Central mammalian synapses release synaptic vesicles in dedicated structures called docking/release sites. It has been assumed that when voltage-dependent calcium entry is sufficiently large, synaptic output attains a maximum value of one synaptic vesicle per action potential and per site. Here we use deconvolution to count synaptic vesicle output at single sites (mean site number per synapse: 3.6). When increasing calcium entry with tetraethylammonium in 1.5 mM external calcium concentration, we find that synaptic output saturates at 0.22 vesicle per site, not at 1 vesicle per site. Fitting the results with current models of calcium-dependent exocytosis indicates that the 0.22 vesicle limit reflects the probability of docking sites to be occupied by synaptic vesicles at rest, as only docked vesicles can be released. With 3 mM external calcium, the maximum output per site increases to 0.47, indicating an increase in docking site occupancy as a function of external calcium concentration. The sensitivity of the exocytosis step to Cai results from the change of conformation of the SNARE complex following rapid Ca2+ binding to a series of Ca2+ binding sites residing on synaptotagmins  at mammalian central synapses is a complex process that comprises two series of events. Firstly, SVs move to a small part of the presynaptic terminal, the active zone (AZ), where they bind to specific proteins such as RIM and Munc13, and undergo a number of maturation steps including docking and priming . At the otagmins . Experimkinetics . The cori values .2+-dependent SV movement towards the plasma membrane, and they suggest some degree of integration between preparatory steps and exocytosis  tethered state (at a distance of <100 nm from the plasma membrane), followed by shorter-lived (about 0.3 s long) docked, and docked/primed states, before exocytosis . These rocytosis .2+ uncaging stimulations display trial to trial fluctuations  whtuations ; (iii) stuations . In spit2+-dependent exocytosis confirms the notion that docking site occupancy is incomplete at rest, and that partial site occupancy limits the synapse output at high release probability. Furthermore, our results suggest that docking site occupancy grows as a function of the external Ca2+ concentration, [Ca2+]o. They further suggest that a given [Ca2+]o, the docking site occupancy can be obtained as the maximal attainable release probability per release site. Finally, our results suggest that previous apparently discrepant results indicating a very high (close to 1) maximal release probability per release site can be explained partially by uncorrected errors in variance-mean analysis, and partially by changes of docking site occupancy with [Ca2+]o.Model simulations indicate that the docking site occupancy at rest strongly influences short-term synaptic plasticity and statistics of SV release . In addiIn this work we examine counts of SV release events measured in simple PF-MLI synapses when increasing the release probability with several methods. Our aim is to identify the source(s) of the increase of release probability in each case, taking advantage of the detailed information provided by SV counting in individual AZs. As further discussed below, we assume for the interpretation of our results that release occurs at a finite set of docking/release sites, and that SVs undergo exocytosis only once they are docked. Within this simple docking/release model, we attempt to distinguish increased probability of docking site occupancy by SVs from increased probability of exocytosis of an occupied docking site .2+ concentration ([Ca2+]o) of 1.5 mM. We then observed a gradual increase in SV counts during a train, reflecting facilitation  before stimulus number i, and pi is the probability of release of a docked SV (sometimes noted pves) following stimulus number i . Variance-mean analysis of SV counts  values, obtained by calculating the ratio of n2/n1, were similar with and without TEA  than in control (for i = 5), but the shapes of the two curves were not strikingly different (compare pink and red curves in 1 obtained in TEA (0.213) is far from the maximum attainable value of 1, leaving room for a significant increase of P linked to facilitation.Despite this marked increase in Pmax in the presence of 1 mM TEA was 0.472, far below the maximum of 1. Therefore, increasing release probability by a combination of synaptic facilitation and of TEA application is not sufficient to bring the release probability to 1.Finally, the value of P2+]o control condition . Globally these results suggest that TEA increases P1 only up to a certain point.A possible interpretation of the results above is that addition of 1 mM TEA was insufficient to bring the release probability of a docked SV (p) to its maximum of 1. To investigate this possibility, we tested 5 mM TEA in a new series of experiments, starting again from the same 1.5 mM o without TEA. In 3 mM [Ca2+]o, P1 was increased compared to 1.5 mM [Ca2+]o , and it reached a mean value similar to that obtained in 1.5 mM [Ca2+]o and TEA (0.28 ± 0.03 vs. 0.21 ± 0.06). Likewise Pmax was larger in 3 mM [Ca2+]o compared to 1.5 mM [Ca2+]o , and was similar in 3 mM [Ca2+]o compared to 1.5 mM [Ca2+]o and TEA (0.46 ± 0.04 vs. 0.47 ± 0.06).We first examined results in 3 mM [Ca1 and Pmax take similar values in 3 mM [Ca2+]o and in 1.5 mM [Ca2+]o plus TEA could reflect a common limit to the strength of the synapse. Alternatively, it could be a sheer numerical coincidence. To distinguish between these two possibilities, we next tested the effects of adding 1 mM TEA starting in 3 mM [Ca2+]o. P1 significantly increased from 0.28 ± 0.03 to 0.51 ± 0.09 , while mean peak miniature EPSC amplitudes did not change significantly . These data suggest that elevating [Ca2+]o results in an elevation of presynaptic [Ca2+]i at PF-MLI synapses. By contrast, we found that TEA (1 mM) did not change miniature EPSC frequency . These results suggest that [Ca2+]o elevation, but not TEA addition, leads to an increase in the presynaptic basal [Ca2+]i. As SV replenishment is enhanced by elevations of [Ca2+]i . This contrasts with many studies reporting P values of 0.9 or larger when performing mean-variance analysis of peak EPSC amplitudes  and of 0.487 with the peak EPSC method ; Pmax values were 0.436 with the SV count method and 0.710 with the peak EPSC method. As illustrated in this example, P estimates were invariably higher with the EPSC amplitude analysis than with the SV count analysis. The discrepancy became larger when correcting the variance for EPSC amplitudes for the variations in q values in the same experiment , as is usually done in peak amplitude variance-mean analysis ; increasing the TEA concentration from 1 mM to 5 mM, starting in a 1.5 mM [Ca2+]o plus 1 mM TEA solution (n = 4); and adding 1 mM TEA in 3 mM [Ca2+]o (n = 5). Group results showed significant increases of Ca2+ entry upon application of 1 mM TEA, both when starting from a 1.5 mM [Ca2+]o solution  and when starting from a 3 mM [Ca2+]o solution  with those in the presence of 5 mM TEA and 1.5 mM [Ca2+]o (purple bar). Whereas P1 is larger in the first solution than in the second , but also on the mode of stimulation (Ca2+ uncaging vs. AP).Current models of SV release assume that a presynaptic AP induces a local I data obtained from 1 as a function of peak global [Ca2+]I , as opposed to indirect release events coming from replacement sites . Also, peak global values from 1, the global release probability in response to the first stimulus in a train. As already pointed out, P1 data points incorporate both δ and p factors, according to the relation P1 = δ p. Clearly, P1 data differ from the curve in 2+]o  has a maximum attainable value that depends on the value of [Ca2+]o. With 1.5 mM [Ca2+]o, the maximum of P1 is 0.22 SV per AP and per release/docking site, whereas with 3 mM [Ca2+]o, the maximum of P1 is 0.47.In the present work we take advantage of the recently developed method of SV counting  to exami1 is limited by the resting docking site probability δ, and that δ is lower in 1.5 mM [Ca2+]o compared to 3 mM [Ca2+]o. The maximum output per site gives an estimate of resting docking site occupancy, a basic parameter of synaptic function that has been difficult to evaluate.Our interpretation of this finding is that when the release probability of docked SVs, p, is close to 1, P2+]o) implies that there is a large discrepancy between the release probability per docking site, P1, and the release probability per occupied docking site, p. By inverting the P1 = δ p relation, we obtain from P1 = 0.089 and δ = 0.22 that p = 0.39 in 1.5 mM [Ca2+]o. At 3 mM [Ca2+]o, we have P1 = 0.28 and δ = 0.47, so that p = 0.60. Therefore both at 1.5 mM [Ca2+]o and at 3 mM [Ca2+]o, the actual release probability of docked SVs is substantially higher than it would appear without the correction. In the presence of TEA, p values calculated from the pooled data of 2+]o: p = 0.85 in 1 mM TEA and p = 0.95 in 5 mM TEA; 3 mM [Ca2+]o: p = 0.89 in 1 mM TEA, value corrected for 2-step release). Therefore in the present preparation, P1 values in the presence of TEA give reasonably good approximations of δ.Our results confirm earlier suggestions that δ is less than 1 at PF-MLI synapses . In addi2+ entry, indicating high p values  and are facilitating, indicating that they belong to the first category, called 'tonic synapses'  synapses. Apart from using high [Ca2+]o conditions, another factor that may have contributed in some studies to high apparent P values is uncorrected synaptic jitter and receptor saturation, as documented above  following the animal care guidelines of Paris Descartes University . Recordings were obtained from either basket or stellate cells in the molecular layer; these cells were collectively called molecular layer interneurons (MLIs). The composition of the extracellular solution was (in mM): 130 NaCl, 2.5 KCl, 26 NaHCO3, 1.3 NaH2PO4, 10 glucose, 2 CaCl2, and 1 MgCl2 (osmolarity: 300 mosm). This solution was equilibrated with 95% O2 and 5% CO2 (pH 7.4). The internal recording solution contained (in mM): 144 K-gluconate, 6 KCl, 4.6 MgCl2, 1 EGTA, 0.1 CaCl2, 10 HEPES, 4 ATP-Na, 0.4 GTA-Na; pH 7.3 (osmolarity: 300 mosm). Recordings were at 30‒34°C.Sagittal slices (200 μm thick) were prepared from the cerebellar vermis of Sprague-Dawley rats −2-amino-5-phosphonopentanoic acid  and gabazine (15 μM). Two alternative procedures were used to find an appropriate location for electrical stimulation. In the first procedure, an electrical stimulation pipette was located in the molecular layer, above the dendritic field of the recorded MLI, and the pipette position and stimulation strength were adjusted to obtain minimal stimulation  a dWe determined occurrence times of individual EPSCs based on deconvolution analysis, as detailed in In TEA experiments, we checked somatic potassium currents by applying 3 ms voltage step to 0 mV until TEA reduced potassium current amplitudes to a stable level, and then we started to collect data.2+ imaging experiments, sagittal (200 μm) slices were prepared using a modified extracellular saline, as detailed in ‒34°C, with the 3 mM extracellular Ca2+ saline including APV and gabazine). Granule cells were loaded under whole-cell recording with a solution containing (in mM): 140 K-gluconate, 5.4 KCl, 4.1 MgCl2, 9.9 HEPES, 0.36 Na-GTP, 3.6 Na-ATP, 500 μM of Oregon green 488 BAPTA-6F  and 20 μM Alexa-594 (Invitrogen). Imaging was performed with a custom-built 2-photon system, with 820 nm excitation provided by a MaiTai-Sapphire laser . In order to visualize the granule cell axon, large raster scans were performed while acquiring the Alexa 594 fluorescence with a red channel photomultiplier . Single varicosity imaging was performed using raster scans of 5 by 2 μm dimensions at dwell times of 2 ms. The granule cells were kept under current clamp conditions. We found that keeping a hyperpolarized holding potential improved recording stability, so that resting membrane potential was kept around ‒90 mV. APs were evoked by 1 ms steps of 350‒500 pA. Stimulation protocols were 4 or 8 APs at 100 Hz and were repeated every 1 min. Calcium signaling was analyzed in the pixels encompassing the varicosity in terms of fluorescence changes relative to pre-stimulus values  with software written in the Igor Pro programming environment .For Ca2+ entry, with equal contributions for each AP , which is identical to l+ x f5. f is a factor determining the increase in vesicle fusion rate upon Ca2+ binding. The fusion rate at V0Ca-4Ca is l+ x f0-4. b is a cooperativity factor . If, as expected, probability of fusion = 1.0 with TEA alone, further increases in calcium by blocking additional K-channels with 4-AP is not expected to change the response. Such an experiment would give more confidence that under the TEA conditions indeed maximal calcium concentrations are achieved as claimed.All reviewers agree that the manuscript is interesting, experimentally well done, and allows for further insights into the physiological foundation of the classical synaptic release parameters, thus deserving publication in Reviewer #1:2+-dependent but argue that Ca2+ influx increased by K channel blocker (TEA increase from 1mM to 5 mM) has no effect on δ. The references quoted (subsection “TEA and [Ca2+]o increase P by two different mechanisms”) do not seem to provide a support this argument. At least a spatial model to distinguish Ca2+ increases, by elevation of external Ca2+ concentration and by TEA, would be necessary to propose this hypothesis.1) Authors assume that both p and δ are Ca2+, 0 TEA) are very different between two sets of experiments, therefore this comparison does not make sense unless the ratio to controls in individual experiments are compared. Likewise, at in subsection “Combining measurements of Ca2+ 334 entry with P measurements”, Ca2+ influx is compared between two conditions to be similar  vs 5 mM TEA / 1.5 mM Ca2+ (purple), with no controls across two sets of experiments.2) In subsection “Saturation of P as a function of TEA concentration”, P1 values in 1 mM TEA and 5 mM TEA are compared, but Figure 1E indicates that control values  Rationale. It is still not clear what conclusions had been made in previous manuscripts, and what were the shortcomings of these publications that this manuscript corrected. At first it seems like well-trod ground for the Marty lab – docking is limiting, increases in docking underlie facilitation. In the end, I had to reread all of the previous manuscripts to determine what had been learned here. In fact, docking values were slightly more  – so what has been learned? They should state succinctly what was previously known, describe the limitations in these previous manuscripts, specifically why it was difficult to precisely determine the docking site occupancy in their previous studies. And underscore the novel result, that is, that calcium increases the number of docked vesicles even before stimulation.eLife reader, even in the Abstract – the significant take-home messages are lost in comments about jitter and saturation. The general reader will not understand the mode of action or the significance of TEA treatment. I appreciate the specific numbers; the mean N value should also be cited.2) Abstract. The authors do not attempt to reach out to the general 3) Acronyms. In the rest of the document, the English style is simple and direct, which is an achievement in itself. Acronyms such as 'AP', 'MLI', 'PF', and 'PPR', are not defined. I think the reader will also need an occasional reminder to distinguish 'P' from 'p' from "release probability" as they read on.o data for 1mM TEA & 1.5 mM Ca2+ varied dramatically between two comparisons in Figure 3. They were more convincingly demonstrated in the 2016 paper. Nevertheless, they achieve significance and the data are consistent with previous experiments; they can stand in my opinion.4) TEA. The conclusion that docking is limiting hangs on the argument that they have maximized p with 1mM TEA – 5mM TEA does not increase P1. But that claim is not fully supported. The alternative is that 1mM TEA saturates calcium influx. The experiment demonstrating that TEA has not saturated calcium influx does not come until Figure 3. In the meantime, I was convinced they were wrong. Rearranging the figures might improve the logic of the argument or noting that this will be demonstrated below. Moreover, the ΔF/F5) Probability of release. subsection “Effect of adding 1 mM TEA on P”: What are the estimated p values for these values of P1? It would useful to include the SV count, δ and p1 that went into these values for {plus minus} TEA. I managed to screw up my math from my back-of-the-envelope calculations when I was checking those values from the data.6) Calcium channels. What does p=1 mean for our models of the active zone? These data suggest that every docked vesicle can be released. That suggests:- All SVs docked near calcium channels and all calcium channels open, or- Calcium domains are as large as the active zone and access all SVs.7) Replenishment. Won't 'crash fusions' from the replacement pool  interfere with their SV count after the first stimulation in the train. These will be included in their occupancy numbers.Reviewer #3:1) I would guess that changing bath calcium alters resting intracellular calcium, thus affecting δ. Did the authors' calcium measurements show such changes?2) One might imagine that TEA could depolarize the terminal and also lead to a change in calcium, yet the authors believe there is no effect of TEA on δ. Some comment about the effect of TEA is needed to help the reader understand its singular action on the spike waveform.2+ entry with P measurements”, highly sensitive TO Ca.3) In subsection “Combining measurements of Ca2+]o' that gave a P of 0.9, given that physiological Ca/Mg levels were not used in that work?4) Subsection “Comparison with other preparations” 'notable exception' of Silver's paper. What is 'normal o increase P by two different mechanisms”2+ increases, by elevation of external Ca2+ concentration and by TEA, would be necessary to propose this hypothesis.) do not seem to provide a support this argument. At least a spatial model to distinguish Ca2+]o to 3 mM and adding 1 mM TEA have similar effects on P1, the first manipulation severely decreases facilitation but the second does not. As shown in Pulido, 2018, docking site models predict that short term synaptic plasticity does not only depend on p, but also on δ, as well as on r, the probability of replenishment of an emptied docking site during one interpulse interval. Specifically, PPR = (1 – p) + pr + (1/ δ – 1) r . As can be seen from this equation, when p increases to its maximum of p = 1, the PPR decreases to a minimum of r/ δ, which is larger than 1 (facilitating) if r > δ. Therefore, certain synapses with low δ values remain facilitating even if p = 1. When on the other hand δ increases to its maximum of 1, the PPR decreases to 1 – p + pr, which is always smaller than 1 . Therefore, all synapses become depressing if δ increases to its maximum of δ = 1. We have changed the text to make the argument clearer.The reference Pulido, 2018 helps explaining that, while increasing [Ca2+]o.The new data with 4-AP in addition to TEA shows that in these conditions, P1 remains low while facilitation remains prominent . This confirms that blocking voltage dependent K channels, either with TEA or with 4-AP, has different effects on synaptic function than increasing [Ca2+]o and adding TEA on miniature current frequencies. These experiments show that elevated [Ca2+]o increases mini frequency, but that addition of TEA does not have any such effect. They are consistent with our suggestion that the basal presynaptic Cai is increased following the first maneuver, but not following the second one. Also, the new experiments mixing TEA and 4-AP  gave results consistent with the hypothesis that TEA (or other K channel blockers) act on p, not on δ.To address the issue of differences in mechanism, we have now included new data contrasting the effects of elevated o' that gave a P of 0.9, given that physiological Ca/Mg levels were not used in that work?4) Subsection “Comparison with other preparations” 'notable exception' of Silver's paper. What is 'normal [CaIndeed, several external concentrations of calcium and magnesium were used in Silver's work. When looking at Figure 9A of this paper however, it seems that in 2 mM calcium and 1 mM magnesium, the parabola is almost complete, indicating P near 0.9. Or did we misunderstand this figure?5) Are there data on how 1 and 5 mM TEA affect the spike in granule cells?We have added a figure  to illustrate the effects of 1 mM TEA on spikes.


Diff): 118 N, p < 0.001), greater work , and greater pectoralis major muscle activation  compared to the corresponding traditional loading schemes. However, there was little evidence of potentiation of the mechanical variables or muscle activity during the subsequent upward phases caused by the AEL schemes. It is possible that the use of weight releasers may disrupt lifting technique particularly during low AEL schemes thereby diminishing any benefits.We compared the effects of accentuated eccentric loading (AEL) on mechanical variables and agonist muscle activation using low (30% 1-repetition maximum (1RM)) and high (80% 1RM) upward-phase loading with AEL (100% 1RM during downward phase) to traditional loading schemes (T) in the bench press. Twelve resistance-trained men  performed sets of two repetitions with three-minute intervals using loading schemes of 30AEL, 30T, 80AEL, and 80T. AEL was applied using weight releasers while force plates and a 3D motion-analysis system were used to measure mechanical variables. Electromyographic activity of the pectoralis major and triceps brachii muscles was also recorded. The greater downward-phase loads experienced during the AEL conditions allowed greater overall mean vertical forces (mean difference ( The acute mechanical, metabolic, and hormonal responses to resistance training exercises have been proposed to drive neuromuscular adaptations that support the long-term enhancement of muscular strength and power ,2,3. FurThere are a variety of methods available to overload the downward phase of resistance training exercises including tempo eccentric training, flywheel inertial training methods, plyometric training, and accentuated eccentric loading methods ,10. Of tFew of the previous investigators have detailed the mechanical differences during the downward phase of the bench press exercise, limiting their analyses to the upward phase and only measuring vertical variables . Previous authors have stipulated that AEL protocols should impose minimal interruption to the natural mechanics of the exercise to provide an effective stimulus  and so aThe present study employed a cross-sectional design to compare the mechanical and agonist muscle activation differences between two AEL (30AEL and 80AEL) and two traditional loading schemes (30T and 80T) during the bench press exercise performed with free weights on a flat bench. The loads for 30AEL and 80AEL were 30% and 80% 1RM, respectively, for the upward phase with a constant load of 100% 1RM applied during the downward phase using mechanical weight releasers. The traditional loading sets used 30% (30T) and 80% (80T) 1RM throughout the downward and upward phases. The two loads used during the upward phases of the exercise reflect the range of loads recommended to improve muscular power output  as well Twelve resistance-trained men  volunteered to participate in this study, which was approved by the Institutional Research Board of East Stroudsburg University. Inclusion criteria for participation in the study was a 1RM in the bench press of 1.2 times body mass, a minimum of one-year resistance training at least 3 times per week, and being free from musculoskeletal injury for the 6 months prior to data collection. Each participant that met the inclusion criteria signed an informed consent prior to any testing session. None of the participants had any previous experience using weight releaser devices during the bench press exercise.Each participant attended five testing sessions across a three-week period. The 1RM for each participant was assessed in the first testing session using the two-point method, outlined by Garcia-Ramos et al. . The parFollowing at least 48 hours after the 1RM testing session, the participants were required to return to the laboratory and perform their first of the four different loading schemes , the order of which was counterbalanced across the participants. Each of these testing sessions was separated by a minimum of 48 hours and the participants were required to avoid any vigorous upper-body resistance training exercises during this time and to maintain their normal diet. A standardized warm-up comprising 10 min of dynamics stretches was completed at the beginning of each testing sessions prior to performing four sets of the bench press  separated by 2 min. The barbell was then loaded with the requisite masses associated with the 30AEL, 80AEL, 30T, or 80T protocols and two lifts were completed by the participants with 3 min of passive recovery between each repetition.During both of the repetitions performed under each of the four loading conditions the participants were required to bring the barbell to the chest in a controlled manner  before lifting it as fast as possible to a position with the elbows fully extended. Spotters were placed at either end of the barbell throughout each repetition and were able to assist with the removal and return of the barbell to the rack during the 30T and 80T protocols. The spotters assisted the participants with the removal of the barbell from the rack during the 30AEL and 80AEL protocols and they ensured that the weight releaser devices were stationary before the lift commenced during the time that the participant was required to maintain the barbell in a locked-out position. However, it was ensured that the spotters did not interfere with the release of the devices from the barbell. The weight releasers contacted a raised area comprising wooden boards  covered with layers of foam  during the downward phase to initiate the release from the barbell. These raised areas were implemented in order to minimize the vibrations associated with the release of the devices that may interfere with the signal recorded from the force platforms. The participant was required to remain on the lifting bench between the two repetitions.The lifting bench was placed on two force platforms  to allow the recording of the ground reaction forces (GRF) during each repetition. An 8-camera 3-dimmensional motion analysis system  that was synchronized with the force platforms was used to record kinematic data associated with a retroreflective marker placed in the middle of the barbell. The 3-dimmensional reference frame that was used defined three axes as vertical (perpendicular to the laboratory floor), anteroposterior , and mediolateral . The raw position data associated with the retroreflective marker were smoothed using a generalized cross-validated quintic spline procedure prior to analysis using the Nexus software . The initiation of the downward phase of each repetition was established as the first negative vertical velocity of the barbell marker with the phase ending when the vertical velocity first became positive. The upward phase of each repetition was defined as the event between the first appearance of positive vertical velocity of the barbell marker and the time of the maximum vertical position attained by the barbell marker. The following mechanical variables were then calculated:The position of the retroreflective marker placed on the barbell along the vertical axis during each repetition was recorded from the motion analysis system and the vertical displacement of the barbell during the downward and upward phases was calculated. The values during the downward phase were transformed to positive for ease of interpretation.The duration of the downward and upward phases of each repetition was determined from the motion analysis data using the vertical displacement of the retroreflective marker placed in the center of the barbell.The position of the retroreflective marker along the three axes was differentiated with respect to time using the first central difference method to provide the velocity of the barbell. The velocity along the vertical axis was averaged during the downward and upward phases of each repetition to provide mean vertical velocity .The position of the retroreflective marker along the vertical axis was double differentiated with respect to time using the second central difference method to provide the acceleration of the barbell. The positive vertical acceleration towards the end of the downward phase of the exercise was averaged to provide the mean vertical acceleration during the downward phase. This value represents the average rate of change in vertical velocity as the barbell descended to the lowest point of the lift, reflecting the application of vertical force to the load. The positive vertical acceleration from the beginning of the upward phase until the first negative acceleration of the barbell was also averaged to provide the mean vertical acceleration during the upward phase. The value reflected the application of force to the load to initiate the upward motion of the barbell.The forces collected from the two force platforms were summed along the vertical axis to provide the reaction to the vertical force applied to the barbell during each repetition. The weight of the lifting bench and that of the participant were removed from the vertical component of the GRF by collecting a separate trial with the participant lying stationary on the bench at the beginning of each testing session. The vertical force was then averaged during the downward and upward phases to provide the mean vertical force.The average rate of the rise in the vertical component of the GRF was calculated during the period of positive vertical acceleration of the barbell towards the end of the downward phase of the exercise as the ratio of the change in vertical force to the change in time. The rate of force development (RFD) value represents the average rate at which the vertical force was applied to the barbell as it descended to the lowest point of the lift. Note that the corresponding RFD was not calculated during the period of positive vertical acceleration observed during upward phase of the repetitions as the vertical force applied to the barbell decreased during this period.The instantaneous barbell velocities along the vertical, anteroposterior, and mediolateral axes were multiplied by the instantaneous forces along the corresponding axes during each repetition to provide the instantaneous mechanical power output. These were then integrated using the trapezoid rule to provide the work performed along each of the three axes. The work was then summed along all axes to provide total mechanical work, which was then divided by the duration of each phase to provide the mean mechanical power output during the downward and upward phases of each repetition .The instantaneous mechanical power output along the vertical, anteroposterior, and mediolateral axes was integrated using the trapezoid rule to provide the mechanical work performed along the axes during each repetition. The work performed along each axis was summed and the mechanical work performed during the downward and upward phases of each repetition. The electromyographic signal from the pectoralis major muscle and the long head of the triceps brachii muscle on the dominant arm (as determined by the participant) was recorded using a wireless system . Data from the anterior deltoid muscle was also collected but due to sampling problems it was not possible to analyze this data for a number of the participants. The signals were synchronized with the motion analysis system. The skin was prepared and bipolar electrodes (11 mm diameter with a 20 mm interelectrode distance) were placed according to previous recommendations . The datp ≤ 0.05. Pairwise comparisons with Bonferroni corrections were used to establish where any significant differences occurred, while the 95% confidence limits (95% CL) of the differences were calculated as were the effect sizes (ES) using Cohen’s d. The ES were interpreted as: ≤0.19 = trivial; 0.20–0.49 = small; 0.50–0.79 = medium; 0.80–1.29 = large; ≥1.30 = very large [All statistical analyses were performed using the IBM SPSS software package . Measures of central tendency and spread of data were represented as means and standard deviations. Statistical differences in the average downward RFD between the loading schemes were assessed by a one-way ANOVA with repeated measures. The differences in the remaining mechanical variables and average agonist muscle activation were assessed using a three-way ANOVA with repeated-measures . Alpha was set at ry large .The results for the kinematic variables are shown in p < 0.001) that was caused by the increase in vertical displacement from the downward to the upward phase during the AEL schemes being greater than the increase during the traditional schemes . A significant load × lifting phase interaction (p < 0.001) was due to the vertical displacement being greater under the low load conditions (30AEL and 30T) compared to the high load conditions . A significant scheme × load × lifting phase interaction was also revealed (p = 0.009). This was caused by the vertical displacement during the downward phase of 30T being greater than 30AEL . For the upward phase, the vertical displacement during 30T was greater than 80T  while that during 30AEL was greater than 80T and 80AEL .There was a significant Scheme × Lifting phase interaction . This was due to the 0.48 s increase in the durations of the phases in 30AEL compared to 30T being less than the 0.08 s increase in the durations from 80T compared to 80AEL , although the ES was medium (0.72). A significant load × lifting phase interaction (p = 0.001) was caused by the 0.51 s decrease in the duration of the upward compared to the downward phase observed during the low load conditions (30AEL and 30T) being greater than the 0.19 s reduction observed during the high load conditions .There was a significant scheme × load interaction for the duration of the downward and upward phases (p < 0.001). Further analyses revealed that the duration of the upward phase during 30T was less than that during both 80AEL and 80T . Finally, the duration of upward phase during 30AEL was less than that during 80AEL and 80T .A significant scheme × load × lifting phase interaction was found for the duration of the downward and upward phase (The percentage of each repetition comprising of the downward and upward phases under the four different loading schemes are presented in p = 0.004) for mean vertical velocity was caused by the 0.11 m/s greater velocities attained during 30T compared to 30AEL schemes being greater than the difference observed between the 80AEL and 80T schemes . There was also a significant load × lifting phase interaction (p < 0.001) caused by the 0.47 m/s increase from the downward to the upward phases observed under the low load conditions (30AEL and 30T) being greater than the 0.06 m/s increase observed under the high load conditions . A significant scheme × load interaction (p = 0.002). Specifically, the mean vertical velocity during the downward phase of 30T was greater than that during 30AEL, 80T, and 80AEL . For the upward phase, the mean vertical velocity during 30T was greater than that during 80T and 80AEL , while the mean vertical velocity during the upward phase of 30AEL was greater than that during 80T and 80AEL . There was a significant scheme × load × lifting phase interaction for mean vertical velocity (p = 0.007). This was caused by the 0.82 m/s2 increase in the positive vertical acceleration from the downward to the upward phases during the 30T and 80T schemes being different from the 0.72 m/s2 decrease from the downward to the upward phases observed under the AEL conditions .A significant scheme × lifting phase interaction was found for the mean vertical acceleration (The results for the kinetic variables are shown in p < 0.001) caused by the 181 N higher force produced during the 30AEL compared to the 30T schemes being greater than the 54 N difference observed between the 80AEL and 80T schemes . A significant scheme × lifting phase interaction (p < 0.001) was produced by a greater reduction in force between the downward and upward phases under the AEL schemes compared to the traditional schemes , while a significant load × lifting phase interaction (p < 0.001) was caused by a greater reduction in force from the downward to the upwards phases of the low load conditions (30AEL and 30T) compared to the high load conditions .There was a significant scheme × load interaction for mean vertical force (p < 0.001). This was caused by the mean force during the downward phase of 30T being less than that during 30AEL, 80AEL, and 80T  while the mean vertical force during the downward phase of 30AEL was lower than both 80AEL and 80T . The mean vertical force during the downward phase of 80AEL was greater than 80T  although the ES was small (0.40). During the upward phase, the mean vertical force was lower in the 30T scheme compared to both 80AEL and 80T  while the mean force during 30AEL was lower than that during 80AEL and 80T .There was a significant scheme × load × lifting phase interaction for mean vertical force . There were no statistically significant main effects or interactions identified for the average downward RFD despite greater values being produced under the AEL schemes (p = 0.020) was caused by the 156 W increase in mean power output from the downward phase to the upward phase under the low load conditions (30AEL and 30T) being greater that the 74 W increase observed under the high load conditions .A significant load × lifting phase interaction (p = 0.001) was caused by the 120 J greater amount of work performed during 30AEL compared with 30T being substantially more than the difference of 52 J observed between the 80AEL and 80T schemes  while a significant scheme × lifting phase interaction (p < 0.001) resulted from the reduction in work between the downward and upward phases of the AEL schemes (30AEL and 80AEL) being different from the increase across the phases observed under the two traditional schemes . A significant load × lifting phase interaction (p < 0.001) was caused by the 29 J reduction in work from the downward to the upward phases during the low load conditions (30AEL and 30T) being different from the 17 J increase in work observed under the high load conditions .A significant scheme × load interaction (p < 0.001). Specifically, less work was performed during the downward phase of 30T compared to 30AEL, 80AEL, and 80T . Less work was also performed during the downward phase of 30AEL compared to both 80AEL and 80T , while that performed during the downward phase of 80T was less than that during 80AEL  with a small ES (0.38). For the upward phase, more work was performed under 30AEL compared to 30T  although the ES was small (0.20), while more work was performed during the upward phase of 80T compared to 30AEL and 30T . Finally, more work was performed during the upward phase of 80AEL compared to 30AEL and 30T .A significant scheme × load × lifting phase interaction was found for the mechanical work performed during the repetitions  was caused by the AEL (30AEL and 80AEL) schemes producing greater average activation compared to the traditional schemes. A significant main effect for Load (p = 0.001) was caused by the high load conditions (80AEL and 80T) producing greater average activation compared to the low load conditions (30AEL and 30T), and a significant main effect for Lifting phase (p = 0.002) was caused by greater average activation during the upward phases compared to the downward phases.For the pectoralis major, a significant main effect for scheme (p= 0.005) for the average activation of the pectoralis major muscle was caused by the 42 µV higher activation during 30AEL compared to the 30T scheme being greater than the 13 µV difference observed between the 80T to the 80AEL schemes . There was also a significant scheme × lifting phase interaction (p = 0.020) for the average activation of the pectoralis major muscle. This was caused by the increase in activation from the downward to the upward phase recorded under the two AEL schemes (30AEL and 80AEL) being lower than the difference recorded under the two traditional schemes , although the ES was small (0.43).A significant scheme × load interaction ( p = 0.004) for the triceps brachii muscle that was caused by the high load conditions (80AEL and 80T) producing greater average activation compared to the low load conditions (30AEL and 30T); a significant main effect for the lifting phase (p = 0.001) was caused by greater average activation during the upward phases compared to the downward phases.There was a significant main effect of load (p = 0.007). This was due to the increase in the activation from the downward to the upward phases during the traditional schemes (30T and 80T) being greater than that recorded during the AEL schemes , although the ES was medium (0.68). Furthermore, a significant scheme × load × lifting phase interaction (p = 0.050) was caused by the increase in the average activation from the downward to the upward phase during 30AEL being less than the increase recorded during 80T .A significant scheme × lifting phase interaction was found for the average activation of the triceps brachii muscle . While the same effect was found in the comparison of 80AEL with 80T (Diff: 109 N) the ES was small (0.40). However, the mean vertical forces applied to the barbell during the upward phases of the lifts were not significantly different between the AEL and traditional schemes performed with the same upward loads. Previous researchers have reported a decrement in the force during the upward phase when applying supramaximal accentuated loads during the downward phase  with loads equivalent to 100% 1RM during the upward phase of the bench press exercise [The purpose of the present study was to investigate the differences in mechanical output and muscle activation between AEL and traditional loading schemes during the bench press exercise performed with low (30% 1RM) and high (80% 1RM) loads during the upward phase. We hypothesized that only the low AEL protocol would result in enhanced mechanical output and agonist muscle activation during the upward phase of the exercise. Our hypothesis was not supported despite the greater magnitudes of the differences in mechanical variables reported in comparisons between 30AEL and 30T, with the comparisons of 80AEL with 80T revealing only small effect sizes. The differences identified were largely confined to the downward phase of the repetitions. For example, the mean vertical force applied to the barbell was greater during the 30AEL scheme compared to that during 30T only during the downward phase of the lift . This small increase was unlikely to be due to changes in the work done to raise the barbell given similar vertical forces and vertical displacements during the upward phase under the 30AEL and 30T conditions. This leaves an increase in the work performed on the barbell along the horizontal axis as the likely explanation of the small difference observed between the 30AEL and 30T schemes. Although previous researchers have not assessed the effects of AEL on mechanical work, Sheppard and Young  reportedOur hypothesis that the low AEL protocol would result in enhanced agonist muscle activation during the upward phase of the exercise was not supported. An analysis of the pectoralis major and triceps brachii muscles revealed that the load conditions exerted a greater effect on the average activation of these agonists compared to the AEL schemes, with greater activation associated with the high load conditions (80AEL and 80T). Previous authors have also reported limited effects of supramaximal and submaximal AEL protocols on muscle activation ,13. IndeWe were unable to establish evidence of potentiation of the either the mechanical variables or agonist muscle activation during the upward phase of the AEL caused by the greater loads during the downward phase in the present study. Previous authors have noted that the effectiveness of AEL protocols is likely to be predicated on minimal disruption to the natural mechanics of the movement . AlthougDiff: 388 N; ES: 1.40). Specifically, the force during the 80AEL scheme was equivalent to a load of approximately 118 kg (90% 1RM for the present participants), while that during the 30AEL scheme was equivalent to a load of approximately 80 kg (60% 1RM) despite both schemes using the same initial load (100% 1RM). The lower mean load experienced during 30AEL compared to 80AEL is due to the proportion of the downward phase with a lower load (30% 1RM vs. 80% 1RM) after the weight releasers had detached from the barbell. Previous researchers have suggested that stronger participants require greater loads applied during the downward phase in order to potentiate the mechanical aspects of the bench throw exercise [The mechanical analysis used in the present study allows for the identification of a number of issues that could confound the effects of AEL protocols. For example, an analysis of the mean vertical force during the downward phases of the AEL schemes demonstrates that neither protocol produced a load that was equivalent to 100% 1RM during the entirety of the phase. This was due to the requirement that the weight releasers detach prior to the completion of the downward phase. Moreover, the mean vertical force during the descent of the barbell was actually significantly different between the two AEL protocols . Since the mean vertical force applied to the barbell during the downward phase of the 30T condition was lower than that during 30AEL both produced comparable mean power outputs. However, the differences in the velocity of the barbell’s descent may have obscured any mechanical advantage produced by the AEL loading scheme. Previous researchers have demonstrated that greater downward velocities resulted in enhanced mechanics during the subsequent upward phases of both bench press and back squat exercises [The mean vertical velocity during the downward phase of 30T was significantly greater than that during 30AEL  or the high  AEL protocols used in the present study were able to effectively enhance the mechanics or agonist muscle activation during the upward phase of the bench press exercise. However, the administration of AEL through the use of weight releaser devices may disrupt the lifting technique and also produce loads during the descent of the barbell below those expected, particularly during the low AEL scheme where the contrast in the loads is largest, thereby diminishing any benefits.


However, it has been suggested that the rapid drop in blood pressure that occurs with a slow decrease in [Ca2+] preceding early diastolic filling is related to the mechanism of rapid sarcomere lengthening associated with spontaneous tension oscillation at constant intermediate [Ca2+]. Here, we analyzed a new type of oscillation called hyperthermal sarcomeric oscillation. Sarcomeres in rat neonatal cardiomyocytes that were warmed at 38–42 °C oscillated at both slow (~ 1.4 Hz), Ca2+-dependent frequencies and fast (~ 7 Hz), Ca2+-independent frequencies. Our high-precision experimental observations revealed that the fast sarcomeric oscillation had high and low peak-to-peak amplitude at low and high [Ca2+], respectively; nevertheless, the oscillation period remained constant. Our numerical simulations suggest that the regular and fast rthythm is maintained by the unchanged cooperative binding behavior of myosin molecules during slow oscillatory changes in [Ca2+].The heart rhythm is maintained by oscillatory changes in [Ca However, the Frank-Starling law—which specifies that more blood returning to the ventricle causes more blood to be ejected during the next systole—is thought to be dependent on sarcomere structure and independent of [Ca2+]8. The rapid distention of the ventricular myocardium during early diastolic filling is also thought to be a mechanism that does not depend on [Ca2+]10. These facts suggest that the heart rhythm is controlled by both [Ca2+]-dependent and [Ca2+]-independent regulatory mechanisms.The cardiac cycle is synchronized physiologically by oscillatory changes in [Ca2+] (~ pCa 6) in cardiomyocytes stripped of their membranes and in myofibrils purified from cardiomyocytes15. The frequencies of SPOC in cardiomyocytes purified from the hearts of cows, pigs, dogs, rabbits and rats are proportional to the beat frequencies of the corresponding hearts16. Living cardiomyocytes treated with ionomycin (a selective Ca2+ ionophore) have been found to exhibit SPOC at constant intercellular [Ca2+] due to inhibition of Ca2+ channels and pumps in the sarcoplasmic reticulum18. These results suggest that cardiomyocytes exhibit oscillatory contraction even at constant [Ca2+] and that this contraction might be coupled to the physiology of the heartbeat in vivo.In fact, spontaneous oscillatory contraction (SPOC) of cardiomyocytes is observed even at constant [Ca2+], the characteristics of oscillatory behavior at elevated temperatures in association with time-dependent [Ca2+] transients should provide further insights into the mechanism of oscillation and its contribution to heart function. Transient contractions of living adult cardiomyocytes have been detected upon a rapid temperature increase from 36 to 41 °C19. In that study, the [Ca2+] as measured by a calcium indicator (Fluo-4) was the same at 41 °C as in relaxed cells at 36 °C, suggesting that the contraction system of cardiomyocytes is activated at high temperatures even under conditions of very low [Ca2+]19. This activation has also been confirmed in an in vitro motility assay using thin filaments reconstituted with α-tropomyosin and troponin20. These filaments were found to be able to slide at a rate of approximately 30% of full activity at a temperature of approximately 37 °C even at a low [Ca2+]20. Adult cardiomyocytes do not show SPOC with temperature jumps, but neonatal cardiomyocytes exhibit oscillatory contraction at high frequencies (6–12 Hz) under conditions of low-frequency Ca2+ oscillation (~ 1.4 Hz)18. The frequency of 6–8 Hz observed at high temperature is close to the neonatal heart rate, while the frequency of ~ 1 Hz Ca2+ oscillation coexisting with the high-frequency contractile oscillation is close to the frequency of spontaneous cardiomyocyte oscillation at 37 °C. The frequency of sarcomeric oscillations  is elevated at high temperature but rapidly returns to the original frequency when the temperature is lowered, indicating the reversibility of the change in oscillation frequency18.In addition to SPOC at constant [Ca2+]. However, sarcomere lengthening during HSOs is observed at high [Ca2+] as well as at low [Ca2+]. The occurrence of sarcomere lengthening even at high [Ca2+] indicates that the mechanism of SPOC exerts a certain effect on the relaxation of cardiomyocytes in a beating heart with standard time-dependent [Ca2+] transients. In previous work, the waveforms of HSOs with frequencies of 7–10 Hz could not be analyzed effectively because the length changes during HSO were observed at a low frame rate of 33 frames per second (fps) and with low spatial precision (~ 8 nm)18. The observation of HSOs at higher spatiotemporal accuracy (500 fps and 4 nm) in the present study enabled detailed analysis of the oscillatory behavior of sarcomeres. In addition, the obtained results were integrated with the effect of temperature in our previous numerical model21 to simulate the stochastic behavior of the actomyosin ATPase cycle, including binding control by the troponin-tropomyosin complex. HSOs had large and small amplitudes at low and high [Ca2+], respectively. The frequencies of HSOs remained constant independent of the amplitudes of the oscillations and of [Ca2+]. Analysis with the numerical simulation model suggest that the regular rhythm of HSOs comes from the unchanged cooperative behavior of active myosin molecules under conditions of slow oscillatory changes in [Ca2+]. Our work helps clarify the molecular mechanism of diastolic heart failure.Sarcomeres usually contract strongly and shorten rapidly at high [Ca2+] transients  state to the weak binding (WB) state by an additional factor of 2  gradually decreased and increased, respectively, with an increase in average strain in the Rig state . Most of the myosin that transitioned to the Pre state generated tension by transitioning to the Post state according to the transition rate < k+3 > (Pre → Post) and suppressed the extension of the half-sarcomere . This <ere Fig. g–k. Whenion Fig. a. Theref2+]  increased under constant [Ca2+]  . For fast oscillations, the period of each cycle was kept constant  increased such that HSOs were observed after heating even at low [Ca2+]20 . Surprisingly, partial HSOs were also observed at ~ 37 °C before heating  generated by single myosin molecules27 so that a small imbalance of numbers of active myosin between sarcomeres explains the long sarcomere length.The amplitude (~ 0.2 µm) and maximum sarcomere length (~ 2.0 µm) of the slow component of oscillation Fig. e were coSOs Fig.  are larg21. Several reaction rates in the crossbridge cycle in the model are known to change with temperature. The binding affinity of troponin and tropomyosin for actin filaments decreases with increasing temperature, and then the sensitivity of [Ca2+] increases32. The speed of the crossbridge cycle of myosin and the stroke reversal rate in skeletal muscle myosin increase with increasing temperature35. When these changes were applied in the model simulation, the HSOs were reproduced reasonably well; the amplitudes of HSOs changed greatly while the period remained constant  and from the Post to the Pre state (k−3). At the end of the shortening phase, the population of myosin with high strain was large, and then an avalanche of power stroke reversals and < k−4 > could be easily triggered by minor sarcomere lengthening  was triggered by a small increase in the sarcomere length  of HSOs is associated with stabilization of heartbeat frequency. The left ventricle of the heart dilates rapidly in early diastole even under conditions of slow  profile similar to that observed in our previous work18 before heating. The [Ca2+] profile after heating was adjusted so that the amplitudes of HSOs were reproduced. The influence of [Ca2+] was taken into account in the state transition of the T/T unit  represented the number of neighboring myosin heads in the WB state or the three binding states . In the binding state, the rate constants In this study, we applied the numerical single-myofibril model Fig.  proposednit Fig. d, which All statistical tests were performed with a library of statistical software tools (R language). The normality and homoscedasticity of the amplitudes, periods and times shown in Fig. Supplementary Video 1.Supplementary Information 1.


Recent work shows the pro-proliferative role of the Ca2+ activated Cl− channel TMEM16A (anoctamin 1), and demonstrates the essential contribution of TMEM16A to CFTR-dependent Cl− secretion. The present data demonstrate an increase in intracellular Ca2+ ([Ca2+]i) signals and Cl− secretion by TMEM16A, in renal collecting duct principle cells from dog (MDCK) and mouse (M1) as well as primary tubular epithelial cells from PKD1−/− knockout mice. M1 organoids proliferated, increased expression of TMEM16A, and secreted Cl− upon knockdown of endogenous polycystin 1 or 2 , by retroviral transfection with shPKD1 and shPKD2, respectively. Knockdown of PKD1 or PKD2 increased basal intracellular Ca2+ levels and enhanced purinergic Ca2+ release from endoplasmic reticulum. In contrast, ryanodine receptors were found not to be expressed in mouse renal epithelial cells and caffeine had no effects on [Ca2+]i. Ca2+ signals, proliferation, and Cl− secretion were largely reduced by knockdown or blockade of TMEM16A. TMEM16A may be therefore important for enhanced Ca2+ release from IP3-sensitive Ca2+ stores in polycystic kidney disease.Polycystic kidney disease (PKD) leads to continuous decline of renal function by growth of renal cysts. Enhanced proliferation and transepithelial chloride secretion through cystic fibrosis transmembrane conductance regulator (CFTR) and Ca• ADPKD leads to continuous decline of renal function by growth of renal cysts.• Knockdown of PKD1 or PKD2 increases TMEM16A expression.2+ signals, Cl− secretion, and proliferation.• TMEM16A enhanced intracellular Ca• TMEM16A contributes to cyst growth in ADPKD.The online version of this article (10.1007/s00109-020-01894-y) contains supplementary material, which is available to authorized users. To measure the ciliary Ca2+ changes, 5HT6-GECO was excited at 485/405 nm, and the emission was recorded between 535 ± 12.5 nm. The results for [Ca2+]cilium and [Ca2+]cyt were obtained at 485/405 nm changes and given in ratio. Measurement of the global cytosolic Ca2+ changes was performed as described recently [2+]i was calculated from the 340/380 nm fluorescence ratio after background subtraction. The formula used to calculate [Ca2+]i was [Ca2+]i = Kd × (R-Rmin)/(Rmax-R) × (Sf2/Sb2), where R is the observed fluorescence ratio. The values Rmax and Rmin (maximum and minimum ratios) and the constant Sf2/Sb2 (fluorescence of free and Ca2+-bound Fura-2 at 380 nm) were calculated using 1 μmol/l ionomycin , 5 μmol/l nigericin, 10 μmol/l monensin (Sigma), and 5 mmol/l EGTA to equilibrate intracellular and extracellular Ca2+ in intact Fura-2-loaded cells. The dissociation constant for the Fura-2·Ca2+ complex was taken as 224 nmol/l. ER Ca2+ signals were detected in Ca2+ sensor ER-LAR-GECO1 . Cells recently . In brie MA, USA ,) expresMaterials and statistical analysis: All compounds used were of highest available grade of purity. Data are reported as mean ± SEM. Student’s t test for unpaired samples and ANOVA were used for statistical analysis. p < 0.05 was accepted as significant difference. We are grateful to Prof. Dr. Dorien J.M. Peters  for providing us with animals with a floxed PKD1 allele. We acknowledge the support by Ms. Ana Fonseca.ESM 1(PDF 80 kb).ESM 2(PDF 3998 kb).


The stereotypical vergence response to a step stimulus consists of two dynamic components: a high velocity fusion initiating component followed by a slower component that may mediate sustained fusion. The initial component has been well-studied and is thought to be controlled by an open-loop mechanism. Less is known about the slow, or fusion sustaining component except that it must be feedback controlled to achieve the positional precision of sustained fusion. Given the delays in disparity vergence control, a feedback control system is likely to exhibit oscillatory behavior. Vergence responses to 4 deg step changes in target position were recorded in eight subjects. The slow component of each response was isolated manually using interactive graphics and the frequency spectrum determined. The frequency spectra of all isolated slow vergence movements showed a large low frequency peak between 1.0 and 2.0 Hz and one or more higher frequency components. The higher frequency components were found to be harmonics of the low frequency oscillation. A feedback model of the slow component was developed consisting of a time delay, an integral/derivative controller and an oculomotor plant based on Robinson’s model. Model simulations showed that a direction dependent asymmetry in the derivative element was primarily responsible for the higher frequency harmonic components. Simulations also showed that the base frequencies are primarily dependent on the time delay in the feedback control system. The fact that oscillatory behavior was found in all subjects provides strong support that the slow, fusion sustaining component is mediated by a feedback system. The control processes that mediate eye movements face two major challenges: the need for a rapid response despite substantial processing delays, and the need to attain accurate positioning despite errors inherent in neural and muscular mechanisms. These two challenges are best met with different control strategies. Feedback can produce extremely accurate responses, but if delays are present in the feedback loop, response speed must be reduced to keep the system stable. Conversely, open-loop  control can generate rapid responses irrespective of processing delays, but these responses will have limited accuracy. Both version and vergence control systems achieve speed and accuracy by combining the two strategies. In version, the two control strategies manifest as separate movements: preprogrammed saccades and feedback-controlled pursuit movements. In vergence, the two control components are less obvious as they merge into a single coordinated response. Nonetheless, considerable evidence supports a “dual mode” control strategy  that conThe neural structures behind these eye movement control components were originally inferred from behavioral data , but havA number of sophisticated models have been developed that expand on the simple structure illustrated in The Erkelens’ model has demonstrated appropriate simulations to both step and sinusoidal stimuli, while the model of Maxwell, Tong, and Schor , has accThe slow, or fusion sustaining, component has not been as well-studied as the fusion initiating, component.We know that feedback control systems will exhibit instabilities in the form of oscillatory behavior if the loop gain or loop delay exceed certain limits. Such limits depend on the dynamic characteristics of the process that is being controlled, in this case the oculomotor plant. The essential dynamics of the oculomotor plant have been experimentally determined and can be represented by a second-order system with a relatively long major time constant in the range of 0.2 to 0.4 sec . Given tEight subjects  between 46 and 72 years of age (57 ± 11.2 years) participated in this study. The average near point of convergence was 9.8 ± 1.9 cm measured from the bridge of the nose while viewing a high acuity target as described in our previous publications . The neaLeft and right-eye movements were recorded using an infrared video-based ISCAN  eye tracker with a reported accuracy of 0.3° over a ±20° horizontal and vertical range. Symmetrical convergence step stimuli of from 2.0 to 6.0 deg angular vergence demand  were produced using vertical lines projected on two computer screens placed 40 cm from the subject arranged as a haploscope. These lines were driven to produce a step change in vergence demand by a custom software package . The stiVergence responses were computed as the difference between separately calibrated left and right eye movements using the calibration data taken before and after each response. A typical ensemble of vergence movements is shown in Isolating the slow component from the total vergence response is the first step in our analysis. While methods have been developed using independent component analysis to separate the initial and slow components, they operate on a group, or ensemble of movements, and identify component averages across the group , 29–31. Independent component analysis has shown that the early initiating component is much faster than the sustained component , 32, 33.To estimate when the slow component becomes dominant, we search the velocity trace for a point where the smooth downward curve of the velocity trace either reverses or changes slope, As the isolated segments may contain eye movement positional drift that will produce artifacts in our frequency analysis, we detrend the isolated segments using a quadratic function. A least-squares analysis is used to determine the quadratic function that best fits the isolated segment. This function is then subtracted from the segment. This will reduce the influence of drifts but will not affect oscillatory behavior.To obtain slow component frequency characteristics, the Fourier transform was applied to the isolated, detrended segments. The discrete Fourier transform implicitly assumes that the data consist of one cycle of a periodic signal. Discontinuities at the two end-points will produce artifacts in the resulting spectrum. Accordingly, it is common to apply a tapering window to truncated data before applying the Fourier transform. We used the Tukey window shown in Simulations of a basic feedback control system were used to aid in the interpretation of the slow component frequency characteristics. The model shown in The controller was taken from an early model of the vergence system by Krishnan and Stark (1977) and contains both a derivative element and a feedforward gain in addition to a time delay representing the response latency. The derivative element contains a direction dependent asymmetry that is responsible for most of the higher harmonics found in the data. This combination essentially produces a pulse signal that is proportional to the delayed vergence error. The derivative component and its asymmetry could be implemented using internal feedback. The feedforward gain, derivative gain, and response latency could be varied during simulations.The large primary peaks found in all responses ranged between 1.0 and 2.0 Hz and they indicate the presence of an oscillatory process. These oscillations are likely due to the marginal stability of the slow component feedback system. Examination of the secondary peak frequencies show that they are closely related to the primary peak frequency. A summary of the data in There are several additional measurements that can be extracted from the spectral data. The fundamental peaks indicate the frequency of the primary oscillatory behavior and the value of this frequency is summarized in Isolated slow component frequency characteristics always included harmonics of the fundamental frequency. The peaks representing these harmonics were smaller than the fundamental peak. The ratio of the magnitude of the second harmonic with respect to the fundamental varied as shown in The amplitude of these oscillations is small. In To show that these spectral features can be produced by a feedback control system compatible with vergence components, we compare simulations of the model with our experimental results. The range of averages for the four variables shown in The slow component feedback model input takes as its input the vergence error at the end of the initial component, the “Initial Component Error” in sf = 400 Hz) and simulation time responses were windowed using the Tukey window before applying the Fourier transform. The detrend operator was also applied to simulation responses, but since these responses contained no noise or drift, this operation had no effect.The responses obtained through model simulations were analyzed in a manner identical to that used on experimental data. The simulation routine used the same sampling frequency  was surprising. While direction dependent nonlinearities did produce harmonics, the magnitude of these harmonics was less than that seen in some subjects. An asymmetrical derivative element was the only element that was found to produce a substantial second harmonic, although it is possible there are other, untried nonlinearities that are as effective.An asymmetrical derivative element may seem like an unusual element in the vergence control pathway, but in fact it has the same action as the pulse signal assumed to drive the fusion initiating response. In the slow component neurological control system, small vergence errors would be translated into small unidirectional pulses by the derivative and the amplitude of such pulses would likely be different for convergent versus divergent pulses. While the model used here featured continuous, smoothly varying signals, it is probable that the slow component, like the initial component, is driven through a pulse-like signal. A more realistic model developed around pulse-like signals is another concept for future work.An extensive search was made for correlations between parameters. For example, the subject with the largest oscillatory behavior, NAC  had the Oscillatory behavior was found in the isolated slow component segments of all responses in all subjects. A frequency analysis of this behavior showed fairly consistent spectral characteristics with a large lower frequency peak indicating a fundamental frequency and one or more higher frequency peaks. A significant correlation was found between the frequency of the higher frequency peaks and the fundamental frequency indicating that the higher frequency peaks were harmonics of the fundamental frequency. The various spectra differed only in the frequency of the fundamental component and the relative magnitude, and occasionally the number of harmonics.A simple feedback control model was able to represent the basic spectral features found experimentally. The fundamental frequency was found to be largely determined by the delay element and the harmonics to be generated by an asymmetrical derivative element in the model’s controller. Many candidate nonlinearities were evaluated for production of harmonic frequency components and while many produced some harmonics, the magnitude of these harmonics did not match that found experimentally. The asymmetrical derivative element functions essentially as pulse generator in response to small vergence errors. Such a component might be found in any neurological control system and may, in fact, be a major component in the vergence slow component control system.The authors declare that the contents of the article are in agreement with the ethics described by the Journal and that there is no conflict of interest regarding the publication of this paper.


They either release Ca2+ from endogenous Ca2+ stores, e.g., endoplasmic reticulum or acidic organelles, or evoke Ca2+ entry by directly activating a Ca2+ channel in the plasma membrane. For 25 years, Professor Barry Potter has been one of the major medicinal chemists in this topical area, designing and contributing numerous analogues to develop structure–activity relationships (SAR) as a basis for tool development in biochemistry and cell biology and for lead development in proof-of-concept studies in disease models. With this review, I wish to acknowledge our 25-year-long collaboration on Ca2+-mobilizing adenine nucleotide second messengers as a major part of Professor Potter’s scientific lifetime achievements on the occasion of his retirement in 2020.Ca A ‘genius’ is defined as, “very great and rare natural ability or skill, especially in a particular area such as science or art, or a person who has this” ; truly P“Ca2+ signaling controlled by Ca2+-mobilizing adenine nucleotide second messengers—that’s a cool innovative idea that should be pursued—it just needs an interesting cell system and novel tools developed by medicinal chemistry”, Barry Potter said while having lunch in a small Italian–Swiss restaurant in Hamburg in fall of 1994. Indeed, our over-lunch conversation turned out to be the starting point of a very fruitful 25-year-long collaboration with the first paper, showing Ca2+ release by cyclic adenosine diphosphoribose (cADPR) in T-lymphocytes, published in 1995 [2+-mobilizing adenine nucleotide second messenger discovered in the 1990s: another even more potent compound, nicotinic acid adenine dinucleotide phosphate (NAADP), was reported in 1995 by Professor Hon Cheung Lee’s laboratory [2+-mobilizing adenine nucleotide second messenger, adenosine diphosphoribose (ADPR), discovered as an agonist of the cation channel transient receptor potential, melastatin subfamily type 2  as a basis for tool development for biochemistry and cell biology and for lead development in proof-of-concept studies in disease models, such as experimental autoimmune encephalomyelitis (EAE).2+-mobilizing adenine nucleotide second messengers, but I wish to highlight the milestone studies developed and published in collaboration with Barry.Without being able to fully estimate what our collaboration has meant to Barry’s career, I can say that it influenced my own scientific curriculum significantly. Therefore, this review is not meant to systematically cover the world of Cad-myo-inositol 1,4,5-trisphosphate (IP3) as a principal second messenger evoking Ca2+ release from intracellular stores in 1983 [2+ signaling has been filled with more and more scientific results and conclusive concepts. This was the case, since rapidly the IP3 signaling pathway was defined by the discovery of the key proteins involved: phospholipase C, IP3 kinase and IP3 phosphatase, as well as the IP3 receptor(s). Further, IP3, as a signal to connect the stimulation of plasma membrane (PM) receptors with intracellular Ca2+ signaling, fueled research regarding other small molecules having similar functions in cells.Since the discovery of Indeed, cADPR and NAADP were soon discovered and structurally defined ,6, follo2+ signaling, besides other signaling pathways (T cells are activated during an infection by antigenic peptides presented by antigen-presenting cells (APC), such as dendritic cells, macrophages or B-lymphocytes. In auto-immunity, auto-antigenic peptides replace the foreign peptides. The term ‘T cell activation’ relates to all biochemical and cell biology processes following the formation of a juxtacrine intimate contact between an APC and the T cell, termed the immunological synapse or immune synapse. Upon immune synapse formation, we can distinguish between different subsequent stages of T cell activation . Signal pathways . Ultimatpathways . 2+ signaling can be further subdivided into NAADP- and ORAI1-dependent local Ca2+ signals [3-dependent global Ca2+ signaling, and cADPR and ryanodine receptor (RYR)-dependent global Ca2+ signaling  signal onset was delayed, and both peak and plateau amplitudes of Ca2+ signaling were decreased partially [3Rs and RYRs by local Ca2+ increases evoked normally by NAADP. Consistently, downstream T cell activation events, such as nuclear translocation of NFAT-1 or IL-2 expression, were also partially inhibited by BZ194. Importantly, the partial inhibition of proliferation by BZ194 was bypassed using a low concentration of ionomycin together with anti-CD3/anti-CD28 monoclonal antibodies. Here, the missing local, NAADP-evoked Ca2+ signals were restored by Ca2+ ionophore ionomycin, resulting in full proliferation [The significant drop of initial Ca2+ signaling in T cell-mediated autoimmunity of the central nervous system (CNS) [BZ194 was then used to characterize the role of NAADP-evoked Caem (CNS) . NAADP aem (CNS) .2+ signaling in T cells  [2+ entry (SOCE) can continue in the absence of IP3-evoked Ca2+ release, since SOCE is mechanistically triggered by a decrease in the luminal ER-Ca2+ concentration, usually resulting from second messenger-evoked Ca2+ release [As introduced above, IPy 15 min . This re release .3 at later time points of T cell activation, e.g., approximately >15 min post stimulation. For this, we characterized cADPR-evoked Ca2+ release in permeabilized T cells [2+ entry in intact T cells [2+ release that subsequently activated SOCE [2+ imaging experiments a major role for cADPR in sustained Ca2+ entry, in the expression of T cell activation markers, and in T cell proliferation [2+ entry was also demonstrated by combined microinjection and Ca2+ imaging experiments with antagonists developed and synthesized by Barry, Potter, e.g., cADPR antagonist 8-OCH3-cADPR and IP3 antagonist inositol 1,4,6-phosphorothioate [2+ release via RYR2 and/or RYR3, activates SOCE in T cells ; moreover, he convinced brilliant chemists, like Professor Satoshi Shuto , who spent time as a guest scientist in Barry’s lab at the University of Bath, to make further complicated analogues, e.g., the carbocylic and thioribose derivatives at the northern ribose of cADPR.Collaborating on the cADPR structure–activity relationship with Barry Potter was an ultimate pleasure. Not only did he make numerous analogues, many of which proved to be very helpful to decipher T cell Ca2+ signaling, the molecule ADPR. Initially, ADPR was simply seen as degradation product of NAD and cADPR, formed by NAD-glycohydrolase/ADP-ribosyl cyclase CD38. However, Peraud et al. demonstrated in 2001 that TRPM2 contained a domain with homology to the nudix family of nucleotide hydrolases, termed the NUDT9-homology domain [As mentioned in the first paragraph, besides NAADP and cADPR, another endogenous adenine nucleotide was shown to be a second messenger in Cay domain . The NUDy domain .+ outward currents at +15 mV were used to quantify antagonism of ADPR-evoked TRPM2 activation [50 of approximately 10 μM [50 = 3 μM) [In 2008/09, Barry Potter, his co-workers, and members of my lab discussed synthesis of ADPR analogues for a full structure–activity relationship of ADPR. In a systematic approach, the synthesis of > 30 ADPR derivatives with modifications in each of the four motifs, (i) adenine base, (ii) adenosine ribose, (iii) pyrophosphate bridge, and (iv) terminal ribose, was carried out . Using rtivation . ADPR anly 10 μM . This an = 3 μM) . Modific = 3 μM) .Then, an almost identical set of ADPR analogues was used in the same experimental setting to explore the agonist structure–activity relationship of ADPR . Since rCd38−/− T cells suggest the formation of this molecule by NAD-glycohydrolase activity of CD38 [A second aspect of this study was the possible 2nd messenger role of 2′-deoxy-ADPR: using HPLC and mass spectrometry, endogenous 2′-deoxy-ADPR was not only detected in unstimulated Jurkat T-lymphocytes, but a T cell receptor/CD3-mediated increase in 2′-deoxy-ADPR was discovered for the first time . Here, B of CD38 .Further studies on the structure–activity relationship of ADPR followed, showing a critical role for OH-groups at C1”, C2” and C3” at the terminal ribose for agonist properties of ADPR  and demo2+ signaling. While IP3 followed by IP3-initiated/mediated activation of SOCE was regarded as the whole story in the 1990s, we added both NAADP and cADPR signaling as essential components of T cell activation. Further, we discovered 2′-deoxy-ADPR as a novel 2nd messenger activating TRPM2 much more effectively than ADPR.In a true scientific collaboration, Barry Potter, myself and both our teams drastically changed the view of T cell CaLooking back to the past 25 years with the many personal interactions Barry and I had, either in Bath, Hamburg or during scientific meetings and conferences, I am delighted to say that he has been one of the most important collaborators I ever had during my scientific career.I well remember one of our last personal meetings during the International Association for Cellular Coenzymes (IACC) Inauguration Symposium  at Dillon’s Coffee located at the corner of Gower Street/Torrington Place in summer 2018. Sitting outside in one of the congress breaks in bright sunshine with a cappuccino, Barry said: “Really fortunate that we met and started our collaboration many years ago…with excellent results, but more important with a lot of fun and friendship!”


The major aim of this study was to gain a better knowledge on the NAGly-dependent impairment of SOCE in neurons of the central nervous system (CNS) from mice. First, we examined the expression of genes encoding for putative lipid sensing GPCRs using transcriptomic data publicly available. This analysis showed that the most abundant GPCRs transcripts present in the cerebral cortices of embryonic brains were coding for lysophosphatidic acid (LPA) and sphingosine-1 phosphate (S1P) receptors. Next, the presence of functional receptors was assessed with live-cell calcium imaging experiments. In primary cortical cells S1P and LPA mobilize Ca2+ from internal stores via a mechanism sensitive to the S1P and LPA receptor antagonists Ex26, H2L5186303, or Ki16425. However, none of these compounds prevented or attenuated the NAGly-dependent impairment of SOCE. We found no evidence for the requirement of lipid sensing GPCRs in this inhibitory process, indicating that NAGly is an endogenous modulator interfering with the core machinery of SOCE. Moreover, these data also raise the intriguing possibility that the depression of SOCE could play a role in the central effects of NAGly.N-arachidonoyl glycine (NAGly) is an endogenous lipid deriving from the endocannabinoid anandamide (AEA). Identified as a ligand of several G-protein coupled receptors (GPCRs), it can however exert biological responses independently of GPCRs. NAGly was recently shown to depress store-operated Ca This endogenous bioactive molecule influences pain perception and displays analgesic properties5. This led to the hypothesis that NAGly could be a natural modulator of pain6. The analgesic actions of NAGly have been studied principally on dorsal root ganglia (DRG) neurons and dorsal horn neurons from spinal cord slices7. For instance, NAGly inhibits low threshold voltage-gated Ca2+ channels (Cav3) in DRG neurons8 and the glycine uptake transporter GLYT2 in dorsal horn neurons, which contributes to enhance inhibitory glycinergic synaptic transmission in these cells7. NAGly has also been shown to depress excitatory NMDA-dependent synaptic transmission7. The effects of NAGly on neurons of the brain have however been much less characterized. In primary cortical neurons NAGly releases Ca2+ from the endoplasmic reticulum (ER), potentiates the passive leakage of Ca2+ out of the ER, and impairs the store-operated Ca2+ entry (SOCE)9.N-arachidonoyl glycine (NAGly) is a lipid deriving from the endocannabinoid anandamide (AEA). It is naturally present in various tissues and organs like the brain10, GPR5511, and GPR9212. It is however worthy of note that NAGly can influence the activity of some effectors without the requirement of GPCRs. This is for instance the case for the NAGly-dependent regulation of voltage-gated Ca2+ channels and Na+/Ca2+ exchanger activity14.One key issue when addressing the question of the cellular effects of NAGly is to determine whether it interferes directly with the activity of its targets or recruits dedicated G-protein coupled receptors (GPCRs) linked to downstream intracellular signaling cascades. NAGly has been proposed to act as an agonist of some orphan GPCRs like GPR182+ route of the plasma membrane. It is activated in response to the depletion of the ER Ca2+ stores15 and involves distinct actors: stromal interacting molecules (STIM1–2) and Orai1–3 channels18. STIM are ER resident proteins that seem to function as Ca2+ sensors19. The depletion of the ER Ca2+ stores governs the molecular interaction between STIM and plasma membrane Ca2+ channels of Orai type that are responsible for the SOCE response18. Recent studies identified NAGly as a potent inhibitor of SOCE in various cell lines  and in primary cultured neural cells21. Two hypotheses were put forward to explain the NAGly-dependent depression of SOCE20: (1) direct disruption of the coupling between STIM and Orai, or (2) recruitment of an intracellular signalling cascade activated downstream to NAGly-sensitive receptors and regulating negatively SOCE activity. The aim of the present work was to verify whether a lipid sensing GPCR is contributing to the NAGly-induced impairment of SOCE in cortical neurons. First, we analyzed a recent publicly available transcriptomic dataset obtained by RNAseq22 to characterize the expression of genes encoding for putative lipid sensing GPCRs in the cerebral cortices of embryonic mice. The most abundant transcripts were coding for lysophosphatidic acid (LPA) and sphingosine-1 phosphate (S1P) receptors. After having checked the presence of functional receptors, the contribution of LPA and S1P receptors to the NAGly-dependent inhibition of SOCE was evaluated using a pharmacological approach.SOCE is an important physiological CaC57Bl6/J (stock #000664) mice were obtained from the Jackson Laboratory (USA). They were housed in a temperature-controlled room under a 12 h light–12 h dark cycle with ad libitum access to food and water. An environmental enrichment consisting in the addition of tunnels was provided for all animals in accordance with the Animal Welfare Committee of the CEA Grenoble. Experimental procedures were approved by the animal care committee of the CEA’s Life Sciences Division . Experiments were conducted in compliance with the French legislation and the European Community Council Directive of 24 November 1986 (86/609/EEC).24. Briefly, tissues were placed in a 1.5 mL Eppendorf tube containing 1 mL of an ice-cold Ca2+- and Mg2+-free Hank’s solution supplemented with 33 mM glucose, 4.2 mM NaHCO3, 10 mM HEPES, and 1% penicillin/streptomycin. Cells were isolated by a mechanical trituration of the medium containing the cerebral cortices. The cell suspension was filtered through a 40-µm cell strainer before plating the cells on 16 mm ∅ glass coverslips. They were kept in a Neurobasal medium supplemented with B27 (2%) and glutamine (500 µM) and maintained in a 5% CO2 atmosphere at 37 °C. All the experiments were conducted on cells kept 2 or 3 days in vitro.Cells were dissociated from cerebral cortices collected from embryonic (E13) mice  according to2, 2 CaCl2, 5.5 glucose, 10 HEPES . LPA- and S1P-induced Ca2+ responses were analyzed with Fluo-4. Cells were incubated with 5 μM Fluo-4/AM for 20 min following procedures described previously24. Images were obtained by a CCD CoolSnap HQ2 camera  mounted on an inverted Zeiss A1 microscope . Cells were excited at 470 nm and emission was collected at 525 nm using a DG-4 wavelength switcher . MetaFluor  was used for image acquisition and analysis. All experimental procedures were conducted at room temperature. Time-lapse changes in Fluo-4 fluorescence intensity were collected at a frequency of 0.2 Hz from 30–45 cell bodies per dish and analyzed off-line by defining regions of measurements. Results were expressed as F/F0, with F being the fluorescence at each time point and F0 being the mean baseline fluorescence that was monitored at the beginning of each experiment for 1 min before the addition of any substance. Culture dishes were discarded at the end of the recording and never re-used. A positive LPA (or S1P)-induced calcium response was determined as one F/F0 greater than 0.02 that develops within 50 s upon the application of the agonist. Fluo-4 responses were measured as area under curve (AUC).The culture medium was removed and replaced by a saline containing (in mM) 150 NaCl, 5 KCl, 1 MgCl2+ probe Fura-2 was used to study store-operated Ca2+ entry (SOCE). The experimental conditions and setup were as above except that cells were incubated with 2.5 µM Fura-2 for 20 min at room temperature. They were then washed twice and kept in a Fura-2-free saline solution for >12 min at room temperature. A dual excitation at 340 and 380 nm was used and emission was collected at 515 nm. Images were acquired at a frequency of 0.2 Hz and analyzed off-line. The classical “Ca2+ add-back” protocol was used to study SOCE. Cells were bathed in a nominally Ca2+-free saline containing (in mM) 150 NaCl, 5 KCl, 3 MgCl2, 5.5 glucose, 10 HEPES . SOCE activation was triggered by depletion of the ER Ca2+ pool with 200 nM thapsigargin, which induced a transient elevation in intracellular Ca2+ concentration before re-admission of 2 mM external Ca2+. SOCE responses were analyzed in cells generating a rapid Ca2+ rise upon the application of a depolarizing saline containing 90 mM KCl. In cultures of embryonic cortical cells, KCl responding cells are identified as neurons whereas KCl-unresponding cells are considered as non-neuronal cells25. The depolarizing (K+ rich) medium had the following composition (in mM): 65 NaCl, 90 KCl, 1 MgCl2, 2 CaCl2, 5.5 glucose, 10 HEPES . Ca2+ changes as a function of time were expressed as delta ratio F340/F380 whereas total Ca2+ responses were measured as area under curve (AUC).The fluorescent Ca2+ signals, at least at the concentrations used.Stock solutions of Ex26, Ki16425, and BTP2 were prepared in dimethyl sulfoxide (DMSO). Methanol and ethanol were used for preparing stock solutions of S1P and NAGly, respectively. These stock solutions were diluted at least 1000-fold into the recording saline immediately before use so that the final concentration of vehicle never exceeded 0.1%. Control experiments were performed with DMSO, ethanol and methanol alone. None of the solvent altered cytosolic Ca22. Raw fastq files are publicly available and can be found on the GEO repository under accession number: GSEXXX.The RNASeq gene expression data derive from2+ imaging experiments, all experiments were done ≥3 times  using distinct dishes from different batches of cells (e.g. from distinct pregnant mice). Data are presented as means ± standard error of the mean (SEM) with n being the number of biological replicates. SigmaPlot  and SigmaStat  were used for plotting graphs and statistical analysis, respectively. Differences between several groups of cells were tested using one-way analysis of variance (ANOVA) followed by a Bonferroni’s post hoc test. A P value < 0.05 was considered statistically significant.Each experimental condition as well as its appropriate control were tested on the same batch of primary neuronal cell cultures. For the CaFluo-4/AM, Fura-2 and tissue culture media were from Molecular Probes . N-arachidonoyl glycine (NAGly) was from Tocris . All the other reagents were obtained from Sigma-Aldrich (France).30. The transcriptomic data were extracted from a recent RNAseq study22. The expression pattern of putative lipid sensing GPCRs was analyzed at 3 embryonic ages: E11, E13 and E17. Only genes for which the number of transcripts per million (TPM) was >2 were considered as significantly expressed31, therefore when the number of transcripts was <2 TPM, the gene was eliminated from the analysis. This resulted in the selection of 14 genes encoding for putative lipid sensing GPCRs  (Cnr1 gene), the orphan receptor GPR12, lysophosphatidic acid (LPA) and sphingosine-1 phosphate (S1P) receptors  and possess voltage-gated Ca2+ channels38, indicating that most cells display a post-mitotic neuronal phenotype. First, the presence of functional LPA and S1P receptors was assessed in the entire cell population. LPA  and ⁓13% (39 out of 303) of the cells tested, respectively. The LPA-induced Ca2+ signals were partially blocked by 10 µM H2L5186303, a selective LPA2/3 receptor antagonist39, and nearly completely suppressed by 10 µM Ki16425, a LPA1/2/3 receptor antagonist42 43 reduced the peak amplitude of the S1P-induced Ca2+ signals and diminished the number of responsive cells with only 12 cells out 220 tested (⁓5%) generating a Ca2+ signal in response to 10 µM S1P  responding to high-K+ whereas non-differentiated cells are not high-K+ sensitive. In cultures of embryonic cortical cells, KCl responding cells are identified as neurons whereas KCl-unresponding cells are considered as non-neuronal cells25. Overall, only 10 of 67 LPA sensitive cells (⁓15%) generated an intracellular Ca2+ rise in response to KCl. These data are consistent with a previous report showing that in the embryonic cerebral cortex LPA receptors are predominantly expressed by neural precursor cells with only a small minority of neurons responding to LPA35. On the other hand, 5 of 25 S1P sensitive cells (20%) were KCl-responsive cells. This indicates that the S1P-sensitive cells are also mainly found in KCl-insensitive cells34. Taken together, LPA or S1P mobilizes Ca2+ from the ER in a subset of cells (<20%). These functional LPA- and S1P-sensitive receptors are essentially expressed by non-neuronal cells44.Previous reports showed that LPA and S1P receptors are mainly found in proliferative regions of the immature cerebral cortex, with few post-mitotic neurons responding to LPA and S1P2+ probe Fura-2 was used to analyze SOCE in cells that responded to the KCl challenge (i.e. post-mitotic neurons). Cells, bathed in a nominally Ca2+-free medium, were challenged with thapsigargin to deplete ER Ca2+ stores. A subsequent re-admission of external Ca2+ was followed by an intracellular elevation of Ca2+  or Ki16425    Fig. . The SOC+-rich saline (90 mM KCl) before recording SOCE responses in neurons (i.e. in KCl-responsive cells). Figure 9, NAGly exerts complex actions on neuronal Ca2+ signalling: (i) it induces a release of cations (Ca2+ and Zn2+) that develops prior to thapsigargin addition ; and (iii) reduces the amplitude of SOCE (phase ③). Even in the presence of 1 µM Ex26 (gray up triangles) or 10 µM Ki16425 . The NAGly-induced inhibition of SOCE (phase ③) was also not affected by Ex26 or Ki16425  observed without NAGly (white bars), with NAGly (gray bars), NAGly + Ki16425 (black bars), NAGly + Ex26 (black/white bars), and NAGly + BTP2 (hatched bars) were analyzed as area under curve (AUC) . These blockers also did not influence the potentiation of the thapsigargin-induced Ca2+ signal induced by NAGly (phase ➁) and the NAGly-dependent depression of SOCE (phase ③). It is proposed that NAGly inhibits a BTP2-sensitive Ca2+ entry pathway without recruiting LPA or S1P-sensitive receptors.The Fura-2 CaUC) Fig. . In conc20. This impairment has been observed in every cell type and cell line tested so far like fibroblasts, neurons, EA.hy926 , INS-1 832/13 (rat pancreatic β-cell line), and RBL-2H3 cells (rat basophilic leukemia cell line)21. However, the mechanism by which NAGly alters SOCE is unclear. In the present study we addressed the question of the contribution of lipid sensing GPCRs as targets of NAGly with the aim to gain a better knowledge on neuronal SOCE functioning and regulation. To reach that goal, we took advantage of a recent transcriptomic analysis of the whole murine genome by RNA-seq.22. This allowed us to consider the mRNA expression of 60 putative lipid sensing GPCRs30. Overall, transcripts of 14 genes (⁓25%) were detected. Their abundance varied during embryonic development with 7 genes being induced  and 7 genes being repressed . At E13, age at which cerebral cortices were collected to perform the Ca2+ imaging experiments9, the most abundant mRNAs were those coding for CB1 and S1P1 receptors, followed by LPA2 and LPA6 receptors. Since the cannabinoid receptor CB1 does not seem to mediate the NAGly-dependent impairment of SOCE9, only the contribution of S1P and LPA receptors in the NAGly-mediated modulation of SOCE was investigated.NAGly inhibits SOCE1–5). They belong to the group of GPCRs and mediate most of the biological actions of the bioactive sphingolipid S1P30. Embryonic cerebral cortices displayed a high mRNA level of S1P1 receptors that declined during embryonic brain development. In addition, cultured cortical cells expressed functional receptors coupled to the release of Ca2+ from the ER and sensitive to the S1P1 antagonist Ex26. These findings are in line with previous reports showing that S1P1 is the major S1P receptor of the murine embryonic brain, followed by S1P2 and S1P3 receptors. It is detected as early as E14, highly expressed in proliferative regions (neurogenic ventricular zone) but its expression decreases at E16 and E1847. The activation of S1P1 receptors is coupled to the mobilization of Ca2+ 33.Five subtypes of S1P receptors are known , and via a mechanism insensitive to the pertussis toxin9. It is worth recalling that NAGly regulates voltage-gated Ca2+ channel activity without acting on GPCRs13. Although we cannot exclude the possibility that NAGly acts on an orphan lipid sensing GPCR that was not considered in the present study, our report suggests that NAGly disturbs the coupling of the core components of the SOCE machinery (STIM-Orai)20. This inhibitory process does not seem to develop in response to an intracellular signalling cascade. These past9 and present data show that the phytocannabinoid cannabidiol, the endocannabinoid AEA and its derivative NAGly are potent inhibitors of neuronal SOCE. This indicates that NAGly and endocannabinoids are endogenous SOCE modulators, and raises the possibility that the depression of SOCE could play a role in the neuro-behavioural effects of cannabinoids and signalling lipids.In conclusion, our data show that NAGly inhibits a BTP2-sensitive Ca