It is often assumed that only stably docked synaptic vesicles can fuse following presynaptic action potential stimulation. However, during action potential trains docking sites are increasingly depleted, raising the question of the source of synaptic vesicles during sustained release. We have recently developed methods to reliably measure release latencies during high frequency trains at single synapses between parallel fibers and molecular layer interneurons. The latency distribution exhibits a single fast component at train onset but contains both a fast and a slow component later in the train. The contribution of the slow component increases with stimulation frequency and with release probability and decreases when blocking the docking step with latrunculin. These results suggest that the slow component reflects sequential docking and release in immediate succession. The transition from fast to slow component, as well as a later transition to asynchronous release, appear as successive adaptations of the synapse to maintain fidelity at the expense of time accuracy. Traditionally, synaptic release latency has been viewed as constant. Here, at parallel fiber-interneuron  synapses, authors see that with sustained release emerges a slow component of increased latencies, which can be simulated with a 2-step model comprised of vesicle docking followed by release. This jitter reflects the short period of time when the presynaptic calcium concentration ([Ca2+]) transient in the vicinity of SVs is large enough to elicit release, as well as the subsequent fusion steps4, and potentially contains key information on synaptic function. To make precise estimates of the distribution of SV release latencies, deconvolution methods have been applied, taking the mean miniature current waveform as kernel5. This method showed that the latency distribution displays a single narrow peak, with a half amplitude duration near 400 μs at room temperature at the calyx of Held synapse6.Following action potential (AP) stimulation, the release of synaptic vesicles (SVs) occurs with a jitter that was estimated around 1 ms at the frog neuromuscular junction at room temperature8. Accordingly, only modest changes of latency distributions have been reported in the calyx of Held9 and endbulb of Held10 during train stimulations. However, central synapses display marked differences in performance during train stimulations11. Many central synapses respond to AP bursts at high frequency14, raising the question of how the potentially conflicting demands of time accuracy and fidelity are reflected at the level of latency distributions during a train.Apart from a scaling factor according to release probability or to previous synaptic activity, synaptic latencies have classically been considered constant2+ from release-inducing reaction sites is fast and if the time course of local [Ca2+] transients at release sites is constant15. On the other hand, long identified reasons to deviate from this scheme include frequency-dependent AP broadening during trains (review: ref. 16) and broadening of local [Ca2+] following accumulation of Ca2+ and/or saturation of fast endogenous buffers (review: ref. 17). Another potentially relevant factor is the relation between release kinetics and the distance between SV docking site (DS) and voltage-gated Ca2+ channels (VGCCs)19. If this distance varies among docked SVs, latencies gradually grow during trains due to an increasing participation of low release probability, slow SVs20 (review: ref. 21). In addition, recent evidence from statistical analysis of release22 and flash-and-freeze experiments23 indicates ultrafast SV recruitment to DSs, occurring within <10 ms. Such ultrafast docking may elicit a distinct component of release latencies depending on the proportion of docked and undocked SVs. Yet another possible mechanism for latency changes is a slowing of the release step following alterations of the presynaptic structure associated with previous synaptic release24 (review: ref. 25).The classical view that the shape of latency distributions is nearly constant applies if dissociation of Ca26. Asynchronous release, like synaptic facilitation, depends on the accumulation of residual Ca2+ during the AP train, and the two processes may share a common underlying mechanism27. In spite of these similarities between facilitation and asynchronous release, it has been suggested that facilitation and asynchronous release each rely on a specific Ca2+ sensor , but so far no consensus has emerged concerning the molecular nature of these potential sensors26.A well-documented phenomenon involving a strong change in synaptic latency is ‘delayed release’, also called ‘asynchronous release’, where release extends for 10 s of ms to 10 s of s following the end of an AP train30). In this model, latency broadening, synaptic facilitation, and asynchronous release rely on a unique sequence of SV docking and release.In the present work, we investigate the timing of SV release at individual small synapses. During high frequency trains, we find a broadening of release latencies that is much more marked than previously reported at giant synapses, particularly under high release probability conditions. We suggest that latency slowing is due to the insertion of an ultrafast docking step before exocytosis. More generally, the results support a model of SV release involving the sequential transition of SVs to two consecutive states before exocytosis (2-step model: refs. 31). Variance-mean analysis has shown that each simple synapse has 2–8 DSs31. Using deconvolution, we determined the latency of individual SV release events31  and postsynaptic molecular layer interneurons (MLIs) in cerebellar brain slices Fig. . When st2+], the latency distribution exhibits a rapid rise for about 200 μs, followed by an exponential decay with a time constant of 362 ± 10 μs , giving a maximum local [Ca2+] slightly >40 μM . We used an allosteric Ca2+-sensor model for release41 . Figure To investigate cellular mechanisms underlying jitter, we simulated SV release in single PF-MLI varicosities. These simple synapses involve a single AZ but several DSs AP Fig. . The amo μM Fig. . By compude Fig. , right;  AZ Fig. . We furte41 Fig.  and the  DS Fig. . This da22 , both paired-pulse ratio (PPR) and covariance should follow the recovery of δ as a function of inter-AP interval, displaying the same time course. In accord with this prediction, PPR and covariance displayed similar kinetics , distributions for facilitated responses to the second AP are biexponential, and they display in addition to τfast a slower component with time constant τslow = 6.81 ms , indicating that a monoexponential model is better. By contrast, when examining the response to a second AP, we found a substantially better (more negative) score with a biexponential fit (BIC: −576.2) than with a monoexponential fit . The present finding of a slow latency component for responses to the second AP is consistent with a previous report of a slower average EPSC decay after the second AP than after the first AP45. Also in agreement with previous results on EPSC kinetics45, the amplitude of the slow component fell off with interstimulus interval in parallel with the extent of facilitation  to induce high SV turnover ; a satisfactory fit was achieved by keeping common values for f increases due to a cumulative increase in global [Ca2+]. If the τfast component arises from docked SVs, its contribution should decrease together with δ. Meanwhile if the τslow component reflects 2-step docking/fusion events, its contribution should increase as Rf and free DSs' availability increase. In accordance with these expectations, we found that the amplitudes of τfast and τslow components varied in opposite directions, so that the τslow component exceeded the τfast component near the end of a train , we also found 2 kinetic components . Amplitudes of fast and slow components varied in opposite directions during AP trains, and the τslow component became dominant at the end of a train , we considered the possibility that the  rats, wef (ref. 22). If the τslow component is due to 2-step release, it should be affected by inhibiting Rf. In agreement with this prediction, we found that application of LatB (15 μM) resulted in a sharp reduction of the τslow component , the τfast to τslow crossover occurred later in a train, shifting from i = 3 under control conditions to i = 6 in LatB  = 2.46, p = 0.027; Fig. τslow component and asynchronous release rely on the docking step Rf.Next, we assessed the effect of the actin polymerization inhibitor latrunculin B (LatB) that blocks facilitation by inhibiting the docking step RatB Fig. . Interes2+ entry per AP on τfast and τslow components. If the τslow component is due to 2-step release, it should vary with the amount of Ca2+ entry as Rf increases with global [Ca2+]. In agreement with this prediction, we found that reducing the external [Ca2+] from 3 to 1.5 mM decreased the contribution of τslow  = 2.55, p = 0.037; Fig. We next examined the impact of changing the amount of Calow Fig. . Meanwhi= 6 Fig. . As expeτslow component relies on Ca2+-dependent Rf increase, its amplitude should decrease when reducing the stimulation frequency, as the global [Ca2+] is then reduced. In agreement with this prediction, reducing the AP frequency from 200 Hz 46. With 100 Hz stimulation, the crossover point shifted back to the left, to i = 3  = 5.36, p = 0.001; Fig. If the  Hz Fig. , left to Hz Fig.  led to a= 7 Fig. , left. T= 3 Fig. , right, τfast ~0.5 ms) to another one displaying a slow time constant (τslow ~2 ms). Blocking SV docking with LatB, decreasing Ca2+ entry and decreasing stimulation frequency all delay the transition from τfast to τslow during an AP train. Conversely, increasing Ca2+ influx or increasing stimulation frequency accelerates this transition. Altogether, latency broadening increases with the intensity of previous synaptic activity and with release probability and depends on an intact cytoskeletal structure. All of these changes support the notion that the τslow component results from 2-step release. In addition, asynchronous release follows the same trends as the percentage of τslow component, suggesting that slow release and asynchronous release share a common underlying mechanism.In summary, latency distributions become markedly slower during AP trains, gradually shifting from a process exhibiting a fast time constant . To model the change of synaptic function following previous synaptic activity, that we call ‘fatigue-induced slowing of release’ below, we altered the parameters describing the release of docked SVs, making the activation steps slower .Like facilitation, asynchronous release depends on global [Ca2+ entry associated with one AP results in a sizable global [Ca2+] elevation  with asynchronous release counts in the following 30 ms time window (time window: 5–35 ms after the last AP). If phasic and asynchronous release are independent of each other, the covariance between these two counts should be nil. If on the other hand the two types of release compete for a common limited resource, for example by sharing the same release sites, the covariance should be negative43. We found a negative covariance between s8 and asynchronous release, both in 3 mM [Ca2+] at 200 Hz and in 1.5 mM [Ca2+] and 1 mM TEA at 100 Hz . By contrast, no correlation was found between s8 and asynchronous release measured in a later window  than for replacement SVs (τRS). Average τDS and τRS values across an AP train are close to the τfast and τslow values determined in Fig. τfast and τslow components, respectively, represent direct release from DSs and indirect release from replacement sites. In addition, both τDS and τRS increase about two-fold during an AP train  and DS (δ) are illustrated . However, our simulations indicate that if the Ca2+ sensitivity of release is kept constant, the distances needed to explain the observed τslow values are >150 nm  that would differ in their release probability, as proposed for primed vs. superprimed SVsτfast and τslow reflects the extra time that is taken by the SV to first move to the DS before being released. For isolated stimuli under standard conditions, the movement of replacement SVs towards the DS occurs too late to overlap significantly with the AP-induced [Ca2+] transient, so that only a τfast component is observed. But if Rf increases due to strong or repeated Ca2+ entry and/or if the [Ca2+] transient is prolonged by combination with earlier transients, newly docked SVs may find a large enough tail of [Ca2+] to undergo exocytosis, giving rise to the τslow component.The appearance of a second component during AP trains suggests two kinetic processes in series. We propose that, whereas fast release events represent the classically envisaged release of docked SVs, slow release events represent sequential docking-fusion events (2-step release) involving SVs that were bound to the replacement site prior to the response-triggering AP. In this view, the difference between τslow component. The dependence of the τfast to τslow crossover point on stimulation frequency  enhances both δ decrease and Rf increase, resulting in an acceleration of the crossover  of vesicles54. The FRP can be subdivided into a ‘superprimed’ pool and a normally primed one53, the former releasing during an AP with high probability (≈0.6) and being replaced quite slowly. If, as suggested here for PF-MLI synapses, the progression from normally primed to superprimed is seen as a maturation process, the question arises as to why release latencies change only very little during AP trains in the calyx of Held. A likely explanation may reside in the relationship between AP-induced [Ca2+] transients and replacement kinetics. Later in the train, release will be dominated in the calyx by vesicles that reach the mature state not during [Ca2+] transients but in the inter-AP interval. Their latencies will reflect the time course of [Ca2+] transients. By contrast in PF-MLI synapses, the τslow component may be seen as direct recruitment and subsequent release during [Ca2+] transients. Thus the higher prominence of τslow release in PF-MLI synapses compared to the calyx of Held could simply reflect faster replacement kinetics (Rf) in the former preparation. This difference between PF-MLI and calyx of Held synapses may reflect different adaptations to distinct functional constraints. The entire sensory pathway driven by cerebellar mossy fibres, including mossy fibre-granule cell and PF-MLI synapses, handles relatively short AP bursts at high frequency55. As argued in previous studies56, maintenance of synaptic strength during these bursts relies on very fast SV replenishment. The present study suggests that this maintenance of transmission occurs at the expense of some loss of time precision. By contrast, the hearing sensory pathway including the calyx of Held synapse relies on precisely timed signals during prolonged, high frequency trains, and in this case, time precision is more important than global synaptic strength.The critical need of an overlap between docking and [Car following local [Ca2+] accumulation, from an increase in the τslow component and from an increase of δ following fast SV recruitment22. This facilitation decays as a function of interpulse interval within 80 ms . Docking reversibility has been proposed on the basis of FM 1-43 imaging experiments57 and is consistent with recent evidence indicating reversibility of some of the steps leading to exocytosis (‘de-priming’: ref. 58). While we assume an Rb step leading back from DS to replacement site, we cannot exclude that docked SVs leave the DS directly into the cytosol instead of returning to the replacement site. We prefer the option of returning to the replacement site because the returned SV can then immediately be reused for docking, whereas a SV that would drift in the cytosol would require a longer recycling route.According to our simulations, the facilitation observed in paired stimulations results from a combination of an increase in P ms Fig. , raising2+] accounts for the observation of a simultaneous return of PPR and covariance values to baseline as a function of interpulse interval  and supply from the reserve pool (asynchronous release). The transition from fast to slow release, as well as that from slow release to asynchronous release, appear as successive adaptations of the synapse to maintain its output during prolonged stimulations, at the expense of a loss of time accuracy following DS depletion and AZ cluttering. At each transition, some degree of time accuracy is abandoned for the sake of maintaining synaptic responsiveness.Even though fast, slow and asynchronous release are readily distinguishable, our results and simulations indicate that these three modes of release are closely related. In particular, our latrunculin results Fig.  suggest 59 has been challenged by subsequent reports that it rather inhibits synaptic facilitation60 or asynchronous release61, leading to the speculation that synaptotagmin 7 may target different processes in different synapses. Very recent studies suggest, however, that synaptotagmin 7 exerts a mixed action on replenishment, facilitation and asynchronous release in the same synapses62, and such a mixed action occurs at PF-MLI synapses63. These intriguing results may be explained within the framework of the 2-step release model if the primary synaptotagmin 7 target is the Ca2+-sensitive transition between replenishment site and DS59 (Rf). In such a case, synaptotagmin 7 ablation would affect synaptic facilitation and asynchronous release in addition to SV replenishment, since the Rf step participates in all three processes.The present proposal that a single release apparatus is responsible for several aspects of synaptic function including initial fast release, facilitation-associated slowing of release and asynchronous release may help to interpret the functional role of certain synaptic proteins. In particular, the initial suggestion that genetic ablation of synaptotagmin 7 in central synapses selectively inhibits SV replenishment3, 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 32‒34 °C.Sagittal slices (200 μm thick) were prepared from the cerebellar vermis of Sprague-Dawley rats (P12‒P16 and P26‒P29) following the animal care guidelines of Paris Descartes University . Recordings were from MLIs, comprising about 2/3 of basket cells and 1/3 of stellate cells. The composition of the extracellular solution was (in mM): 130 NaCl, 2.5 KCl, 26 NaHCOA receptors were blocked by inclusion of D(‒)-2-amino-5-phosphonopentanoic acid (50 μM) and gabazine (15 μM). Procedures to find an appropriate location for electrical stimulation were as described38. Briefly, we puff-applied the internal solution including 150 mM K+ from a pipette using small pressure steps while moving the pipette in the granule cell layer. When we found a burst-like EPSC response in the postsynaptic cell, we reduced the pressure of puffing to better define the spot for stimulation. Then we switched to electrical stimulation, using the same pipette, and we adjusted stimulation intensity to fire a connected granule cell under minimal stimulation conditions. Definitive acceptance of the experiment as a usable simple synapse recording occurred after analysis and depended on three criteria31: (i) a decrement of EPSC amplitudes of second events in a pair, reflecting activation of a common set of receptors belonging to one postsynaptic density; (ii) a Gaussian distribution of EPSC amplitudes with a coefficient of variation (CV) <0.5; and (iii) stability of the overall responsiveness over time. Specifically, in our experiments amplitude occlusion of first EPSC pairs (ω) ranged from 0.264 to 1 (median = 0.648). The time constant of recovery from the occlusion ranged from 0.266 to 7.22 ms (median = 1.50 ms). CV for the distribution of EPSC amplitudes ranged from 0.22 to 0.39 (mean ± sd = 0.307 ± 0.050). A slope of plots of the number of events in a train as a function of sweep number ranged from −0.137 to 0.099 events per train (mean ± sd = −0.026 ± 0.064). Single stimulations and trains of two or eight stimulation pulses were applied repetitively with intervals of 10 s between sweeps. Statistical data were derived from sequences of 10‒30 trains.Recordings of simple PF-MLI synapses were obtained under voltage clamp at −60 mV. NMDA receptors and GABA31, and we built latency distributions by averaging the occurrence times across experiments. We briefly describe the analysis here. First, we made an average of single EPSCs obtained during asynchronous release to obtain a template in a given synapse. Then the average miniature EPSC (mEPSC) was fit by triple-exponential function with five free parameters . Next, mEPSC and individual data traces were deconvolved using the five parameters. The deconvolved mEPSC resulted in a narrow spike, and the deconvolved data traces resulted in sequences of spikes. Finally, we fit a given deconvolved trace by a sum of scaled narrow spikes in order to obtain the timing of each event. The amplitude parameter was free because the peak EPSC amplitude varied during a train due to receptor saturation and desensitization. The above procedure had a detection limit that caused a failure of separation of two events occurring within 0.2 ms. To correct for missed events, we split into two the events having amplitude 1.7 times larger than the average amplitude obtained during asynchronous release.We determined occurrence times of individual EPSCs based on deconvolution analysis, as detailed in Malagon et al.In LatB experiments, data were collected at least 5 min after switching the bath solution to the test solution. 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 Brenowitz and Regehr42. Experiments were conducted in the same conditions as in the electrophysiology experiments . 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 . 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 signalling was analysed in the pixels encompassing the varicosity in terms of fluorescence changes relative to prestimulus values  with software written in the IGOR-Pro programming environment .For Cao values into intracellular [Ca2+] is explained in detail in Miki et al.22. This procedure gave a slope of 0.32 μM per AP in 3 mM extracellular [Ca2+]  or 5–10 ms after each AP (at 100 Hz). These remaining data were filtered, and gaps were filled in using connecting straight lines  method with a fixed time step (0.03 μs) and an elementary integration volume (i.e. voxels). Simulation voxel was a 10 × 10 × 10 nm3 cube. The total simulation volume was a 0.9 (x) × 0.5 (y) × 0.5 (z) μm3 cuboid, matching the size of the bouton volume64. Simulation voxel was a 10 × 10 × 10 nm3 cube. The four surfaces were set to reflect all diffusants. A single Ca2+ entry site was placed in the centre of a single surface (z = 0) bounding this volume. The Ca2+ entry site was composed of three channel clusters, each having nine channels. The nearest neighbour distance between channel clusters (centre to centre) was 126 nm, and the nearest neighbour distance between channels within a cluster was 20 nm , the amplitude of Ca2+ influx through each channel during an AP was set at 0.2 pA, considering a single channel current of Cav2.2 at −65 mV of 0.33 pA (ref. 65) and a fraction of channel open probability during an AP of ~0.7 (ref. 66). We assumed a Gaussian-shaped Ca2+ influx with a half-amplitude duration of 0.34 ms (ref. 67). Other model parameters were obtained from previous recordings from PF boutons if possible or else from other presynaptic terminals as follows: Ca2+ diffusion coefficient DCa = 220 μm2 s−1 (ref. 68), resting free [Ca2+] = 50 nM, endogenous buffer concentration = 2 mM, endogenous buffer KD = 50 μM, and endogenous buffer forward binding rate constant Kon = 2 × 108 M−1 s−1 (ref. 67). We also included 200 μM free ATP in the simulation19. The values for Ca2+ binding to ATP were: KD, Ca = 200 μM, Kon, Ca = 5 × 108 M−1 s−1 (ref. 69), DATP = 220 μm2 s−1. Ca2+ extrusion via active transport was included on all surfaces at a rate of 0.9 Ca2+ ms−1 (ref. 70). To match the decay of measured (Ca2+ imaging) and simulated global [Ca2+], we adjusted the concentration of calretinin, which is a major endogenous buffer in PF boutons39. Although 30 μM best reproduced Ca2+ recovery data, we adopted a calretinin concentration of 100 μM, as release rates were almost identical within 100 ms after AP train onset for 30 and 100 μM calretinin. In these simulations, the additional buffering capacity associated with the presynaptic pipette solution (OGB-6F) were taken into account. Kinetics of Ca2+ binding to calretinin is from Fass et al.71. Simulation parameters for standard conditions (unmodified presynaptic solution) are summarized in Supplementary Table  nm Fig. . This ‘sSimulation of 2-step model: We calculated the release rate by numerical integration of differential equations for single and paired-pulse experiments using Igor Pro with a time interval of 0.01 ms , which is identical to l+ × f5. f is a factor determining the increase in vesicle fusion rate upon Ca2+ binding. The fusion rate at V0Ca−4Ca is given l+ × f0‒4. b is a cooperativity factor41. This procedure was based on the local [Ca2+] obtained from Ca2+ simulation at 40-nm distance from the nearest Ca2+ channels.ms Figs.  and 12. 1.3 Fig. . γ is thr. Nevertheless, as the average release probability values for the first AP were slightly different for the experiments of Fig. f to the simple model following a Michaelis–Menten reaction having two free parameters, Vmax and Kd, as shown in Fig. max value of 800 s−1 and a Kd of 2 μM for the forward reaction rate (Rf). The backward reaction rate (Rb) was set to keep a δ value of 0.3 at a resting [Ca2+] of 50 nM.In Fig. 22, the possibility was kept open that SVs would occupy both one DS and its associated replacement site simultaneously. As for Rf (see above), we defined the recruitment step Sf using a Michaelis–Menten reaction having two free parameters, Vmax and Kd, that were determined in order to fit experimental data while keeping the parameter values for Pr, Rf and Rb . The backward reaction rate (Sb) was set to keep a δ value of 0.3 and a δ value of 0.9 at a resting [Ca2+] of 50 nM. To improve fitting results for late responses in trains, we introduced a slow Pr having 10 times reduced Kon and 2.5 times reduced Koff compared to normal Pr .In the simulations for eight pulse experiments Figs. ‒5, we usr, Rf, Rb, Sf and Sb were first calculated from simulated [Ca2+] and from the initial occupancy of DS and replacement site as above. The amount of release and occupancies in 4 DSs were obtained with a time interval of 0.01 ms. We performed the calculation 5000 times in a given condition and used the averaged value as result.In Monte Carlo simulation, PAll relevant codes are available from the authors.Supplementary InformationPeer Review File


Inhibition of NMDARs by ATL was found to be dependent upon external Ca2+ concentration ([Ca2+]) in a voltage-independent manner, with an IC50 of 0.72 μM in 4 mM [Ca2+]. The ATL IC50 value increased exponentially with decreasing [Ca2+], with an e-fold change observed per 0.69 mM decrease in [Ca2+]. Loading neurons with BAPTA abolished Ca2+-dependent inhibition, suggesting that Ca2+ affects NMDARs from the cytosol. Since there is one known Ca2+-dependent process in gating of NMDARs, we conclude that ATL most likely promotes Ca2+-dependent desensitization. We also found ATL to be a trapping open-channel blocker of NMDARs with an IC50 of 220 µM at 0 mV. An e-fold change in ATL IC50 was observed to occur with a voltage shift of 50 mV in 0.25 mM [Ca2+]. Thus, we disclose here a robust dependence of ATL potency on extracellular [Ca2+], and demonstrate that ATL bound in the NMDAR pore can be trapped by closure of the channel.Although the tricyclic antidepressant amitriptyline (ATL) is widely used in the clinic, the mechanism underlying its high therapeutic efficacy against neuropathic pain remains unclear. NMDA receptors (NMDARs) represent a target for ATL and are involved in sensitization of neuropathic pain. Here we describe two actions of ATL on NMDARs: 1) enhancement of Ca Use of antidepressants as an adjuvant therapy for ameliorating chronic pain is a promising treatment strategy for patients displaying both neuropathic pain and depression2. However, the mechanisms underlying analgesic effects of many psychotropic drugs are currently unknown. Antidepressants represent a wide variety of compounds with diverse molecular structures, making it difficult to attribute their analgesic properties to action on specific targets. Among antidepressants with analgesic properties, amitriptyline (ATL) has long been successfully used in treatment of pain3.More than half of patients with chronic neuropathic pain also suffer from depression+, K+ and Ca2+ channels [for review see4]. Additionally, ATL inhibits Ca2+ transport by Na+/Ca2+-exchanger (NCX) in synaptosomes and also demonstrates voltage- and Mg2+-dependent open-channel block of NMDA receptors (NMDARs)6. ATL is metabolized by the liver to nortriptyline, which further expands its wide set of molecular targets8.ATL, a tricyclic antidepressant, demonstrates a broad profile of pharmacological actions. ATL effects considered to be relevant for antidepressant therapy include: 1) inhibition of monoamine uptake such as norepinephrine, dopamine, serotonin etc., 2) considerable M-cholinolytic, antihistamine and α-adrenolytic activities, and 3) block of Na10. In particular, injury of the dorsal root ganglion and spinal superficial dorsal horn neurons increases expression of α2δ-111, a voltage-activated Ca2+ channel subunit that also interacts with NMDARs, promoting synaptic and surface expression of α2δ-1-NMDAR complexes and sensitization of neuropathic pain12. We recently described that NMDARs are upregulated by NCX in physiological conditions and that inhibition of Ca2+ export from neurons by the NCX inhibitor (2-[4-[(4-nitrophenyl)methoxy]phenyl]ethyl ester, methanesulfonate, KB-R7943) or lithium enhances Ca2+-dependent desensitization of NMDARs14. Modulation of NMDAR activity by ATL may potentially contribute to its efficacy in treating neuropathic pain. Indeed, other drugs acting directly on NMDARs  or indirectly modulating NMDAR activity  possess analgesic potential17.It is well established that excitatory glutamatergic synaptic transmission is involved in sensitization of neuropathic pain. Nerve injury potentiates NMDAR activity at both pre- and postsynaptic sites19 inhibit integral NMDA-activated currents less effectively than trapping channel blockers22. Relatively large ATL concentrations (15–60 µM) are required for the NMDAR channel block5. However, this somewhat contradicts clinical observations that neuropathic pain relief by ATL treatment can be rapidly achieved with dosages that are lower than those required for treatment of depression23.Despite our knowledge of the molecular targets of ATL, the high therapeutic efficacy of ATL against neuropathic pain remains a puzzle. While ATL blocks open channels of NMDARs, the precise mechanism of the blockade is still unclear. In general, compounds satisfying the “sequential” model of open-channel block24 forming diheteromeric and triheteromeric NMDARs26. We also utilize electrophysiological recordings and structural modeling of NMDARs to investigate the mechanism of ATL binding to the NMDAR channel. We disclose here a novel mode of pharmacological action of ATL, a robust dependence of ATL potency on extracellular Ca2+ concentration, and demonstrate that ATL causes trapping open-channel block of NMDARs, binding to a site similar to MK-801 and memantine binding sites within the NMDAR channel pore.To gain insights into the mechanism of ATL action on NMDARs, we used patch-clamp electrophysiology to study the effects of ATL on whole-cell NMDAR currents in primary cultures of rat cortical neurons, which express GluN1, GluN2A, and GluN2B subunits2+ in the external solution, caused block of NMDAR currents that depended on membrane voltage. Fractions of blocked currents  were considerably larger at −70 mV than at −30 mV . Indeed, 10 μM ATL inhibited NMDAR currents in a Ca2+-dependent manner, with inhibition by ATL being far more pronounced in 2 mM Ca2+- than in 0.25 Ca2+- containing media  ATL concentrations appeared to be facilitated in 2 mM Ca2+ Fig.  compared 2+ Fig. , suggestdia Fig. .2+, as a permeant ion of NMDAR channels, could affect open-channel block. It is well established that the parameters of channel block of NMDARs by a blocking molecule27 and/or Mg2+29 are strongly affected by permeant ions. In addition, several binding sites for Ca2+ were found in the ion pathway of NMDARs31 that also likely interact with blocking molecules within the ion pore of NMDARs. A lack of information on this subject forced us to perform experiments with N--1,5-pentanediamine dihydrobromide (IEM-175432), a “pure” open-channel blocker that can bind to a shallow binding site in the NMDAR channel, preventing closure of the channel, as well as a deeper binding site, where it can be trapped in the channel27. Effects of 10 μM IEM-1754 on NMDA-activated currents were examined in the presence of 2 mM Ca2+ and 0.25 mM Ca2+ in the external solution. Figure 2+ and 0.25 mM Ca2+, suggesting that parameters of general open-channel block are not affected by extracellular Ca2+ within this [Ca2+] range. This later observation raises a question about the origin of the discrepancy we observed between the effects of low  and high (100 μM) ATL concentrations on NMDARs - how could Ca2+ be contributing to inhibition by ATL?In general, it cannot be excluded that Ca2+] on inhibition by ATL, we studied concentration-inhibition relationships to estimate the concentration that causes half-maximal inhibition (IC50) in the presence of different [Ca2+]s in the bathing solution. In these experiments, increasing concentrations of ATL were sequentially applied during NMDA-activated currents at steady state , where e-fold change value.To quantify the effect of external . We observed a peculiarity in the discrepancy between the effects of low (10 μM) and high (100 μM) ATL concentrations. Notably, 10 μM ATL in the presence of 1 mM Ca2+ in the bathing solution inhibited NMDAR currents in a voltage-independent manner, and changes of extracellular [Ca2+] in any direction considerably altered magnitude of inhibition by ATL. In contrast, blockade of NMDAR currents by 100 μM ATL was voltage-dependent, which pointed toward ATL acting as an open-channel blocker, in agreement with a previous study5. To address whether these contradictory observations could be explained in terms of the open-channel block, we tested how external [Ca2+] could influence NMDAR inhibition by IEM-1754, a “pure” open-channel blocker without any other known mechanism of action19 on NMDARs. Blockade of NMDAR currents by IEM-1754 was resistant to a 4-fold increase of [Ca2+] in the external solution, suggesting a lack of Ca2+ effects on blockade. Similarly, the NMDAR channel blocker ketamine21 shows no sensitivity to changes in external [Ca2+] while memantine22 slows the rate of recovery from desensitization of GluN1/GluN2A NMDARs (but not GluN1/GluN2B receptors) in a Ca2+-dependent manner, suggesting that memantine stabilizes a Ca2+-dependent desensitized state of GluN1/GluN2A receptors49. In general, NMDAR open-channel block by a variety of structurally different compounds is not affected by external [Ca2+] to the extent displayed by ATL. Considering the strong dependence of the IC50 value of ATL on external [Ca2+], with a 1 mM increase of external [Ca2+] inducing a roughly 5-fold decrease of the IC50 for ATL inhibition of NMDAR currents, we conclude that Ca2+-dependent inhibition of NMDARs by ATL represents a mode of action that differs from canonical open-channel block. Since Ca2+-dependent desensitization is currently the only known Ca2+-dependent characteristic of NMDAR kinetics, we suggest that ATL promotes the Ca2+-dependent desensitization of NMDARs and this particular mechanism of action dominates at low ATL concentrations.To clarify the mechanism of ATL effects on NMDARs, here we studied the dependence of ATL inhibition of NMDARs on membrane voltage and extracellular  (2–4 mM), ATL seems to enhance Ca2+-dependent desensitization of NMDARs, displaying much more potent IC50 values ranging from 0.7–7 µM. This concentration range is within the therapeutic window for treatment of both depression as well as neuropathic pain. Considering that open-channel block and Ca2+-dependent inhibition of NMDARs by ATL represent two independent processes, the experimentally measured NMDAR block is determined by additive contributions of both mechanisms. Within the physiological membrane voltages and extracellular [Ca2+]s, the experimentally measured value . The a value, which is zδF/RT, and the b value estimated from experiments were 0.021 and 0.63, respectively. When these parameters were optimized by fitting all bunch of the data  on glass coverslips coated with poly-D-lysine and were used for experiments after 10–15 days in culture67.All manipulations on animals were performed in accordance with the guide of the Federation for Laboratory Animal Science Associations and were approved by the Animal Care and Use Committees of Sechenov Institute. Briefly, 16 days pregnant Wistar rats  were placed in a plastic box connected to a CO13. Unless otherwise specified, the external bathing solution contained (in mM): 140 NaCl; 2.8 KCl; 1.0 CaCl2; 10 HEPES, at pH 7.2–7.4, osmolarity 310 mOsm. The intrapipette solution contained (in mM): 120 CsF, 10 CsCl, 10 EGTA, and 10 HEPES, osmolarity 300 mOsm14, with pH adjusted to 7.4 with CsOH. Patch pipettes of 2–4 MΩ were pulled from Sutter BF150-89-10 borosilicate capillaries. Experiments were performed at 23–25 °C. Under control conditions, neurons were voltage clamped at −70 mV. Data are reported without corrections for liquid junction potential, which was measured as −11 mV. To activate NMDARs, 100 µM NMDA and 30 µM glycine were co-applied. Some experiments were performed on neurons loaded with BAPTA. To load BAPTA, cortical neuron cultures were incubated in bathing solution containing 10 µM BAPTA-AM for 2 hours.Whole-cell currents were recorded from cultured rat cortical neurons using a MultiClamp 700B patch-clamp amplifier with Digidata 1440 A controlled by pClamp v10.2 software (Molecular Devices). Recordings were 8-order low-pass filtered at 200 Hz and the acquisition rate was 20000 samples per second. Solution exchange was performed by means of a fast solution application system as described earlierN--1,5-pentanediamine dihydrobromide] was synthesized by Dr. V. E. Gmiro at the Institute of Experimental Medicine, Russian Academy of Medical Sciences, St. Petersburg.Compounds were acquired from Sigma-Aldrich, St. Louis, MO, USA. The adamantane derivative IEM-1754 ). Amplitudes of currents measured in the presence of blocker were normalized to maximal current response in control  or offset . From these measurements, an estimation of rate constants of block (kon) and unblock (koff) was estimated. kon was obtained from τon measured at 10 μM and 100 μM ATL (C1 and C2 respectively) using the equation:NMDAR current relaxations during block or unblock by ATL were fit with the single exponential equation:off the reciprocal to measured τoff:The kd) for ATL was calculated as follows:The equilibrium dissociation constant  and δ were set as free parameters. IC50(0 mV) is the half-maximal inhibition concentration at Vm = 0 mV, δ is the fraction of the membrane voltage field exerting force on ATL at its binding site, z = 1 is an ATL molecule electric charge. For Faraday constant, gas constant and room temperature of 25 °C the RT/F ≈ 25.7 mV.To obtain the voltage dependence of NMDAR block by ATL, its concentration-response curves were generated at three membrane holding potentials V1 and I2 are amplitudes of the exponential and τ1 and τ2 are time constants of the exponential and C equals I1 + I2 + Ib.The rise time of currents activated by the testing NMDA + Gly application in trapping channel block experiments was fit in ClampFit  using the double exponential function:t-test. Statistical significance is reported in the figures according to the following symbols *** and ****, which indicate p values below (<) 0.001 and 0.0001, respectively. Curve fitting was performed using OriginPro software (OriginLab Corp.).Data are presented as representative measurements as well as mean values ± standard error of the mean (S.E.M.). Sample number (n) refers to the number of recorded cells. Groups were compared using ANOVA with Bonferroni correction and Student’s two-tailed 68. Acid dissociation constants (pKa) of the compound and the percentage of different protonation states at neutral pH were calculated by MarvinView program 17.3.13.0 . There are several three-dimensional models of NMDAR in Protein Data Bank69. For our molecular modeling experiments, we used a model of NMDAR of African frog Xenopus laevis, with a molecule of dizocilpine (MK-801) bound inside the receptor channel. The code of the structure is 5UN170.The three-dimensional model of ATL was generated from pdb entry 3APV59.The chosen receptor model 5UN1 consists of two molecules of NMDAR in the crystalized unit containing the following polypeptide chains: chains A,C,E,G (GluN1 subunits) and chains B,D,F,H (GluN2B subunits). Subunits were expressed lacking the amino-terminal domains (∆ATD receptor) to facilitate structural analysis and crystallized with glycine, glutamate and MK-801. Its three-dimensional structure was determined by diffraction method with the resolution of 3.5 Å70 code for A0A1L8F5J9 GluN1 and code A7XY94 for GluN2B subunits. We removed all other amino acids and ligands from the structure. Missing atoms were added using the VMD software71 and the structure was optimized by energy minimization using NAMD software72.For the molecular modeling experiments, we used one of the two NMDAR molecules of 5UN1 structure: chains A, C (GluN1) and B, D (GluN2B). Only the moieties of the receptor subunits that form the channel pore were used for simulation: amino acids 600–653 of GluN1 subunits and amino acids 598–653 of GluN2B subunits. Hereinafter, the numbering corresponds to the primary sequence of the receptor according to UniProt database:73. The coordinates of the nitrogen atom of MK-801 in 5UN1 was used as the center of the Autodock search area. The search area size was set to 15 × 15 × 40 Å3, which covers the entire channel. The “exhaustiveness” parameter, which represents the amount of computational effort, was set to 24. The parameter “energy range”, characterizing the maximum scatter of energy values of conformations in the output file, was set to 3 kcal/mol. The number of the most optimal conformations in the output file (num_modes) was set to 10. The conformation with the best value of the binding energy was chosen for further optimization by molecular dynamics (MD) simulations.Molecular docking of ATL into the NMDAR channel was performed using the Autodock Vina 1.1.2 software package72 using CHARMM27 force filed73. Partial charges of atoms in the ATL molecule used in the CHARMM force field are presented in Fig. 74. 0.15 mol/L of NaCl was added to make the system neutral. The resulting structure had a total of 103 POPC lipid molecules, 20 sodium ions, 21 chloride ions and 7153 water molecules.The structure obtained by molecular docking was optimized with a 2 ns MD simulation with the help of NAMD software75 with nonbonded cutoff of 12 Å was applied to compute long-range electrostatic interactions. Periodic boundary conditions were used.The MD simulation was preceded by a three-stage relaxation of the system. At the first stage, all atoms were fixed except the tails of the POPC molecules, which were relaxed by 1000 steps of conjugate gradient energy minimization followed by 500 ps of equilibration. At the second stage, NMDAR and ATL molecules were constrained, the energy of the system was minimized, and the system was equilibrated for 500 ps. At the final stage of the relaxation procedure, all molecules were released, and the system was equilibrated for 500 ps. Following the preparation stages, a 2 ns MD simulation was run with a time step of 2 fs. Temperature (310 K) and pressure (1 bar) were kept constant using a Langevin thermostat and piston methods. The particle-mesh Ewald method (PME)supplementary information


Treatment of the cells with 2-aminoethoxydiphenyl borate  abolished the elevation of both intracellular Ca2+ and ENaC activity. Treatment of the cells with apocynin (an NADPH oxidase inhibitor), dithiothreitol/NaHS (reducing agents), or LY294002 (a phosphoinositide 3-kinase (PI3K) inhibitor) prevented palmitate-induced ENaC activity, whereas thimerosal (an oxidizing agent) mimicked the effects of palmitate on ENaC activity. However, these treatments did not alter the levels of intracellular Ca2+, indicating that elevation of reactive oxygen species (ROS) and activation of PI3K are downstream of the signaling cascade. Since we have shown that ROS stimulate ENaC by activating PI3K, these data together suggest that palmitate first elevates intracellular Ca2+, then activates an NADPH oxidase to elevate intracellular ROS and PI3K activity, and finally increases ENaC activity via the activated PI3K.Previous studies indicate that the epithelial sodium channel (ENaC) in the kidney is upregulated in diabetes mellitus. Here, we show that ENaC single-channel activity in distal nephron cells was significantly increased by palmitate, a free fatty acid which is elevated in diabetes mellitus. We also show that palmitate increased intracellular Ca Na+ transport across the nephrons is critical for Na+ homeostasis and thus plays a vital role in maintaining salt balance and systemic blood pressure. Other more frequently observed pathological factors that alter ENaC activity may have much greater clinical significance treating hypertension. Hypertension is a common complication found in diabetes mellitus. Previous studies have shown that ENaC is activated in diabetic patients with nephropathy due to the elevation of ENaC-activating enzymes in the urine [The epithelial sodium channel (ENaC), which is expressed primarily in the apical membrane of the epithelial cells lining the distal segment of nephrons, lung airways, alveoli, the descending colon, and endothelial cells , plays ahe urine . It has he urine . These she urine . FFAs arhe urine . However2+ efflux from the endoplasmic reticulum (ER) [2+, somehow, stimulates mitochondria to produce reactive oxygen species (ROS) [3), which is a potent activator of ENaC [2+, ROS, and PI3K activity and that the stimulation can be corrected by hydrogen sulfide.FFAs generally refer to nonesterified fatty acids that are present in the blood. Palmitate, which is the main component of FFAs, can induce Calum (ER) , 7. The es (ROS) , 9. Sevees (ROS) , 11. Bot of ENaC . These s of ENaC . Therefo of ENaC , which mXenopus laevis and constitute an appropriate cell model for studying ENaC. A6 cells were purchased from the American Type Culture Collection  and grown in medium consisting of 3 parts DMEM/F-12 (1 : 1) medium  and 1 part H2O, with 15 mM NaHCO3 , 2 mM L-glutamine, 10% fetal bovine serum , 25 units/ml penicillin, and 25 units/ml streptomycin, as previously described. A6 cells were cultured in plastic flasks in the presence of 1 μM aldosterone at 26°C and 4% CO2. After the cells reached 70% confluence, they were subcultured on the polyester membranes of Transwell inserts  for confocal microscopy analysis or Snapwell inserts  for cell-attached patch-clamp experiments. To allow them to be fully polarized, cells were cultured for at least 2 to 3 weeks before performing the experiments [A6 cells are an established renal cell line derived from distal nephron segments of 2, 1 MgCl2, and 10 HEPES, adjusted to pH 7.4 with NaOH. This NaCl solution was used as the bath solution for recordings and to fill the electrodes. Reagents were added to the bath solution. The borosilicate glass electrodes had a tip resistance of 7–10 MΩ when filled with the NaCl solution. Experiments were conducted at room temperature (22–25°C). Data were acquired by applying a 0 mV pipette potential, sampled at 5 kHz and low-pass filtered at 1 kHz using Clampex 10.2 software . Prior to analysis, the single-channel traces were further filtered at 30 Hz. The total number of functional channels in the patch was determined by observing the number of peaks detected on the current amplitude histograms during a recording period of at least 10 min. The open probability (PO) values of ENaCs before and after chemical application were calculated using Clampfit 10.2 . Control ENaC activity was recorded for 2 min after the cell-attached mode was established and ENaC activity stabilized. A single patch was typically recorded for at least 30 min before any experimental manipulation.ENaC single-channel currents were recorded using the cell-attached patch-clamp configuration with an Axopatch-200B amplifier  as described previously . A6 cellμM 5-(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA), a membrane-permeable ROS-sensitive fluorescent probe  that becomes fluorescent when oxidized. Prior to the application of palmitate, A6 cells were treated with an iron chelator, 50 μM 2,2′-dipyridyl, for 3 min [2+ levels, A cells were incubated with 5 μM Fluo-3, AM, a fluorescent Ca2+ indicator, for 60 min [Confocal microscopy  studies were performed as previously described , 17. A6 r 60 min . ConfocaUnless otherwise noted, all chemicals and reagents were purchased from Sigma-Aldrich . All solutions were premade and stored in a −20°C freezer or made fresh before use. Palmitic acid was purchased from Sigma, and BSA (FFA-free) was purchased from Roche. Palmitic acid was dissolved in 0.1 M NaOH at 70°C and then complexed with 10% BSA at 55°C for 10 min to achieve a final palmitate concentration of 0.3 mM. Stock solutions of 3 mM palmitate with 10% BSA and of a 10% BSA control were prepared 1 day before the experiments.t-tests were used to compare pre- and posttreatment activities. Analysis of variance (ANOVA) was used to perform multiple comparisons among various treatment groups. Differences were considered statistically significant at P < 0.05.Data are presented as the mean ± S.E. Statistical analysis was performed using SigmaPlot and SigmaStat Software . Student's in vivo mode of plasma palmitate delivery. Single-channel ENaC currents were recorded for at least 30 min in each experiment. Addition of palmitate (0.3 mM) to the basolateral bath significantly increased ENaC PO from 0.29 ± 0.01 (control before the addition) to 0.59 ± 0.04   significantly increased intracellular Ca2+ within approximately 20 s, followed by a gradual decline, but the increase remained significantly higher than the levels before the treatment for at least 3 min. In contrast, the exposure of cells to 2% BSA did not alter intracellular calcium levels (μM) , having an EC50 of 330.5 ± 0.62 μM for palmitate to activate ENaC (n = 6 for each data point). Therefore, 300 μM of palmitate (a dose close to EC50) was used for the rest of the experiments.To investigate whether palmitate alters ENaC activity, we performed cell-attached patch-clamp experiments. Because plasma palmitate levels are elevated in diabetic patients , palmitates ENaC . Therefom levels . ENaC acels (μM) . The cal2+ on palmitate-induced ENaC activity, we treated A6 cells with BAPTA-AM (a membrane-permeable Ca2+ chelator) for 5 min before the addition of palmitate. Basolateral addition of palmitate no longer increased ENaC activity in the presence of BAPTA-AM, albeit BAPTA-AM slightly but significantly decrease ENaC activity. ENaC PO was 0.32 ± 0.02 (control), 0.25 ± 0.03 (BAPTA-AM), and 0.23 ± 0.02  , we treated A6 cells with 2-APB, an inhibitor of IP3 receptors, for 5 min before the addition of palmitate. The data show that palmitate no longer alters ENaC activity in the presence of 2-APB. ENaC PO was 0.27 ± 0.02 (control), 0.30 ± 0.02 (2-APB), and 0.27 ± 0.02  . In the presence of palmitate, application of 0.1 mM NaHS to the basolateral bath reversed the effect. ENaC PO was reduced, from 0.78 ± 0.02 to 0.33 ± 0.03 . Conversely, in the presence of 0.1 mM NaHS, palmitate failed to increase ENaC activity. ENaC PO remained unchanged, 0.25 ± 0.03 (NaHS) vs. 0.23 ± 0.03  . However, NaHS only slightly decreased ENaC Po, 0.31 ± 0.02 (control) vs. 0.25 ± 0.03 (NaHS) . These results suggest that H2S exerts a strong protective effect against the palmitate-induced ENaC activity in A6 cells by reducing oxidative stress induced by palmitate. To determine if elevation of intracellular Ca2+ occurs before the oxidative stress, we pretreated the cells with BAPTA-AM and then applied palmitate to the cells. The data show that BAPTA-AM abolished palmitate-induced elevation of intracellular ROS , 0.34 ± 0.05 (apocynin), and 0.33 ± 0.03  , 0.36 ± 0.02 , and 0.34 ± 0.02  , ENaC activity was increased, but addition of palmitate did not cause any additive effects, and 0.1 mM NaHS did not reverse the effects of thimerosal. ENaC PO was 0.58 ± 0.03 , 0.60 ± 0.05 , and 0.65 ± 0.04  , 0.51 ± 0.02 , and 0.30 ± 0.02  , palmitate no longer affects ENaC activity. Addition of NaHS (0.1 mM) also did not affect ENaC activity. ENaC 3 via activation of PI3K [μM LY294002, a PI3K inhibitor, before addition of 0.3 mM palmitate to the basolateral bath. As shown in Figures PO was slightly decreased from 0.35 ± 0.02 to 0.32 ± 0.03 by LY294002, whereas palmitate failed to increase ENaC activity in cells pretreated with LY294002. ENaC PO remained unchanged, 0.32 ± 0.03 (LY294002) vs. 0.29 ± 0.03 . However, LY294002 did not affect palmitate-induced elevation of intracellular Ca2+ P of PI3K , 17. To 2+ and ROS, (2) NaHS reverses the effects of palmitate on ENaC activity by reducing the palmitate-induced accumulation of intracellular ROS, (3) the inhibitory effect of NaHS on palmitate-induced ENaC activity is exerted through its reducing action, and (4) palmitate stimulates ENaC by increasing PI3K activity.Our major findings in this study are as follows: (1) palmitate stimulates ENaC by elevating intracellular Caβ-cell apoptosis [2+ can form pores in the membrane for Ca2+ influx [2+-permeable pores. However, we do not favor this possibility because our data show that the increases in intracellular Ca2+ can be abolished by 2-APB, an inhibitor of the IP3 receptor which is located in the ER membrane. Therefore, we argue that a high concentration may be required for palmitate to pass through the basolateral membrane of A6 cells to finally target the ER membrane to cause Ca2+ release from ER. Previous data suggest that elevation of intracellular Ca2+ inhibits ENaC by activating protein kinase C [2+ near the apical membrane inhibits ENaC through purinergic signaling via the P2Y2 receptor on the apical membrane [2+ near the basolateral membrane stimulates ENaC via mitochondria sequester intracellular Ca2+, creating intracellular Ca2+ signaling microdomains [Previous studies have shown that palmitate is able to induce poptosis . However+ influx . It is pkinase C . Howevermembrane . In contodomains .2+ causes an NADPH oxidase-dependent elevation of ROS. This is not surprising because previous studies have already shown that there is an interaction between ER Ca2+ and mitochondrial ROS in pulmonary arterial smooth muscle cells [2+ also stimulates NADPH oxidase 4 (NOX-4) in the mitochondria [3, a product of PI3K, is a strong activator of ENaC [2+-initiated elevation of intracellular ROS and the downstream activation of PI3K because LY294002 did not affect palmitate-induced elevation of intracellular Ca2+. Although there is no direct evidence to show Ca2+ stimulate PI3K, our data have shown that palmitate mediates elevation of intracellular Ca2+ and subsequently causes ROS elevation. Other's data shows that ROS increased the levels of PI3K activity [β and γ ENaC subunits are modified by Cys palmitoylation through fatty acid-exchange chemistry experiment and β-subunit palmitoylation is associated with an increase in channel activity [Here, we show that the released Cale cells . Since Cchondria , we havechondria . We arguchondria . Therefo of ENaC –15. We factivity . Previouactivity . Our dat2+, ROS via an NADPH oxidase, and PIP3 via PI3K to elevate blood pressure (Overall, we proposed the underlying mechanism by which PA upregulates ENaC probably via a sequential pathway associated with elevation of intracellular Capressure . The imp2+-dependent activation of NADPH oxidase, production of ROS, and activation of PI3K. The palmitate-induced stimulation of ENaC can be reversed by NaHS.Palmitate stimulates ENaC activity in A6 cells via Ca


This decrease in density occurred predominantly in the t-tubules, with no change in total ICa, and was therefore a consequence of the increase in membrane area. Cav-3 KO had no effect on L-type Ca2+ channel expression, and C3SD peptide, which mimics the scaffolding domain of Cav-3, had no effect on ICa in KO myocytes. However, inhibition of PKA using H-89 decreased ICa at the surface and t-tubule membranes in both KO and wild-type myocytes. Cav-3 KO had no significant effect on Na+/Ca2+ exchanger current or Ca2+ release. These data suggest that Cav-3 KO causes cellular hypertrophy, thereby decreasing t-tubular ICa density.Caveolin-3 (Cav-3) is a protein that has been implicated in t-tubule formation and function in cardiac ventricular myocytes. In cardiac hypertrophy and failure, Cav-3 expression decreases, t-tubule structure is disrupted, and excitation-contraction coupling is impaired. However, the extent to which the decrease in Cav-3 expression underlies these changes is unclear. We therefore investigated the structure and function of myocytes isolated from the hearts of Cav-3 knockout (KO) mice. These mice showed cardiac dilatation and decreased ejection fraction in vivo compared with wild-type control mice. Isolated KO myocytes showed cellular hypertrophy, altered t-tubule structure, and decreased L-type CaNEW & NOTEWORTHY Caveolin-3 (Cav-3) is a protein that inhibits hypertrophic pathways, has been implicated in the formation and function of cardiac t-tubules, and shows decreased expression in heart failure. This study demonstrates that Cav-3 knockout mice show cardiac dysfunction in vivo, while isolated ventricular myocytes show cellular hypertrophy, changes in t-tubule structure, and decreased t-tubular L-type Ca2+ current density, suggesting that decreased Cav-3 expression contributes to these changes in cardiac hypertrophy and failure. ICa triggers Ca2+ release from adjacent sarcoplasmic reticulum (SR) via Ca2+ release channels [ryanodine receptors (RyRs)]. This Ca2+-induced Ca2+ release (CICR)  (Excitation-contraction coupling (ECC) in cardiac myocytes is initiated by the action potential, which activates sarcolemmal L-type Cae (CICR)  produces+ sparks ] that su-tubules , 34, 40. release , and thuer (NCX) .ICa to the t-tubules (ICa density at the t-tubules (2+ release (Caveolin-3 (Cav-3) is a cholesterol-binding protein that is critical to the formation of caveolae and has been implicated in t-tubule formation  and loca-tubules . C3SD pe-tubules , 22, dec-tubules , which i release , 11.ICa . Indeed, global genetic knockout (KO) of Cav-3 results in a progressive cardiomyopathy characterized by ventricular hypertrophy and dilation as well as reduced fractional shortening . AdditioAdult (12 wk) male wild-type (WT) C57Bl/6 and homozygous Cav-3 KO mice, produced as previously described , were usIn vivo cardiac structure and function were monitored using echocardiography. Animals were anesthetized (1–3% isoflurane), heart rate was monitored, and measurements of contractile performance were made from M-mode images acquired from the parasternal short axis view using a Vevo 3100  and MX550D transducer.Ventricular myocytes were isolated using standard enzymatic digestion via Langendorff perfusion as previously described  and usedProtein samples  were run on 4–15% gradient SDS-PAGE gels and transferred onto Immobilon-P membranes. Blots were probed with antibodies against Cav-3 , JPH-2 , BIN-1 , LTCCs , and GAPDH . Protein bands were visualized and images were captured using horseradish peroxidase-conjugated secondary antibodies , chemiluminescence, and a G:BOX Chemi XT4 imaging system . The density of the bands was measured using ImageJ  and normalized to GAPDH.Cell width and length were measured from bright-field images of isolated myocytes obtained using a 0.5 numerical aperture, ×16 oil-immersion objective; the large field of view enabled many cells to be captured in a single image. Cell volume was calculated from these measurements as previously described .1/P0). t-Tubule density and orientation were obtained by first processing the volumetric data with a tubule-enhancing three-dimensional (3-D) filter, segmenting using an Otsu threshold in MATLAB R2015a , and converting to a skeleton using ImageJ. The skeleton was used to calculate t-tubule density [length per unit volume (in μm/μm3)] and local Eigenvectors for t-tubule angles. Tubule orientation is expressed relative to the z-disk plane, so that 0° corresponds to a transverse tubule and 90° corresponds to a tubule that is orthogonal to the z-disk, extending along the sarcomere . Branch points were calculated using Skeletonize (2-D/3-D) in ImageJ.Surface and t-tubular cell membranes were labeled by incubating cells with 5 μmol/l di-8-ANEPPS (ThermoFisher) for 10 min. Image volumes were obtained using a LSM 880 confocal microscope  in Airyscan “superresolution” mode with a 1.2 numerical aperture, ×40 water-immersion objective, sampled at 40 nm in plane and 180 nm along the optical axis. The regularity of t-tubule staining was quantified by applying a two-dimensional (2-D) fast Fourier transform to an offset-subtracted square region of the interior of the cell, and the power of the first harmonic was normalized to that of the average image intensity (P2). DT efficiency (in %) was then defined as 1 – (DT cell t-tubule density/intact cell t-tubule density) × 100. DT efficiency was similar to that observed in previous studies and was not significantly different between cell types .DT efficiency was quantified by comparing t-tubule density in intact and DT cells labeled with di-8-ANEPPS, as previously described (+ current) followed by steps to potentials between −50 and +80 mV for 500 ms before repolarization to the holding potential, at a frequency of 0.2 Hz. Absolute ICa amplitude (in pA) in intact myocytes was measured as the difference between peak inward current and current at the end of the depolarizing pulse; absolute ICa in the t-tubular and surface membranes was calculated from measurements of ICa and membrane capacitance in intact and DT myocytes with correction for incomplete DT (DT efficiency) as previously described  to calculate ICa density (in pA/pF). ICa density in the t-tubule membrane was calculated from the loss of membrane current and capacitance after DT; ICa density in the surface membrane was calculated from currents measured in DT myocytes with correction for incomplete DT as previously described . Pipette resistance was typically 1.5–3 MΩ when filled with pipette solution (see below), and pipette capacitance and series resistance were compensated by ~70%. Currents were activated from a holding potential of −80 mV by step depolarization to −40 mV for 200 ms (to inactivate NaINCX), BAPTA was omitted from the pipette solution (see below) and replaced with fluo-4 pentapotassium salt (ThermoFisher) to allow simultaneous measurement of intracellular Ca2+ and membrane current. Recordings were made 5 min after breaking into the cell to allow time for dialysis. Pipette resistance was typically 1.2–2 MΩ when filled with pipette solution. After a series of conditioning pulses (a 500-ms ramp from −80 to −40 mV followed by a step to 0 mV for 100 ms) at 1 Hz to steady state, stimulation was stopped and intracellular Ca2+ and INCX were recorded during application of 10 mM caffeine as previously described  and the voltage-sensitive dye di-4-AN(F)EPPTEA . Data normality was assessed using the Shapiro-Wilk test, and subsequent testing was performed using a Student’s t-test or Mann-Whitney test, or one-way ANOVA or Kruskal-Wallis, as appropriate. ICa density-voltage relationship curves were analyzed using repeated-measures ANOVA with voltage and intervention (i.e. Cav-3 KO) as factors. Single myocyte properties, including those elicited by a step depolarization to a single voltage, were analyzed with two-way ANOVA; post hoc tests used Bonferroni correction. The errors in derived variables  and the subsequent statistical analysis (unpaired Student’s t-test) were calculated using propagation of errors from the source measurements. The limit of statistical confidence was taken as P < 0.05.Data are expressed as means ± SE . Cav-3 KO was associated with a 26% decrease in fractional shortening . W. W48). Wortening C and an ortening D, with sortening E and sysortening F left veN = 9) and Cav-3 KO (N = 8) mice also showed no significant differences in body weight , tibia length , heart weight-to-tibia length ratio , or lung weight-to-tibia length ratio . Thus, Cav-3 KO was associated with ventricular dilation but not cardiac hypertrophy or overt failure at 12 wk of age.Despite the mild impairment of cardiac function, echocardiography showed no significant change in the mass  or diastolic  or systolic  posterior wall thickness of the left ventricle. WT .Despite no evidence of hypertrophy at the organ level, myocytes isolated from Cav-3 KO hearts were increased in width A and thutop) and Cav-3 KO (bottom) myocytes; the latter showed more prominent gaps in the network and an increase in the number of axial tubules. Quantification of t-tubule structure using 2-D fast Fourier transform analysis showed that t-tubule regularity, assessed as P1/P0 (see methods), was decreased in Cav-3 KO myocytes (top). More detailed 3-D analysis of image stacks revealed that this decrease was due to decreased t-tubule density (bottom) and changed tubule orientation .Staining the the surface membrane using di-8-ANEPPS revealed that t-tubule organization was also altered in Cav-3 KO myocytes. myocytes C, top. M density C, bottomentation D; there ICa, we investigated whether Cav-3 KO altered expression of these proteins; however, JPH-2 and BIN-1 expression levels were not significantly different between WT and KO cardiac tissue  higher at the surface of Cav-3 KO myocytes , although previous work has shown that tonic Cav-3-dependent stimulation of ICa is inhibited by C3SD in WT myocytes  and DT  myocytes, so that ICa distribution was qualitatively similar in the absence and presence of H-89 in the two cells types ; however, in contrast to the data obtained in the absence of H-89, and as shown in ICa at the surface membrane was no longer significantly larger  and absolute t-tubular ICa was now significantly smaller in KO myocytes . These data suggest significant tonic stimulation of ICa by PKA at both the surface and t-tubule membranes in WT and KO myocytes, which is slightly greater in KO myocytes, helping to maintain absolute ICa.We also investigated whether activity . Figure m intact B and DT m intact C myocyte compare E and 4D.ICa density, we also investigated the effect of Cav-3 KO on the main Ca2+ efflux pathway, INCX. 2+ caused by application of 10 mM caffeine to intact WT and KO myocytes (top) and the accompanying inward currents , showing no difference between the two cell types. 2+ in intact and DT WT and Cav-3 KO myocytes , suggesting a redistribution of INCX from the cell surface to the t-tubules after Cav-3 KO.Since Cav-3 KO decreased t-tubular myocytes C, suggesescribed , is simiescribed , and wasescribed D. HoweveICa and SR Ca2+ content are key determinants of SR Ca2+ release, we investigated the effect of Cav-3 KO on Ca2+ release. 2+ transients recorded from WT and Cav-3 KO myocytes, showing that Cav-3 KO had little effect on Ca2+ transient amplitude or time course. Mean data show that Cav-3 KO did not significantly change Ca2+ transient amplitude, time to peak, or time to half decay (Since t-tubular lf decay B.top) and Cav-3 KO (bottom) myocytes. Measurement of the latency from the start of the action potential upstroke (yellow lines) to the initial Ca release along the t-tubule (red lines) revealed that Cav-3 KO had almost no effect  nor the variability in latency along the t-tubule -induced hypertrophy and HF are associated with decreased cardiac Cav-3 expression . AlthougThe left ventricular dilatation and reduced fractional shortening and ejection fraction observed in vivo in Cav-3 KO mice confirm previous work using a different Cav-3 KO mouse line . These c1) it occurred predominantly at the t-tubules, 2) loss of caveolae would decrease capacitance, and 3) cholesterol depletion appears to have no effect on either t-tubule or surface membrane capacitance  that the decreased expression of these proteins observed in HF  that the effects of Cav-3 KO cannot be ascribed to changes of JPH-2 and BIN-1 expression and thus that Cav-3 KO itself is responsible for the changes in t-tubule morphology. This may be the result of cellular hypertrophy without a commensurate increase in t-tubular growth or secondary to other changes, for example, mechanical changes , which appear to include increased PKA activity  was unchanged. It could be due to loss of Cav-3-dependent stimulation of ICa (above) without the compensatory increase in stimulation by PKA but may reflect relocation of LTCCs. Together, however, the data show that the decrease in t-tubule, and thus whole cell, ICa density in Cav-3 KO myocytes is due to the increase in t-tubule area, partly offset by increased stimulation by PKA.The reason for the smaller absolute t-tubular ICa, Cav-3 KO did not alter INCX density in the intact cell, nor did it significantly alter INCX at surface and t-tubule membranes. However, Cav-3 KO significantly decreased the ratio of INCX at the cell surface to INCX at the t-tubules, suggesting redistribution of INCX from the cell surface to the t-tubular membrane. This redistribution is the reverse of that observed for ICa, so that Cav-3 appears to have reciprocal effects on the distributions of ICa and INCX, although the mechanism of regulation of INCX distribution is unknown. However, the lack of effect of Cav-3 KO on INCX density in the intact cell implies some upregulation of INCX so that its density remains unchanged despite the cellular hypertrophy induced by Cav-3 KO.In contrast to ICa density was reduced in Cav-3 KO myocytes, particularly at the t-tubules, neither the amplitude nor time course of the whole cell Ca2+ transient amplitude was altered. More detailed examination of CICR by measuring the latency and heterogeneity of local Ca2+ release along a t-tubule also revealed no effect of Cav-3 KO.Although 2+ release was maintained with no significant change in SR Ca2+ content and may be explained by redundancy in t-tubular ICa in ventricular myocytes (1) dyadic ICa was maintained, since absolute ICa was unchanged in KO myocytes; 2) a larger population of LTCCs is stimulated by PKA in KO myocytes (above), helping to maintain CICR; 3) Ca2+/calmodulin-dependent protein kinase, which was inhibited by BAPTA for the measurement of ICa, helps maintain ICa in intact myocytes; and 4) RyR sensitivity increased because of increased PKA-dependent phosphorylation.Camyocytes . However2+ release compared with the increased latency and heterogeneity of Ca2+ release at the t-tubule observed in HF suggests that other factors may be affecting Ca2+ release in HF. One possibility is dyadic disruption in HF as a result of decreased expression of JPH-2 and BIN-1, which have been implicated in dyad formation  frequencies. However, the decrease occurred in the presence of increased heart size; since wall tension will increase with dilation, the law of Laplace shows that a reduced ejection fraction does not necessarily imply reduced contractility. Impaired cardiac function in KO mice is also associated with an increase in extracellular matrix and fibrosis  and RG/12/10/29802 ; National Institutes of Health HL091071 (to H. H. Patel), HL066941 (to H. H. Patel and D. M. Roth), andAG052722 (to H. H. Patel); and Veterans Affairs Merit BX001963 (to H. H. Patel) and BX000783 (to D. M. Roth).No conflicts of interest, financial or otherwise, are declared by the authors.S.M.B., C.H.T.K., J.J.W., H.C.G., D.M.R., H.H.P., M.B.C., A.F.J., and C.H.O. conceived and designed research; S.M.B., C.H.T.K., J.J.W., and H.C.G. performed experiments; S.M.B., C.H.T.K., J.J.W., and H.C.G. analyzed data; S.M.B., C.H.T.K., J.J.W., H.C.G., A.F.J., and C.H.O. interpreted results of experiments; S.M.B., C.H.T.K., and H.C.G. prepared figures; S.M.B., C.H.T.K., and C.H.O. drafted manuscript; D.M.R., H.H.P., M.B.C., and A.F.J. edited and revised manuscript; C.H.O. approved final version of manuscript.


Whilst optical glutamate sensors have enabled detection of synaptic discharges, understanding presynaptic machinery requires simultaneous readout of glutamate release and nanomolar presynaptic Ca2+ in situ. Here, we find that the fluorescence lifetime of the red-shifted Ca2+ indicator Cal-590 is Ca2+-sensitive in the nanomolar range, and employ it in combination with green glutamate sensors to relate quantal neurotransmission to presynaptic Ca2+ kinetics. Multiplexed imaging of individual and multiple synapses in identified axonal circuits reveals that glutamate release efficacy, but not its short-term plasticity, varies with time-dependent fluctuations in presynaptic resting Ca2+ or spike-evoked Ca2+ entry. Within individual presynaptic boutons, we find no nanoscopic co-localisation of evoked presynaptic Ca2+ entry with the prevalent glutamate release site, suggesting loose coupling between the two. The approach enables a better understanding of release machinery at central synapses.Information processing by brain circuits depends on Ca How neurotransmitter release relates to presynaptic calcium dynamics is not fully understood. Here the authors develop an approach based on FLIM and optical quantal analysis for simultaneous imaging of calcium dynamics and glutamate release at presynaptic boutons. However, in many central circuits glutamate release occurs with a low probability and a high degree of inter-synaptic heterogeneity involving multiple aspects of synaptic organisation2. Therefore, understanding presynaptic function requires reliable monitoring of presynaptic action potentials (APs), presynaptic Ca2+ dynamics, and release of glutamate quanta in individual synapses. The optical quantal analysis method went some way toward this goal by providing quantification of release probability at individual synapses in brain slices4. In parallel, advances in the imaging techniques suited to monitor membrane retrieval at presynaptic terminals have enabled detection of synaptic vesicle exocytosis in cultured neurons5. Recently developed optical glutamate sensors7 have drastically expanded the sensitivity and the dynamic range of glutamate discharge detection in organised brain tissue9. However, such methods on their own cannot relate neurotransmitter release to presynaptic Ca2+ dynamics, which is the key to unveiling presynaptic release machinery. Direct examination of this relationship has hitherto been feasible only in classical studies of giant synapses that permit direct electrophysiological access and probing of presynaptic Ca2+ dynamics12.Stochastic, Ca2+ imaging, we have recently employed fluorescence lifetime imaging microscopy (FLIM) of the Ca2+ indicator OGB-114. The OGB-1 FLIM readout provided nanomolar-range Ca2+ sensitivity, with little influence of light scattering, dye concentration, focus drift, or photobleaching. However, OGB-1 emission is chromatically inseparable from that of the existing glutamate sensors, thus prohibiting simultaneous imaging of glutamate release9.Furthermore, conventional imaging methods based on fluorescence intensity readout face multiple challenges when applied in turbid media such as organised brain tissue. Detected emission intensity can be affected by focal drift, photobleaching, or any experimental concomitants impacting on light scattering. To overcome such difficulties in Ca2+ indicators and discover that the lifetime of red-shifted Cal-590, an indicator successfully used in deep-brain imaging15, is also sensitive to low nanomolar Ca2+. In parallel, we develop a glutamate sensor variant SF-iGluSnFR.A184S, which has a suitable dynamic range but a slower off-rate than the existing SF-iGluSnFR.A184V, and thus permits monitoring of glutamate release simultaneously at multiple sites. In organotypic hippocampal slices, we register concurrent, chromatically separated signals reporting evoked glutamate release and presynaptic Ca2+ kinetics, at individual or multiple axonal release sites  traced from the soma. This multiplex imaging approach establishes some basic relationships between presynaptic resting Ca2+, AP-evoked Ca2+ entry, and synaptic release efficacy. At a sub-microscopic level of individual axonal boutons, we employ this method to evaluate localisation of glutamate release sites and how they are related to presynaptic Ca2+ entry, an issue fundamental to neurobiology18. The present approach helps to understand better the mechanism of synaptic release, its use-dependent changes, and the underpinning Ca2+ machinery in identified brain circuits in situ.To deal with this challenge, here we systematically explore FLIM properties of Ca2+ indicators, we tested and excluded the classical rhodamine-based dyes: they appear highly lipophilic, which would restrict imaging in axonal boutons away from the soma. In contrast, the fluorescein-based, red shifted Cal-590 was previously found to provide reliable Ca2+ monitoring for deep brain imaging in vivo15. We therefore set about testing the Ca2+ sensitivity of its fluorescence lifetime, particularly for two-photon excitation, alongside the green glutamate sensor SF- iGluSnFR7. We found a clear dependence between the Cal-590 lifetime and [Ca2+] using a series of Ca2+-clamped solutions, as described previously14  method in which photon counts are integrated, as the area under the lifetime decay curve, over the Ca2+-sensitive interval , and the result is related to the peak fluorescence value , we employed the bright fluorescence of the optical glutamate sensor SF-iGluSnFR; two sensor variants, A184V and A184S, provided similarly bright expression labelling. We achieved sparse cell expression by biolistically transfecting SF-iGluSnFR (Methods), patch-loaded SF-iGluSnFR-expressing CA3 pyramidal cells with Cal-590 (300 µM), and traced their axonal boutons for at least ~200 microns from the soma, towards area CA1 , and next following a 500-ms 100-Hz spike train evoked at the soma  for at least 10–15 ms. Fortuitously, this requirement is normally met by high-affinity Ca2+ indicators, including Cal-59015.We next asked whether the kinetic features of the SF-iGluSnFR sensors were suited for simultaneous imaging of multiple axonal boutons. This was important because the laser beam has to dwell for 1–2 ms at each bouton to achieve signal-to-noise ratio comparable to that obtained under tornado scanning of individual boutons7 had a much lower off-rate than the faster variant SF-iGluSnFR.A184V  but also an evaluation of the quantal properties (vesicular content) of release, trial-to-trial, at several synapses simultaneously. Quantal analyses were carried out by adapting a classical approach in which the signal amplitude histogram (SF-iGluSnFR.A184S ΔF/F readouts) was systematically fitted with multiple Gaussians, with the fitting parameters constrained by the noise  and by the initial condition range of peak locations21 from where it carried on unsupervised (Methods). The method revealed multiple, nearly equally spaced peaks in amplitude histograms, thus indicating the numbers and occurrences of released vesicles .First, we set out to monitor glutamate release and presynaptic Ca590 Fig. . To explile Fig. , as detaals Fig. , whereasals Fig. , both chΔF/F signal histogram revealed quantal numbers and occurrences, suggesting up to four vesicles per release  and the corresponding quantal content of release were positively correlated with the magnitude of concurrent AP-evoked [Ca2+] increments . To gauge these potential concomitants, we first imaged a sub-set (n = 7 cells) of axonal boutons at relatively short times after whole-cell break-in, when Cal-590 had not yet equilibrated throughout the axon. Thus, during 20–22 recording trials (1 min apart), the axonal Cal-590 concentration, hence local Ca2+ buffering capacity and its related effects, continued to rise, up to 2–3-fold, as clearly indicated by the rising total photon count of Cal-590 emission  evoked by either a single AP or a four-AP train depended on presynaptic resting [Ca2+] across the experiments  is considered the key to release probability control26. What this distance is, whether it varies trial to trial , or whether it depends on mode of release remain debated27.Neurotransmitter release is triggered by rapid Ca2+ indicator  plane of view and 0.8–1.5 µm in the z direction. Mechanical fluctuations add to the blur. In these settings, a spiral (tornado) laser scan provides roughly uniform excitation across the bouton, with 0.2–0.4-µm resolution, in the focal plane, while integrating the signal in the z direction: in other words, the imaged bouton reflects its projection on the x–y plane  glutamate sensor and a cytosolic (red) Cator Fig. . The inhane Fig. .Fig. 6Na29. Thus its fluorescent signal generated by SF-iGluSnFR.A184V is expected to show a nanoscopic hotspot with a spread reflecting glutamate escape. However, because this signal originates from the three-dimensional bouton it is important to understand how it projects onto the x–y plane. The bouton projection that is registered by the circular tornado scan   and the spherical surface (radius a) is unlikely to exceed ~5%  and a arcsin (yi/a), respectively, to form a corrected geodesic surface map  Fig. , left. O/b) Fig. , right cmap Fig. .ΔF/F SF-iGluSnFR.A184V signal, which should effectively nullify this bias. However, because this correction could prove useful in other cases, we have detailed its theory and validated it using a micro-vesicle with a fluorescent shell  revealed clear hotspots for the eak Fig. . The fac2+ entry and the glutamate release site . Nonetheless, Ca2+ entry hotspots (>2SD above the noisy background) were detected in at least nine synaptic boutons . These boutons provided data to assess whether the juxtaposition pattern for Ca2+ entry and glutamate release occurred purely by chance. To this end, we ran a straightforward statistical test in which the experimental scatter of distances between Ca2+ and glutamate hotspots was compared with the scatter of distances among arbitrary pairs of points randomly scattered over a similar circular area , a critical advantage of FLIM readout is that it does not depend on the dye amount or concentration, light scattering, focus fluctuations, and other concomitants of live imaging14. The latter is particularly important for imaging in awake or freely moving animals. Having a relatively more complicated procedure to record and analyse FLIM recordings compared to standard fluorometric monitoring14 appears a small price to pay for such advantages.In addition to high  levels are controlled by active [Ca2+]-sensing mechanisms rather than by passive Ca2+ buffers33. At the same time, the effect of Ca2+ indicators on rapid Ca2+ dynamics and release probability (both average Pr and instantaneous Pr/v) could be significant, as we found previously with OGB-136. Nonetheless, Cal-590 has a relatively low affinity (Kd ~ 560 nM) compared to OGB-1 (Kd ~ 170–260 nM)37, and therefore should have a lower Ca2+ buffering effect. Indeed, we found no detectable influence of Cal-590 on resting presynaptic [Ca2+], spike-evoked (bouton volume-equilibrated) Ca2+ entry, or the average Pr  are too small to influence neurotransmitter release. While this speaks in favour of the method, the physiological implications of this observation require a separate study: one plausible scenario is that, with or without added Ca2+ indicators, presynaptic Ca2+ transients ‘saturate’ the release machinery on its Ca2+-sensing side. Reassuringly, it was earlier reported that bolus-loading application of Cal-590 was fully compatible with intense network spiking activity in the visual cortex in vivo15. Furthermore, Ca2+ indicators with higher affinity, such as OGB-1 or some genetically encoded Ca2+ indicators, have been widely used to monitor neural network activity in freely moving animals, without apparent behavioural implications38. A systematic examination of axons loaded with Cal-590 at different concentrations is required to quantitatively assess potential subtle effects of the dye.Because Ca Pr Fig. . It appe2+ dynamics and glutamate release at small central synapses in situ is important for at least two reasons. First, glutamate signal readout provides monitoring of stochastic release (and thus release probability) under varied physiological conditions. At the same time, axonal Ca2+ monitoring is a reliable indicator of presynaptic spikes: the efficiency of (bolus-applied) Cal-590 in registering APs in deep brain imaging settings has recently been demonstrated15. Thus, comparing the occurrence of spikes in Ca2+ channel with the statistics of stochastic glutamate release events in the SF-iGluSnFR channel at axonal boutons should provide a quantitative gauge of synaptic transmission fidelity.Simultaneous readout of presynaptic Ca2+ entry and vesicle release probability Pr/v and how this relationship changes during use-dependent synaptic plasticity in individual synapses. To address such fundamental questions, classical studies in giant  synapse preparations combined presynaptic Ca2+ imaging with patch-clamp recordings in presynaptic and/or postsynaptic structures, which were large enough to allow access39. By being able to probe and manipulate presynaptic mechanisms in individual synapses and thus to avoid multiple concomitant factors contributing to inter-synaptic variability of release, such studies have been pivotal for understanding the basics of Ca2+-dependent release machinery. Our multiplexed imaging method enables a similar type of probing in common small synapses.Second, the method provides insights into the mechanistic, albeit statistical, relationship between Ca2+] as well as in the AP-evoked Ca2+ entry affect release efficacy. The role of presynaptic depolarisation-induced rises in basal [Ca2+] for neurotransmitter release has previously been shown in giant calyceal synapses40. Our findings suggest that the relationship between basal [Ca2+] and release efficacy is maintained in small central synapses, in quiescent tissue conditions in situ. Whether the observed fluctuations in presynaptic resting [Ca2+] occur purely stochastically or are driven by the external (network) factors remains to be ascertained. Similarly, the observation that fluctuations in AP-evoked Ca2+ entry are also correlated with glutamate release makes such fluctuations a significant contributor to release variability, in addition to other presynaptic mechanisms.The present study reveals that fluctuations in basal presynaptic  calibration protocol was similar to the standard calibration method provided by the Invitrogen Ca2+ calibration buffer kit manual. The practical procedure in large part replicated the previously described FLIM calibration protocol for OGB-114. In brief, to match the Ca2+ buffering dynamics to that of Cal-590 more closely, the standard 10 mM chelating agent EGTA was replaced with 10 mM BAPTA, and the solution constituents were replaced with the experimental intracellular solution (see above). pH was adjusted using KOH, and the KCl concentration was adjusted accordingly, to keep ion constituents in the solution unchanged. The calculated [Ca2+] was therefore slightly different from the standard Invitrogen’s calibration set and was finely adjusted using Chris Patton’s WEBMAXC program . In the control tests, excitation wavelength was varied between 800 and 950 nm and temperature was varied from 20 °C to 37 °C (using a Scientific Systems Design PTC03 in-line heater).The Cal-590 [Ca14. The fluorescent decay time course was first normalised to its peak value and then integrated (area under curve) over ~3 ns post-peak . The output of Cal-590 lifetime at different clamped [Ca2+] values produced almost noiseless decay traces  where t and T stand for the lifetime decay timing and the  experiment-wise timing, respectively. The corresponding off-line data sets were analysed using the custom-written analysis software (https://github.com/zhengkaiyu/FIMAS). For bouton-average Cal-590 FLIM measurements, the x, y, and z data sets were summed along their respective axes. In contrast, to obtain FLIM readout maps within individual boutons, the recorded photon counts in all relevant co-ordinates (tornado scanning mode) were summed across trials (T) to produce count numbers sufficiently high for the reliable NTC measure.FLIM linescan data were collected as previously described21. In such histograms, amplitude fluctuations around zero (failure) responses reflect the signal noise inherent to the particular recording settings. Establishing the noise fluctuation using a straightforward unsupervised Gaussian fitting  will thus constrain (approximately) the key noise parameter σ, which should apply to all quantal sizes . In the present study, once σ has been constrained, we set up initial parameter values (peak centres xi), their margins (±15% of the mean), σi margins (±15% of the mean), and employed the unsupervised Levenberg–Marquardt iteration algorithm for multi-Gaussian fitting . The outcome produced individual Gaussians that correspond to the peaks of quantal amplitude and the summated distribution , the maps were plotted as ΔF/F SF-iGluSnFR.A184V heat maps (collected during the period between first AP and 15 ms after the fourth AP). The maps represent averaged signals between the time points 2 ms before and 10 ms after the AP-induced fluorescence intensity peaks, thus excluding the values in troughs of the signal waveform. Because the registered glutamate signal hotspot shapes were generally asymmetrical , to localise the estimated centres we first used spatial filtering (~100 nm range) and registered the area that has top 5% brightness in the hotspot; in the case of symmetrical hotspots, this procedure generates exactly the same outcome as the standard Gaussian PSF-based localisation.Spatial localisation of the glutamate release site was carried out in the average Supplementary InformationPeer Review FileReporting Summary Checklist