.05; when significant, the exact p value is indicated in each figure. In  This paper shows compelling experimental evidence for a novel mechanism for analog transmission of synaptic information from dendrites to distal nerve terminals in CNS interneurons. Brief subthreshold depolarizations caused by small EPSPs in dendrites are capable of activating the voltage sensors of presynaptic Ca2+ channels in nerve terminals, thus leaving them in a transient "primed" state. A subsequent suprathreshold action potential can then open these Ca2+ channels more efficiently leading to transmitter release facilitation. The effect decays within a few milliseconds and it is not dependent on residual Ca2+ levels or changes in presynaptic action potential waveform. public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Gary Westbrook as the Senior Editor. The reviewers have opted to remain anonymous. The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Thank you for submitting your article "Analogue signaling of somato-dendritic synaptic activity to axon enhances GABA release in young cerebellar molecular layer interneurons" for consideration by Essential revisions:No new experiments are required but more analysis and modeling is recommended to support the conclusions of the paper. Please address the following two items:1) Provide more detail on Rs values and the quality of the voltage clamp of Ca currents. For example, provide a plot of Ca current peak amplitudes (or current charge) against Rs to determine if these are correlated or not. Summarize this analysis in the methods or as a new supplementary figure.2) Further modeling  should be attempted to demonstrate the plausibility of the Ca channel gating scheme as an explanation for the facilitation of the release. Provide further evidence that the short depolarizations can indeed drive the several closed states of the Ca channel to an open state. Otherwise, the conclusions need to be toned down and perhaps Figure 6 should be removed if the authors cannot provide it with further modeling support.Reviewer #1 (Recommendations for the authors):Based on the outstanding technical quality, the convincing conclusions, and the novel and innovative proposed mechanism, we strongly support publication of the manuscript. However, we have the following comments:Time-dependent modeling of calcium channel gating should be provided to demonstrate that the proposed mechanism can indeed explain the experimental data. We would suggest to analyze the gating of calcium channels during APs (1.) with pre-APs, (2.) without pre-APs, and (3.) with pre-APs with a 3-ms-gap.Reviewer #2 (Recommendations for the authors):The following points may be considered when revising the manuscript.1) The time resolution of the traces is not so fast in Figure 2. It will be good to show sub-threshold responses in a ms time scale, so that one can see the delay and attenuation by eye.2) It is a bit pity that most critical experiments  have been done in culture. However, it is technically demanding and clamp is difficult in slices. The authors should explain more explicitly why these sets of experiments are done in culture and others in slices.3) The authors cannot exclude the two possibilities, perhaps. (a) Ca dependent facilitation of Ca channels. They can be relatively short-lived, and sensitive only to very concentration of Ca buffers (expression of high endogenous Ca buffers). (b) Some sort of voltage clamp problem. The authors assume perfect clamp at the terminal. Maybe activation, deactivation kinetics of Ca currents, and the clamp speed  can be reanalyzed from the existing data.Reviewer #3 (Recommendations for the authors):1) I don't think the term "analog-digital coupling" is very self-explanatory. Please consider finding a better description for example "analog and AP coding of axons" or "analog coding via subthreshold somatic potentials" etc.2) The author frequently use the qualifiers "axon" or "axonal" or even "axonal presynaptic terminal" when in fact referring to presynaptic compartments, presynaptic ICa(V) or presynaptic AP waveform. The authors never directly recorded from axons which likely have very different properties than boutons/terminals. Please change your terminology for clarification, i.e. use 'presynaptic ICa(V)' and 'presynaptic AP' etc.3) When recording in whole-cell configuration from presynaptic boutons, the authors use high resistance pipettes (18 or 25 MOhm) which results in very high access resistance (83 MOhm on average). This is to some degree unavoidable when recording from such tiny structures as MLI varicosities but it nevertheless impacts the recordings. Therefore, the whole issue deserves some more detailed description and discussion. The authors correctly emphasize that because of the 10fold smaller capacitance of varicosities compared to somata, adequate voltage clamp speed may still be achieved, especially because the recorded whole-terminal currents are generally quite small. What was the upper threshold in terms of Rs for accepting recordings? Was there any series compensation applied during voltage clamp and if so how much and what was the resulting uncompensated Rs? Current-clamp recordings are also expected to be heavily low-pass filtered unless 100% bridge compensation was used with the HEKA amplifiers. Perhaps such filtering can partially explain the large scatter of AP widths in Figure 1D?4) During the experiments shown in Figure 1C, both soma and boutons were held in voltage-clamp. But likely only the soma was depolarized. If so, please move the command-voltage waveform on top of the somatic recording and call the INa(V) recorded in the bouton an escaping sodium current as it escaped the presynaptic voltage clamp at Vhold.5) Please supply some more motivation for performing the Glu uncaging experiments illustrated in Figure 2E,F. What additional information is gained by the uncaging that could not be gained by simply injecting depolarizing current waveforms in to somata of recorded cells or by recordings such as those illustrated in Figure 2A-D. At present, the reader is a bit left alone with figuring out why Glu uncaging near dendrites was performed for studying axonal signaling of subthreshold somatic potentials to boutons.6) In Figure 4 F-H, no second AP was fired in response to two somatic depolarizations in very close succession (<2.2 ms in the example in panel F) presumably due to refractoriness. For ease of understanding, that refractory period should also show up in panels G and H as a blank space (i.e. missing numbers) and the time axis should start at 0 ms inter-stimulus interval.7) In Figure 5B, an average increase in presynaptic ICa(V) following small depolarizations of ~55% was calculated from 4 cells showing very little increase (~30%) and two other cells showing a large increase >100%. If the ICa(V) facilitation is due to an effect on channel gating, as the authors suggest, one would expect a very reproducible phenomenon in voltage-clamp recordings. This does not seem to be the case. Why? Different subtypes of presynaptic VGCCs? Different classes of MLI boutons?8) In Figure 6C , the authors measure variance in order to support their argument that VGCCs do not open during the small conditioning depolarization. The mean current does not reveal any increase. Why do the authors think that current variance would reveal channel opening which does however not show up in the mean current trace?9) In Figure 6E,F, the authors present model fits for the ICa(V) I-V curve and presumed state occupancies of a kinetic scheme depicted in panel D. It is not clear to me how the authors produced these traces. Are these simulations using a kinetic scheme such as that presented in Li, Bischofberger and Jonas (2007) J.Neurosci.. If so, the model parameter should be presented. If not, the authors should implement such kinetic model, fit it to their data and demonstrate that such scheme is able to reproduce the ICa(V) facilitation in response to small conditioning prepulses which they observe experimentally. That would strongly strengthen the authors' conclusion about the proposed mechanism of IC(V) facilitation. Without such kinetic simulations, it remains speculation.10) In the paragraph about "Ideas and speculation" that authors seem to suggest that the phenomenon they studied is vanishing with maturation. Even if that is the case, it would be more comforting for a reader to be given a 'more positive outlook'. The authors further suggest that strong somato-axonal coupling may play a role during maturation of GABAergic contacts but fail to indicate how that could work.11) Finally, I strongly advice referencing and discussion additional previous work, especially work on presynaptic calyx terminals which seems strangely completely absent from the manuscript. Strong candidates include (but are not limited to) for example:(i) regarding propagation of subthreshold depolarizations to terminalsParadiso and Wu (2008) Nature Neurosci. "Small voltage changes at nerve terminals travel up axons to affect action potential initiation" (i) regarding presynaptic ICa(V)Borst and Sakmann (1998) J.Physiol. "Calcium current during a single action potential in …"Borst and Sakmann (1998) J.Physiol. "Facilitation of presynaptic calcium currents in …"2+ Channels in …"Li, Bischofberger and Jonas (2007) J.Neurosci. "Differential Gating and Recruitment of P/Q-, N-, and R-Type ca2+ inﬂux and vesicle exocytosis at the mouse endbulb of Held …"Lin, Oleskevich and Taschenberger (2011) "Presynaptic ca(ii) regarding presynaptic AP waveform and its relationship to release probabilitySabatini and Regehr (1997) J.Neurosci. "Control of Neurotransmitter Release by Presynaptic Waveform at …"Borst and Sakmann (1999) PhilTransRSocLondB "Effect of changes in action potential shape on calcium currents and transmitter release in …"Taschenberger and von Gersdorff (2000) J.Neurosci. "Fine-Tuning an Auditory Synapse for Speed and Fidelity …"2+ Channels in …"Li, Bischofberger and Jonas (2007) J.Neurosci. "Differential Gating and Recruitment of P/Q-, N-, and R-Type caBoudkkazi, Fronzaroli-Molinieres and Debanne (2011) J.Physiol. "Presynaptic action potential waveform determines …" Essential revisions:No new experiments are required but more analysis and modeling is recommended to support the conclusions of the paper. Please address the following two items:1) Provide more detail on Rs values and the quality of the voltage clamp of Ca currents. For example, provide a plot of Ca current peak amplitudes (or current charge) against Rs to determine if these are correlated or not. Summarize this analysis in the methods or as a new supplementary figure.++ pre and the series resistance values.We have performed further analysis on the relationship between Rs and Ca currents, as suggested. Furthermore, we have extended the discussion and we have given more explanation in the Materials and Method section. Finally, we have incorporated a new Supplementary Figure  where we show that there is no correlation between the kinetics of the ICa2) Further modeling  should be attempted to demonstrate the plausibility of the Ca channel gating scheme as an explanation for the facilitation of the release. Provide further evidence that the short depolarizations can indeed drive the several closed states of the Ca channel to an open state. Otherwise, the conclusions need to be toned down and perhaps Figure 6 should be removed if the authors cannot provide it with further modeling support.We thank reviewers for pointing out an important simulation analysis. As suggested, we have performed time-dependent simulation of Ca channel activation upon an AP or a large square pulse depolarization, with or without coupling with various patterns of pre-pulses. The results are now summarized in a new Figure 6. Our simulation clearly supports our idea that brief subthreshold depolarization partially activate some of Cav voltage sensors, priming the opening of Cav upon the following suprathreshold depolarization. Importantly, such an effect disappears very quickly when an interval of several ms is given between the pre-pulse and the AP (or large depolarization pulse).Reviewer #1 (Recommendations for the authors):Based on the outstanding technical quality, the convincing conclusions, and the novel and innovative proposed mechanism, we strongly support publication of the manuscript. However, we have the following comments:We appreciate the very supportive positive comments by the reviewer. We have responded to all the helpful comments provided by three reviewers, as described below.Time-dependent modeling of calcium channel gating should be provided to demonstrate that the proposed mechanism can indeed explain the experimental data. We would suggest to analyze the gating of calcium channels during APs (1) with pre-APs, (2) without pre-APs, and (3) with pre-APs with a 3-ms-gap.According to the constructive suggestion, we have performed simulation of Ca channel activation with time-dependent change upon an AP with or without pre-pulses, and the results are now shown in a new Figure 6. Our results support the idea that the subthreshold depolarization right before the AP tends to ‘prime’ the channels by bringing some of the voltage sensors to the activated state. This priming rapidly disappears in several milliseconds.Reviewer #2 (Recommendations for the authors):The following points may be considered when revising the manuscript.1) The time resolution of the traces is not so fast in Figure 2. It will be good to show sub-threshold responses in a ms time scale, so that one can see the delay and attenuation by eye.Following the reviewer’s recommendation, we have added a new panel in Figure 2D showing examples of individual, simultaneous somatic and axonal EPSPs in a ms time scale.2) It is a bit pity that most critical experiments  have been done in culture. However, it is technically demanding and clamp is difficult in slices. The authors should explain more explicitly why these sets of experiments are done in culture and others in slices.As it is mentioned in the text, the Ca current data shown in Figure 6A and B does include recordings from acute slices. Data were similar in culture and slice, so they are pooled together in the analysis. In the text, we have added more explanation to specifically mention this situation . Concerning the results presented in Figure 5, we tried hard to obtain paired recordings from a presynaptic varicosity and its postsynaptic partner in the slice. Unfortunately, however, we never succeeded in getting a stable basket cell bouton>Purkinje cell recording and thus turned to the primary culture model. We added a short sentence to specifically mention this .3) The authors cannot exclude the two possibilities, perhaps. (a) Ca dependent facilitation of Ca channels. They can be relatively short-lived, and sensitive only to very concentration of Ca buffers (expression of high endogenous Ca buffers). (b) Some sort of voltage clamp problem. The authors assume perfect clamp at the terminal. Maybe activation, deactivation kinetics of Ca currents, and the clamp speed  can be reanalyzed from the existing data.Thank you for pointing out our insufficient explanation. Regarding the possibility (a) of, that is, Ca-dependent Ca channel facilitation, here the subthreshold pre-pulse did not produce any Ca influx, but the Ca current upon the immediately following AP was nevertheless augmented. Thus, the predominant mechanism working here is different from Ca-dependent Ca current facilitation. Rather, our results may imply that the Ca current facilitation and the resultant synaptic facilitation observed during the repetitive high-frequency presynaptic AP firings might be partly mediated by the Ca channel ‘priming’ mechanism in addition to the Ca-mediated Ca current facilitation. We thank the reviewer for giving us the insightful comment to think about that possibility. As for the second possibility (b), the reviewer’s concern is reasonable. Therefore, we have added explanation about the potential concern arising from the incomplete voltage-clamp condition in the text (p24-25). Concerning the additional simulation, we have carefully analyzed the Ca current activation and de-activation to fit the biophysical model of Ca channels, and the model simulation nicely captures the essential experimental results, mainly the ‘priming’ effect of sub-threshold depolarization on Ca channels. Thus, now we believe that our data, with support of the new simulation results, demonstrate the mechanism of the analogue signaling involves a modulation of Ca channel states that affect release upon an AP arriving at the presynaptic terminal.Reviewer #3 (Recommendations for the authors):1) I don't think the term "analog-digital coupling" is very self-explanatory. Please consider finding a better description for example "analog and AP coding of axons" or "analog coding via subthreshold somatic potentials" etc.We are sorry that our explanation about the term “analog-digital coupling” was not sufficient in the previous manuscript. However, the term is widely used to unify the different mechanisms that have been described in the literature to highlight that the subthreshold somatodendritic activity transmitted to the axon can affect AP-dependent release. To make it clearer, following the reviewer’s comments, we have nevertheless added explanations in Introduction  and Discussion (p22) to clarify this point.2) The author frequently use the qualifiers "axon" or "axonal" or even "axonal presynaptic terminal" when in fact referring to presynaptic compartments, presynaptic ICa(V) or presynaptic AP waveform. The authors never directly recorded from axons which likely have very different properties than boutons/terminals. Please change your terminology for clarification, i.e. use 'presynaptic ICa(V)' and 'presynaptic AP' etc.The reviewer is right that we never recorded from the axon proper, sometimes also called main axonal trunk, but from presynaptic axonal boutons; both structures have certainly different properties, both anatomically and physiologically. We have changed the terminology all along the text in order to follow the reviewer’s suggestion.3) When recording in whole-cell configuration from presynaptic boutons, the authors use high resistance pipettes (18 or 25 MOhm) which results in very high access resistance (83 MOhm on average). This is to some degree unavoidable when recording from such tiny structures as MLI varicosities but it nevertheless impacts the recordings. Therefore, the whole issue deserves some more detailed description and discussion. The authors correctly emphasize that because of the 10fold smaller capacitance of varicosities compared to somata, adequate voltage clamp speed may still be achieved, especially because the recorded whole-terminal currents are generally quite small. What was the upper threshold in terms of Rs for accepting recordings? Was there any series compensation applied during voltage clamp and if so how much and what was the resulting uncompensated Rs? Current-clamp recordings are also expected to be heavily low-pass filtered unless 100% bridge compensation was used with the HEKA amplifiers. Perhaps such filtering can partially explain the large scatter of AP widths in Figure 1D?The quantification of the presynaptic AP width has been done exclusively from recordings in the cell-attached configuration  to avoid the errors associated with the pipette capacitance in small structures . The large scatter of presynaptic AP widths in the young age group has already been described by Rowan et al. and Begun et al. (with electrophysiology and voltage dye imaging) and is probably due to a lack of maturation of voltage-dependent conductances at this age range. With maturation the variability goes down, which is also an indication that the large scatter in the presynaptic AP width it is not an artifactual finding. However, we agree with the reviewer that the voltage-clamp speed and the associated space-clamp deserves more explanation. We have added a paragraph in the Discussion section (p24 and 25). The average, uncompensated series resistance value was 82 Mohm, with a SD of 31 Mohm. Rs values were compensated by 30%. The recordings from the boutons usually lasted less than 5 to 6 minutes, and the Rs values remained constant during these time. However, only the experiments in which the Rs values did not change more than 20% were used. These points have also been explained in the Methods .4) During the experiments shown in Figure 1C, both soma and boutons were held in voltage-clamp. But likely only the soma was depolarized. If so, please move the command-voltage waveform on top of the somatic recording and call the INa(V) recorded in the bouton an escaping sodium current as it escaped the presynaptic voltage clamp at Vhold.We have changed the sentence (p4) and figure, as suggested.5) Please supply some more motivation for performing the Glu uncaging experiments illustrated in Figure 2E,F. What additional information is gained by the uncaging that could not be gained by simply injecting depolarizing current waveforms in to somata of recorded cells or by recordings such as those illustrated in Figure 2A-D. At present, the reader is a bit left alone with figuring out why Glu uncaging near dendrites was performed for studying axonal signaling of subthreshold somatic potentials to boutons.Based on the results published in 2012 by Abrahamsson et al. (Neuron), who showed that in more mature MLIs the dendritic filtering of synaptic potentials is not negligible, we decided to perform glutamate uncaging because this is the only way to activate physiologically the dendritic postsynaptic receptors in situ and therefore to accurately quantify the distance-dependent reduction of dendritic EPSPs while they propagate down the dendrites and the axon. The results presented here in the younger age group indicate that the amount of dendritic filtering at this age range is negligible; however, this was not known beforehand. In addition, our data exhibited, more importantly, that such different sizes of EPSPs similarly reached the axonal presynaptic boutons. Following the reviewer’s suggestion, we have added to the manuscript sentences explaining the reasons to perform Glu uncaging experiments .6) In Figure 4 F-H, no second AP was fired in response to two somatic depolarizations in very close succession (<2.2 ms in the example in panel F) presumably due to refractoriness. For ease of understanding, that refractory period should also show up in panels G and H as a blank space (i.e. missing numbers) and the time axis should start at 0 ms inter-stimulus interval.We have changed the time scale of Figure 4G and H, according to the reviewer’s suggestion.7) In Figure 5B, an average increase in presynaptic ICa(V) following small depolarizations of ~55% was calculated from 4 cells showing very little increase (~30%) and two other cells showing a large increase >100%. If the ICa(V) facilitation is due to an effect on channel gating, as the authors suggest, one would expect a very reproducible phenomenon in voltage-clamp recordings. This does not seem to be the case. Why? Different subtypes of presynaptic VGCCs? Different classes of MLI boutons?As the reviewer correctly points out, the ICa++ and PSCs  measured in 2 of the cells show a dramatic increase when comparing the test (with a pre-pulse depolarization before AP) vs control conditions, while in 4 of the cells the increase is more subtle. Similar diversity was also observed at the pre-pulse-caused PSC augmentation in slice . We do not know the reason for such a heterogeneity and we can only speculate. As our new simulation results indicate, the slow kinetics of Ca channels is very critical for the ICa augmentation . Thus, even slight difference of the Ca channel modulation and/or subtypes may largely affect how the augmentation operates. As for the diverse effects, we have added explanation in the text .Previous work indicates that the amount of vesicles in the RRP is limited  and that the amount of postsynaptic receptor saturation is high . These 2 aspects set an upper limit to the increase in the PSC amplitude. However, we have recorded synapses where postsynaptic receptor saturation does not seem to be present . We tend to think that this may partly explain the heterogeneity observed in Figure 5C. Concerning the VGCC, their exact nature has only been studied in MLI presynaptic terminals with 2P calcium imaging, but not with electrophysiology. According to these studies, different types of VGCC contribute to release from these terminals ] but their exact role under different paradigms of stimulation and their distribution are not known.8) In Figure 6C , the authors measure variance in order to support their argument that VGCCs do not open during the small conditioning depolarization. The mean current does not reveal any increase. Why do the authors think that current variance would reveal channel opening which does however not show up in the mean current trace?As pointed out by the Reviewing editor (item 2) and the reviewers , this work lacks direct experimental evidence for the priming of the Ca channels. Our work proposes a new mechanism by which subthreshold depolarizations reaching the presynaptic terminals can affect release in MLIs. To test this idea, according to the reviewers’ recommendation, we have performed additional simulation, and observed such a ‘priming’ effect of Ca channels by sub-threshold depolarization . Thus, we have omitted the variance data which was shown in the previous version of manuscript, because it was used to indirectly support the idea of some ‘priming’ phenomenon just by emphasizing the lack of direct channel activation.9) In Figure 6E,F, the authors present model fits for the ICa(V) I-V curve and presumed state occupancies of a kinetic scheme depicted in panel D. It is not clear to me how the authors produced these traces. Are these simulations using a kinetic scheme such as that presented in Li, Bischofberger and Jonas (2007) J.Neurosci.. If so, the model parameter should be presented. If not, the authors should implement such kinetic model, fit it to their data and demonstrate that such scheme is able to reproduce the ICa(V) facilitation in response to small conditioning prepulses which they observe experimentally. That would strongly strengthen the authors' conclusion about the proposed mechanism of IC(V) facilitation. Without such kinetic simulations, it remains speculation.According to the constructive suggestion, we have performed simulation of Ca channel activation, and the results are now shown in new Figure 6. Our results support the idea that subthreshold depolarization tends to ‘prime’ the channels by putting some of voltage sensors in an activated state, which is rapidly gone in several milliseconds.10) In the paragraph about "Ideas and speculation" that authors seem to suggest that the phenomenon they studied is vanishing with maturation. Even if that is the case, it would be more comforting for a reader to be given a 'more positive outlook'. The authors further suggest that strong somato-axonal coupling may play a role during maturation of GABAergic contacts but fail to indicate how that could work.We have done experiments in the mature age group that show that the EPSP coupling is significantly reduced and we are currently performing experiments to understand the mechanism  of such a reduction. Nevertheless, we have followed the reviewer’s advice and have rewritten that section (p25-26) in order to give a “more positive outlook” in the “ideas and speculations” section, by highlighting the potential role in developmental maturation of GABAergic synapses.11) Finally, I strongly advice referencing and discussion additional previous work, especially work on presynaptic calyx terminals which seems strangely completely absent from the manuscript. Strong candidates include (but are not limited to) for example:We thank the reviewer for these suggestions and apologize for not making a thorough citation of appropriate literature. We have made an effort to cite important articles throughout the text and the discussion.


In September 2020, 15 861 SARS-CoV-2 case records failed to upload from the Second Generation Surveillance System (SGSS) to the Contact Tracing Advisory Service (CTAS) tool, delaying the contact tracing of these cases. This study used CTAS data to determine the impact of this delay on population health outcomes: transmission events, hospitalisations and mortality. Previously, a modelling study suggested a substantial impact.Observational study.England.Individuals testing positive for SARS-CoV-2 and their reported contacts.Secondary attack rates (SARs), hospitalisations and deaths among primary and secondary contacts were calculated, compared with all other concurrent, unaffected cases. Affected SGSS records were matched to CTAS records. Successive contacts and cases were identified and matched to hospital episode and mortality outcomes.Initiation of contact tracing was delayed by 3 days on average in the primary cases in the delay group (6 days) compared with the control group (3 days). This was associated with lower completion of contact tracing: 80% (95% CI: 79% to 81%) in delay group and 83% (95% CI: 83% to 84%) in control group. There was some evidence to suggest increased transmission to non-household contacts among those affected by the delay. The SAR for non-household contacts was higher among secondary contacts in the delay group than the control group . There did not appear to be a significant difference between the delay and control groups in the odds of hospitalisation (crude OR: 1.1 (95% CI: 0.9 to 1.2)) or death (crude OR: 0.7 (95% CI: 0.1 to 4.0)) among secondary contacts.Our analysis suggests that the delay in contact tracing had a limited impact on population health outcomes; however, contact tracing was not completed for all individuals, so some transmission events might not be captured. Shows empirical data on the health impact of an event leading to a delay in contact tracing so can test hypotheses generated by models of the potential impact of a delay in contact tracing.Estimates the extent of further transmission and odds of increased hospitalisation or mortality in up to the third generation of cases affected by the event.The event acts as a natural experiment to describe the possible impact of contact tracing, comparing a group affected by chance by delayed contact tracing to a control group who experienced no delay.Contact tracing was not completed for all individuals, so the study might not capture all affected contacts or transmissions.As of October 2023, there have been over 22 million cases and 200 000 deaths from COVID-19 reported in the UK.1The event did not affect the notification of test results to patients or affect the results of any specific testing sites, geographical areas or population groups. However, the delay in the upload of case records to CTAS resulted in a delay in the contact tracing of these cases, and therefore a potential delay in instructing their contacts to self-isolate, which could have resulted in further transmission of SARS-CoV-2, and potentially more COVID-19-related hospitalisations and deaths. We expect that health outcomes of the initial group of cases (primary cases) whose records failed to upload to CTAS would not have been affected, as they received their test results and advice to isolate in the usual timeframe, and transmission events from primary cases to their contacts (primary contacts who may become secondary cases) would have most likely occurred prior to the isolation of the case. However, these primary contacts may not have been aware that they were a close contact of a case or advised to isolate in a timely manner, which may have increased the risk of transmission to their contacts (secondary contacts who may become tertiary cases). The outcomes of secondary contacts are therefore the most likely to have been impacted by the delay in contact tracing. In addition, there could potentially be delays in secondary cases seeking healthcare support, increasing their risk of hospitalisation or mortality.A published preliminary model suggested that this event may have been associated with an increase of up to a third additional infections and 30–40% additional deaths.6In this study, we assess directly the health impact of the event, in terms of transmissions, hospitalisations and deaths, using data on affected and concurrent cases and their contacts.The aims of this study are to:Describe the nature of any contact tracing delay experienced by cases affected by the event.Assess the extent of further transmission, by calculating attack rates from primary cases to their contacts and from secondary cases to their contacts.Assess the impact of the delay on health outcomes of primary and secondary contacts, in terms of hospitalisations and deaths.The list of SGSS records affected by the event was provided by the UK Health Security Agency (UKHSA). SGSS contains demographic and diagnostic information from laboratory test reports for patients in England who test positive for SARS-CoV-2.910.1136/bmjopen-2022-064982.supp1Supplementary dataContact records were linked to the case who reported them and assigned to the same group (delay or control group) as their associated case. Secondary cases were defined as (1) individuals reported as a contact by a primary case and (2) having a contact event with the primary case between 2 and 14 days inclusive prior to the onset of symptoms of the secondary case (or test date if no symptom onset available). If the secondary case was a household contact of the primary case, the date of interaction was taken to be the date of symptom onset (or test date if no symptom onset available) of the primary case. Secondary cases may be exposed to more than one primary case; therefore, only one transmission event was chosen to link a secondary case to the primary case who most likely infected them based on the following hierarchy: (1) a household transmission event was prioritised above all other types of contact event (due to the higher risk of transmission in this setting); (2) where multiple events of the same priority occurred, the most recent exposure was selected as the transmission event. Individuals who had taken multiple SARS-CoV-2 tests generated a CTAS record for each positive test result. These were identified by matching on name, date of birth, NHS number and contact details. Only the record with the earliest test date was retained, as contact tracing should have been undertaken on receipt of the first positive result.Contact tracing records were linked to mortality data provided by UKHSA which describe death in people with a positive SARS-CoV-2 test. Matching was performed using NHS number. Two definitions of mortality consistent with SARS-CoV-2 surveillance were used: (1) death of a person with a laboratory-confirmed positive SARS-CoV-2 test and whose death was within 60 days of the first specimen date or more than 60 days after the first specimen date if COVID-19 is mentioned on the death certificate and (2) death of a person with a laboratory-confirmed positive SARS-CoV-2 test and whose death was within 28 days of the first positive specimen date.10Contact data sets were also linked to the UKHSA hospital-onset COVID-19 data set (extracted on 22 November 2021), which pulls from identifiable daily feeds of data on hospitalisations: Secondary Uses Services ; Emergency Care Data Set ; and COVID-19 testing data. The data set provides one hospital spell per case if they had a hospital admission or discharge in the 14-day period before or after their first positive SARS-CoV-2 test, including people already in hospital at the time of test. Matching was performed using NHS number, date of birth, name and sex.Analysis was performed in R V.1.3.1056. Investigators LF, LP, CT, AW and CC had access to the study data. Demographic information and contact tracing outcomes were described for the case and contact data sets. Time taken for contact tracing was estimated using different dates in the contact tracing process: dates of test, laboratory report, SGSS record creation, CTAS record creation and contact tracing completion . Time taTo understand the effect of the event on transmission, adjusted secondary attack rates (SARs) and adjusted ORs of infection with SARS-CoV-2 among primary and secondary contacts were estimated via postestimation from a logistic regression model. The predictor under investigation was whether the exposing case was in the delay or the control group. After review of crude SARs stratified by contact setting (household or non-household), effect modification of the predictor (delay or control group) by the contact setting (household or non-household) was allowed for in the model. The potential confounders included in the model were age group, sex, geographic region  of the contact and whether the contact completed contact tracing.To understand the effect of the event on health outcomes, ORs for experiencing hospitalisation or mortality among primary and secondary contacts were calculated using logistic regression with Firth penalisation, comparing individuals in the delay and control groups. When individuals had contact episodes with multiple cases, the earliest episode per individual was retained. A very small number of mortality events was reported; therefore, unadjusted, crude ORs are presented to avoid overfitting the regression model, and the covariate balance between the delay and control groups suggests any major confounding is unlikely. As a sensitivity analysis, health outcomes of contacts in delay group were compared with a subgroup of contacts in the control group who were contact traced rapidly, defined as within 3 days of the test date of the associated case.Patients or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research.Overall, 15 861 SGSS records were identified as affected by the event and 15 467 (98%) were matched to a CTAS record . FollowiFor primary cases in the delay group, it took on average 2 days longer for their records to be uploaded to CTAS, initiating contact tracing, after the laboratory report date of their positive test result . PrimaryThe proportion of cases for whom contact tracing was completed was slightly lower for primary cases in the delay group  than the control group , a difference of −3.5% (95% CI: −4.2% to −2.8%) . There wThere was no evidence of a difference between the delay and control groups in the overall adjusted SARs among primary or secondary contacts . HoweverCOVID-19 death was a rare outcome in both groups with no evidence of a difference in 28-day or 60-day mortality rates among primary or secondary contacts in the delay and control groups . Using tThe proportion of contacts admitted to hospital as inpatients within 14 days of their first SARS-CoV-2 test was almost identical in the delay and control groups, for primary contacts (delay 3.3% (95% CI: 3.1% to 3.5%), control 3.1% (95% CI: 3.0% to 3.2%)) and secondary contacts (delay 3.4% (95% CI: 3.0% to 3.9%), control 3.2% (95% CI: 3.0% to 3.5%)) . There wThe contact tracing of 15 861 SARS-CoV-2 cases affected by the event was delayed by an average of 3 days. There was evidence that the delay was associated with a slight decrease in the proportion of cases and contacts who completed contact tracing. Despite the delay, the mean and median number of overall contacts and non-household contacts made per case were similar in the delay and control groups, for primary, secondary and tertiary cases, and the median number of household contacts per case was near-identical.As expected, we observed a higher SAR from secondary to tertiary cases, for non-household contact events in the delay group. There was no evidence of a difference in the SARs or odds of infection for secondary to tertiary cases overall or for household contact events. We found no evidence of a difference in hospitalisation or mortality among primary or secondary contacts in the delay or control group using the standard definitions for COVID-19-related hospitalisation and mortality used for surveillance purposes in the UK.The event acted as a natural experiment whereby one group of individuals by chance experienced delayed contact tracing, enabling a comparison of their outcomes with concurrent, unaffected cases and contacts to assess the impact of contact tracing more generally. Having a single national system collating all test results and a single national contact tracing system facilitated the study. Through record linkage, we could identify successive generations of contacts and cases and describe key health outcomes associated with the delay in contact tracing; however, some outcomes were rare and it is possible that we did not have the power to detect a small difference in hospitalisation or mortality.Secondary transmission could only be estimated using contacts reported by cases who met the contact definition.It is important to note that there was an inherent lag in the initiation of contact tracing for all cases; therefore, the delay group was not compared with a perfect example of contact tracing, which may explain the lack of difference in observed health outcomes. To assess this, we conducted sensitivity analyses comparing the delay group to a subset of the control group who initiated contact tracing most quickly, but we still did not detect a difference in health outcomes and results were consistent with the main analysis. This could be partly because secondary and tertiary contacts in the delay group did not appear to experience a significant delay in initiating or completing tracing compared with the control group. Additionally, some cases had multiple CTAS records, for example, if they tested several times; we retained only the earliest episode. It is possible that some cases in the delay group reported subsequent positive tests soon after their first test which were not affected by the event . Therefore, these people might still have been contact traced in a timely manner.Finally, there is likely to be high uncertainty when tracing the complex network of contacts from the community transmission stage of the pandemic. The actual risk of infection among contacts is likely to vary significantly depending on degree of contact, which was not described. Some transmissions were likely yielded by superspreading events with a disproportionate impact on the pandemic, which were not captured in our comparison of SARs. Notably, primary cases were defined as being either affected by the event (delay group) or from the same time period (control group). Because the event occurred over a period of a small number of days, during which time transmission events occurred, primary cases generated some secondary and tertiary cases which were also classed as primary cases themselves and may have been directly affected by the event. Therefore, transmission chains may have been affected at different stages. Additionally, with regard to household transmission chains, it is worth noting that because many secondary cases were exposed to the virus in their household, their onward household attack rates could be lowered as their exposer would not become a case again within the time period studied.A previous modelling exercise using aggregate published data projected substantial adverse health outcomes, describing a drastic rise in SARS-CoV-2 infections and deaths in areas most affected by the event and estimating over 125 000 additional infections and 1500 additional COVID-19-related deaths.15 16Contacts of SARS-CoV-2 cases are at increased risk of infection, and this risk is higher among household contacts.There was no difference in the number of contact events. This may reflect that people have generally maintained low levels of contacts throughout the pandemic.Contact tracing is an effective tool to control transmission of infection.We did not find a significant impact on population health outcomes in individuals affected by a delay in contact tracing.


The long-term outcomes of patients with disorders of consciousness after traumatic brain injury (TBI) is unclear. We investigated the long-term outcomes over 20 years in patients who were in a persistent vegetative state (VS). We conducted a retrospective cohort study using a review of medical records and collected data by telephone and written interviews with patients and their families. We included patients who were treated for TBI at our hospital, between October 1996 and January 2003 and who were in a persistent VS, defined as a Disability Rating Scale (DRS) score of ≥22 at 1 month after TBI. The DRS was administered at 1 month, 6 months, 1 year, and then annually out to 20 years. We evaluated their clinical course until July 2021 with the DRS. We analyzed 35 patients in a persistent VS attributable to TBI. We were able to confirm the 20-year outcomes for 26 of the 35 patients (74%); at 20 years post-TBI, 19 (54%) patients were found to be deceased and 7 (20%) were alive. Over the 20-year study period, 23 of the 35 patients (65.7%) emerged from a persistent VS. Among the 35 patients in a persistent VS at 1 month post-TBI, 20 (57%) emerged from a persistent VS within 1 year, and 3 patients (8.6%) emerged from a persistent VS after more than a year after injury. DRS scores improved up to 9 years post-injury, whereas the change in DRS scores from 10 to 20 years post-injury was within ±1 point in all patients. We found that patients with persistent VS attributable to TBI may show improvement in functional disability up to 10 years post-injury. On the other hand, no substantial improvement in functional disability was observed after the 10th year. A vegetative state (VS) that lasts for more than 1 month post-TBI is called a persistent VS, and that which lasts for more than 1 year post-TBI is called a chronic VS, which used to be called permanent VS.8 However, long-term outcomes of patients with DoC ≥10 years after TBI has not been reported. We conducted this study to examine the hypothesis that functional recovery in patients with a persistent VS may continue beyond 10 years post-injury. If functional recovery continues beyond 10 years post-injury, this may suggest that continued functional assessment and care plan updates over the very long term may be beneficial to the patient. The objective of this study was to evaluate the long-term trajectory of outcomes using the Disability Rating Scale (DRS) of patients with TBI in a persistent VS over a 20-year period by retrospective data analysis.Recent reports suggest that it may be in the patient's best interest to continue functional assessment and updated care plans for patients with DoC for at least 10 years post-TBI.9 This study was approved by the ethics committee of Osaka University Hospital, which agreed with the opt-out consent process used .We conducted a retrospective cohort study using a review of medical records and collected data by telephone and written interviews with patients and their families. We used the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guidelines in preparing this article.This study was conducted at the Department of Traumatology and Acute Critical Medicine, Osaka University Graduate School of Medicine, which is a tertiary care center. The center specializes in severe trauma and acute critical care and is staffed by neurosurgeons, trauma surgeons, emergency physicians, and intensive care physicians. It is equipped with computerized tomography (CT) scanning equipment and an operating room that enables the rapid diagnosis and treatment of TBI. The center has 20 intensive care beds for the provision of acute care, and once a patient's condition stabilizes, the patient is discharged home or transferred to another hospital for rehabilitation or recuperative treatment.All patients with TBI who were treated at the Department of Traumatology and Acute Critical Medicine, Osaka University Graduate School of Medicine, between October 1996 and January 2003, and who were in a VS based on the DRS at 1 month after admission, were included in this study. Dr. Nakamura (the corresponding author) and Dr. Shiozaki (the second author) reviewed the medical records. For patients discharged from the hospital within 1 month post-injury, Dr. Shiozaki visited the patient 1 month after their injury to evaluate and record the DRS. The diagnosis of TBI is based on the presence of intracranial hemorrhage, cerebral contusion, or diffuse axonal injury findings on head CT or a magnetic resonance imaging (MRI) scan. In this study, we diagnosed VS with a DRS score of ≥22 and defined a DRS of <22 as recovery from a VS. Patients who died within 1 month of admission, those who recovered from a VS within 1 month, and those younger than 15 years of age at admission were excluded.10 and is reported to reflect the improvement effect of rehabilitation treatment more accurately and assess the clinical course of patients with severe TBI on a more sensitive scale than the Glasgow Outcome Scale,12 so we considered it useful as an index for evaluating long-term outcomes in this study.We evaluated patient background, Glasgow Coma Scale (GCS) on admission, injury mechanism, imaging findings including the Traumatic Coma Data Bank (TCDB) CT scan classification, length of stay in the hospital, and treatment details based on the medical records. We used the DRS as the indicator of long-term outcomes. The DRS consists of eight items  and is used to assess the level of disability for severe TBI . The DRSIn our study, the DRS was administered by trained and qualified emergency physicians or neurosurgeons at 1 month, 6 months, 1 year, and then annually until 2016, with the last administration in 2021. They evaluated the DRS in person or by telephone interviews with family members, caregivers, or medical social workers. For those without 20-year outcomes because of a loss to follow-up, we treated them as censored, whereas patients who died during the follow-up period and those whose DRS could be evaluated at the 20th year were treated as having completed follow-up. For each case, we evaluated whether the DRS follow-up outcomes were confirmed for all epochs, the duration of follow-up, the DRS trend, history of inpatients' rehabilitation treatment, and the cause of death. Patients with a DRS of ≥22 until the first year post-injury were defined as chronic VS and those with a DRS <22 were defined as non-chronic VS.Patient characteristics are reported using descriptive statistics. Continuous variables are presented as medians and interquartile range (IQR), and categorical variables are presented as counts and percentages. A line chart was used to visualize the trend in DRS over time. Data were analyzed using JMP Pro 16.2 statistical analysis software .There were 329 patients with TBI subjected to chart review during the study period. Of these, 35 patients with a persistent VS at 1 month were identified through the review of medical records and were included in the study . Their cAll patients were transferred to other hospitals, and none were discharged home. After transfer from our hospital, 15 patients (43%) were found to be undergoing rehabilitation treatment. Of these, 2 (13%) were chronic VS patients and 13 (87%) were non-chronic VS patients.The median DRS at 1 month after admission for the patients in this study was 26 (24–27). Among the 35 patients in a persistent VS at 1 month, we had complete DRS follow-up data at all years in 26 patients (74%), of whom 7 (20%) were alive and 19 (54%) had died. Nine patients (26%) were lost-to follow up and were treated censored patients. The median follow-up period for all 35 patients was 7 (4–17) years. The median follow-up period for the 26 patients who had DRS data for all epochs was also 7 (4–20) years, whereas that for the 9 censored patients was 13 (6–17) years .The 26 patients who had DRS data for all epochs had a median survival of 7 (4–20) years.We confirmed that 19 patients (54%) died in the study period, with 16 dying by the 10th year. Causes of death were pneumonia in 11, asphyxia in 1, pyelonephritis in 1, renal failure in 1, sepsis in 1, enteritis in 1, stroke in 1, and unknown in 2 patients.The course of DRS during the first 10 years is shown in We describe the progress of the 2 patients who returned to society. Case 1 was a teenage male suffering multiple trauma attributable to a traffic accident . The patThe patient continued to show functional recovery, improving to DRS 1 at 6 years post-injury and has remained at DRS 1 at 14 years post-injury. The item of his DRS scores that remained at 1 was Employability. During this period, he was able to lead a normal social life, including driving a car and getting married. Case 2 was a male in his twenties who suffered from multiple trauma attributable to a traffic accident . His levIn this retrospective cohort study, we used the DRS to evaluate long-term outcomes over a 20-year period in patients with so-called persistent VS who remained in a VS at 1 month post-TBI. Patients who remained in a VS after 1 year of injury had a poor outcome thereafter, whereas those who recovered from a VS within 1 year of injury showed improvement in the DRS thereafter.1 Patients in a VS post-TBI are expected to recover consciousness within 1 year, whereas recovery after 1 year is rare.3 A systematic review of outcomes of patients in a VS post-TBI also reported that 78% of patients with a persistent VS recovered consciousness by 1 year.13 In our study, more than half of patients with a persistent VS recovered from VS within 1 year, which is consistent with previous reports.In 1994, the Multi-Society Task Force on persistent VS defined patients who remain in a VS for more than 1 month post-TBI as being in a persistent VS and those who remain in a VS for more than 1 year as being in a “permanent VS.” Currently, the term permanent VS is not recommended, and it is recommended that the term “chronic VS” be used, accompanied by the duration of VS.1 and some even gained the ability to communicate and participate in social activities.14 Another study evaluating the long-term outcomes of TBI patients recovered from VS after 1 year post-injury has shown that significant recovery continues for 2 years post-injury and, to a more modest degree, for 5 years post-injury.15 Unfortunately, in this study we were unable to identify any patients in a chronic VS who achieved significant functional recovery as previously reported, with many of these patients having died as an outcome.In addition, three of the patients in our study also improved from a chronic VS. However, they only improved from a VS to a state of extremely severe disability, which did not contribute to the functional improvement of these patients in the chronic phase. It has been reported that the majority of patients in a chronic VS do not improve from the VS, but a few patients have recovered consciousness,8 The present study also showed that although improvement in the DRS can be expected for ∼10 years post-injury in patients in a persistent VS who did not progress to a chronic VS, DRS trends plateaued after 10 years and did not appear to improve. We believe that our results strengthen the argument that the functional recovery of patients with DoC attributable to TBI can be expected in the first 10 years, but we did not demonstrate substantial functional recovery beyond the 10th year. We believe that persistent VS patients who recover from VS within 1 year can be expected to gain further improvement in the DRS, which may provide a rationale for continued rehabilitation treatment for patients in a persistent VS.Recent studies evaluating the long-term functional outcomes of patients with DoC attributable to TBI, including VS, over a 10-year period have shown that an increasing number of patients achieve functional independence up to 10 years post-injury.As one limitation of this study, we were unable to follow the trend of the DRS in 9 patients until the end of the study. In addition, 3 of these 9 patients (including 1 with persistent VS) were lost to follow-up within 10 years, so we could not confirm the long-term follow-up of all 35 patients.16There are also limitations regarding the accuracy of the diagnosis of VS. It is difficult to accurately diagnose VS and evaluate its course by neurobehavioral evaluation, and evaluation using a variety of imaging studies and the Coma Recovery Scale-Revised (CRS-R) is recommended.17 this study was initiated before such diagnostic criteria were established. Therefore, we used the previously established criteria of diagnosing DRS ≥22 as a VS and defined recovery from a VS if the DRS was ≤21. Therefore, it must be acknowledged that there are limitations regarding the accuracy of the diagnosis of a VS in terms of the accuracy of the differentiation between VS and MCS.Although the CRS-R has also been reported to be useful in differentiating a minimally conscious state (MCS) from a VS,It was not possible to assess exactly what rehabilitation treatment was given to patients in this study. From the DRS survey records, 15 patients were identified as having received rehabilitation treatment, most of whom were non-chronic VS patients. Therefore, we cannot rule out the possibility that there was a difference in rehabilitation treatment intervention between patients who recovered from a VS and those who did not. The effectiveness of rehabilitative treatment for patients in a VS cannot be definitively discussed in this study.We followed a cohort of patients who were in a persistent VS at 1 month post-TBI and evaluated their functional outcomes by the DRS up to year 20.More than half of the patients in a persistent VS at 1 month post-TBI recovered from the VS within 1 year. These patients showed functional improvement for up to 10 years, although a substantial improvement in the DRS beyond that time was not observed. In contrast, most of the chronic VS patients died within 10 years of their injury, and none were alive at 20 years.


In the digitalized world, there is a need for developing new online teaching and learning methods. Although audio and video recordings are increasingly used in everyday learning, little scientific evidence is available on the efficacy of new online methods. This randomized trial was set out to compare the learning outcomes of online and classroom teaching methods in training healthcare students to diagnose breathing difficulties in children.In total, 301 students of medicine (N = 166) and nursing (N = 135) volunteered to participate in this total sampling study in 2021–2022. The students were randomized into four groups based on teaching methods: classroom teaching , streamed classroom teaching , audio recording  and video recording . Each 45-minute lesson was taught by the same teachers and used the same protocol. The students participated an online test with their own electronic device at three distinct time points: prior to any teaching (baseline), immediately after teaching , and five weeks later (long-term memory test). The test consisted of 10 multiple-choice questions on recognizing breathing difficulties from real-life videos of breathing difficulties in pre-school age. The test results scale ranged from − 26 to 28 points. Statistical analyses were performed using ANOVA multiple comparison and multiple regression tests.The mean scores (SD) of the final tests were 22.5 (5.3) in the vodcast, 22.9 (6.1) in the live, 20.0 (5.6) in the podcast (p < 0.05 vs. live) and 20.1 (6.8) in the live-stream group. The mean difference of test scores before and after the lesson improved significantly (p < 0.05) in all study groups, with 12.9 (6.5) in the vodcast, 12.6 (5.6) in the live, 10.9 (7.0) in the live-stream and 10.4 (6.9) in the podcast group. The improvement in test scores was significantly higher in the vodcast (p = 0.016) and the live (p = 0.037) groups than in the podcast group. No significant differences were found between the other groups. However, there was a nonsignificant difference towards better results in the vodcast group compared to the live-stream group.While the new online teaching methods produce learning, only video learning is comparable to team teaching in classrooms. The COVID-19 pandemic in 2020 permanently changed learning challenges and opportunities around the world by forcing schools and universities to fully transition to distance learning in just a few months .Now, after the pandemic, distance learning methods seem to have become a permanent part of our education also in the fields of health care, medicine, and nursing , 6. HoweDespite the at least partial shift to distance learning, the principles of learning have not changed. Motivation to learn remains vital and should be promoted, for example by the MUSIC model constructed by Professor Jones in 2016. The model is based on five valid dimensions underlying the student’s motivation: eMpowerment, Usefulness, Success, Interest and Caring –11. MnemIn this study, we wanted to promote the identification of breathing problems in children and to study the effectiveness of new distance teaching methods, vodcast and podcast, compared to live and streamed teaching. We chose the topic because learning the mechanism of breathing is multidimensional and complicated in practice and especially remotely , 14. BreAs early as the turn of the millennium, it has been shown that video recordings produced with high quality can be useful in learning . CurrentSeveral studies have suggested video recordings to be a higher quality pre-material for on-site learning in comparison with textbooks and conventional online learning methods , 18. HowA podcast is a digitally implemented audio file that can be uploaded to a smart device by the listener or listened to directly from a playback service –21.While podcasts are frequently used in medical education, there is sparse data on the efficacy of podcasts in medical learning. A recent Canadian study found no significant difference in learning by podcast or blog writing (blog) , whereasIn addition to new learning methods (vodcast and podcast), on-site team teaching has also received good feedback. In this method, two teachers give a lesson together in the same room. The method has been recently shown to be an efficient way to engage students , 25 by mAnother way to promote long-term learning is to provide information in multiple ways during a single lesson. This sort of multimedia learning involves providing combinations of content in different formats such as text, audio, images, animations, or videos in a single interactive presentation. While this is an effective learning method, it can also overwhelm the learner with cognitive processing requirements. The cognitive theory of multimedia learning (CTML) relies on an understanding of how people learn from words and images, which is particularly important in the context of medical education , 25, 27.Streamed teaching means that the learners follow live classroom teaching via the internet either simultaneously or later using a video recording. This method has gained popularity in recent years, although research data showed controversial results on the effect of the method on students’ academic performance already before the COVID-19 pandemic. An Australian review that included 71 articles (2016) on the learning of high school students showed that the impacts on learning outcomes were not positive. The results were evaluated within four result-efficient dimensions: academic, social, psychological and teacher’s mediation .However, live-streamed learning methods may have some positive outcomes, as evidenced in two Canadian studies. In a study by Grafton-Clarke and colleagues (2022), live streaming was used in medical education in the context of a real-life patient interview situation. The students evaluated the method in terms of quality and found it effective (9.7/ 10 points) . AnotherThe purpose of the present study was to compare the learning results between online and classroom teaching methods in teaching health care students to identify and treat breathing difficulties in children.In this study, we wanted to combine team teaching with the new methods and create modern vodcasts, podcasts and activating lessons, and to compare their effectiveness. We chose a child’s respiratory distress lesson for our approach as it is a challenging task even for experienced physicians , 31. IdeAs far as we know, there are no studies in the medical education field comparing vodcast, podcast and live-stream didactics as learning methods, at least in the context of learning to identify respiratory distress in child patients. Thus, we deemed it relevant to compare the efficacy and experience of such learning methods for the identification of acute respiratory distress in children.The study consisted of eight separate teaching events organised during the academic year 2021–2022. Each event included 40 students randomly assigned to four small groups (n = 10) that received teaching by different methods: teaching in the classroom (live), streamed classroom teaching (live-stream), audio recording (podcast) and video recording (vodcast). Each student participated in the lesson only once.The study design and participant flow are described in Fig. A total of 325 students  were recruited to participate in the trial. All medical students were in their fifth year of studies at the University of Eastern Finland and participated in an eight-week paediatric course implemented between August 2021 and May 2022. Additionally, paramedic nurse  students from the Savonia University of Applied Sciences in Kuopio (Finland) participated in the study during their paediatric course at the same time as the medical students. Both medical and nurse student groups were invited to participate in this randomized controlled trial. In this study, we refer to these professional groups as medical students and nurse students.The participants were randomized into four groups: classroom teaching, streamed teaching, podcast and vodcast groups Fig. .. A totaAll fifth-year medical students (n = 175) and all nurse students from each of the four classes (n = 150) were asked to participate in the study. Participation in the study was voluntary, but the examined teaching was a part of the medical students’ curriculum concerning respiratory distress in children.https://clincalc.com), the change in the test scores from baseline to final test was considered as the main outcome and a difference of 1.5 points between the groups was identified as an important difference. With this assumption and α-error of 0.05 and power (1- β) of 0.80, the result of the sample size calculation was 28 students per group.A pilot study was carried out among 20 paramedic nurse students to estimate baseline mean values and standard deviations in the classroom teaching, podcast or vodcast groups. For sample size calculation .The students were randomized into four equally sized teaching method groups using a computer program (The teaching took place simultaneously in four teaching groups. The students in the classroom teaching group (n = 72) participated in an on-site class facilitated by the teachers. The students in the remote teaching groups participated in the teaching intervention at their chosen locations using their own computers or smart devices. The podcast (n = 79) and vodcast (n = 73) groups participated in the teaching activity by listening to or watching recorded material. The stream teaching group (n = 77) viewed a live-taught course through Zoom webconferencing software, and had an opportunity to communicate with the teachers, either by speaking through their computer’s microphone or by texting their comments in Zoom’s chat feature.The the podcast and vodcast groups did not have such an opportunity.The same script and protocol were used in all the teaching groups. The script was jointly prepared by three clinical teachers in paediatrics and evaluated before recording the material. For example, the vodcast was recorded and edited beforehand by adding subtitles. Subsequently, the audio file was isolated from the vodcast into a separate recording, which was subsequently made into a podcast. Therefore, the vodcast and the podcast were the exact same recording with the exception that there was no video content included in the podcast.In all the teaching groups, the lesson was based on the MUSIC  theory and followed the principles of multimedia teaching –11 to prThe content of the lesson was similar in all the teaching groups and consisted of the identification and medical treatment of the most common respiratory problems in children Table ..The primary outcome was how much the learning results improved in the identification of breathing problems in children. Learning was measured using the same Webropol e-Test that was sent to the participants via e-mail on three separate occasions . The test was time-constrained (15 min) and consisted of 10 multiple-choice questions: five theoretical questions and five video clips of children with respiratory distress. The number of correct response alternatives was one or more in each question: Every correct choice accounted for plus 2 points and every wrong choice for 1 minus point. The maximum score was plus 28 points, and the minimum was minus 26 points.Learning efficacy was defined as the difference between the final and baseline test scores.The IBM SPSS statistical software 27.0 was used to compare and analyze the results of the study.As the test scores followed the normal distribution, the ANOVA multiple comparison test was used for assessing differences between teaching groups, and post hoc tests for paired comparison were performed using the Least Significant Difference (LSD) test. Multiple regression analysis was used to test the effect of background variables on the results. The level of statistical significance was set at p < 0.05.All fifth-year medical students (UEF) and all paramedic nurse students (Savonia) were invited to participate in the study.At the outset of the baseline test, students were requested to provide their consent for participation in the study. The students had the right to participate in each of the teaching interventions even if they opted out of participating in the tests of the present study. The data were collected with a pseudo-descriptive method. The data were collected and analyzed anonymously.The research plan was evaluated by the Research Ethical Committee of UEF, although its authorization was not required under Finnish law.A total of 301 92.6%) of the invited 325 students participated in the study, including 176 (58.3%) women and 125 (41.7%) men. There were 166 medical students and 135 nurse students, and 50 participants had completed previous education in health care , were 9.5 in the vodcast (5.8), 10.25 in the live (6.1), 9.6 in the podcast (6.0) and 9.1 in the live-stream (4.9) groups (non-significant). The mean scores (SD) in the final tests were: 22.5 5.3) in vodcast, 22.9 (3.7) in live, 20.0 (5.6) in podcast (p < 0.05 vs. live) and 20.1 (6.8) in live-stream groups (Table  in vodcaThe difference in the test scores before and after the lesson improved significantly (p < 0.05) in all the study groups and was 12.9 (6.5) in the vodcast, 12.6 (5.6) in the live teaching session, 10.9 (7.0) in the live-stream and 10.4 (6.9) in the podcast groups Table  and 4. IThere was a statistically significant difference in the final test scores between the methods, favouring the vodcast method compared with the podcast method (p = 0.016). In addition, a significant difference was confirmed between attending live teaching session vs. listening to the podcast p = 0.037). There was also a trend indicating that the students preferred vodcasts compared to streamed teaching (p = 0.055)  demonstrated that access to vodcasts prior to and after traditional medical classroom teaching promotes both short-term and long-term learning . SimilarIt should be emphasized that while the vodcast group had good efficacy in our study, this finding cannot be directly applied to video learning via YouTube. The quality and accuracy of videos used to teach medicine on YouTube varies, which makes it necessary to develop high-quality videos with scientifically approved content for health care learning . Our expA Belgian review of 33 peer-reviewed research articles shows that team teaching supports teachers’ ability to achieve better learning outcomes in cooperation with teachers and learners. Team teaching can create “rich and varied lessons” appreciated by students . In our Effective medical education is underpinned by an understanding of the efficacy of various visual and auditory inputs that form the training material. Retention and transfer tests are used to measure students’ recall and practical application skills post-learning. Mayer has studied the cognitive theory of multimedia learning (CTML) and concluded that information provided via multimedia strengthens long-term learning. In planning teaching, care is required to ensure the learners’ cognitive ability is not overwhelmed by cognitive processing requirements , 25.Our research is one of the first randomized controlled trials where a podcast is compared to other teaching methods. Some recent studies have suggested that podcasts could be as effective as other e-learning tools , 37, sucAlthough students tend to prefer prerecorded short podcasts that they can listen to during their daily activities to traditional classroom lessons and express the effectiveness of the method on their skills, their learning grades do not seem to reflect this when compared to on-site teaching . In our Our well-prepared podcast seemed to promote effective learning with results equal to streamed teaching. Even though the results were inferior to live teaching or vodcasts, the effectiveness was unexpectedly good, given that the participants did not see the picture and videos of breathing difficulties at all. Maybe using only one sense improves concentration and the ability to absorb details from the material.Live streaming is a currently widely used teaching method , but ourWhen evaluating the effect each method had on long-term learning, we observed a difference in the learning outcomes during the five to seven–week follow-up period among the medical and nurse students. Among the nurses, the scores of the long-term memory test were lower than in the final test in all the teaching groups. In contrast, among medical students, the learning results improved in all groups from the final test to the long-term memory test. This was most likely due to additional teaching (2 h) of breathing difficulties during the course, enabling the students to observe children’s breathing in real life and from video recordings.Interestingly, the live teaching and video recording groups still tended to get better learning results than the live-stream and podcast groups, despite the provided additional learning for all the medical student groups. The results suggest that live-streamed teaching is not a preferable teaching method if there are other options available.The results may have been affected by a large number (one-third) of dropouts after the final test. The number of respondents decreased most in the live-stream (42%) and podcast groups (41%). Learning results were also poorer in these groups. The decrease in participation rates in the long-memory test was more evident among the nurse students (54%) than among the medical students (16%).Our lesson was based on team teaching, socio-constructivism  and multAs studies on multimedia , 25 learThe strengths of our study were a randomized design (RCT), a sufficient number of participants based on power calculation and a high participation rate. The test used to measure learning was sufficiently difficult and the results were statistically normally distributed. We also managed to follow a 20-principle approach to all teaching, so the methods and settings were comparable with each other. Our teaching was in line with the principles of meaningful learning , 25 and The large number of dropouts in the long-term memory test for nurses may have affected the comparison of the teaching groups and reduced the accuracy of statistical analyses.Even though we managed to follow the 20 principles programme of teaching, the live lessons may have been somewhat different on each occasion due to a varying social atmosphere and learner activity. The long-term learning of the medical students was affected by 2 h of additional training they received on breathing difficulties, which affected the results of the long-term memory test.Both activating team teaching and well-prepared pre-recorded video teaching (vodcast) promote learning effectively. Podcasts also promote learning, but they are not as efficient as the aforementioned methods (live and vodcast). In our comparison, the live-stream method of teaching was found to be the weakest learning method and using it should be therefore carefully considered.